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EFFECTS OF FR-91 ON HUMAN TUMOR CELL LINES

DICIEMBRE 2009 | Nº 4 | P.V.P 5,00€

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A CIENCIA no puede esconderse ni permanecer ajena a la crisis económica mundial; porque la ciencia tiene que vivir inmersa en el mundo real. La ciencia, de hecho, no ignora la crisis porque gran parte de la ciencia oficial es mercenaria de presupuestos públicos y prebendas privadas, que se desmoronan con la crisis. Los oficialistas de la ciencia contribuyen a la burbuja, llamada del ladrillo, por su falta de compromiso con las ne-cesidades de la sociedad. La ortodoxia científica no puede vivir agazapada en su atalaya subterrá-nea y sólo sacar la cabeza cuando se le tocan los presupuestos de supervivencia. Los obsesos de la trampa estadística son cómplices de la crisis; porque la crisis no es sólo económica. La crisis que nos afecta a todos es el resultado del fracaso del modelo de sociedad que hemos estado crean-do sobre la base de un crecimiento insostenible, asimétrico e injusto, en el cual se ignora al 80% y en el cual un ciudadano que trabaja mantiene a cinco que no lo hacen, entre los cuales abundan caraduras y vagonetas impertérritos. La crisis de valores arrastra a la educación, la justicia, la sani-dad, el trabajo, la familia, la política, la moral… y acaba midiéndose en parámetros económicos. Si educación, justicia, sanidad, trabajo, familia, polí-tica y moral se desvían hacia principios erráticos, es lógico que se venga abajo la economía; des-pués de todo, como diría mi amigo Manuel Gago, la moderna “neuroeconomía”, basada en los estu-dios pioneros de Camerer, Loewenstein y Prelec, recientemente revisada por Kenning y Plassmann (o George Soros), y ferozmente criticada por Ariel Rubinstein o Faruk Gul y Wolfgang Pesendorfer, no es otra cosa que neurociencia, economía y psi-cología aplicadas a entender las tomas de decisio-nes humanas. Si fallan los valores que gobiernan nuestra conducta cotidiana, tarde o temprano se nos vendrá abajo el andamio.

Las crisis no aparecen por casualidad; ocurren porque o se las provoca o porque revienta el mo-delo que las alimenta. Resulta paradójico que las aparentemente privilegiadas mentes de los que dirigen el destino de los pueblos no se hayan dado cuenta de que en el último siglo el mundo se ha transformado, la pirámide de la población se ha invertido, las patologías humanas han pasado de ser infecciosas a degenerativas (excepto en el mundo subdesarrollado) y los ciudadanos hoy tienen libre acceso al mundo del conocimiento del que se veían privados antes de la era Internet. Han cambiado tantas cosas que pretender soste-ner el modelo sociopolítico y socioeconómico de

comienzos del siglo XX en plena efervescencia del XXI parece propio de dirigentes muy limitados. El modelo no sirve y hay que cambiarlo, pero nadie quiere poner el cascabel al gato. La responsabi-lidad (o irresponsabilidad) de científicos e inte-lectuales radica en que han dejado en manos de “opinadores” a sueldo el futuro de la sociedad y se han escondido en los despachos y laboratorios que mantiene el dueño público, confundiendo que quien paga su silencio cómplice no es el partido gobernante sino la ciudadanía a la que debieran servir con lealtad. Los sostenedores de la ciencia, la tecnología y la medicina actuales son cómpli-ces cuando anteponen el salario personal a la responsabilidad profesional, cuando la miseria de la supervivencia está por encima de los prin-cipios, cuando la individualidad mata el interés de la colectividad, cuando se utiliza lo público para el beneficio privado, cuando se sacrifica la colabora-ción por mantener un liderazgo vacío.

En medio de esta trama de putrefacción colectiva está la clase política, preocupada por la próxima legislatura, por sus intrigas internas, por el ene-migo externo, por desenterrar muertos y cultivar odios, por las tramas de corrupción institucional encubiertas y por el aplauso de los adeptos, más que por el análisis de los críticos bienpensantes; todo ello cuidadosamente retroalimentado por los núcleos de poder mediático al servicio del color que más nutre. En medio está la población des-orientada, adoctrinada por los predicadores dia-rios, confundida por la contradicción informativa, atónita ante el descalabro social, la desaparición de puestos de trabajo, la huida de los inversores hacia cloacas más favorables, la hipocresía de sus dirigentes, la debilidad del principio de auto-ridad en la escuela, el silencio de la universidad, la pasividad comprada de los sindicatos, el hedor a estiércol de las instituciones.

Este mundo en crisis viene a recordarnos que el dinero no lo es todo; que el beneficio económico debe ser el resultado justo del esfuerzo, del sacri-ficio, del trabajo bien hecho, de la honradez, de la corresponsabilidad, de la profesionalidad, de la co-rrecta gestión de lo propio y de lo ajeno, del respeto a lo público y a lo privado, de garantizar los lindes de la libertad individual sin violar las fronteras que delimitan el derecho de los otros, de mensurar el crecimiento sostenible, de garantizar el progreso personal y colectivo mediante la colaboración, de entender que el valor de las ideas y los principios está por encima del precio de las cosas.

Este mundo en crisis nos viene a recordar que el tornillo del progreso a fuerza de golpes y talonario, prevaricación, chantaje o fraude, se pasó de ros-ca, y hay que reponerlo; hay que taladrar la pla-taforma del progreso y fortalecerla con puntales más sólidos para soportar el peso del desarrollo futuro. Hay que empezar a crear un nuevo modelo de sociedad donde el peso de las ideas y de los principios suplanten al hormigón del inmovilismo anacrónico, al pladur de las asimetrías ficticias y a las ventanas de cristal oscuro que adulteran la transparencia. Hay que empezar a creer en el cambio de las estructuras de poder, sin perder de vista que la rueda ya está inventada y que la histo-ria de la humanidad evoluciona en ciclos.

Hay que dar paso a la cultura del conocimiento. Los cimientos sobre los que asienta el edificio del conocimiento son el sentido común y la experien-cia. Lo que distingue al sabio es su capacidad de discernimiento y su habilidad para no instalarse en falsos postulados de verdades inquebranta-bles. Dice un proverbio americano que la prospe-ridad descubre el vicio y la adversidad la virtud. En las escuelas anglosajonas se enseña que el peor enemigo del conocimiento es la ignorancia; y en la cultura hindú se conserva el dicho de que el conocimiento inútil es como una antorcha en las manos de un ciego. En los pasillos de Wall Street y de la CIA es frecuente oír que knowledge is power. Pero no basta con adquirir sabiduría, como decía Cicerón; es preciso además saber usarla; y ser humilde. Confucio predicaba: “El sabio sabe que ignora. El sabio teme la bonanza; pero cuando descarga la tempestad camina sobre las olas y desafía los vientos. Lo que quiere el sabio lo busca en sí mismo; el vulgo lo busca en los demás”. Para Lao-Tsé saber creyendo no saber era algo excelso y no saber creyendo saber era una enfermedad. Y en esta aldea global, hasta Demócrito está de moda: “Toda la tierra está al alcance del sabio, ya que la patria de un alma elevada es el universo”. La megalomanía científica también tiene crítica en los pensamientos de Remy de Gourmont: “Saber lo que todo el mundo conoce es como no saber nada. El saber comienza allí donde el mundo co-mienza a ignorar. La verdadera ciencia está más allá de la verdadera ciencia” porque las verdades absolutas de hoy son sólo verdades relativas de mañana. A los que perdieron el rumbo, Sócrates les dice que el saber es la parte principal de la felicidad; y a los que se mueven en las ciénagas de la envidia ibérica, Solón les diría “guárdate bien de decir todo lo que sabes”.

Paso a la Cultura del Conocimiento

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EDITORIALRamón Cacabelos

rcacabelos@gen-t.es

diciembre 2009 3

A modo de Presentaciónpor Ciprián Rivas

EditorialEuroEspEs publishing info@euroespespublishing.com

PatrocinaFundación EuroEspEs

Editor-Jeferamón cacabElos

Direcciónciprián rivas

Dirección administrativa JaviEr sánchEz gladys bahamondE administración@gen-t.es

Dirección de Producción antonio bErmo

Producción Gráfica EuroEspEs publishing

Gen-Tsanta marta dE babío s/n

15165 bErgondo

coruña, España

tEléFono: 981 780 505

ISSN1888-7937

Depósito Legalc 713-2007copyright 2009

gEn-t no sE rEsponsabiliza dE las opinionEs y critErios Emitidos por los autorEs, rEsErvándosE la propiEdad dE los trabaJos publicados. QuEda ExprEsamEntE prohibida la rEproducción parcial, litEraria o iconográFica dE cualQuiEr contEnido sin prEvia autorización dEl Editor.

Gen-T Número 4 Diciembre 2009

en-T, The EuroEspes Journal, acude de nuevo a la cita con sus lectores. Y lo hace coincidiendo con la celebración, el 19 de diciembre, de la IV Conferencia Anual EuroEspes. Este año, está dedicada a los avances en medicina genómica. Expertos de diversas especialidades disertarán ante un público con ganas de intercambiar, madurar y aumentar su conocimiento en una materia de vanguardia y también de enorme actualidad. El esfuerzo realizado para la puesta en marcha de esta actividad, de alto nivel científico, por el Comité Organizador, es la respuesta al interés de los asistentes de las conferencias que previamente se han celebrado. La III Conferencia realizada el pasado año es objeto de un breve análisis en nuestras páginas.

Uno de los objetivos marcados por el Comité Organizador de la IV Conferencia, que organiza la Funda-ción EuroEspes, es educar y difundir -entre todo el colectivo sanitario - los beneficios de la medicina genómica. Hoy, la utilidad clínica de la farmacogenómica es ya una realidad en la optimización del uso de medicamentos y en la personalización del tratamiento farmacológico.

En este número de Gen-T pueden leer a algunos de los ponentes. Se deben destacar todos los trabajos publicados, realizados con gran rigurosidad científica, pero nos gustaría incidir en dos de ellos. Uno lo firma el director de la Conferencia, el doctor Ramón Cacabelos y su equipo de investigación: “Personalized medicine of Dementia. Pharmacogenomics of Alzheimer’s disease”.

El otro ha sido elaborado por el profesor J.A. García-Agúndez de la Universidad de Extremadura. Se titula “Farmacogenómica de los AINEs. Personalizar el tratamiento”. En este trabajo nos informa que más de 30 millones de personas son tratadas diariamente con antiinflamatorios no esteroideos (AINEs) y cerca del 25 por ciento de la población ha experimentado alguna vez en su vida reacciones adversas causadas por AINEs que han requerido tratamiento médico.

Gen-T, por primera vez, se convierte en un medio de comunicación bilingüe, vertiendo en sus páginas artículos en lengua inglesa, como el ya mencionado, escrito por el editor de la revista y los pertene-cientes a los liderados por el Dr. Andreas Pfützner y el Dr. Valter Lombardi.

En otro orden de cosas, el Grupo EuroEspes celebra con su presidente el recién creado Máster en Bio-tecnología de la Salud de la Universidad Camilo José Cela de Madrid. La Agencia Estatal de Evaluación de la Calidad y Acreditación (ANECA) ha informado favorablemente sobre este Plan de Estudios que se impartirá a partir del curso académico 2010/11.

Ramón Cacabelos dirigirá también el doctorado en Medicina Genómica de la Universidad Camilo José Cela. Este programa fue aprobado recientemente por el Ministerio de Educación. Son dos actividades educativas de alto nivel que se imparten desde la Cátedra EuroEspes que hace años viene apostando por la innovación en el campo de la medicina.

Gen-T es una revista con clara vocación científica para uso de científicos e investigadores. Es un com-promiso adquirido convertirse en un medio que tiene como misión integrar la medicina genómica en la sociedad a través de estudios rigurosos que se pueden ver en formato papel o bien a través de la web de la propia publicación, amplificando de esta manera su difusión y aproximación a la ciudadanía.

Somos una revista única en su género en el campo editorial español y, por ello, nos obligamos a ser críticos con la obra que tiene en sus manos en este momento. Conocemos de antemano que estamos ante una labor difícil, pero apostamos por este reto con la fuerza que da un grupo empresarial que tiene como misión la innovación y el desarrollo en aras de la salud y del bienestar del ser humano.

Ciencia y divulgación

A modo de Presentaciónpor Ciprián Rivas

Optimize sus defensas naturales

DefenVid ® (E-JUR-94013 ®)es el primer nutracéutico biomarino con estructura lipoproteica natural que presenta propiedades de inmunopotenciación y regulación metabólica e inmunoglobulínica

19 de Diciembre de 2009, Bergondo, La Coruña

SEDE: Centro de Investigación Biomédica EuroEspes, Bergondo, Coruña, España | ORGANIZA: Fundación EuroEspes

EUROESPES

FUNDACIÓN

8:30 RECEPCIÓN

9:00 Acto inaugural

9:30 Sesión Plenaria-I Farmacogenómica de los trastornos metabólicos Prof. Dr. Andreas Pfützner PharmGenomics, Mainz, Alemania

10:15 Estrategias terapéuticas en la enfermedad de Alzheimer

Dr. X. Antón Álvarez Departamento de Farmacología Clínica y Experimental Centro de Investigación Biomédica EuroEspes, Coruña

10:45 Genómica clínica de los trastornos del movimiento Dra. Lucía Fernández-Novoa Departamento de Genómica Médica EuroEspes Biotecnología (Ebiotec), Coruña

11:15 DESCANSO

11:30 Sesión Plenaria-II Medicina personalizada en el abordaje clínico de la

demencia: Farmacogenómica de la enfermedad de Alzheimer

Prof. Dr. Ramón Cacabelos Departamento de Neurociencias Clínicas Centro de Investigación Biomédica EuroEspes, Coruña

12:15 Farmacogenética de los tratamientos anticoagulantes

Dra. Ruth Llovo Departamento de Farmacogenética EuroEspes Biotecnología (Ebiotec), Coruña

12:45 Genómica de la patología cerebrovascular Dr. Juan Carlos Carril Departamento de Genómica e Identificación Humana EuroEspes Biotecnología (Ebiotec), Coruña

14:00 COMIDA

16:00 Sesión Plenaria-III Farmacogenética de los AINEs Prof. Dr. J.A. García-Agúndez Departamento de Farmacología y Psiquiatría Facultad de Medicina, Universidad de Extremadura,

Badajoz

16:45 Modelos transgénicos en enfermedades neurodegenerativas

Dr. Iván Carrera Departamento de Neurociencias EuroEspes Biotecnología (Ebiotec), Coruña

17:15 Neurodegeneración y cáncer Dr. Salvador Harguindey Instituto de Biología Clínica y Metabolismo Vitoria, Álava

17:45 DESCANSO

18:00 Aplicaciones actuales de la farmacogenética en terapias del cáncer

Dr. Stefan Prause PharmGenomics GmbH Mainz, Alemania

18:30 Fibronectina en la enfermedad de Alzheimer Prof. Dr. Jerzy Leszek Medical University of Wroclaw Wroclaw, Polonia

19:00 Acto de clausura Presidido por D. Augusto Silva Director General Terapias Avanzadas y Trasplantes Ministerio de Sanidad y Consumo, Madrid

diciembre 2009 7

SUMARIO

Personalized medicine of dementiaPharmacogenomics of Alzheimer´s disease 18-48

Farmacogenómica de los AINEs 62-68

Effects of FR-91 on humantumor cell lines

Genómica de la patología cerebrovascular

50-60

70-84

Opinión03 Editorial

04 A modo de presentación

Ciencia08 Pharmacogenomics of

Metabolic Disease

18 Personalized medicine of dementia: Pharmacogenomics of Alzheimer´s disease

50 Effects of FR-91on human tumor cell lines

62 Farmacogenómica de los AINEs

70 Genómica de la patología cerebrovascular

Sociedad86 Cooperación multisectorial

para impulsar el desarrollo de Galicia

88 III conferencia anual EuroEspes

Noticias94 Noticias EuroEspes

Suscripción98 Boletín de suscripción

8

Pharmacogenomics of Metabolic Disease1. IKFE - Institute for Clinical Research and Development, Parcusstr. 8, D-55116 Mainz, Germany 2. PharmGenomics GmbH, Parcusstr. 8, D-55116 Mainz, Germany

Andreas Pfützner1,2, Stefan Prause2, Moritz Eidens2, Alexander Weise1, Thomas Forst1

diciembre 2009 9

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Sumary

he prevalence of metabolic diseases, such as atherosclerosis, diabetes mellitus and cardio-metabolic syndrome, has reached pandemic di-mensions. The increasing incidence in emerg-ing countries, which is linked to an improved access to processed food, will make these dis-eases the major burden for the affected health care systems. Orchestrated by a complex interac-tion of the joint underlying pathophysiological deteriorations (insulin resistance, ß-cell dysfunc-tion, visceral adipogenesis, and chronic systemic inflammation), the disease presents with a vari-ety of clinical phenotypes, characterized by dif-ferent compositions and severities of the most common symptoms: hypertension, dyslipidemia, hyperglycemia and atherosclerosis. The disease diagnosis is usually defined by the major symp-toms and (e.g.) many patients with normoglyce-mic vascular insulin resistance die from a mac-rovascular event without ever being treated with a drug affecting insulin resistance. These drugs are normally only prescribed in case of overt diabetes with hyperglycemia. On the contrary, even well controlled patients with diabetes die from macrovascular events because the interna-tionally accepted therapy guidelines target high blood glucose levels and HbA1c only and disre-gard the macrovascular risk induced by insulin resistance and the inflammatory activity of the visceral lipid tissue.

All these findings underline the immediate ne-cessity to develop diagnostic approaches for in-dividualized assessment of the diverse contribu-tions of the underlying disease components to the patient risk profile. Such approaches would help to improve the major challenges in this in-dication: identifying patients at risk of disease development, monitoring the efficacy of pre-ventive measures, identifying the most optimal therapeutic approaches and monitoring their effectiveness in patients with overt disease, and identifying patients at a very high risk for macro-vascular events justifying intense and expensive treatment interventions.

Appropriate diagnostic options can be identi-fied at all levels of cellular activity starting with DNA markers for risk identification based on de-termination of candidate gene mutations (usu-ally linked to ß-cell function or atherosclerosis), assessment of increased mRNA expression (e.g. as a measure of macrophage activation), and de-termination of plasma protein levels of biomark-ers specifically associated with the related disor-ders. Modern laboratory platforms, such as the MutaChip technology, allow for economic and specific determination of DNA, mRNA, and pro-tein biomarker panels to increase the efficacy of individually selected therapeutic interventions,

and will help to avoid the otherwise unavoidable progression of metabolic syndrome, cardiomet-abolic syndrome or type 2 diabetes mellitus to finally end in macrovascular death.

Introduction

Type 2 diabetes mellitus is one of the most fre-quent diseases with a worldwide prevalence of 4-5 % and a 10 % annual incidence rate. The ma-jor pathophysiological drivers are a hereditary or metabolic insulin resistance in combination with the inability of the pancreas to augment in-sulin secretion to the required amount1. About 40 % of the US population are overweight and develop insulin resistance. In the majority of these cases, the pancreas is able to counterbal-ance the increasing insulin need by an appro-priately increased secretion. However, in a sig-nificant minority of about a third of these cases, a concomitant ß-cell dysfunction leads to the development of a metabolic syndrome and to diabetes mellitus2. Insulin resistance, however, is also associated with an increased cardiovascular risk and the majority of the patients finally die from myocardial infarction or other macrovas-cular complications3, 4.

The disease is commonly regarded as chronic progressive and general treatment guidelines are available to guide physicians how to best reach normoglycemia, which is commonly defined via a hemoglobin A1c value in the target range of < 6.5 % (Europe) or < 7.0 % (USA)5, 6. However, recent outcome studies have demonstrated that HbA1c is only a very moderate surrogate marker with limited or almost no prognostic value for the prediction of cardiovascular outcome.

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Pharmacogenomics of Metabolic Disease

genetic predisposition or other environmental factors including excess food uptake and devel-opment of obesity. Increased insulin resistance demands for increased insulin secretion, which is normally provided by the ß-cells in a compen-satory manner to lower blood glucose levels into the normal range. However, insulin is the only physiological hormone known to induce growth of lipid tissue, and an increase in body weight will most likely occur in the presence of suffi-cient food supply. It has recently been shown that growth of visceral adipose tissue results in differentiation of mesenchymal stem cells to pre-adipocytes, which finally become mature lipid cells. The pre-adipocyte, however, is the source of a substantial number of different cytokines and hormones (referred to as “adipokines”) known to support insulin resistance and thus a circle is closed leading to constantly increasing insulin resistance and obesity (Fig. 1)14.

As long as the ß-cells are not compromised by a ß-cell dysfunction, the situation may be meta-bolically under control and can be reversed by reduction of food intake and increased physical exercise. However, in patients developing type 2 diabetes, ß-cell dysfunction will occur and will further accelerate the disease progression. Dys-function of the ß-cell in type 2 diabetes is com-prised of three components: secretion timing disorder, quantitative disorder and qualitative disorder. An indicator for the secretion timing disorder in early stages of type 2 diabetes is the loss of first phase insulin response, an important inhibitory signal for hepatic glucose release15. In later stages, loss of pulsatile insulin release can also be observed as additional failure in secre-tion timing16. The quantitative disorder starts when the ß-cell increases the volume of insulin release based on the increased external demand. In later stages, increasing exhaustion of the pro-duction capacity may result in complete loss of insulin secretion17. An increased quantitative secretion of proinsulin, and - in parallel - also of other pro-hormones, like pro-islet amyloid polypeptide (which is processed by the same conversion enzymes as proinsulin18) can finally lead to a deterioration of the secretion product composition.

When new assays for assessment of unprocessed intact proinsulin became available, they helped to understand previous findings regarding el-evated fasting proinsulin levels in the plasma of non-diabetic patients19. In the natural develop-ment of type 2 diabetes, proinsulin may only be elevated in the case of a significant insulin re-sistance, and we were thus able to demonstrate that elevated fasting morning intact proinsulin is indeed a highly specific indicator for insulin resistance20. Proinsulin is able to lower glucose levels but shows only 10-20 % of the efficacy of

In these trials, achievement and maintenance of the target HbA1c range for several years had no pronounced influence on the incidence and out-come of cardiovascular events7, 8. While elevated blood glucose is certainly a contributor to the increased cardiovascular risk, even normaliza-tion of HbA1c leaves the patient with substantial further event risk9. It has to be concluded that current treatment guidelines address the under-lying pathophysiology only in a suboptimal way and that new and more individualized treatment targets may be required to effectively improve the vascular prognosis of the affected patients. A closer investigation of the pathophysiology of-fers surprising insights into this complex disease and offers attractive ways for identification of suitable biomarkers for more effective and indi-vidualized interventions.

Pathophysiology of the cardiometabolic syndrome

The close relation between insulin resistance and ß-cell dysfunction has long been established and confirmed in large epidemiological stud-ies10-13. Insulin resistance may occur based on

Fig. 1 5 The pathophysiological link between insulin resistance, ß-cell dysfunction and visceral adipogenesis.

insulin resistance

adipogenesisß-Celldysfuntion

anti-insuline hormones

adiponectin

insulinrequirement

insulinproinsulin

diciembre 2009 11

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crease in intranuclear NF-κB binding, a decrease in IκB-ß and an increase in the transcription of proinflammatory genes regulated by NF-κB, in-cluding migration inhibitory factor (MIF), IL-6, TNFα, and matrix metalloproteinase 9 (MMP-9)9. It is believed that the crosstalk between the pre-adipocytes and other tissues contributes to a general up-regulation of the immune system, including an activation of circulating monocytes and macrophages, resulting in an increased risk for atherosclerosis and vascular disease32, 33.

The integrated knowledge about these com-plex pathophysiological conditions and rela-tions enable the introduction of new diagnostic tools, which may help to identify the underly-ing genetic risk, may be helpful to describe the acute proinflammatory risk and may also allow identification of the most optimal therapeutic interventions by introducing concepts leading to individualized medicine approaches. These diagnostic opportunities can be found on the DNA, mRNA and protein level.

insulin21. Unfortunately, it has similar adipoge-netic effects and, therefore, a five- to ten-fold higher driving force for development of obesity is prevalent in the affected patients22.

In many patients, proinsulin action is sufficient to maintain blood glucose at normal levels, while the other pathophysiological drivers are still de-teriorating. This may explain the findings from several groups that normoglycemic patients with elevated fasting proinsulin levels have an excess mortality from stroke and myocardial in-farction23-25. In our opinion, the ultimate proof of a harmful action of proinsulin was provided when an interventional phase II trial testing subcutaneous injection of proinsulin vs. insulin for preprandial treatment of diabetes had to be stopped prematurely due to an elevated number of cardiovascular events which happened exclu-sively in the proinsulin arm21.

In the presence of sufficient food supply, the metabolic situation deteriorates when insulin and proinsulin induce a major visceral adipogen-esis, which leads to an increase in body weight. This process, based on the differentiation of mesenchymal stem cells to adipocytes26 is usu-ally accompanied by an elevation in the tissue expression and plasma levels of proinflamma-tory cytokines, such as tumor necrosis factor-α (TNFα)27, interleukin-6 (IL-6)28, 29, plasminogen activator-inhibitor-1 (PAI-1)30 and others31. This protein expression profile indicates the preva-lence of a chronic systemic inflammation. It has been demonstrated by Ghanim and coworkers that circulating mononuclear cells in obese pa-tients are in a proinflammatory state with an in-

In many patients, proinsulin action is sufficient to

maintain blood glucose at normal levels

12

Pharmacogenomics of Metabolic Disease

DNA Markers for diabetes prediction and cardiovascular risk assessment

There is strong evidence that genetic factors play an important role in the development of insulin resistance and ß-cell dysfunction. The majority of the single nucleotide polymorphisms (SNPs) of genes associated with an increased risk of type 2 diabetes is hypothesized to influence B-cell func-tion34. It has been concluded from linkage stud-ies, candidate gene approaches, and genome-wide association studies that single nucleotide polymorphisms (SNPs) within up to 10 genes are associated with an increased risk of type 2 diabetes35-38. The suspected SNPs are located in the regions encoding the following proteins: Transcription factor 7-like 2 (TCF7L2), Cyclin-dependent kinase inhibitor-2A (CDKN2A) and CDKN2B, Human hematopoietically expressed homeobox (HHEX), CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1), Solute carrier family 30 (zinc transporter) member 8 (SLC30A8), and Potassium inwardly-rectifying channel subfamily J member 11 (KCNJ11)34. Each of these proteins plays a major role in in-sulin processing or insulin secretion. A list of the candidate mutations is provided in Table 1.

However, it has been suggested that the SNPs within or near these genes most likely do not alter their function or expression and empha-size a lack of influencing pancreatic B-cell de-velopment, regeneration, and function in the etiology of type 2 diabetes. It is also possible that there is an inherited reduction of the mitochon-drial content in skeletal muscle of the insulin-resistant pre-diabetic offspring of parents with type 2 diabetes. This reduction may be respon-sible for decreased oxidative phosphorylation in skeletal muscle39. In addition, a recent study suggests a link between mitochondrial function and glucose transporter trafficking. Impairment of respiratory chain function leads to impaired insulin-stimulated glucose transport in adipose cells40. It was demonstrated that the insulin sensitizer pioglitazone stabilizes the outer mito-chondrial membrane protein mitoNEET, which is expressed in many insulin-responsive tissues and plays a key role in regulating the maximal capacity for electron transport and oxidative phosphorylation41. It is therefore highly possible that a hereditary mitochondrial defect, and not an inherited B-cell defect, plays the critical role in the onset of type 2 diabetes. Further work is required to elucidate the genetic origin of the observed reduction in mitochondrial ATP syn-thesis, which may add to existing concepts to ex-plore the genetic basis of type 2 diabetes.

Use of mRNA for individual cardiovascular risk assessment in patients with type 2 diabetes

As mentioned above, it has been demonstrat-ed by Ghanim and coworkers that circulating mononuclear cells in obese patients are in a proinflammatory state with increased expres-sion of intranuclear NF-κB protein, and sub-sequent up-regulation in the transcription of proinflammatory genes regulated by NF-κB9. The same group was able to demonstrate that an increased plasma concentration of MIF and an increased transcription of MIF mRNA in mononuclear cells, which was related to the body-mass index and hsCRP concentrations, could be reduced by a six week treatment with metformin in eight non-diabetic patients with obesity. The authors concluded that metformin might have beneficial effects on cardiovascu-lar mortality in patients with type 2 diabetes42, which is in part confirmed by the few currently existing larger outcome trials on this topic43, 44. It has been shown in randomized prospective trials that treatment with pioglitazone, an ago-nist to the peroxisome proliferators-activated receptor γ, may improve clinical and laboratory surrogate markers for atherosclerosis and car-

Confirmed loci for risk of type 2 diabetes development 34

Chro

mos

ome

SNP

Nucl

eotid

e nu

mbe

r

Gene

Posi

tion

bp Mut

atio

n

Refe

renc

e

6p rs7754840

rs10946398

20,769,229

20,769,013

CDKAL1 INTRON20,757,587- 20,847,729

35,37

36

8q rs13266634 118,253,964 SLC30A8 EXON-10118,253,956–114,789,774

Arg325Trp 35, 37

9p rs10811661 22,124,094 CDKN2B 21,992,902–

21,999,280

35-37

10q rs1111875

rs5015480

94,452,862

94,455,539

HHEX 94,439,690–

94,445,383

35, 37

36

10q rs7903146

114,748,339

114,748,339 TCF7L2 INTRON-3

114,714,373–

114,789,774

35, 37

11 rs5219

KCNJ11

17,366,148 KCNJ11 EXON

17,365,042–

17,366,214

Glu23Lys 35, 37

Table 1

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tion of appropriate therapeutic interventions. Recent studies have indicated that “intact pro-insulin” measured with newly developed assays for the complete protein [19] may serve as an excellent indicator of a clinically relevant later stage ß-cell dysfunction and as a highly specific laboratory marker for insulin resistance20. Fast-ing intact proinsulin levels and the HOMA-score for insulin resistance can be used for a clinically useful staging of ß-cell dysfunction, which has been measured and validated in multiple large cross-sectional and prospective controlled inter-ventional studies47, 60, 61. The characteristics and interpretation of fasting intact proinsulin as a marker for ß-cell dysfunction and insulin resis-tance are provided in Table 2.

diovascular risk, such as intima-media thickness, hsCRP, or MMP-9 independent from glycemic control45-47, and that it may even improve mac-rovascular outcome in type 2 diabetic patients when used in secondary prevention48-50. The anti-inflammatory and anti-thrombotic effects of glitazones occur very rapidly and significantly earlier as compared to the metabolic and glyce-mic effects of the drugs51, 52. In a recent study, we explored the short-term effects of the addition of pioglitazone (vs. placebo) to an existing ef-fective oral anti-diabetic therapy with metformin and/or sulfonylurea on the proinflammatory ac-tivation of circulating mononuclear cells in well controlled patients with type 2 diabetes mellitus and elevated risk for atherosclerosis. For this purpose, we investigated the mRNA expression of the inhibitors to NF-κB (IκB-α and IκB-β)53, p105 (precursor to the p50 subunit) and Rel-A (p65 subunit) as measures for of the quantity of intranuclear NF-κB54, and several proinflam-matory mediators and markers known to be modulated by NF-κB, such as TNFα, IL-6, MIF, and MMP-928, 55, 56 before and after four weeks of treatment in relation to a housekeeping gene. We found significant reduction of NF-κB expres-sion, and the expression of the modulated cytok-ines by pioglitazone, while no change occurred with placebo. We concluded that the TZD down-regulated macrophage activation, which could be visualized by quantification of macrophage mRNA57, 58.

In conclusion, mRNA quantification of distinct mRNA markers in peripheral macrophages may be a way to determine the acute macrophage acti-vation, which is an indicator of acute atherogen-ic action in the vasculature. Assessment of these markers by means of more efficacious and less costly diagnostic tools, e.g. macro-arrays, may al-low determination of an acute atherogenic activ-ity in the vasculature and may become a future tool to monitor the efficacy of anti-inflammatory therapeutic interventions.

Protein biomarkers for selection and monitoring of individualized treatment interventions

The determination of clinical and laboratory routine markers (glucose, HbA1c, lipids, BMI, blood pressure) provides only insufficient infor-mation regarding the severity of the underlying pathophysiology, but new laboratory markers may provide the means to classify the acute in-dividual metabolic and cardiovascular risk situ-ation. The determination of ß-cell dysfunction may provide additional information with regard to disease stage and may be helpful for the selec-

Characteristics of the marker panel for assessment of metabolic and cardiovascular risk in patients with type 2 diabetes mellitus

Marker Ranges Interpretation

Intact Proinsulin

≤ 11 pmol/l

> 11 pmol/l

normal value, no severe insulin resistance, ß-cell dysfunction stage I-IIelevated, clinically relevant insulin resistance,stage III ß-cell dysfunction, elevated cardiovascular risk

Adiponectin 10 - 12 mg/l8 – 10 mg/l

> 10 mg/l7 – 10 mg/l< 7 mg/l

normal reference range in womennormal reference range in men

no elevated riskunclear result, no risk determination possibleelevated risk, insulin resistance

hsCRP 0 - 1 mg/l>1 – 3 mg/l>3 – 10 mg/l> 10 mg/l

low cardiovascular risk moderate cardiovascular riskhigh cardiovascular riskunspecific inflammation, no conclusion regarding cardiovascular risk elevation possible

Table 2

New laboratory markers may provide the means to

classify the acute individual metabolic and cardiovascular

risk situation

14

Pharmacogenomics of Metabolic Disease

10 mg/l may arise due to other unspecific in-fections and inflammations and cannot be used for assessment of the chronic systemic vascular inflammatory process causing atherosclerosis. A reduction in the hsCRP concentrations is as-sociated with the reduction of the cardiovascu-lar risk profile67.

Perspectives

As of now, the laboratory determination of HbA1c, glucose, cholesterol, and triglycerides and the clinical assessment of blood pressure and the body-mass-index are used to obtain a basic and crude understanding of the degree and severity of insulin resistance, ß-cell dysfunc-tion and cardiovascular risk. Currently multiple studies investigate the effects of different thera-pies on chronic systemic inflammation and on protein biomarkers, such as intact proin-sulin, adiponectin and hsCRP concentrations. However, assessment of multimarker panel is still a costly undertaking. Modern laboratory platforms, such as the MutaChip technology (PharmGenomics, Mainz, Germany), allow for specific but economically sound determination of DNA, mRNA, and protein biomarker panels to increase the efficacy of individually selected therapeutic interventions, and will help to avoid the otherwise unavoidable progression of meta-bolic syndrome, cardiometabolic syndrome or type 2 diabetes mellitus to finally end in macro-vascular death.

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Pharmacogenomics of Metabolic Disease

14

Another laboratory indication for the actual metabolic situation is provided by the total plas-ma adiponectin concentrations. Values below 7 mg/dl have been associated with an increased cardiovascular risk in controlled clinical trials62,

63. While adiponectin may have less value for the initial diagnosis of insulin resistance than intact proinsulin64, it appears to provide substantial information regarding the overall metabolic situation that reacts very sensitively to success-ful therapeutic measures. Different high and low molecular weight isoforms of adiponectin have been detected. However, their distinct determination does not seem to provide addi-tional information in the context of metabolic risk screening. An increase in total adiponectin concentrations after intervention indicates an improvement in the metabolic and cardiovascu-lar risk profile (Table 2)62, 63.

A detailed analysis of the Framingham study co-hort by Ridker et al. showed that CRP concen-trations in the near normal range (> 10 mg/dl) allow for an independent stratification of the cardiovascular risk into three risk groups, when measured with a highly sensitive assay method (hsCRP, see Table 2)65, 66. This marker has been globally accepted and has become part of the risk assessment guidelines of many scientific associations, including the American Heart Association and the American Diabetes Association. Values below 1 mg/l describe a low cardiovascular risk, 1 – 3 mg/l indicate a moderate cardiovascular risk, and 3 – 10 mg/l describe a high risk population. Values above

Dr. Andreas Pfützner andreasp@ikfe.de

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16. Matthews D.R., Lang D.A., Burnett M.A., Turner R.C. Control of pulsatile insulin secretion in man. Diabeto-logia 1983; 24:231-237.

17. Porte D., Kahn S.E. Beta-cell dysfunction and failure in type 2 diabetes: potential mechanisms. Diabetes 2001; 50(Suppl.1):S160-S163.

18. Clark A, Nilsson MR. Islet amyloid: a complication of islet dysfunction or an aetiological factor in Type 2 Diabetes. Diabetologia 2004; 47:157-169.

19. Pfützner A, Kunt T, Löbig M, Knesovic M, Forst T: Clin-ical and laboratory evaluation characteristics of new chemiluminescence assays for intact and total proin-sulin. Clin Chem Lab Med 2003; 41:1234-1238.

20. Pfützner A. Kunt T., Hohberg C, Mondok A, Pahler S, Konrad T, Lübben G, Forst T.: Fasting intact proinsulin is a highly specific indicator of insulin resistance in type 2 diabetes. Diabetes Care 2004; 27:682-687.

21. Galloway JA, Hooper SA, Spradlin CT, Howey DC, Frank BH, Bowsher RR, Anderson JH: Biosynthetic human proinsulin. Review of chemistry, in vitro and in vivo receptor binding, animal and human pharma-cology studies, and clinical trial experience. Diabetes Care 1992; 15:666-692.

22. Pfützner A, Pansky A, Maiworm A, Matthey M, Lück-erath K, Roitzheim B, Forst T, Tobiasch E. Mesen-chymal stem cell differentiation into adipocytes is equally induced by insulin and proinsulin in vitro. Diabetologia 2006; 49(Suppl.1):P-707.

23. Lindahl B, Dinesen B, Eliasson M, Roeder M, Jansson JH, Huhtasaari F, Hallmans G: High proinsulin con-centration precedes acute myocardial infarction in a nondiabetic population. Metabolism 1999; 48:1197-1202.

24. Lindahl B, Dinesen B, Eliasson M, Roder M, Hallmans G, Stegmayr B: High proinsulin levels precede first-ever stroke in a nondiabetic population. Stroke 2000; 31:2936-2941.

25. Zethelius B, Byberg L, Hales CN, Lithell H, Berne C. Proinsulin is an independent predictor of coronary heart disease: report from a 27-year follow-up study. Circulation 2002; 7:2153 – 2158.

26. Fain JN, Madan AK, Hiler ML, Cheema P, Bahouth SW. Comparison of the release of adipokines by adi-pose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans. Endocrinology 2004; 145:2273-82.

27. Dandona P, Weinstock R, Thusu K, Abdel-Rahman E, Aljada A, Wadden T, Tumor necrosis factor-alpha in sera of obese patients: fall with weight loss. J Clin. Endocrinol Metab 1998; 83:2907-10.

28 Bastard JP, Jardel C, Delatore J, Hainque B, Bruck-ert E, Oberlin F. Evidence for a link between adipose tissue interleukin-6 content and serum C-reactive protein concentrations in obese subjects. Circulation 1999; 99:2221-22.

29. Teramoto S, Yamamoto H, Ouchi Y. Increased C-reactive protein and increased plasma interleukin-6 may synergistically affect the progression of coro-nary atherosclerosis in obstructive sleep apnea syn-drome. Circulation 2003; 107:E40-40.

30. Samad F, Loskutoff DJ. Tissue distribution and regu-lation of plasminogen activator inhibitor-1 in obese

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58. Forst T, Karagiannis E, Lübben G, Hohberg C, Schöndorf T, Dikta G, Drexler M, Morcos M, Dan-schel W, Borchert M, Pfützner A. Pleiotrophic and anti-inflammatory effects of pioglitazone precede the metabolic activity in type 2 diabetic patients with coronary artery disease. Atherosclerosis 2008; 197:311-317.

59. Pfützner A, Weise A, Hähnel H, Musholt P, Lübben G, Karagiannis E, Forst T. Pioglitazone down-regulates the mRNA Expression and Secretion of Inflammatory Proteins by Monocyte/Macrophages in Patients with Type 2 Diabetes independent from glycemic control. Diabetes 2007; 56(Suppl.1):A188.

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48. Mazzone T, Meyer PM, Feinstein SB et al. Effect of pioglitazone compared with glimepiride on carotid intima-media thickness in type 2 diabetes: a ran-domized trial. JAMA 2006; 296:2572-81.

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51. Wilcox R, Bousser MG, Betteridge DJ et al. Effects of Pioglitazone in Patients With Type 2 Diabetes With or Without Previous Stroke. Results From PROactive (PROspective pioglitAzone Clinical Trial In macroVas-cular Events 04). Stroke 2007; 38:865-73.

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Ramón Cacabelos, Rocío Martínez-Bouza, Lucía Fernández-Novoa, Juan Carlos Carril, Ruth Llovo, Iván Tellado, Lola Corzo, Carmen Fraile, Natalia Cacabelos, Amanda Bello, Adam McKay, Antonio Bermo, Iván Carrera, Antón Álvarez, Valter Lombardi

Abstract:

ecenT advances in genomic medicine can con-tribute to accelerating our understanding on the pathogenesis of dementia, improving diag-nostic accuracy with the introduction of novel biomarkers, and personalizing therapeutics with the incorporation of pharmacogenetic and pharmacogenomic procedures to drug develop-ment and clinical practice. Most neurodegen-erative disorders, including Alzheimer’s disease (AD), share some common features, such as a genomic background in which hundreds of genes might be involved, genome-environment interactions, complex pathogenic pathways,

poor therapeutic outcomes and chronic disabil-ity. The main aim of a cost-effective treatment is to halt disease progression via a modification of the functional cascade involving AD genom-ics, transcriptomics, proteomics and metabolo-mics. Unfortunately, the drugs available for the treatment of dementia are not cost-effective. The pharmacological treatment of dementia accounts for 10-20% of direct costs, and less than 20% of the patients are moderate respond-ers to conventional drugs, some of which may cause important adverse drug reactions. Future anti-dementia drugs must address the complex pathogenic niche of the disease from a multifac-torial perspective. Pharmacogenetic and phar-

Correspondence: Prof. Dr. Ramón Cacabelos

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diciembre 2009 19

macogenomic factors may account for 60-90% of drug variability in drug disposition and phar-macodynamics. In addition to anti-dementia drugs, patients with AD, or with other forms of dementia, need concomitant medications for the treatment of diverse disorders of the central nervous system (CNS) associated with progres-sive brain dysfunction. Approximately 60-80% of drugs acting on the CNS are metabolised via enzymes of the CYP gene superfamily, and 10-20% of Caucasians are carriers of defective CYP2D6 polymorphic variants that alter the metabolism of many psychotropic agents. Only 26% of the patients are pure extensive metabo-lisers for the trigenic cluster integrated by allel-

ic variants of the CYP2D6, CYP2C19, CYP2C9 in combination. Although many genes have been suggested to be associated with AD, with the exception of APOE, most polymorphic variants of potential risk exhibit a very weak association with AD. APOE-4/4 carriers exhibit a dramatic biological disadvantage in comparison with oth-er genotypes, and AD patients harbouring this homozygous condition are the worst respond-ers to conventional drugs. The incorporation of pharmacogenetic/pharmacogenomic protocols into AD research and clinical practice can fos-ter the optimisation of therapeutics by helping to develop cost-effective biopharmaceuticals and improving drug efficacy and safety.

20

Personalized Medicine of Dementia

Introduction

The lack of accurate diagnostic markers for early prediction and of effective therapy of dementia are the two most important prob-lems to efficient diagnosis and halting of progression of the disease. About 10-20% of the costs in dementia are attributed to pharmacological treatment, including an-ti-dementia drugs, psychotropics (antide-pressants, neuroleptics, anxiolytics), and other drugs currently prescribed in the el-derly (antiparkinsonians, anticonvulsants, vasoactive compounds, anti-inflammatory drugs, etc)1. During the past 25 years, over 300 drugs have been partially or totally developed for Alzheimer’s disease (AD) with poor repercussion in public health. Despite a considerable research effort and high expenditure over more than two de-cades, only 5 drugs (tacrine, donepezil, rivastigmine, galantamine, memantine) with moderate-to-poor efficacy and ques-tionable cost-effectiveness have been ap-proved in developed countries, and less than 20% of the patients can benefit from current anti-dementia drugs1-3.

Common features in CNS disorders include the following: (a) polygenic/complex dis-orders in which genomic and environmen-tal factors are involved; (b) deterioration of higher activities of the CNS; (c) multifacto-rial dysfunctions in several brain circuits; and (d) accumulation of toxic proteins in the nervous tissue in cases of neurodegen-eration. For instance, the neuropathologi-cal hallmark of Alzheimer’s disease (AD)(amyloid deposition in senile plaques, neu-rofibrillary tangle formation, and neuronal loss) is but the phenotypic expression of a pathogenic process in which different gene clusters and their products are potentially involved4.

Drug metabolism, and the mechanisms underlying drug efficacy and safety, are also genetically regulated complex traits in which hundreds of genes cooperatively participate. Structural and functional ge-nomics studies demonstrate that genomic factors, probably induced by environmen-tal factors, cerebrovascular dysfunction, and epigenetic phenomena, might be re-sponsible for pathogenic events leading to premature neuronal dysfunction and/or death.

Pharmacogenetic and pharmacogenomic factors may account for 60-90% of drug variability in drug disposition and pharma-codynamics. About 10-20% of Caucasians are carriers of defective CYP2D6 polymor-phic variants which alter the metabolism of many psychotropic agents. The incor-poration of pharmacogenetic/pharma-cogenomic protocols into dementia re-search and clinical practice can foster the optimization of therapeutics by helping to develop cost-effective pharmaceuticals and improving drug efficacy and safety5-11.

Dementia Phenotype and Biomarkers

Alzheimer’s disease is the most common form of dementia (50-60%), followed by vascular dementia (20-30%) and mixed dementia (10-20%), which has become the most prevalent type of dementia in individu-als older than 75 years of age. There are over 100 different types of dementia with a com-mon phenotypic denominator composed of cognitive and mental deterioration, psycho-motor dysfunction, behavioural changes, and progressive functional decline1.

AD is a complex disorder in which mul-tiple pathogenic mechanisms may be in-volved giving rise to a common phenotype. From a didactic point of view, it has been established that primary pathogenic events in AD are represented by genetic factors (mutations, susceptibility SNPs) and pro-grammed neuronal death, since neurons start to die 30-40 years before the onset of the disease. Secondary pathogenic events are associated with the phenotypic expres-sion of senile plaques (amyloid deposi-tion) and neurofibrillary tangles (NFT), together with synaptic loss, dendritic de-sarborization, and neuronal death, as the major hallmarks of AD pathology. Tertiary and quaternary pathogenic events are re-flected by neurotransmitter deficits, neu-roinflammatory reactions, oxidative stress phenomena and free radical formation, excitotoxic reactions, alterations in cal-cium homeostasis, deficit of neurotrophic factors, and cerebrovascular perturbations, among many other neurochemical phe-notypes12. All these pathogenic elements configure the AD phenotype which differs from that of the healthy elderly population. The phenotypic features of the disease represent the biomarkers to be modified with an effective therapeutic intervention (Fig.1). Important differences have been

Fig.1 6The process of pharmacogenomics intervention in CNS disorders and dementia

Therapeutic Intervention

Functional GenomicsTranscriptomicsProteomics Metabolomics

GenotypeGenomic Profile

Ethnic backgroundFamily HistoryDisease GenotypePharmacogenetic GenotypePharmacogenomic GenotypeNutrigenetic GenotypeNutrigenomic Genotype

Disease PhenotypeAge and GenderAge at onsetDisease Stage and SeverityConcomitant PathologyGenotype-Phenotype CorrelationsNutritional Conditions

Disease Phenotype Modification Biochemical changes Neurochemical changes Neuropsychological changes Mood Behavior Cognition Functioning Neuroimaging changes Brain Function Cerebrovascular changes Gene Expression Profile Transcriptomics Proteomics Metabolomics

PhenotypeCNS Disorder

Pharmacogenomicoutcome

Genotype-RelatedDrug Metabolism Pharmacokinetics Pharmacodynamics

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found in the AD population as compared with healthy subjects in different biologi-cal parameters, including blood pressure, glucose, cholesterol and triglyceride levels, transaminase activity, haematological pa-rameters, metabolic factors, thyroid func-tion, brain haemodynamic parameters, and brain mapping activity (Table 1; Fig.2)

7-10,12-14. Blood pressure values, glucose lev-els and cholesterol levels are higher in AD than in healthy elderly subjects (Table 1). Approximately 20% of AD patients are hypertensive, 25% are diabetics, 50% are hypercholesterolemic (Fig.2a), and 23% are hypertriglyceridemic. Over 25% of the patients exhibit high GGT activity (Fig.2b), 5-10% show anaemic conditions, 30-50% show an abnormal cerebrovascu-lar function characterized by poor brain perfusion (Fig.2c), and over 60% have an abnormal electroencephalographic pat-tern, especially in frontal, temporal, and parietal regions, as revealed by quantitative EEG (qEEG) or computerized mapping (Table 1; Fig.2d) 14-16. Significant differences are currently seen between females and males, indicating the effect of gender on the phenotypic expression of the disease. In fact, the prevalence of dementia is 10-15% higher in females than in males from 65 to 85 years of age. All these parame-ters are highly relevant when treating AD patients because some of them reflect a concomitant pathology which also needs therapeutic consideration. On the other hand, they can also represent general bio-markers together with regional brain atro-phy and perfusion and cognitive function, which may serve as therapeutic outcome measures. Other biomarkers of potential interest include cerebrospinal fluid (CSF) and peripheral levels of Aß42, protein tau, histamine, interleukins, and some other candidate markers4,13-16.

The molecular mechanisms underlying β-amyloid deposition in brain tissue and blood vessels, as well as abnormalities in tau protein leading to NFT formation, have been elucidated over the past 20 years by a number of groups all over the world defining the fundamentals for promising therapeutic strategies oriented towards in-hibiting the formation of amyloid deposits or reducing senile plaque burden. Not-withstanding, the complexity of the patho-genic cascade in AD invites the prediction that many other genetic factors and patho-genic mechanisms may be involved in the etiology of AD, together with epigenetic phenomena, cerebrovascular dysfunction, and environmental events12(Fig.3).

Genomics

Structural Genomics:Approximately 5% of the human genome is structurally variant in the normal popu-lation, involving more than 800 genes17. There are roughly 7-10 million positions in the human genome that can show vari-ability among individuals, and differences in the DNA sequence are the genetic basis of human variability and complex traits. The spectrum of variation in the human genome includes: (a) single changes (sin-gle nucleotide polymorphisms (SNPs), point mutations)(1 bp), (b) small inser-tions/deletions (binary insertion/dele-tion events of short sequences)(1-50 bp), (c) short tandem repeats (microsatellites)(1-500 bp), (d) fine-scale structural varia-tion (deletions, duplications, tandem re-peats, inversions)(50 bp – 5 kb), (e) retro-element insertions (SINEs, LINEs, LTRs, ERVs)(300 bp – 10 kb), (f) intermediate-scale structural variations (deletions, du-plications, tandem repeats, inversions)(5 kb – 50 kb), (g) large-scale structural variation (deletions, duplications, large tandem repeats)(50 kb – 5 Mb), and (h) chromosomal variations (euchromatic variations, cytogenetic deletions, dupli-cations, translocations, inversions, and aneuploidy)(>5 Mb)17,18. Segmental du-plications of low copy repeats are blocks of DNA ranging from 1-400 kb in length which occur at multiple sites within the genome and typically share a high level (>95%) of sequence identity17. Segmen-tal duplications frequently mediate poly-morphic rearrangements of intervening sequences via non-allelic homologous recombination (NAHR) with major im-plications for human disease. SNPs and insertion (I)/deletion (D) events are the most frequent types of structural varia-tion. I/D polymorphisms of several genes with functions in enzymatic pathways or in drug metabolising enzymes (e.g. CYP2D6) may drastically influence a variety of com-mon phenotypes with pathogenic and/or pharmacogenetic relevance. The dif-ferential expression of common variants is a major source of genetic variation with important repercussions in human diver-sity and disease heterogeneity. Prior to the completion of the Human Genome Proj-ect and the emergence of dense genetic maps, scientists used linkage studies and positional cloning to identify DNA muta-tions in rare diseases, but in the past two decades association study designs became more powerful compared with linkage

Fig.2 5Comparison of biological parameters between patients with Alzheimer’s disease and the general population.

a. Total cholesterol, LDL-cholesterol, and HDL-choles-terol.

b. Transaminase activity: GOT/AST, GPT/ALT, GGT.c. Brain hemodynamic parameters: Systolic velocity

(Sv), Diastolic velocity (Dv) and Mean velocity (Mv)d. Brain mapping activityAD: Alzheimer’s disease; FD: Frontal Delta; FT: Frontal

Theta; GP: General population; PD: Parietal Delta; PT: Parietal Theta; TD: Temporal Delta; TT: Temporal Theta; OA: Occipital Alpha.

Adapted from Cacabelos42

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study designs in identifying susceptibility loci and SNP variation18. Currently, over 10 million DNA sequence variations have been uncovered in the human genome18.

It has been observed that the genetic varia-tion rate (GVR) is higher in AD patients than in the general population12,14,19. Kary-otype anomalies appear in less than 3% of the cases, with no differences as compared with the general population. The variabil-ity of bigenic, trigenic, tetragenic and poly-genic genotypes of AD-related genes is cur-rently higher in AD than in controls, with an absolute genetic variation (AGV) of 40-

60% and a relative genetic variation (RGV) of 0.85-1.89% depending on the number of genes included in the haplotype-like clus-ter. Approximately, 40% of AD cases exhib-it a GVR higher than 1% as compared to controls when a trigenic cluster integrated by combinations of APOE+PSEN1+PSEN2 polymorphic variants is examined14. In-creased GVR in AD might indicate that the over-representation of a series of genes in-volved in brain maturation and in the main-tenance of higher activities of the CNS has surpassed a natural selection threshold (ex-cessive genome complexity, genomic over-diversification), constituting a Darwinian disadvantage which shortens life span in humans12,19. Recent observations support the contention that serial segmental dupli-cation events might have orchestrated pri-mate evolution by the generation of novel fusion/fission genes as well as potentially

by genomic inversions associated with de-creased recombination rates facilitating gene divergence20. Recent studies with alignments of 10,238 human genes have identified protein-coding sequences with an accelerated rate of base substitutions along the human lineage. Exons evolving at a fast rate in humans have a tendency to contain clusters of AT-to-GC biased substi-tutions. Accelerated exons occur in regions with elevated male recombination rates and exhibit an excess of non-synonymous substitutions relative to the genomic aver-age. These findings might indicate that a recombination-associated process (biased gene conversion) is driving fixation of CG alleles in the human genome. This process can lead to accelerated evolution in coding sequences and excess amino acid replace-ment substitutions, thereby contributing to positive or negative selection21. Genes that

Fig.3 5Pathogenetic mechanisms potentially involved in Alzheimer’s disease.(Adapted from http://www.genome.jp/. Kyoto University Bio-informatics Center, Kyoto, Japan; Kanehisa Laboratories).

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have undergone positive selection during species evolution are indicative of func-tional adaptations which drive differences between species. In has been shown that genes predicted to have been subject to positive selection pressure during human evolution are implicated in diseases such as epithelial cancers, schizophrenia, autoim-mune diseases and AD22.

Extensive molecular genetics studies car-ried out during the past two decades have demonstrated that most CNS disorders are multifactorial, polygenic/complex disor-ders in which hundreds of genes distrib-uted across the human genome might be involved12,23. Many of these genetic associa-tions could not be replicated in different settings and different populations due to a number of complex (methodological, technological) factors12,24. Furthermore, the same genomic defect can give rise to apparent diverse phenotypes, and different genomic defects can converge in an appar-ently common phenotype, this increasing the complexity of genomic studies.

The genetic defects identified in AD can be classified into 3 main categories: a. Mendelian or mutational defects in genes directly linked to AD, including (i) 32 mutations in the amyloid beta (Aβ)(ABP) precursor protein (APP) gene (21q21)(AD1); (ii) 165 mutations in the presenilin 1 (PSEN1) gene (14q24.3)(AD3); and (iii) 12 mutations in the presenilin 2 (PSEN2) gene (1q31-q42)(AD4)12,23,25. PSEN1 and PSEN2 are important determinants of γ-secretase activity responsible for prote-olytic cleavage of APP and NOTCH recep-tor proteins. Mendelian mutations are very rare in AD (1:1000). Mutations in exons 16 and 17 of the APP gene appear with a fre-quency of 0.30% and 0.78%, respectively, in AD patients. Likewise, PSEN1, PSEN2, and microtubule-associated protein Tau (MAPT)(17q21.1) mutations are present in less than 2% of the cases. Mutations in these genes confer specific phenotypic pro-files to patients with dementia: amyloidoge-neic pathology associated with APP, PSEN1 and PSEN2 mutations; and tauopathy asso-ciated with MATP mutations, representing the two major pathogenic hypotheses for AD12,26-28.

b. Multiple polymorphic variants of risk characterized in more than 200 different genes can increase neuronal vulnerabil-ity to premature death12 (Table 1). Among these genes of susceptibility, the apolipo-protein E (APOE) gene (19q13.2)

Biological parameters in patients with Alzheimer’s disease versus general population.

ParameterGeneral

populationAlzheimer’s

diseaseP value

Abnormal Ratein AD

Blood PressureSystolic (SBP) (mm Hg)Diastolic (DBP) (mm Hg)

127.46 ± 21.6076.49 ± 10.81

138.42 ± 20.4679.47 ± 10.34

<0.001<0.001

SBP>160: 17.92%DBP>85: 28.52

Glucose (mg/dL) 94.97 ± 23.10 101.02 ± 27.75 <0.001 Glucose>105: 25.89%

Cholesterol Total-Cholesterol (mg/dL)HDL-Cholesterol (mg/dL)LDL-Cholesterol (mg/dL)

210.19 ± 46.4852.76 ± 17.03136.54 ± 40.23

220.26 ± 45.5453.32 ± 14.22144.31 ± 40.02

<0.001<0.001<0.001

T-CHO>220: 50.15%HDL-CHO<45: 29.69%LDL-CHO>140: 47.18%

Triglycerides (mg/dL) 106.07 ± 72.70 114.09 ± 65.23 <0.001 TG>140: 23.25%

Transaminase acivity GOT/AST (IU/L) GPT/ALT (IU/L) GGT (IU/L)

22.15 ± 15.89 24.49 ± 20.1428.74 ± 36.11

22.21 ± 17.9223.66 ± 18.8030.84 ± 37.64

0.590.46<0.001

GOT/AST>30: 9.09%GPT/ALT>30: 18.91%GGT>30: 28.55%

Red Blod Cells (RBC) (x106/mm3)

4.66 ± 0.46 4.61 ± 0.44 <0.001 RBC<4: 7.58%

Hematocrit (Ht)(%) 42.01 ± 4.26 41.93 ± 4.22 0.60 Ht<35: 3.94%Ht>45: 21.04%

Hemoglobin (Hb)(g/dL) 14.07 ± 2.03 13.98 ± 1.38 0.24 Hb<12: 6.47%Hb>15: 22.45%

Iron (Fe)(µg/dL) 87.82 ± 40.81 86.79 ± 43.66 0.44 Fe<50: 12.04%

Ferritin (Fer)(ng/mL) 106.58 ± 126.64 127.11 ± 147.74 <0.001 Fer<30: 15.26%Fer>300: 8.09%

Folate (Fol)(ng/mL) 7.14 ± 4.20 7.03 ± 3.99 <0.01 Fol<3: 4.98%

Vitamin B12 (pg/mL) 493.62 ± 254.88 505.81 ± 289.74 0.89 B12<300: 19.17%

TSH (µIU/mL) 1.55 ± 1.99 1.50 ± 2.63 <0.001 TSH<1: 41.05%Tsh>5: 1.59%

Tiroxin (T4) 0.88 ± 0.35 0.91 ± 0.44 <0.001 T4<0.6: 3.05%T4>1.5: 1.14%

Hemodynamic Parameters in the Left Middle Cerebral Artery (LMCA)

Mean blood velocity (Mv) (cm/sec)Systolic blood velocity (Sv)(cm/sec)Diastolic blood velocity (Dv)(cm/sec)Pulsatility Index (Units)Resistance Index (Units)

51.58 ± 16.44

81.33 ± 24.82

33.70 ± 12.10

0.94 ± 0.230.58 ± 0.07

42.77 ± 12.86

69.11 ± 19.52

26.66 ± 9.03

1.01 ± 0.240.61 ± 0.08

<0.001

<0.001

<0.001

<0.001<0.001

Mv<40: 30.05%

Sv>60: 43.28%

Dv<25: 37.45%

PI>1: 63.60%RI>0.5>72.04%

Brain Mapping ActivityFrontal Delta (%)Parietal Delta (%)Temporal Delta (%)Frontal Theta (%)Parietal Theta (%)Temporal Theta (%)Occipital Alpha (%)

4.30 ± 2.053.69 ± 1.803.55 ± 2.072.82 ± 1.482.60 ± 1.392.36 ± 1.443.54 ± 3.68

5.21 ± 3.654.38 ± 3.284.43 ± 3.742.97 ± 1.892.78 ± 1.892.56 ± 1.892.72 ± 1.59

<0.001<0.001<0.0010.11<0.05<0.002<0.001

68.30%73.56%75.82%40.24%69.54%71.65%83.12%

Source: R. Cacabelos, EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine (2009)General Population: N=3301; Alzheimer’s disease: N=1364 (Females: 777; Males: 587); Race: Caucasians.

Adapted from Cacabelos42

Table 1

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(AD2) is the most prevalent as a risk fac-tor for AD, especially in those subjects harbouring the APOE-4 allele, whereas carriers of the APOE-2 allele might be pro-tected against dementia12. APOE-related pathogenic mechanisms are also associ-ated with brain aging and with the neuro-pathological hallmarks of AD. Other genes of this category include the following: AD5 (Alzheimer’s disease 5)( 12p11.23-q13.12), AD6 (10q24), AD7 (10p13), AD8 (20p), AD9 (19p13.2), AD10 (7q36), APBB2 (Amyloid beta A4 precursor protein-bind-ing, family B, member 2, FE65L1)(4p14), HFE (Hemochromatosis gene, HLA-H, HFE1, MVCD7)(6p21.3), eNOS3 (Nitric oxide synthase 3, endothelial cell)(7q36), PACIP1 (PAX transcription activation domain-interacting protein 1, PAXIP1L, PTIP)(7q36), PLAU (Plasminogen acti-vator, urokinase, URK)(10q24), SORL1 (Sortilin-related receptor, L(DLR class) A repeats-containing, LR11, SORLA)(11q23.2-q24.2), A2M (Alpha-2-macro-globulin)(12p13.3-p12.3), BLMH (Bleo-mycin hydrolase, BMH)(17q11.2), ACE

(Angiotensin I converting enzyme, dipep-tidyl carboxypeptidase-1, DCP1, ACE1, MVCD3)(17q23), MPO (Myeloperoxidase)(17q23.1), FOS (V-Fos FBJ murine osteo-sarcma viral oncogene homolog)(14q24.3), MTHFR (Methylenetetrahydrofolate re-ductase)(1p36.3), CETP (Cholesteryl ester transfer protein, Lipid transfer protein 1)(16q21), AGT (Angiotensinogen, Serpina 8)(1q42-q43), BDNF (Brain-derived neu-rotrophic factor)(11p13), CH25H (Cho-lesterol 25-hydroxylase)(10q23), CHRNB2 (Cholinergic receptor, nicotinic, beta poly-peptide-2, EFNL3)(1q21), CST3 (Cystatin C, ARMD11)(20p11.2), CTSD (Cathep-sin D, lysosomal aspartyl protease, CPSD, CLN10)(11p15.5), DAPK1 (Death-associ-ated protein kinase-1)(9q34.1), DHCR24 (24-dehydrocholesterol reductase, KIAA0018)(1p33-p31.1), IL1B (Interleu-kin-1, beta)(2q14), LMNA (Lamin A/C, LMN1, EMD2, FPLD, CMD1A, HGPS, LG-MD1B)(1q21.2), MYH13 (Myosin, heavy polypeptide 13, skeletal muscle)(17p13.1-p12), PCK1 (Phosphoenolpyruvate car-boxykinase-1)(20q13.31), PRNP (Prion protein)(20pter-p12), SORCS1 (SORCS receptor 1)(10q23.3), TFAM (Transcrip-tion factor A, mitochondrial, TCF6L2, TCF6L1, TCF6L3, MTTF1, TCF6)(10q21), TNK1 (Tyrosine kinase, nonreceptor, 1)(17p13.1), CALHM1 (Calcium homeosta-sis modulator 1, FAM26C)(10q24.33), and

some other candidate genes recently incor-porated to the AD-related gene pipeline, such as CLU (Clusterin (complement lysis inhibitor, SP-40,40; sulfated glycoprotein 2; testosterone-repressed prostate message-2; apolipoprotein J))(8p21-p12) and CR1 (Complement component (3b/4b) recep-tor-1)(1q32) and PICALM (Phosphati-dylinositol-binding clathrin assembly pro-tein)(11q14)12,23,25,29,30,31 (Table 1). One of the newest members of the AD-gene family is SORL1, a gene which encodes a mosaic protein with a domain structure which sug-gests it is a member of both the vacuolar protein sorting-10 (Vps10) domain-con-taining receptor family and the low density lipoprotein receptor (LDLR). Inherited variants of the SORL1 neuronal sorting receptor are associated with late-onset AD. Polymorphisms in two different clusters of intronic sequences within the SORL1 gene may regulate tissue-specific expression of SORL1, which directs trafficking of APP into recycling pathways. When SORL1 is underexpressed, APP is sorted into Aß-gen-erating compartments leading to amyloid accumulation in neuronal tissues32. As with many other potential AD-related genes, the association of SORL1 with AD32,33 could not be replicated in other stud-ies34. Another interesting gene is DHCR24 (3ß-hydroxysterol-δ-24-reductase) or Se-ladin-1, a key element in the cholester-ologenic pathway in which the DHCR24 enzyme catalyses the transformation of desmosterol into cholesterol35,36. Seladin-1 was originally identified as a gene whose expression was down-regulated in the AD brain, demonstrating a neuroprotective effect against neurodegeneration. Recent studies indicate that Seladin-1/DHCR24 is an LXR (liver X nuclear hormone recep-tor) target gene potentially involved in the regulation of lipid raft formation35. Anoth-er gene, with potential therapeutic interest as a tau kinase, might be the GSK3 gene. Analysis of the promoter and all 12 exons revealed that an intronic polymorphism (IVS2-68G>A) occurred at more than twice the frequency among patients with fronto-temporal dementia (10.8%) and patients with AD (14.6%) than in aged healthy sub-jects (4.1%). This is the first evidence that a gene known to be involved in tau phos-phorylation is associated with risk for pri-mary neurodegenerative dementias37. Pro-moter polymorphisms modulating HSPA5 expression might also increase susceptibil-ity to AD. Endoplasmic reticulum chaper-one heat shock 70 kDa protein 5 (HSPA5/GRP78) is known to be involved in APP metabolism and neuronal death in AD.

Fig.4 5Different polymorphic variants in genes distributed across the human genome are responsible for pharmacokinetic and pharmacodynamic variability through pharmacogenetic / pharmacogenomic mechanisms influencing drug efficacy and safety.

Genetic Polymorphisms

METABOLISM

Safety Efficacy

DISEASE

Pharmacokinetics Absortion Distribution Metabolism Excretion

Pharmacodynamics Receptors Ion channels Enzymes Proteins

Pathogenic mechanisms

TranscriptomicsProteomics

Metabolomics

DRUGSDDRRUUUGGGGSSSSS

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Locus Symbol Title/Gene MIM

1p32 ZFYVE9SARAMADHIP

Zinc finger, FYVE domain containing 9SMAD anchor for receptor activation MADH-interacting protein

603755

1p32 PRNPIP, PINT1 Prion protein-interacting protein 609917

1p33-p31.1 DHCR24, KIAA0018

24-dehydrocholesterol reductase 606418

1p34 LRP8APOER2

Low-density lipoprotein receptor-related protein 8

602600

1p36 AD7CNTP Alzheimer disease neuronal thread protein (ADNTP)

607413

1p36.3 MTHFR Methylenetetrahydrofolate reductase 236253104300

1q21 S100A S100 Calcium-binding protein A1 176940

1q21 CHRNB2, EFNL3 Cholinergic receptor, nicotinic, beta polypeptide-2

118507

1q21.2 LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B

Lamin A/C 150330

1q21-q23 APCS Serum amyloid P component 104770

1q23 NCSTNAPH2

Nicastrin 605254

1q25 SOAT1STATACAT

Acyl-CoA:Cholesterol acyltransferaseSterol O-acyltransferase 1

102642

1q31-q42 AD4PSEN2STM2

Presenilin-2 600759104300

1q32 CR1, C3BR Complement component (3b/4b) receptor-1

120620

1q42-q43 AGT, SERPINA8 Angiotensinogen 106150

Chr. 1 APH1A C. elegans anterior pharynx defective homolog

607629

2p14-p13 RTN4 NOGO Neurite outgrowth inhibitor (reticulon 4) 604475

2p25 ADAM17TACE

A desintegrin and metalloproteinase domain 17Tumor necrosis factor-alpha converting enzyme

603639

2q14 IL1A Interleukin-1-Alpha 147760

2q21.1 CSENDREAMKCNIP3

Calsenilin 604662

2q21.2 LRP1B Low density lipoprotein receptor-related protein 1B

608766

3q13.3 GSK3B Glycogen synthase kinase 3-beta 605004

3q26.1-q26.2

BCHE Butyrylcholinesterase 177400

3q26.2-qter APOD Apolipoprotein D 107740

3q32.3-q34 CREB1 cAMP response element-binding protein 123810

4p14 APBB2FE65L1

Amyloid beta-A4 precursor protein-binding, family B, member 2

602710

Locus Symbol Title/Gene MIM

5q15-q21 CAST Calpastatin 114090

5q31 APBB3FE65L2

Amyloid beta A4 precursor protein-binding, family B, member 3

602711

5q35.3 DBN1 Drebrin E 12660

6p21.3 AGERRAGE

Advance glycosylation end product-specific receptor

600214

6p21.3 HFE, HLA-H, HFE1, MVCD7

Hemochromatosis gene 235200

6p21.3 TNFA Tumor necrosis factor-aCachectin

191160

7p21 IL-6IFNB2

Interleukin-6Beta-2 interferon

147620

7q36 AD10 Alzheimer disease-10 609636

7q36 NOS3 Nitric Oxide Synthase-3 163729

7q36 PACIP1, PAXI-P1L, PTIP

PAX transcription activation domain-interacting protein 1

608254

8p21-p12 CLU, CLI, SGP2, TRPM2

Clusterin (complement lysis inhibitor, SP-40,40; sulfated glycoprotein 2; testosterone-repressed prostate messa-ge-2; apolipoprotein J)

185430

8p22 CTSBCPSB

Cathepsin BAmyloid precursor protein secretase

116810

9q13 APBA1X11MINT1LIN10

Amyloid beta-A4 precursor protein-binding, family A, member 1

602414

9q34 HSPA5, GRP78 Heat-shock 70kD protein-5 (glucose-regulated protein, 78kD)

138120

9q34.1 DAPK1 Death-associated protein kinase-1 600831

10p13 AD7 Alzheimer disease-7 606187

10q21 TFAM, TCF6L2, TCF6L1, TCF6L3, MTTF1, TCF6

Transcription factor A, mitochondrial 600438

10q23 CH25H Cholesterol 25-hydroxylase 604551

10q23.3 SORCS1 SORCS receptor 1 606283

10q23-q25 IDE Insulin-degrading enzyme 146680

10q24 AD6 Alzheimer disease-6 6,05526E+11

10q24 PLAUURK

Plasminogen activator, urokinase 191840

10q24.33 CALHM1, FAM26C

Calcium homeostasis modulator 1 612234

11p13 BDNF Brain-derived neurotrophic factor 113505

11p15 APBB1F65

Amyloid beta-A4 precursor protein-binding, family B, member 1

602709

11p15.1 SAA1 Serum amyloid A1 104750

11p15.5 CTSD, CPSD, CLN10

Cathepsin D (lysosomal aspartyl protease) 116840

11q14 PICALM, CALM, CLTH, LAP

Phosphatidylinositol-binding clathrin assembly protein

603025

Table 2

Selected human genes investigated as potential candidate genes associated with dementia and age-related neurodegenerative disorders

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Of the 3 major polymorphisms (-415G/A (rs391957), -370C/T (rs17840761), -180del/G (rs3216733)), the HSPA5-415G/A and -180del/G variants showed sig-nificant differences between AD cases and controls. Subjects harbouring the -415AA/-180GG genotype or the -415A/-180G allele might be less susceptible to develop AD37.

The rs5952C and rs1568566T alleles of the APOD rs5952T/C and rs1568566C/T vari-ants increase the risk for AD, whereas the rs5952T-rs1568566C haplotype reduces it38. ApoD is a lipoprotein associated glycopro-tein which is increased in the hippocam-pus and CSF of AD patients39. CALHM1 encodes a multipass transmembrane glyco-

protein that controls cytosolic Ca2+ concen-trations and Aβ levels. The CALHM1 P86L polymorphism (rs2986017) has been asso-ciated with AD40, but this association could not be replicated in other studies.

Harold et al31 undertook a two-stage genome-wide association study (GWAS)

Locus Symbol Title/Gene MIM

11q23.2-q24.2

SORL1, LR11, SORLA

Sortilin-related receptor, L(DLR class) A repeats-containing

602005

11q23.3 BACE1BACE

Beta-site amyloid beta A4 precursor protein-cleaving enzymeBeta-secretaseMemapsin-2

604252

11q24 APLP2 Amyloid beta-A4 precursor-like protein 2 104776

12p11.23-q13.12

AD5 Familial AD-5 602096

12p12.3-p12.1

IAPPIAPDAP

Islet amyloid polypeptideAmylinDiabetes-associated peptide

147940

12p13.3-p12.3

A2M Alpha-2-Macroglobulin 103950

12q13.1-q13.3

LRP1A2MR

Low density lipoprotein-related protein-1Alpha-2-macroglobulin receptor

107770

14q24.3 FOS FBJ murine osteosarcoma viral (v-fos) oncogene homologOncogene Fos

164810

14q24.3 AD3PSEN1

Presenilin-1 104311

14q32.1 SERPINA3AACTACT

Alpha-1-antichymotrypsin 107280

14q32.1 CYP46CYP46A1

Cytochrome P450Family 46, Subfamily APolypeptide 1Cholesterol 24-hydrolase

604087

Chr. 15 APH1B Homolog of C. elegans anterior pharynx defective 1B

607630

15q11-q12 APBA2X11L

Amyloid beta-A4 precursor protein-binding, family A, member 2

602712

16q21 CETP, HDLCQ10 Cholesteryl ester transfer protein, plasma 118470

16q22 APPBP1 Amyloid beta precursor protein-binding protein 1

603385

17p13.1 TNK1 Tyrosine kinase, nonreceptor, 1 608076

17p13.1-p12

MYH13 Myosin, heavy polypeptide 13, skeletal muscle

603487

17q11.2 BLMHBMH

Bleomycin hydrolase 602403

17q21 STH Saitohin 607067

17q21.1 MAPTMTBT1DDPACMST

Macrotubule-associated protein tau 157140600274168610172700601104

Locus Symbol Title/Gene MIM

17q21-q22 GPSC Familial progressive subcortical gliosis 221820

17q22-q23 APPBP2PAT1

Amyloid beta precursor protein-binding protein 2

605324

17q23 ACE ACE1DCP1

Angiotensin I converting enzymeDipeptidyl carboxipeptidase-1

106180104300

17q23.1 MPO Myeloperoxidase 254600

17q24 FALZFAC1

Fetal Alzheimer antigen 601819

18q11.2-q12.2

TTRPALB

TransthyretinPrealbumin

176300

19p13.2 NOTCH3CADASILCASIL

Drosophila Notch 3 homolog 600276

19p13.2 AD9 Alzheimer disease-9 608907

19p13.3-p13.2

ICAMCD54BB2

Intercellular adhesion molecule 1 147840

19p13.3 APBA3X11L2

Amyloid beta-A4 precursor protein binding, family A, member 3

604262

19q13.12 PEN2 Presenilin enhancer 2 607632

19q13.2 APOE Apolipoprotein E 107741

19q13.2 APOC1 Apolipoprotein C-I 107710

19cen-q13.2

AD2 Alzheimer disease-2 104310

19cen-q13.2

APLP1 Amyloid beta-A4 precursor-like protein 1 104775

19q31-qter APPL1 Amyloid beta-A4 precursor protein-like 1 104740

20p AD8 Alzheimer disease-8 607116

20p11.2 CST3 Cystatin 3 604312

20p11.2 CST3 Cystatin C 604312

20pter-p12 PRNP, PRIP Prion protein (p27-30) 176640

20q13.31 PCK1 Phosphoenolpyruvate carboxykinase-1 (soluble)

261680

21q21 AD1APPAAA CVAP

Amyloid beta (A4) precursor proteinAmyloid of aging and Alzheimer diseaseCerebrovascular amyloid peptideProtease nexin II

104760

21q22.3 BACE2ALP56DRAP

Beta-site amyloid beta A4 precursor protein-cleaving enzyme 2Down syndrome-region aspartic protease

605668

22q11 RTN4R, NOGORHN

NOGO receptor (reticulon 4 receptor)Humanin

605566606120

Continued, Table 2

Adapted from Cacabelos12,42, Cacabelos and Takeda9 and OMIM23

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of AD involving over 16,000 individuals, and found association with SNPs at two loci not previously associated with the disease, at the CLU (Clusterine, APOJ) gene (rs11136000) and 5’ to the PICALM gene (rs3851179). In another GWAS with patients from France, Belgium, Finland, Italy and Spain, Lambert et al30 found association with CLU and with the CR1 gene, encoding the complement compo-nent (3b/4b) receptor 1, on chromosome 1 (rs6656401).

c. Diverse mutations located in mito-chondrial DNA (mtDNA) through hetero-plasmic transmission can influence aging and oxidative stress conditions, conferring phenotypic heterogeneity12,41.

Although APP and PSEN mutations are considered causative factors for AD, the to-tal number of mutations identified in the APP, PSEN1 and PSEN2 genes account for less than 3% of the cases with AD, clearly indicating that neurodegeneration associ-ated with AD pathogenesis cannot be ex-clusively attributed to APP/PSEN-related cascades (amyloid hypothesis). Alterations in the ubiquitin-proteasome system and biochemical disarray in the chaperone machinery are alternative and/or comple-mentary pathogenic events potentially leading to defects in protein synthesis, folding, and degradation with subsequent conformational changes, aggregation, and accumulation in cytotoxic deposits10,12. A more plausible explanation would seem to

be that multiple susceptibility SNPs with a very subtle genetic variation cooperatively contribute, in concert with environmental factors and concomitant CNS vulnerability, to premature neurodegeneration in de-mentia.

We have compared the distribution and frequency of major polymorphic variants of different genes potentially associated with AD (i.e. APOE, PSEN1, A2M-V1001, A2M-I/D, ACE, FOS, AGT-235, AGT-174, eNOS3-E298D, eNOS3-27bpTR, CETP, MTHRF) in the general population, in adults (>45 years) with no family history of dementia, and in patients with dementia, and could not find any significant differ-ences among the three groups except

Gene Polymor-phism

General Population

Adults>45 yrs NFHD

Alzheimer’s disease

N % N % N %

APOE APOE-2/2 6 0,21 1 0,13 1 0,11

APOE-2/3 228 7,94 55 7,37 73 7,68

APOE-2/4 38 1,32 7 0,94 14 1,47

APOE-3/3 1938 67,51 534 71,58 577 60,74

APOE-3/4 600 20,9 136 18,24 250 26,32

APOE-4/4 61 2,12 13 1,74 35 3,68

2871 100 746 100 950 100

PSEN1 PSEN1-1/1 449 27,26 139 25,27 213 27,95

PSEN1-1/2 974 59,14 345 62,73 435 57,09

PSEN1-2/2 224 13,6 66 12 114 14,96

1647 100 550 100 762 100

A2M-V100I

A2M-A/A 769 46,89 251 46,48 359 48,32

A2M-A/G 728 44,39 238 44,07 325 43,74

A2M-G/G 143 8,72 51 9,45 59 7,94

1640 100 540 100 743 100

A2M-I/D A2M-D/D 30 1,81 12 2,22 10 1,33

A2M-I/D 434 26,24 143 26,38 201 26,8

A2M-I/I 1190 71,95 387 71,4 539 71,87

1654 100 542 100 750 100

ACE ACE-D/D 839 35,96 261 38,33 304 34,35

ACE-I/D 1137 48,74 333 48,9 430 48,59

ACE-I/I 357 15,3 87 12,77 151 17,06

2333 100 681 100 885 100

FOS FOS-A/A 733 71,1 252 70 314 71,85

FOS-A/B 259 25,12 94 26,11 107 24,49

FOS-B/B 39 3,78 14 3,89 16 3,66

1031 100 360 100 437 100

Gene Polymor-phism

General Population

Adults>45 yrs NFHD

Alzheimer’s disease

N % N % N %

AGT-235 AGT-M/M 230 10,1 70 10,45 78 8,97

AGT-M/T 1560 68,48 454 67,76 600 69,03

AGT-T/T 488 21,42 146 21,79 191 22

2278 100 670 100 869 100

AGT-174 AGT-M/M 13 0,57 6 0,9 4 0,46

AGT-M/T 477 20,93 137 20,45 194 22,32

AGT-T/T 1789 78,5 527 78,65 671 77,22

2279 100 670 100 869 100

eNOS3-E298D

eNOS3-G/A 4 0,24 3 0,58 0 0

eNOS3-G/G 647 39,33 198 38,37 241 38,94

eNOS3-G/T 776 47,17 250 48,45 299 48,3

eNOS3-T/T 218 13,26 65 12,6 79 12,76

1645 100 516 100 619 100

eNOS3-27bpTR

eNOS3-A/A 45 2,74 14 2,72 12 1,94

eNOS3-A/B 408 24,82 123 23,88 164 26,54

eNOS3-B/B 1191 72,44 378 73,4 442 71,52

1644 100 515 100 618 100

CETP CETP-B1/B1 361 36,54 108 40,6 116 34,32

CETP-B1/B2 497 50,3 124 46,62 173 51,18

CETP-B2/B2 130 13,16 34 12,78 49 14,5

988 100 266 100 338 100

MTHFR MTHFR-C/C 407 42,48 103 40,71 133 40,43

MTHFR-C/T 417 43,53 109 43,08 149 45,29

MTHFR-T/T 134 13,99 41 16,21 47 14,28

958 100 253 100 329 100

Source: R. Cacabelos 42

Distribution and frequency of polymorphic variants of selected Alzheimer’s disease-associated genes in the general population, in adults (>45 years) with no family history of dementia, and in patients with Alzheimer’s disease.

Table 3

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in the case of the APOE gene which ex-hibits a clear accumulation of APOE-3/4 and APOE-4/4 genotypes (overload of the APOE-4 allele) in AD cases42 (Table 3). If we consider that a genetic variation higher than 2% could be of significant value, then several polymorphisms clearly differ in AD as compared with the other two population clusters, including the PSEN1-1/2, ACE-D/D, ACE-I/I, CEPT-B1/B1, and MTHFR-T/T polymorphisms42 (Table 3).

It is also likely that defective functions of genes associated with longevity may influ-ence premature neuronal survival, since neurons are potential pacemakers defin-ing life span in mammals9,12. All these ge-netic factors may interact in genetic net-works which are still unknown, leading to a cascade of pathogenic events charac-terized by abnormal protein processing and misfolding with a subsequent accu-mulation of abnormal proteins (confor-mational changes), ubiquitin-proteasome system dysfunction, excitotoxic reactions, oxidative and nitrosative stress, mitochon-drial injury, synaptic failure, altered metal homeostasis, dysfunction of axonal and dendritic transport, and chaperone misop-eration6-10,12. These pathogenic events may exert an additive effect, converging in fi-nal pathways leading to premature neu-ronal death. Some of these mechanisms are common to several neurodegenerative disorders which differ depending upon

the gene(s) affected and the involvement of specific genetic networks, together with cerebrovascular factors, epigenetic factors (DNA methylation) and environmental conditions (nutrition, toxicity, social fac-tors, etc)6-13 (Fig.3). The higher the num-ber of genes involved in AD pathogenesis, the earlier the onset of the disease, the faster its clinical course, and the poorer its therapeutic outcome6-13.

Functional genomicsIt is very likely that over 80% of the genes which conform the structural architecture of the human genome are expressed in the brain in a time-dependent manner along the lifespan. High throughput mi-croarray gene expression profiling is an effective approach for the identification of candidate genes and associated molecu-lar pathways implicated in a wide variety of biological processes or disease states. The cellular complexity of the CNS (with 103 different cell types) and synapses (with each of the 1011 neurons in the brain hav-ing around 103-104 synapses with a com-plex multiprotein structure integrated by 103 different proteins) requires a very powerful technology for gene expression profiling, which is still in its very early stag-es and is not devoid of technical obstacles and limitations43. Transcripts of 16,896 genes have been measured in different CNS regions. Each region possesses its own unique transcriptome fingerprint which is independent of age, gender and energy in-take. Less than 10% of genes are affected by age, diet or gender, with most of these changes occurring between middle and old age. Gender and energy restriction

have robust influences on the hippocam-pal transcriptome of middle-aged animals. Prominent functional groups of age- and energy-sensitive genes are those encod-ing proteins involved in DNA damage re-sponses, mitochondrial and proteasome functions, cell fate determination and synaptic vesicle trafficking. The systematic transcriptome dataset provides a window into mechanisms of neuropathogenesis and CNS vulnerability44.

Functional genomics studies have demon-strated the influence of many genes on AD pathogenesis and phenotype expression. The study of genotype-phenotype correla-tions is essential for the evaluation of the actual impact of specific polymorphic vari-ants of a particular gene on the clinical manifestation of the disease and/or bio-logical markers reflecting the disease con-dition or different biological states of the individual. It has been demonstrated that mutations in the APP, PSEN1, PSEN2, and MAPT genes give rise to well-characterized differential neuropathological and clinical phenotypes of dementia12,23,25. APP muta-tions are associated with AD1, early-onset progressive autosomal recessive dementia, early-onset AD with cerebral amyloid an-giopathy, and hereditary amyloidosis with cerebral haemorrhage Dutch type, Italian type, or Iowa type. PSEN1 mutations are associated with the phenotypes of familial AD3, familial AD3 with unusual plaques, fa-milial AD with spastic paraparesis and un-usual plaques, familial AD with parapare-sias and apraxia, frontotemporal dementia, Pick’s disease, and dilated cardiomyopathy. MAPT mutations are associated with fron-totemporal dementia, frontotemporal de-mentia with parkinsonism, Pick’s disease, progressive supranuclear palsy, progressive atypical supranuclear palsy, and tauopathy and respiratory failure12,23.

Transgenic animals also reproduce to some extent the neuropathological hallmarks of AD in a sequential manner. The triple trans-genic mouse model of AD (3xTg-AD) har-bours 3 AD-related loci: human PS1M146V, human APPswe, and human MAPTP301L. These animals develop both amyloid plaques and NFT-like pathology in a pro-gressive and age-dependent manner in hip-pocampus, amygdala, and cerebral cortex, the main foci of human AD neuropathol-ogy. The evolution of AD-related transgene expression, amyloid deposition, tau phos-phorylation, astrogliosis, and microglia ac-tivation throughout the hippocampus, en-torhinal cortex, primary motor cortex, and

Fig.5 5Psychotropic drugs acting as major substrates for enzymatic products of CYP genes. (Adapted from Cacabelos 42)

CYP1A2 CYP2B6 CYP2C19 CYP2D6 CYP3A40

20

40

60

80

100(%)

Antidepressants

Neuroleptics

Benzodiazepines

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amygdala over a 26-month period has been immunohistochemically documented. In-tracellular Aß accumulation is the earliest of AD-related pathologies to be detect-able, followed temporally by phospho-tau, extracellular Aß, and finally paired helical filament and NFT pathology45. In the same model, a decrease in neurogenesis directly associated with the presence of amyloid plaques and an increase in the number of Aß containing neurons in the hippocam-pus has been demonstrated46.

Different APOE genotypes also confer spe-cific phenotypic profiles to AD patients. Some of these profiles may add risk or ben-efit when the patients are treated with con-ventional drugs, and in many instances the clinical phenotype demands the adminis-tration of additional drugs which increase the complexity of therapeutic protocols. From studies designed to define APOE-related AD phenotypes6-16,28,42,47-52, several confirmed conclusions can be drawn: (i) the age-at-onset is 5-10 years earlier in ap-proximately 80% of AD cases harbouring the APOE-4/4 genotype; (ii) the serum levels of ApoE are lowest in APOE-4/4, in-termediate in APOE-3/3 and APOE-3/4, and highest in APOE-2/3 and APOE-2/4; (iii) serum cholesterol levels are higher in APOE-4/4 than in the other genotypes; (iv) HDL-cholesterol levels tend to be low-er in APOE-3 homozygotes than in APOE-4 allele carriers; (v) LDL-cholesterol levels are systematically higher in APOE-4/4 than in any other genotype; (vi) triglycer-ide levels are significantly lower in APOE-4/4; (vii) nitric oxide levels are slightly lower in APOE-4/4; (viii) serum Aß levels do not differ between APOE-4/4 and the other most frequent genotypes (APOE-3/3, APOE-3/4); (ix) blood histamine lev-els are dramatically reduced in APOE-4/4 as compared with the other genotypes; (x) brain atrophy is markedly increased in APOE-4/4>APOE-3/4>APOE-3/3; (xi) brain mapping activity shows a significant increase in slow wave activity in APOE-4/4 from early stages of the disease; (xii) brain haemodynamics, as reflected by reduced brain blood flow velocity and increased pulsatility and resistance indices, is signifi-cantly worse in APOE-4/4 (and in APOE-4 carriers, in general, as compared with APOE-3 carriers); (xiii) lymphocyte apop-tosis is markedly enhanced in APOE-4 car-riers; (xiv) cognitive deterioration is faster in APOE-4/4 patients than in carriers of any other APOE genotype; (xv) occasion-ally, in approximately 3-8% of the AD cases, the presence of some dementia-

PathogenicMechanism

Therapeutic Strategy

Genomic disarrayMonogenic-relatedPolygenic-related

Gene therapyRNAi

β-amyloid deposition β-secretase inhibitorsγ-secretase inhibitorsα-secretase activatorsAβ-fibrillization and aggregation inhibitorsAmyloid ImmunotherapyCopper chelating agentsSolubilizers of Aβ aggregatesAPP Production inhibitorsAβ selective regulators

Tau pathology Phosphatase activatorsGSK-3 inhibitorsCdk5 inhibitorsP38 inhibitorsJNK inhibitors

Apoptosis Caspase inhibitorsNeurotrophic agents

Neurotransmission deficits

AcetylcholineEnzymes

Muscarinic receptors

Nicotinic receptors

GABA

GlutamateNMDAAMPA

DopamineNoradrenalineHistamineSerotonin

Acetylcholine-release stimulantAcetylcholine reuptake inhibitorCholinesterase inhibitorsCholine-acetyl-transferase stimulantMuscarinic agonistsMuscarinic antagonistsNicotinic agonists GABA modulatorsInverse GABA-receptor agonistGlutamate agonistsNMDA antagonistsAmpakinesDopamine reuptake inhibitorsAdrenoreceptor modulatorsHistamine H3 antagonists5HT3 receptor agonist5HT1A receptor agonist5HT6 antagonistSerotonin stimulant

Neurotrophic deficit Neurotrophic agentsNGF agonistsGrowth factorsSynthetic neuropeptides

Neuronal loss Neuronal stem cellsGrowth factorsNeurite outgrowth activatorsSynaptogenesis activatorsNogo inhibitors

PathogenicMechanism

Therapeutic Strategy

Neuronal loss MOP inhibitorsGSK-3 inhibitorsJNK inhibitorsP38 inhibitors

Neuroinflammation Cyclooxygenase-1 inhibitorsCyclooxygenase-2 inhibitorsComplement activation inhibitorsP38 inhibitorsCaspase-1 inhibitorseNOS inhibitorsPPARα agonistsPPARγ agonistsNovel NSAIDsCytokine inhibitors

Oxidative stress AntioxidantsCaspase inhibitorsAntioxidating enzyme enhancers

Excitotoxic reactions NMDA antagonistsAmpakinesModulators of glutamate transporters

Calcium dysmetabolism

Calcium channel blockers

Neuronal hypometabolism

PPARγ agonistsGSK-3 inhibitors

Lipid dysfunction HMG-CoA reductase inhibitorsPPARγ agonistsNovel biomarine lipoproteins

Cerebrovascular dysfunction

Vasoactive substancesNO inhibitorsHIF inhibitorsDandrolene-related agentsNovel lipoproteinsLiver X receptor agonists

Neuronal dysfunction associated with nutritional deficiency

NutrigenomicsNutraceuticalsBrain metabolic enhancers

Other pathogenic mechanisms

Estrogen agonistsMAO-B inhibitorsSomatostatin stimulantsInsulin sensitizers ImmunostimulantsMAP kinase inhibitorsProlyl-endopeptidase inhibitorsAnti-neurodegenerative agentsImmunotrophinsEndogenous nucleotidesNeurotrophic AntibiotherapyBenzodiazepine partial inverse agonistOthers

Adapted from Cacabelos42

Table 4

Potential therapeutic strategies in Alzheimer disease and dementia.

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Personalized Medicine of Dementia

related metabolic dysfunctions (e.g. iron, folic acid, vitamin B12 deficiencies) ac-cumulate more in APOE-4 carriers than in APOE-3 carriers; (xvi) some behavioral disturbances (bizarre behaviors, psychotic symptoms), alterations in circadian rhythm patterns (e.g., sleep disorders), and mood disorders (anxiety, depression) are slightly more frequent in APOE-4 carriers; (xvii) aortic and systemic atherosclerosis is also more frequent in APOE-4 carriers; (xviii) liver metabolism and transaminase activ-ity also differ in APOE-4/4 with respect to other genotypes; (xix) blood pressure (hy-pertension) and other cardiovascular risk factors also accumulate in APOE-4; and (xx) APOE-4/4 are the poorest respond-ers to conventional drugs. These 20 major phenotypic features clearly illustrate the biological disadvantage of APOE-4 ho-mozygotes and the potential consequenc-es that these patients may experience when they receive pharmacological treat-ment6-16,28,42,47-54.

Therapeutic Strategies

Modern therapeutic strategies in AD are addressed to interfering with the main pathogenic mechanisms potentially in-volved in AD (Table 4). Major pathogenic events (drug targets) and their respective therapeutic alternatives include the fol-lowing: genetic defects, β-amyloid depo-sition, tau-related pathology, apoptosis, neurotransmitter deficits, neurotrophic deficits, neuronal loss, neuroinflamma-tion, oxidative stress, calcium dysmetabo-lism, neuronal hypometabolism, lipid metabolism dysfunction, cerebrovascular dysfunction, neuronal dysfunction associ-ated with nutritional and/or metabolic deficits, and a miscellany of pathogenic mechanisms potentially manageable with diverse classes of chemicals or biopharma-ceuticals6-16,28,42,47,52(Table 4). Since the early 1980s, the neuropharmacology of AD was dominated by the acetylcholinesterase in-hibitors, represented by tacrine, donepe-zil, rivastigmine, and galantamine2,3,55. Me-mantine, a partial NMDA antagonist, was introduced in the 2000s for the treatment of severe dementia56; and the first clinical trials with immunotherapy, to reduce amy-loid burden in senile plaques, were with-drawn due to severe ADRs57. During the past few years no relevant drug candidates have been postulated for the treatment of AD, despite the initial promises of β- and γ-secretase inhibitors7,10,42 (Table 4).

1*/1*1*xN/1*

1*/2*1*/3*1*/4*1*/5*1*/6*

1*xN/2*1*xN/4*1*xN/5*

3*/3*3*/4*3*/6*4*/4*4*/5*4*/6*5*/5*5*/6*

0 10 20 30 40 50 60

GP-FGP-MAD-FAD-M

Females % Males % AD-F % AD-M %*1/*1 488 57,75147929 459 57,9545455 208 56,5217391 150 54,3478261*1xN/*1 50 5,917159763 50 6,31313131 20 5,43478261 19 6,88405797*1/*2 1 0,118343195 0 0 0 0 0 0*1/*3 18 2,130177515 15 1,89393939 9 2,44565217 5 1,8115942*1/*4 196 23,19526627 177 22,3484848 87 23,6413043 66 23,9130435*1/*5 32 3,786982249 27 3,40909091 11 2,98913043 8 2,89855072*1/*6 10 1,183431953 9 1,13636364 6 1,63043478 7 2,53623188*1xN/*2 0 0 1 0,12626263 0 0 0 0*1xN/*4 12 1,420118343 15 1,89393939 8 2,17391304 5 1,8115942*1xN/*5 1 0,118343195 0 0 1 0,27173913 0 0*3/*3 1 0,118343195 0 0 1 0,27173913 0 0*3/*4 3 0,355029586 3 0,37878788 1 0,27173913 0 0*3/*6 1 0,118343195 0 0 0 0 0 0*4/*4 21 2,485207101 21 2,65151515 12 3,26086957 12 4,34782609*4/*5 4 0,473372781 8 1,01010101 2 0,54347826 2 0,72463768*4/*6 1 0,118343195 4 0,50505051 0 0 1 0,36231884*5/*5 3 0,355029586 3 0,37878788 2 0,54347826 0 0*5/*6 3 0,355029586 0 0 0 0 1 0,36231884

845 100 792 100 368 100 276 100

F(%)

CYP2D6 Genotypes: Gender-Related DifferencesGeneral Population vs Alzheimer’s disease

*1/*1*1xN/*1

*1/*2*1/*3*1/*4*1/*5*1/*6

*1xN/*2*1xN/*4*1xN/*5

*3/*3*3/*4*3/*6*4/*4*4/*5*4/*6*5/*5*5/*6

0 10 20 30 40 50 60

General PopulationAlzheimer's Disease

CYP2D6 N % AD %*1/*1 947 57,84972511 358 55,5900621*1xN/*1 100 6,108735492 39 6,05590062*1/*2 1 0,061087355 0 0*1/*3 33 2,015882712 14 2,17391304*1/*4 373 22,78558338 153 23,757764*1/*5 59 3,60415394 19 2,95031056*1/*6 19 1,160659743 13 2,01863354*1xN/*2 1 0,061087355 0 0*1xN/*4 27 1,649358583 13 2,01863354*1xN/*5 1 0,061087355 1 0,1552795*3/*3 1 0,061087355 1 0,1552795*3/*4 6 0,36652413 1 0,1552795*3/*6 1 0,061087355 0 0*4/*4 42 2,565668907 24 3,72670807*4/*5 12 0,733048259 4 0,62111801*4/*6 5 0,305436775 1 0,1552795*5/*5 3 0,183262065 2 0,31055901*5/*6 6 0,36652413 1 0,1552795

1637 100 644 100F(%)

CYP2D6 GenotypesGeneral Population vs Alzheimer’s disease

General Population Alzheimer's Disease0

10

20

30

40

50

60

70Frequency (%)

Extensive MetabolizerIntermediate MetabolizerPoor MetabolizerUltra-Rapid Metabolizer

CYP2D6 PhenotypesMetabolizer N %EM 973 59,5107034IM 487 29,7859327PM 73 4,4648318UM 102 6,23853211

1635 100

Metabolizer AD %EM 372 57,7639752IM 200 31,0559006PM 34 5,27950311UM 38 5,90062112

644 100

Fig.6 5Distribution and frequency of CYP2D6 genotypes and phenotypes in Alzheimer’s disease and in the general population. Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos42)

Fig.7 5Sex-Related differences in the distribution and frequency of CYP2D6 genotypes in patients with Alzheimer’s disease and in the general population. Source: R. Cacabelos. EuroEspes Biomedical Research CenterInstitute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos42)

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Pharmacogenomics

Pharmacogenetics/Pharmacogenom-ics relates to the application of genomic technologies, such as genotyping, gene sequencing, gene expression, genetic epi-demiology, transcriptomics, proteomics, metabolomics and bioinformatics, to drugs in clinical development and on the mar-ket, applying the large-scale systematic ap-proaches of genomics to speed up the dis-covery of drug response markers, whether they act at the level of drug target, drug metabolism, or disease pathways8-11,42,58.

The potential implications of pharmacog-enomics in clinical trials and molecular therapeutics is that a particular disease could be treated according to genomic and biological markers, selecting medica-tions and diseases which are optimised for individual patients or clusters of patients with a similar genomic profile. For many medications, interindividual differences are mainly due to SNPs in genes encod-ing drug metabolising enzymes, drug transporters, and/or drug targets (e.g., genome-related defective enzymes, recep-tors and proteins, which alter metabolic

pathways leading to disease phenotype expression).

The application of these procedures to CNS disorders is an extremely difficult task, since most neuropsychiatric diseases are complex disorders in which many different genes might be involved4. In addition, it is very unlikely that a single drug be able to reverse the multifactorial mechanisms associated with neuronal dysfunction in most CNS pro-cesses with a complex phenotype affecting mood, personality, behaviour, cognition, and functioning. This heterogeneous

Table 5

DrugsPharmacological

CategoryMajor

SubstrateMinor

SubstrateInhibitors

OtherGenes

Amitriptyline Tricyclic antidepressantTertiary amineBenzodiazepine

CYP2D6 CYP1A2,CYP2B6,CYP2C8/9,CYP2C19,CYP3A4

CYP1A2,CYP2C8/9,CYP2C19,CYP2D6CYP2E1

ABCB1,ADRA1,GNB3,GNAS1,KCNE2,SCN5A,TNF-A

Amoxapine Tricyclic antidepressantSecondary amine

CYP2D6 ADRA1, GnB3GNAS1

Bupropion AntidepressantDopamine-reuptake inhibitor

CYP2B6 CYP1A2,CYP2A6,CYP2C8/9,CYP2D6,CYP2E1,CYP3A4

CYP2D6

Citalopram AntidepressantSelective serotonin reuptake inhibitor

CYP2C19CYP3A4

CYP2D6 CYP1A2,CYP2B6,CYP2C19,CYP2D6

GNB3,GNAS1,HTR2A,MAOA,SLC6A4

Clomipramine Tricyclic AntidepressantTertiary amine

CYP1A2CYP2C19CYP2D6

CYP3A4 CYP2D6 GNB3GNAS1

Desipramine Tricyclic AntidepressantSecondary amine

CYP2D6 CYP1A2 CYP2A6,CYP2B6,CYP2D6,CYP2E1,CYP3A4

Doxepin Tricyclic AntidepressantTertiary amine

CYP1A2CYP2D6CYP3A4

ABCB1GNB3GNAS1

Duloxetin AntidepressantSerotonin/Norepinephrine Reuptake Inhibitor

CYP1A2CYP2D6

CYP2D6

Escitalopram AntidepressantSelective Serotonin Reuptake Inhibitor

CYP2D6CYP3A4

CYP2D6 GNB3,GNAS1,,HTR2A,SLC6A4

Fluoxetine AntidepressantSelective Srotonin Reuptake Inhibitor

CYP2C8/9CYP2D6

CYP1A2,CYP2B2,CYP2C19,CYP2E1CYP3A4

CYP1A2,CYP2B2,CYP2C8/9,CYP2C19,CYP2D6,CYP3A4

GNB3,GNAS1,HTR2A,SLC6A4,MAOA

Fluvoxamine AntidepressantSelective Serotonin Reuptake Inhibitor

CYP1A2CYP2D6

CYP1A2,CYP2B6CYP2C8/9,CYP2C19CYP2D6,CYP3A4

Imipramine Tricyclic AntidepressantTertiary Amine

CYP2C19CYP2D6

CYP1A2,CYP2B6CYP3A4

CYP1A2,CYP2C19CYP2D6,CYP2E1

ABCB1,ADRA1,GNB3,GNAS1KCNE2,SCN5A

Maprotiline Tetracyclic Antidepressant CYP2D6 ABCB1

Mirtazapine AntidepressantAlpha-2 antagonist

CYP1A2CYP2D6CYP3A4

CYP2C8/9 CYP1A2CYP3A4

ADRA1GNB3GNAS1

Antidepressants metabolized via enzymes of the CYP gene family and other gene-related enzymes.

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clinical picture usually requires the utiliza-tion of different drugs administered simul-taneously. This is particularly important in the elderly population. In fact, the average number of drugs taken by patients with de-mentia ranges from 6 to over 10 per day depending upon their physical and mental conditions. Nursing home residents receive, on average, 7-8 medications each month, and over 30% of residents have monthly drug regimes of 9 or more medications, including (in descending order) analge-sics, antipyretics, gastrointestinal agents, electrolytic and caloric preparations, CNS agents, anti-infective agents, and cardiovas-cular agents59. In population-based studies over 35% of patients older than 85 years are moderate or chronic antidepressant users60. Polypharmacy, drug-drug interac-tions, adverse reactions, and non-compli-ance are substantial therapeutic problems

in the pharmacological management of elderly patients61, adding further compli-cations and costs to the patients and their caregivers. More than 25% of elderly indi-viduals receive at least one of more than 30 potentially inappropriate medications in 10 health maintenance organizations (HMOs) of the USA62. Although drug effect is a complex phenotype which depends on many factors, it is estimated that genetics accounts for 20% to 95% of variability in drug disposition and pharmacodynamics63. Under these circumstances, therapeutics optimisation is a major goal in neuropsy-chiatric disorders and in the elderly popula-tion, and novel pharmacogenetic and phar-macogenomic procedures may help in this endeavour4-10,28,42,47-52,64.

The pharmacogenomic outcome depends upon many different determinant factors

including (i) genomic profile, (ii) disease phenotype, (iii) concomitant pathology, (iv) genotype-phenotype correlations, (v) nutritional conditions, (vi) age and gen-der, (vii) pharmacological profile of the drugs, (viii) drug-drug interactions, (ix) gene expression profile, (x) transcriptomic cascade, (xi) proteomic profile, and (xii) metabolomic networking (Fig.1). The dis-section and further integration of all these factors is of paramount importance for the assessment of the pharmacogenomic out-come in terms of safety and efficacy (Fig.4).

The vast majority of drugs in current use and many psychotropics (Tables 5-7; Fig.5) are metabolised by enzymes known to be genetically variable, including: (a) esterases: butyrylcholinesterase, paraoxo-nase/arylesterase; (b) transferases: N-acetyltransferase, sulfotransferase, thiol

DrugsPharmacological

CategoryMajor

SubstrateMinor

SubstrateInhibitors

OtherGenes

Moclobemide AntidepressantReversible MAO inhibitor

CYP2C19CYP2D6

CYP1A2,CYP2C19CYP2D6

MAOA

Nefazodone AntidepressantSerotonin Reuptake Inhibitor/Antagonist

CYP2C8/9CYP3A4

CYP1A2,CYP2B6CYP2D6,CYP3A4

ABCB1,ADRA1,GNB3,GNAS1

Nortriptyline Tricyclic AntidepressantSecondary Amine

CYP2D6 CYP1A2,CYP2C19CYP3A4

CYP2D6CYP2E1

ABCB1,ADRA1,GNB3GNAS1

Paroxetine AntidepressantSelective Serotonin Reuptake Inhbitor

CYP2D6 CYP1A2,CYP2B6CYP2C8/9,CYP2C19CYP2D6,CYP3A4

DRD2,DRD4,GNB3,GNAS1HTR2A,MAOA,SLC6A4TNF-A,TPH2

Protriptyline Tricyclic Antidepressant, Secondary Amine CYP2D6

Sertraline AntidepressantSelective Serotonin Reuptake Inhibitor

CYP2C19CYP2D6

CYP2B6,CYP2C8/9CYP3A4

CYP1A2,CYP2B6CYP2C8/9,CYP2C19CYP2D6,CYP3A4

Trazodone AntidepressantSerotonin Reuptake Inhibitor/Antagonist

CYP3A4 CYP2D6 CYP2D6 ADRA1,GNB3,GNAS1

Trimipramine Tricyclic AntidepressantTertiary Amine

CYP2C19CYP2D6CYP3A4

ABCB1ADRA1GNB3GNAS1

Venlafaxine AntidepressantNorepinephrine/Serotonin Reuptake Inhibitor

CYP2D6CYP3A4

CYP2C8/9CYP2C19

CYP2B6,CYP2D6CYP3A4

Symbols:ABCB1: ATP-Binding Cassette, Subfamily B, Member 1;ACHE: Acetylcholinesterase; ADRA1: Alpha-1-Adrenergic Receptor; ADRB1: Beta-1-Adrenergic Receptor; ADRB3: Beta-3-Adrenergic Receptor; APOE: Apolipoprotein E; CHRNA2: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 2; CHRNA3: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 3; CHRNA4: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 4; CHRNA5: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 5; CHRNA9: Cholinergic Receptor, Neuronal Nicotinic, Alpha Polypeptide 9; CHRNA10: Cholinergic Re-ceptor, Neuronal Nicotinic, Alpha Polypeptide 10; CHRNB2: Cholinergic Receptor, Neuronal Nicotinic, Beta Ppolypeptide 2; CHRNA3: Cholinergic Receptor, Neuronal Nicotinic, Beta Ppolypeptide 3; CHRNA4: Cholinergic Receptor, Neuronal Nicotinic, Beta Ppolypeptide 4; CHRNA7: Cholinergic Receptor, Neuronal Nicotinic, Beta Ppolypeptide 7; COMT: Catechol-O-Methyl Transferase; CYP: Cytochrome P450 Family Genes; DRD2: Dopamine Receptor D2; DRD3: Dopamine Receptor D3; DRD4: Dopamine Receptor D4; GABAR: Gamma-Aminobutyric Acid Receptors; G6PD: Glucose-6-Phosphate Dehydrogenase; GNB3: G-Protein Beta-3 Subunit; GNAS1: Gs Protein Alpha-Subunit; GPIIIA: Glycoprotein IIIa Receptor; HLA-A1: Minor Histocompatibility Antigen HA-1; HRH1: Histamine Receptor H1; HRH2: Histamine Receptor H2; HTR1A: Serotonin Receptor 1A; HTR1B: Serotonin Receptor 1B; HTR1D: Serotonin Receptor 1D; HTR2A: Serotonin Receptor 2A; HTR2C: Serotonin Receptor 2C; HTR6: Serotonin Receptor 6; INPP1: Inositol Polyphosphate 1-Phosphatase; KCNE2: Cardiac Potassium Ion Channel; LTC4S: Leukotriene C4 Synthase; MAOA: Monoamine Oxidase A; MAOB: Monoamine Oxidase B; RGS2: Regulator of G-Protein Signaling 2; SCN5A: Cardiac Sodium Channel; SLC6A2: Solute Carrier Family 6 (Neurotransmitter Transporter, Noradrenaline), Member 2; SLC6A3: Solute Carrier Family 6 (Neurotransmitter Transporter, Dopamine), Member 3; SLC6A4: Solute Carrier Family 6 (Neurotransmitter Transporter, Serotonin), Member 4; TNF-A: Tumor Necrosis Factor-Alpha; TPH2: Tryptophan Hydroxylase.

Adapted from Cacabelos42

Continued, Table 5

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Drugs PharmacologicalCategory

MajorSubstrate

Minor Substrate

Inhibitors OtherGenes

Aripiprazole Atypical antipsychotic CYP2D6,CYP3A4 ADRA1,DRD2,DRD3,HTR1A,HTR2A,HTR2C

Chlorpromazine Phenothiazine Antipsychotic CYP2D6 CYP1A2,CYP3A4 CYP2D6,CYP2E1 ABCB1,ADRA1,DRD2,KCNE2,SCN5A

Clozapine Atypical antipsychotic CYP1A2 CYP2A6,CYP2C8/9,CYP2C19,CYP2D6,CYP3A4

CYP1A2,CYP2C8/9CYP2C19,CYP2D6CYP2E1,CYP3A4

ADRA1,ADRB3,DRD2,DRD3,DRD4,GNB3,GNAS1,RGS2,HLAA1,HRH1,HRH2,HTR1A,HTR2A,HTR2C,HTR6,SLC6A2,SLC6A4, TNF-A

Droperidol Atypical Antipsychotic ADRA1,DRD2,KCNE2,SCN5A

Haloperidol Typical Antipsychotic CYP2D6,CYP3A4 CYP1A2 CYP2D6,CYP3A4 ABCB1,ADR1A,DRD2,DRD3,DRD4,KCNE2,SCN5A

Loxapine Typical Antipsychotic ADR1A,DRD2,KCNE2,SCN5A

Mesoridazine Typical Antipsychotic ADR1A,DRD2,KCNE2,SCN5A

Molindone Typical Antipsychotic ADRA1,DRD2

Olanzapine Atypical Antipsychotic CYP1A2,CYP2D6 CYP1A2,CYP2C8/9CYP2C19,CYP2D6CYP3A4

ADRA1,DRD2,DRD3,HRH1,HRH2,HTR2AHTR2C,HTR6,RGS2,TNF-A

Perphenazine Typical AntipsychoticPhenothiazine

CYP2D6 CYP1A2,CYP2C8/9,CYP2C19,CYP3A4

CYP1A2CYP2D6

ADRA1DRD3

Pimozide Typical Antipsychotic CYP1A2CYP3A4

CYP2C19,CYP2D6CYP2E1,CYP3A4

ADRA1,DRD2,KCNE2,SCN5A

Pipotiazine Typical Antipsychotic CYP2D6,CYP3A4

Prochlorperazine Typical Antipsychotic ABCB1,ADRA1,DRD2

Quetiapine Atypical Antipsychotic CYP3A4 CYP2D6 ADRA1,DRD2,KCNE2,SCN5A

Risperidone Atypical Antipsychotic CYP2D6 CYP2A4 CYP2D6CYP3A4

ABCB1,ADRA1,DRD2,DRD3,DRD4,HTR1A,HTR2AHTR2C,KCNE2,RGS2,SLC6A2SCN5A

Thioridazine Typical AntipsychoticPhenothiazine

CYP2D6 CYP2C19 CYP1A2,CYP2C8/9CYP2D6,CYP2E1

ADRA1,DRD2,KCNE2SCN5A

Thiothixene Typical Antipsychotic CYP1A2 CYP2D6 ADRA1,DRD2,KCNE2,SCN5A

Trifluoperazine Typical AntipsychoticPhenothiazine

CYP1A2 ADRA1DRD2

Ziprasidone Atypical Antipsychotic CYP1A2CYP3A4

CYP2D6CYP3A4

ADRA1,DRD2,DRD3,HTR1A,HTR2A,HTR2CKCNE2,SCN5A

Zonisamide Anticonvulsant CYP3A4

Zuclopenthixol Typical Antipsychotic CYP2D6 ADRA1,DRD2,KCNE2,SCN5A

Symbols: as in table 5

Adapted from Cacabelos42

Neuroleptics metabolized via enzymes of the CYP gene family and other gene-related enzymes

Table 6

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methyltransferase, thiopurine methyltrans-ferase, catechol-O-methyltransferase, glu-tathione-S-transferases, UDP-glucurono-syltransferases, glucosyltransferase, histamine methyltransferase; (c) Re-ductases: NADPH:quinine oxidoreductase, glucose-6-phosphate dehydrogenase; (d) oxidases: alcohol dehydrogenase, aldehy-dehydrogenase, monoamine oxidase B, catalase, superoxide dismutase, trimeth-ylamine N-oxidase, dihydropyrimidine de-hydrogenase; and (e) cytochrome P450 en-zymes, such as CYP1A1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CY-P3A5 and many others4,9,10,42. Polymorphic variants in these genes can induce altera-tions in drug metabolism modifying the ef-ficacy and safety of the prescribed drugs.

Drug metabolism includes phase I reac-tions (i.e. oxidation, reduction, hydrolysis) and phase II conjugation reactions (i.e., acetylation, glucuronidation, sulphation, methylation). The principal enzymes with polymorphic variants involved in phase I reactions are the following: CYP3A4/5/7, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1B1, CY-P1A1/2, epoxide hydrolase, esterases, NQO1 (NADPH-quinone oxidoreductase), DPD (dihydropyrimidine dehydrogenase), ADH (alcohol dehydrogenase), and ALDH (aldehyde dehydrogenase). Major enzymes involved in phase II reactions include the

following: UGTs (uridine 5’-triphosphate glucuronosyl transferases), TPMT (thiopu-rine methyltransferase), COMT (catechol-O-methyltransferase), HMT (histamine methyl-transferase), STs (sulfotransferas-es), GST-A (glutathion S-transferase A), GST-P, GST-T, GST-M, NAT2 (N-acetyl transferase), NAT1, and others4,9,10,42.

Pharmacogenetics of Psychotropic DrugsThe typical paradigm for the pharma-cogenetics of phase I drug metabolism is represented by the cytochrome P-450 enzymes, a superfamily of microsomal drug-metabolising enzymes. P450 enzymes comprise a superfamily of heme-thiolate proteins widely distributed in bacteria, fungi, plants and animals. The P450 en-zymes are encoded in genes of the CYP superfamily and act as terminal oxidases in multicomponent electron transfer chains which are called P450-containing mono-oxigenase systems. Some of the enzymatic products of the CYP gene superfamily can share substrates, inhibitors and inducers whereas others are quite specific for their substrates and interacting drugs.

The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are re-sponsible for the oxidative metabolism of more than 90% of marketed drugs. About 60-80% of the psychotropic agents current-ly used for the treatment of neuropsychiat-ric disorders are metabolised via enzymes of the CYP family, especially CYP1A2, CYP2B6, CYP2C8/9, CYP2C19, CYP2D6 and CYP3A4 (Tables 5-7; Fig.5). CYP3A4 metabolises more drug molecules than all

other isoforms together. Most of these poly-morphisms exhibit geographic and ethnic differences65-68. These differences influ-ence drug metabolism in different ethnic groups in which drug dosage should be adjusted according to their enzymatic ca-pacity, differentiating normal or extensive metabolisers (EMs), intermediate metabo-lisers (IMs), poor metabolisers (PMs) and ultrarapid metabolisers (UMs).

Most drugs act as substrates, inhibitors or inducers of CYP enzymes. Enzyme induc-tion enables some xenobiotics to acceler-ate their own biotransformation (auto-induction) or the biotransformation and elimination of other drugs. A number of P450 enzymes in the human liver are in-ducible. Induction of the majority of P450 enzymes occurs by an increase in the rate of gene transcription and involves ligand-activated transcription factors, aryl hydro-carbon receptor, constitutive androstane receptor (CAR), and pregnane X recep-tor (PXR)69,70. In general, binding of the appropriate ligand to the receptor initi-ates the induction process that cascades through a dimerisation of the receptors, their translocation to the nucleus and bind-ing to specific regions in the promoters of CYPs70. CYPs are also expressed in the CNS, and a complete characterization of consti-tutive and induced CYPs in the brain is es-sential for understanding the role of these enzymes in neurobiological functions and in age-related and xenobiotic-induced neu-rotoxicity71. CYP2D6 mRNA expression is detected in all regions of the human brain where it may be involved in the metabolism of amines and steroids and in the regula-tion of diverse CNS activities72.

There are substantial differences between individuals in the effects of psychotropic drugs in the treatment of neuropsychiat-ric disorders. Pharmacogenetic studies of psychotropic drug response have focused on determining the relationship between variation in specific candidate genes and the positive and adverse effects of drug treatment73-75. Approximately, 18% of neu-roleptics are major substrates of CYP1A2 enzymes, 40% of CYP2D6, and 23% of CYP3A4 (Table 6); 24% of antidepressants are major substrates of CYP1A2 enzymes, 5% of CYP2B6, 38% of CYP2C19, 85% of CYP2D6, and 38% of CYP3A4 (Table 5); 7% of benzodiazepines are major substrates of CYP2C19 enzymes, 20% of CYP2D6, and 95% of CYP3A4 (Table 7; Fig.5) 4,42. About 80% of patients with resistant depression, 60% of patients non-responsive to neuro-

Fig.85Distribution and frequency of CYP2C9 genotypes/phenotypes in the Spanish population.Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain.

*1/*1-EM

*1/*2-IM

*1/*3-IM

*2/*2-PM

*2/*3-PM

*3/*3-PM

0 10 20 30 40 50 60 70

Frequency (%)

D istrib u tio n a n d F re q u e n cy o f C YP 2C 9G e n o typ e N %*1/*1-EM 957 60,8778626*1 /*2-IM 377 23,9821883*1 /*3-IM 160 10,178117*2 /*2-P M 40 2,54452926*2 /*3-P M 34 2,16284987*3 /*3-P M 4 0,25445293

1572 100

Distribution and Frequency of CYP2C9 Genotypes/Phenotypes

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leptics, and 50-70% of patients with para-doxical responses to benzodiazepines are carriers of mutant variants of the CYP2D6, CYP2C9 and CYP3A4 genes, falling within the categories of poor or ultra-rapid me-tabolisers42.

CYPs in Dementia

In dementia, as in any other CNS disor-der, CYP genomics is a very important is-sue since in practice over 90% of patients with dementia are daily consumers of psy-chotropics. Furthermore, some acetylcho-linesterase inhibitors (the most prescribed anti-dementia drugs worldwide) are metab-olised via CYP enzymes4,9,10,28,42,49,50. CYP2D6, CYP2C19, CYP2C9 and CYP3A4/5 deserve special consideration.

The CYP2D6 enzyme, encoded by a gene that maps on 22q13.1-13.2, catalyses the oxidative metabolism of over 100 clini-cally important and commonly prescribed drugs such as cholinesterase inhibitors, an-tidepressants, neuroleptics, opioids, some β-blockers, class I antiarrhythmics, analge-sics and many other drug categories, act-ing as substrates, inhibitors or inducers with which many other drugs may poten-tially interact, this leading to the outcome of ADRs. The CYP2D6 locus is highly poly-morphic, with over 100 different CYP2D6 alleles identified in the general population showing deficient (PM), normal (EM), in-termediate (IM) or increased enzymatic activity (UM)76,77. Most individuals (>80%) are EMs; however, remarkable interethnic differences exist in the frequency of the PM and UM phenotypes among different societies all over the world9,68,78. On the av-erage, approximately 6.28% of the world population belongs to the PM category. Europeans (7.86%), Polynesians (7.27%), and Africans (6.73%) exhibit the highest rate of PMs, whereas Orientals (0.94%) show the lowest rate. The frequency of PMs among Middle Eastern populations, Asians, and Americans is in the range of 2-3%. CYP2D6 gene duplications are rela-tively infrequent among Northern Europe-ans, but in East Africa the frequency of al-leles with duplication of CYP2D6 is as high as 29%79.

The most frequent CYP2D6 al-leles in the European population are as follows: CYP2D6*1 (wild-type)(normal), CYP2D6*2 (2850C>T)(normal), CYP2D6*3 (2549A>del)

(inactive), CYP2D6*4 (1846G>A)(in-active), CYP2D6*5 (gene deletion)(inactive), CYP2D6*6 (1707T>del)(inactive), CYP2D6*7 (2935A>C)(inac-tive), CYP2D6*8 (1758G>T)(inactive), CYP2D6*9 (2613-2615 delAGA)(partially active), CYP2D6*10 (100C>T)(partially active), CYP2D6*11 (883G>C)(inac-tive), CYP2D6*12 (124G>A)(inactive), CYP2D6*17 (1023C>T)(partially active), and CYP2D6 gene duplications (with in-creased or decreased enzymatic activity de-pending upon the alleles involved)4,9,10,77.

In the Spanish population, where the mix-ture of ancestral cultures has occurred for centuries, the distribution of the CYP2D6 genotypes differentiates 4 major catego-ries of CYP2D6-related metabolyzer types: (i) Extensive Metabolisers (EM)(*1/*1, *1/*2,*1/*10); (ii) Intermediate Metabo-lisers (IM)(*1/*3, *1/*4, *1/*5, *1/*6, *1/*7, *10/*10, *4/*10, *6/*10, *7/*10); (iii) Poor Metabolisers (PM)(*4/*4, *5/*5); and (iv) Ultra-rapid Metabolisers (UM)(*1xN/*1, *1xN/*4, Dupl). In this sample we have found 51.61% EMs, 32.26%

IMs, 9.03% PMs, and 7.10% UMs10,18,28,47-

50,52. In a more recent study with 1637 sub-jects and 644 patients with AD (Fig.6) we did not find any significant difference be-tween AD cases and the general population (GP)42 (Fig.6). A variation rate higher than 2% was only found in the EM-*1/*1 geno-type which is more frequent in the GP than in AD. The proportion of EMs was 59.51% in GP and 57.76% in AD; IMs were 29% in GP and 31% in AD; PMs were 4.46% in GP and 5.27% in AD; and UMs were 6.23% in GP and 5.9% in AD42 (Fig.6). No major differences between females and males were found in the GP group; however, in AD, EMs are more frequent in females than in males, and PMs are more frequent in males than in females, indicating that males might be at higher risk for develop-ing ADRs42 (Fig.7).

Association of CYP2D6 Variants with Alzheimer’s Disease-Related GenesWe have also investigated the association of CYP2D6 genotypes with AD-related genes, such as APP, MAPT, APOE,

Drugs MajorSubstrate

Minor Substrate

Inhibitors OtherGenes

Alprazolam CYP3A4

Bromazepam CYP3A4 CYP2E1

Chlordiazepoxide CYP3A4

Clobazam CYP2D6, CYP3A4

Clonazepam CYP3A4

Clorazepate CYP3A4

Diazepam CYP2C19, CYP3A4 CYP1A2, CYP2B6,CYP2C8/9

CYP2C19,CYP3A4

Estazolam CYP3A4

Flurazepam CYP3A4 CYP2E1

Midazolam CYP3A4 CYP2B6 CYP2C9,CYP3A4 ABCB1

Oxacepam CYP3A4

Pinazepam CYP3A4

Prazepam CYP3A4

Quazepam CYP3A4

Temazepam CYP2B6,CYP2C8/9,CYP2D6,CYP3A4

Triazolam CYP3A4 CYP2C8/9

Symbols: as in table 5.Adapted from Cacabelos42

Table 7

Benzodiazepines metabolized via enzymes of the CYP gene family and other gene-related enzymes.

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PSEN1, PSEN2, A2M, ACE, AGT, FOS, and PRNP variants10,42,47-52. Homozy-gous APOE-2/2 (12.56%) and APOE-4/4 (12.50%) accumulate in UMs, and APOE-4/4 cases were also more frequent in PMs (6.66%) than in EMs (3.95%) or IMs (0%). PS1-1/1 genotypes were more frequent in EMs (45%), whereas PS-1/2 genotypes were over-represented in IMs (63.16%) and UMs (60%). The presence of the PS1-2/2 genotype was especially high in PMs (38.46%) and UMs (20%). A mutation in the PS2 gene exon 5 (PS2E5+) was markedly present in UMs (66.67%). About 100% of UMs were A2M-V100I-A/A, and the A2M-V100I-G/G genotype was absent in PMs and UMs. The A2M-I/I genotype was absent in UMs, and 100% of UMs were A2M-I/D and ACE-D/D. Homozygous mutations in the FOS gene (B/B) were also only pres-ent in UMs. AGT-T235T cases were ab-sent in PMs, and the AGT-M174M geno-type appeared in 100% of PMs. Likewise, the PRNP-M129M variant was present in 100% of PMs and UMs. These association studies clearly show that in PMs and UMs there is an accumulation of AD-related polymorphic variants of risk which might be responsible for the defective thera-peutic responses currently seen in these AD clusters10,47-52.

CYP2D6-Related Biochemical and Haemodynamic Phenotypes in Alzheimer’s DiseaseIt appears that different CYP2D6 vari-ants, expressing EMs, IMs, PMs, and UMs, influence to some extent several biochemical parameters, liver function,

and vascular haemodynamic parameters which might affect drug efficacy and safe-ty. Blood glucose levels are found to be elevated in EMs (*1/*1 vs *4/*10) and in some IMs (*4/*10 vs *1xN/*4), whereas other IMs (*1/*5 vs *4/*4) tend to show lower levels of glucose compared with PMs (*4/*4) or UMs (*1xN/*4). The highest levels of total-cholesterol are detected in the EMs with the CYP2D6*1/*10 geno-type (vs *1/*1, *1/*4 and *1xN/*1). The same pattern has been observed with regard to LDL-cholesterol levels, which are significantly higher in the EM-*1/*10. In general, both total cholester-ol levels and LDL-cholesterol levels are higher in EMs (with a significant differ-ence between *1/*1 and *1/*10), inter-mediate levels are seen in IMs, and much lower levels in PMs and UMs; and the op-posite occurs with HDL-cholesterol lev-els, which on average appear much lower in EMs than in IMs, PMs, and UMs, with the highest levels detected in *1/*3 and *1xN/*4. The levels of triglycerides are highly variable among different CYP2D6 polymorphisms, with the highest levels present in IMs (*4/*10 vs *4/*5 and *1xN/*1)10,50,52. These data clearly indi-cate that lipid metabolism can be influ-enced by CYP2D6 variants or that specific phenotypes determined by multiple lip-id-related genomic clusters are necessary to confer the character of EMs and IMs. Another possibility might be that some lipid metabolism genotypes interact with CYP2D6-related enzyme products lead-ing to the definition of the pheno-gen-otype of PMs and UMs. No significant changes in blood pressure values have been found among CYP2D6 genotypes;

however, important differences became apparent in brain cerebrovascular hae-modynamics. In general terms, the best cerebrovascular haemodynamic pattern is observed in EMs and PMs, with higher brain blood flow velocities and lower re-sistance and pulsatility indices, but differ-ential phenotypic profiles are detectable among CYP2D6 genotypes. For instance, systolic blood flow velocities (Sv) in the left middle cerebral arteries (LMCA) of AD patients are significantly lower in *1/*10 EMs, with high total cholesterol and LDL-cholesterol levels, than in IMs (*4/*10); and diastolic velocities (Dv) also tend to be much lower in *1/*10 and especially in PMs (*4/*4) and UMs (*1xN/*4), whereas the best Dv is mea-sured in *1/*5 IMs. More striking are the results of both the pulsatility index (PI=(Sv-Dv)/Mv) and resistance index (RI=(Sv-Dv)/Sv), which are worse in IMs and PMs than in EMs and UMs. These data taken together seem to indicate that CYP2D6-related AD PMs exhibit a poorer cerebrovascular function which might affect drug penetration into the brain with the consequent therapeutic implica-tions10,47-52.

Influence of CYP2D6 Genotypes on Liver Transaminase ActivityIn order to elucidate whether or not CYP2D6-related variants may influence transaminase activity, we have studied the association of GOT, GPT, and GGT activ-ity with the most prevalent CYP2D6 geno-types in AD10,48-50. Globally, UMs and PMs tend to show the highest GOT activity and IMs the lowest. Significant differences ap-pear among different IM-related geno-types. The *10/*10 genotype exhibited the lowest GOT activity with marked dif-ferences as compared to UMs. GPT activ-ity was significantly higher in PMs (*4/*4) than in EMs (*1/*10) or IMs (*1/*4, *1/*5). The lowest GPT activity was found in EMs and IMs. Striking differences have been found in GGT activity between PMs (*4/*4), which showed the highest levels, and EMs (*1/*1; *1/*10), IMs (*1/*5), or UMs (*1xN/*1)50. Interesting enough, the *10/*10 genotype, with the lowest values of GOT and GPT, exhibited the second highest levels of GGT after *4/*4, prob-ably indicating that CYP2D6-related en-zymes differentially regulate drug metabo-lism and transaminase activity in the liver. These results are also clear in demonstrat-ing the direct effect of CYP2D6 variants on transaminase activity10,49,50.

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CYP2D6-Related Therapeutic Response to a Multifactorial Treatment in DementiaNo clinical trials had been performed to elucidate the influence of CYP2D6 variants on the therapeutic outcome in AD in re-sponse to cholinesterase inhibitors or other anti-dementia drugs. We have performed the first prospective study in AD patients who received a combination therapy with (a) an endogenous nucleotide and choline donor, CDP-choline (500 mg/day), (b) a nootropic substance, piracetam (1600 mg/day), (c) a vasoactive compound, 1,6 dim-ethyl 8β-(5-bromonicotinoyl-oxymethyl)-10α-methoxyergoline (nicergoline)(5 mg/day), and (d) a cholinesterase inhibi-tor, donepezil (5 mg/day), for one year. With this multifactorial therapeutic in-tervention, EMs improved their cognitive function (MMSE score) from 21.58±9.02

at baseline to 23.78±5.81 after 1-year treat-ment. IMs also improved from 21.40±6.28 to 22.50±5.07 (r=+0.96), whereas PMs and UMs deteriorate from 20.74±6.72 to 18.07±5.52 (r=-0.97), and from 22.65±6.76 to 21.28±7.75 (r=-0.92), respectively. Ac-cording to these results, PMs and UMs were the worst responders, showing a pro-gressive cognitive decline with no thera-peutic effect, and EMs and IMs were the best responders, with a clear improvement in cognition after one year of treatment. Among EMs, AD patients harbouring the *1/*10 genotype responded better than patients with the *1/*1 genotype. The best responders among IMs were the *1/*3, *1/*6 and *1/*5 genotypes, whereas the *1/*4, *10/*10, and *4/*10 genotypes were poor responders. Among PMs and UMs, the poorest responders were carri-ers of the *4/*4 and *1xN/*1 genotypes,

respectively10,28,42,47-50. In a recent study, Pi-lotto et al80 have confirmed the influence of CYP2D6 variants (rs1080985) on the ef-ficacy of donepezil in AD.

From all these data we can conclude the following: (i) The most frequent CYP2D6 variants in the Southern European popu-lation (Iberian peninsula) are the *1/*1 (57.84%), *1/*4 (22.78%), *1xN/*1 (6.10%), *4/*4 (2.56%), and *1/*3 (2.01%) genotypes, accounting for more than 80% of the population; (ii) the fre-quency of EMs, IMs, PMs, and UMs is about 59.51%, 29,78%, 4.46%, and 6.23%, respectively, in the general population, and 57.76, 31.05%, 5.27%, and 5.90%, respectively in AD cases (Fig.6); (iii) EMs are more prevalent in GP (59.51%) than in AD (57.76%); IMs are more frequent in AD (31.05%) than in GP (29.78%);

Procedure Technology Parametric Data

Clinical history Anamnesis. Pedigree. Physical, neurologic and psychiatric examination

Present conditionsFamily historyPersonal historyPhysical, neurological and psychi-atric information

Laboratory tests ConventionalTest-specific

Blood, urine, cerebrospinal fluid

Neuropsychological Assessment

Neuropsychological testsBatteries

Mood. Behavior. Cognition. Functioning

Cardiovascular Evaluation

ElectrocardiogramEcocardiogramFunctional tests

Heart functionCirculatory function

Imaging Conventional X-Ray Chest, neck, other structures or organs

Structural Neuroimaging

Computerized Tomography (CT-Scan)Magnetic Resonance Imaging (MRI)

Brain structure

Functional Neuroimaging

Single Photon Emission Computer-ized Tomography (PECT) Positron Emission Tomography (PET) CT-Brain Perfusion,Brain Digital Topography

Brain FunctionCerebrovascular functionBrain oxygenation

Brain Electrophysiology

EEG, qEEG, EMG, EP Brain mappingNeuromuscular transmissionEvoked Potentials

Cerebrovascular Assessment

SPECTCT-Brain PerfusionBrain Digital TopographyTranscranial Doppler Ultrasonog-raphy

Brain PerfusionBrain OxygeneationCerebrovascular Hemodynamics

Structural Genomics

Gene mappingLinkage analysisAssociation studiesDNA Microarrays

MutationsDisease-associated genotypesSNPs

Procedure Technology Parametric Data

Functional Genomics

Microarray TechnologyGenotype-Phenotype CorrelationsTranscriptomicsProteomicsMetabolomics

Genotype-associated defects

Pharmacogenetics Genotyping of genes associated with drug metabolism

Prediction of therapeutic responseDrug toxicityADRsSafety issues

Pharmacogenomics Genotyping of genes associated with disease phenotypeHigh Throughput Screening

Drug-induced gene(s) expression and disease phenotype modifica-tionEfficacy issues

Nutrigenetics Genotyping of genes associated with nutrients metabolism

Nutrition-related effectsNutrition benefitsNutrition toxicitySafety issues

Nutrigenomics Genotyping of genes associated with disease induced by nutritional factorsHigh Throughput Screening

Nutrition-related disease analysisNutrition-induced gene expression and disease phenotype modifica-tionEfficacy issues

Data Integration Bioinformatics Data ManagementCorrelation Analysis

Intelligent Assignments

Artificial Intelligence Probabilistic DiagnosisTherapeutic OptimizationNutritional OptimizationPredictive AnalysisIndividual Preventive OptionsRisk EvaluationGenetic Counselling

Adapted from Cacabelos4

Table 8

The EuroEspes Protocol for Genomic Medicine of CNS Disorders

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the frequency of PMs is slightly higher in AD (5.27%%) than in GP (4.46%); and UMs are more frequent in GP (6.23%) than in AD (5.90%)(all these differences are not significant)(Fig.6); (iv) there are differences between females and males in the distribution and frequency of CYP2D6 genotypes which might be of relevance in therapeutic terms and risk of ADRs (Fig.7); (v) there is an accumulation of AD-related genes of risk in PMs and UMs; (vi) PMs and UMs tend to show higher transaminase ac-tivities than EMs and IMs; (vii) EMs and IMs are the best responders, and PMs and UMs are the worst responders to a com-bination therapy with cholinesterase in-hibitors, neuroprotectants, and vasoactive

substances; and (viii) the pharmacogenetic response in AD appears to be dependent upon the networking activity of genes in-volved in drug metabolism and genes in-volved in AD pathogenesis10,28,42,47-50.

CYP Clustering in Alzheimer’s diseaseSince more than half of the available drugs are metabolised via different CYP enzymes and other metabolic pathways, it is convenient to understand the network-ing activity of CYP genes and the genomic profiles of these genes in particular groups of risk. In the case of dementia, 73.71% of AD patients are CYP2C19-EMs, 25.12% IMs, and 1.16% PMs. The distribution and frequency of CYP2C9 genotypes is as fol-lows: *1/*1-EM 60.87%, *1/*2-IM 23.98%, *1/*3-IM 10.17%, *2/*2-PM 2.54%, *2/*3-PM 2.16%, and *3/*3-PM 0.25%, globally representing 60.87% CYP2C9-EMs, 34.16% IMs, and 4.97% PMs42(Fig.8). This is especially important because the CYP2C9-Ile359Leu (CYP2C9*3 allele) and CYP2C9-Arg144Cys (CYP2C9*2 allele) vari-

ants are associated with warfarin sensitivity. Clustering together CYP2C9 and VKORC1 variants, we can estimate that approximate-ly 30% of the elderly population is sensitive to warfarin anticoagulants. Concerning CYP3A4/5 polymorphisms, 82.75% of AD cases are EMs (CYP3A5*3/*3), 15.88% are IMs (CYP3A5*1/*3), and 1.37% are UMs (CYP3A5*1/*1)42.

The construction of a genetic map integrating the most prevalent CYP2D6+CYP2C19+CYP2C9 polymorphic variants in a trigenic cluster yields 82 dif-ferent haplotype-like profiles (Fig.9). The most frequent trigenic genotypes in the AD population are *1*1-*1*1-*1*1 (25.70%), *1*1-*1*2-*1*2 (10.66%), *1*1-*1*1-*1*1 (10.45%), *1*4-*1*1-*1*1 (8.09%), *1*4-*1*2-*1*1 (4.91%), *1*4-*1*1-*1*2 (4.65%), and *1*1-*1*3-*1*3 (4.33%)(Fig.9). These 82 trigenic genotypes repre-sent 36 different pharmacogenetic pheno-types (Fig.10). According to these trigenic clusters, only 26.51% of the patients show a pure 3EM phenotype, 15.29% are 2EM1IM,

Fig.9 5Distribution and frequency of trigenic clusters integrated by combination of CYP2D6, CYP2C19, and CYP2C9 genotypes in patients with Alzheimer’s disease.Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos 42)

1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233343536373838404142434445464748495051525354555657585960616263646566676869707172737475767778798081820

5

10

15

20

25

30

F(%)

Number CYP2D6 CYP2C19 CYP2C9 N % Phenotype1 *1/*1 *1/*1 *1/*1 403 25,7015306 EM+EM+EM2 *1/*1 *1/*2 *1/*2 167 10,6505102 EM+EM+IM3 *1/*1 *1/*3 *1/*3 68 4,33673469 EM+EM+IM4 *1/*1 *1/*4 *2/*2 27 1,72193878 EM+EM+PM5 *1/*1 *1/*5 *2/*3 19 1,21173469 EM+EM+PM6 *1/*1 *1/*6 *3/*3 3 0,19132653 EM+EM+PM7 *1/*1 *1/*2 *1/*1 164 10,4591837 EM+IM+EM8 *1/*1 *1/*2 *1/*2 39 2,4872449 EM+IM+IM9 *1/*1 *1/*2 *1/*3 13 0,82908163 EM+IM+IM

10 *1/*1 *1/*2 *2/*3 1 0,06377551 EM+IM+PM11 *1/*1 *2/*2 *1/*1 10 0,6377551 EM+PM+EM12 *1/*2 *1/*2 *1/*2 1 0,06377551 EM+IM+IM13 *1/*3 *1/*1 *1/*1 16 1,02040816 IM+EM+EM14 *1/*3 *1/*1 *1/*2 4 0,25510204 IM+EM+IM15 *1/*3 *1/*1 *1/*3 2 0,12755102 IM+EM+IM16 *1/*3 *1/*1 *2/*3 1 0,06377551 IM+EM+PM17 *1/*3 *1/*2 *1/*1 6 0,38265306 IM+IM+EM18 *1/*3 *1/*2 *1/*2 3 0,19132653 IM+IM+IM19 *1/*4 *1/*1 *1/*1 127 8,0994898 IM+EM+EM20 *1/*4 *1/*1 *1/*2 73 4,65561224 IM+EM+IM21 *1/*4 *1/*1 *1/*3 35 2,23214286 IM+EM+IM22 *1/*4 *1/*1 *2/*2 7 0,44642857 IM+EM+PM23 *1/*4 *1/*1 *2/*3 7 0,44642857 IM+EM+PM24 *1/*4 *1/*2 *1/*1 77 4,91071429 IM+IM+EM25 *1/*4 *1/*2 *1/*2 20 1,2755102 IM+IM+IM26 *1/*4 *1/*2 *1/*3 8 0,51020408 IM+IM+IM27 *1/*5 *1/*1 *1/*1 18 1,14795918 IM+EM+EM28 *1/*5 *1/*1 *1/*2 15 0,95663265 IM+EM+IM29 *1/*5 *1/*1 *1/*3 3 0,19132653 IM+EM+IM30 *1/*5 *1/*2 *1/*1 15 0,95663265 IM+IM+EM31 *1/*5 *1/*2 *1/*2 1 0,06377551 IM+IM+IM32 *1/*5 *1/*2 *1/*3 4 0,25510204 IM+IM+IM33 *1/*5 *2/*2 *1/*1 1 0,06377551 IM+PM+EM34 *1/*6 *1/*1 *1/*1 9 0,57397959 IM+EM+EM35 *1/*6 *1/*1 *1/*2 1 0,06377551 IM+EM+IM36 *1/*6 *1/*1 *1/*3 2 0,12755102 IM+EM+IM37 *1/*6 *1/*1 *1/*1 6 0,38265306 IM+IM+EM38 *1xN/*1 *1/*1 *1/*1 42 2,67857143 UM+EM+EM39 *1xN/*1 *1/*1 *1/*2 16 1,02040816 UM+EM+IM40 *1xN/*1 *1/*1 *1/*3 15 0,95663265 UM+EM+IM

41 *1xN/*1 *1/*1 *2/*2 3 0,19132653 UM+EM+PM42 *1xN/*1 *1/*1 *2/*3 1 0,06377551 UM+EM+PM43 *1xN/*1 *1/*2 *1/*1 14 0,89285714 UM+IM+EM44 *1xN/*1 *1/*2 *1/*2 5 0,31887755 UM+IM+IM45 *1xN/*1 *1/*2 *1/*3 1 0,06377551 UM+IM+IM46 *1xN/*4 *1/*1 *1/*1 9 0,57397959 EM+EM+EM47 *1xN/*4 *1/*1 *1/*2 6 0,38265306 EM+EM+IM48 *1xN/*4 *1/*1 *1/*3 4 0,25510204 EM+EM+IM49 *1xN/*4 *1/*1 *2/*3 1 0,06377551 EM+EM+PM50 *1xN/*4 *1/*2 *1/*1 5 0,31887755 EM+IM+EM51 *1xN/*4 *1/*2 *1/*2 2 0,12755102 EM+IM+IM52 *3/*3 *1/*1 *1/*2 1 0,06377551 PM+EM+IM53 *3/*4 *1/*1 *1/*1 2 0,12755102 PM+EM+EM54 *3/*4 *1/*1 *1/*2 1 0,06377551 PM+EM+IM55 *3/*4 *1/*1 *2/*3 2 0,12755102 PM+EM+IM56 *3/*4 *1/*2 *1/*1 1 0,06377551 PM+IM+EM57 *3/*6 *1/*2 *1/*2 1 0,06377551 PM+IM+EM58 *4/*4 *1/*1 *1/*1 13 0,82908163 PM+EM+EM59 *4/*4 *1/*1 *1/*2 10 0,6377551 PM+EM+IM60 *4/*4 *1/*1 *1/*3 1 0,06377551 PM+EM+IM61 *4/*4 *1/*1 *2/*2 2 0,12755102 PM+EM+PM62 *4/*4 *1/*1 *2/*3 2 0,12755102 PM+EM+PM63 *4*/4 *1/*1 *3/*3 1 0,06377551 PM+EM+PM64 *4/*4 *1/*2 *1/*1 2 0,12755102 PM+IM+EM65 *4/*4 *1/*2 *1/*2 6 0,38265306 PM+IM+IM66 *4/*4 *1/*2 *1/*3 1 0,06377551 PM+IM+IM67 *4/*5 *1/*1 *1/*1 5 0,31887755 PM+EM+EM68 *4/*5 *1/*1 *1/*2 2 0,12755102 PM+EM+IM69 *4/*5 *1/*1 *1/*3 1 0,06377551 PM+EM+IM70 *4/*5 *1/*2 *1/*1 1 0,06377551 PM+IM+EM71 *4/*5 *1/*2 *1/*3 1 0,06377551 PM+IM+IM72 *4/*5 *2/*2 *1/*1 1 0,06377551 PM+PM+IM73 *4/*6 *1/*1 *1/*1 1 0,06377551 PM+EM+EM74 *4/*6 *1/*1 *1/*2 2 0,12755102 PM+EM+IM75 *4/*6 *1/*1 *2/*2 1 0,06377551 PM+EM+PM76 *4/*6 *1/*2 *1/*1 1 0,06377551 PM+IM+EM77 *5/*5 *1/*1 *1/*1 1 0,06377551 PM+EM+EM78 *5/*5 *1/*2 *1/*1 1 0,06377551 PM+IM+EM79 *5/*5 *2/*2 *1/*1 1 0,06377551 PM+PM+EM80 *5/*6 *1/*1 *1/*1 1 0,06377551 PM+EM+EM81 *5/*6 *1/*1 *1/*2 1 0,06377551 PM+EM+IM82 *5/*6 *1/*2 *1/*1 1 0,06377551 PM+IM+EM

1568 100

Number CYP2D6 CYP2C19 CYP2C9 N % Phenotype

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2.04% are pure 3IM, 0% are pure 3PM, and 0% are 1UM2PM (the worst possible phenotype) 42 (Fig.10).

Taking into consideration the data avail-able, it might be inferred that at least 10-15% of the AD population may exhibit an abnormal metabolism of cholinesterase in-hibitors and/or other drugs which under-go oxidation via CYP2D6-related enzymes. Approximately 50% of this population clus-ter would show an ultrarapid metabolism, requiring higher doses of cholinesterase inhibitors in order to reach a therapeutic threshold, whereas the other 50% of the cluster would exhibit a poor metabolism, displaying potential adverse events at low doses. If we take into account that approxi-mately 60-70% of therapeutic outcomes depend upon pharmacogenomic criteria (e.g. pathogenic mechanisms associated with AD-related genes), it can be postu-lated that pharmacogenetic and pharma-cogenomic factors are responsible for 75-85% of the therapeutic response (efficacy) in AD patients treated with conventional drugs10,42,47-52. Of particular interest are the potential interactions of cholinesterase in-hibitors with other drugs of current use in patients with AD, such as antidepressants,

neuroleptics, antiarrhythmics, analgesics, and antiemetics which are metabolised by the cytochrome P450 CYP2D6 enzyme. Although most studies predict the safety of donepezil and galantamine, as the two principal cholinesterase inhibitors metabo-lised by CYP2D6-related enzymes few phar-macogenetic studies have been performed so far on an individual basis to personalize the treatment, and most studies reporting safety issues are the result of pooling to-gether pharmacological and clinical infor-mation obtained with routine procedures. In certain cases, genetic polymorphism in the expression of CYP2D6 is not expected to affect the pharmacodynamics of some cholinesterase inhibitors because major metabolic pathways are glucuronidation, O-demethylation, N-demethylation, N-oxi-dation, and epimerization. However, excre-tion rates are substantially different in EMs and PMs. For instance, in EMs, urinary me-tabolites resulting from O-demethylation of galantamine represent 33.2% of the dose compared with 5.2% in PMs, which show correspondingly higher urinary ex-cretion of unchanged galantamine and its N-oxide81. Therefore, there are still many unanswered questions regarding the me-tabolism of cholinesterase inhibitors and

their interaction with other drugs (poten-tially leading to ADRs) which require phar-macogenetic elucidation. It is also worth mentioning that dose titration (a common practice in AD patients treated with cholin-esterase inhibitors; e.g., tacrine, donepezil) is an unwise strategy, since approximately 30-60% of drug failure or lack of therapeu-tic efficacy (and/or ADR manifestation) is not a matter of drug dosage but a problem of poor metabolising capacity in PMs. Ad-ditionally, inappropriate drug use is one of the risk factors for adverse drug reactions (ADRs) in the elderly. The prevalence of use of potentially inappropriate medica-tions in patients older than 65 years of age admitted to a general medical or geriat-ric ward ranges from 16% to 20%82, and these numbers may double in ambulatory patients. Overall, the most prevalent inap-propriate drugs currently prescribed to the elderly are amiodarone, long-acting

0 5 10 15 20 25

Frequency (%)

Phenotype N %EM+EM+EM 416 26,513703EM+EM+IM 240 15,2963671EM+EM+PM 49 3,12300829EM+IM+EM 167 10,6437221EM+IM+IM 56 3,56915233EM+IM+PM 1 0,06373486EM+PM+EM 10 0,63734863EM+PM+IM 0 0EM+PM+PM 0 0IM+EM+EM 171 10,8986616IM+EM+IM 135 8,6042065IM+EM+PM 16 1,01975781IM+IM+EE 105 6,69216061IM+IM+IM 32 2,03951562IM+IM+PM 0 0IM+PM+EM 1 0,06373486IM+PM+IM 5 0,31867431IM+PM+PM 0 0PM+EM+EM 25 1,59337157PM+EM+IM 18 1,14722753PM+EM+PM 7 0,44614404PM+IM+EM 8 0,5098789PM+IM+IM 8 0,5098789PM+IM+PM 0 0PM+PM+EM 2 0,12746973PM+PM+IM 0 0PM+PM+PM 0 0UM+EM+EM 43 2,74059911UM+EM+IM 30 1,91204589UM+EM+PM 4 0,25493945UM+IM+EM 14 0,89228808UM+IM+IM 6 0,38240918UM+IM+PM 0 0UM+PM+EM 0 0UM+PM+IM 0 0UM+PM+PM 0 0

1569 100

EM+EM+EMEM+EM+IMEM+EM+PMEM+IM+EMEM+IM+IMEM+IM+PMEM+PM+EMEM+PM+IMEM+PM+PMIM+EM+EMIM+EM+IMIM+EM+PMIM+IM+EEIM+IM+IMIM+IM+PMIM+PM+EMIM+PM+IMIM+PM+PMPM+EM+EMPM+EM+IMPM+EM+PMPM+IM+EMPM+IM+IMPM+IM+PMPM+PM+EMPM+PM+IMPM+PM+PMUM+EM+EMUM+EM+IMUM+EM+PMUM+IM+EMUM+IM+IMUM+IM+PMUM+PM+EMUM+PM+IMUM+PM+PM

CYP2D6+CYP2C19+CYP2C9 Clustered PhenotypesAlzheimer´s Disease

Fig.10 5Frequency of trigenic phenotypes resulting from the combination of CYP2D6, CYP2C19 and CYP2C9 genotypes in patients with Alzheimer’s disease.Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos 42)

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Personalized Medicine of Dementia

benzodiazepines and anticholinergic anti-spasmodics; however, the list of drugs with potential risk also include antidepressant, antihistaminics, NSAIDs, amphetamines, laxatives, clonidine, indomethacin, and several neuroleptics82, most of which are processed via CYP2D6 and CYP3A5 en-zymes. Therefore, pre-treatment CYP screening might be of great help in order to rationalize and optimise therapeutics in the elderly, by avoiding medications of risk in PMs and UMs.

Pharmacogenomics of AD-Related Genes

The pharmacogenomics of AD is still in a very primitive stage. In over 100 clinical trials for dementia, APOE has been used as the only gene of reference for the phar-macogenomics of AD10,42,47-52,83,84. Several studies indicate that the presence of the APOE-4 allele differentially affects the quality and extent of drug responsiveness in AD patients treated with cholinergic en-hancers (tacrine, donepezil, galantamine, rivastigmine), neuroprotective com-pounds (nootropics), endogenous nucle-otides (CDP-choline), immunotrophins (anapsos), neurotrophic factors (cere-brolysin), rosiglitazone or combination therapies10,42,47-52,83-85; however, controversial results are frequently found due to meth-odological problems, study design, and pa-tient recruitment in clinical trials.

In long-term open clinical trials with a mul-tifactorial treatment, APOE-4/4 carriers are the worst responders (Fig.11)10,42,47-52. With a similar therapeutic protocol, PSEN1-1/1 homozygotes are the worst responders and PSEN1-2/2 carriers are the best responders (Fig.11)42. Significant ACE-related therapeu-tic responses to multifactorial treatments have also been reported10,42,48,50. Among ACE-I/D variants, ACE-D/D patients were the worst responders (r=-0.58), and ACE-I/D carriers were the best responders (r=+0.26), with ACE-I/I showing an in-termediate positive response (r=+0.01)10. ACE-related biochemical and haemody-namic phenotypes have been studied in pa-tients with AD9,10,12. ACE-I/I patients tend to be younger than ACE-I/D or ACE-D/D patients at the time of diagnosis and also to show a more severe cognitive deteriora-tion. Serum ApoE, total cholesterol, LDL-cholesterol, HDL-cholesterol, nitric oxide, histamine, and ACE levels are higher in ACE-I/I carriers than in patients with the

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Standard deviation 0,65 0,47 1,16 0,54

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0,09 0,69 0,51 0,52

R-squared 0,009 0,48 0,26 0,27

a Coefficient 22,59 22,56 21,95 22,5

b Coefficient 0,03 0,19 0,35 0,16

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Fig.11 5APOE-Related therapeutic response to a multifactorial treatment in patients with Alzheimer’s disease.Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos 42)

Fig.12 5PSEN1-Related therapeutic response to a multifactorial treatment in patients with Alzheimer’s disease.Source: R. Cacabelos. EuroEspes Biomedical Research Center, Institute for CNS Disorders and Genomic Medicine, Coruña, Spain. (Adapted from Cacabelos 42)

diciembre 2009 41

Pharmacogenomics of Alzheimer’s disease

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other genotypes; in contrast, serum triglyc-eride and VLDL levels are notably lower in ACE-I/I patients compared to patients harbouring the ACE-I/D or ACE-D/D gen-otypes, whereas Aß levels do not show any clear difference among ACE-related geno-types. Cerebrovascular function tends to be worse in ACE-D/D, with lower brain blood flow velocities and higher pulsatility and resistance indices, than in ACE-I/D (inter-mediate cerebrovascular haemodynamics) or ACE-I/I (almost normal cerebrovascular function)9,10,12,50. The correlation between lipid levels and brain haemodynamics is very similar in this study to data observed in that of CYP2D6-related metaboliser profiles in which EM patients with moderate cho-lesterol and lipoprotein levels (as well as relatively high nitric oxide, histamine, ACE, and ApoE levels) tend to show a better cere-brovascular haemodynamic profile than AD patients with lower cholesterol and lipopro-tein levels50. This apparently paradoxical correlation appears to indicate that major influences in cerebrovascular homeostasis and haemodynamic brain blood flow are cholesterol, lipoproteins, nitric oxide, ACE, and histamine, among many other factors, in AD, and that peripheral levels of Aß are indifferent in this concern. On the other hand, it seems likely that low triglyceride lev-els may facilitate cerebrovascular function. It is also worth mentioning that ACE-I/I pa-tients with the highest cholesterol levels are the worst in mental performance. Other in-terpretation of these data might suggest an association between poor cerebrovascular function with ACE-D/D and ACE-I/D, and an association between alterations in lipid metabolism in ACE-I/I10,50.

Both APOE and ACE variants also affect behaviour and the modification of behav-ioural changes (mood, anxiety) in demen-

tia after non-psychotropic pharmacological treatment4,10,48,50,52. At baseline, all APOE variants show similar anxiety and depres-sion rates, except the APOE-4/4 carriers who differed from the rest in significantly lower rates of anxiety and depression. Re-markable changes in anxiety were found among different APOE genotypes. Practi-cally, all APOE variants responded with a significant diminution of anxiogenic symptoms, except patients with the APOE-4/4 genotype who only showed a slight improvement. The best responders were APOE-2/4 ( r=-0.87) > APOE-2/3 (r=-0.77) > APOE-3/3 (r=-0.69) > APOE-3/4 carriers (r=-0.45). The potential influence of APOE

variants on anxiety and cognition in AD does not show a clear parallelism, suggest-ing that other more complex mechanisms are involved in the onset of anxiety in de-mentia. Concerning depression, all APOE genotypes improved their depressive symp-toms with treatment except those with the APOE-4/4 genotype which worsen along the treatment period. The best respond-ers were APOE-2/4 (r=-0.85) > APOE-2/3 (r=-0.77) > APOE-3/3 (r=-0.73) > APOE-3/4 (r=-0.16), and the worst responder was APOE-4/4 (r=+0.31)10,48,50,52. Patients with each one of the 3 ACE-I/D indel variants are equally anxiogenic and depressive at baseline and all of them respond favourably to the multifactorial protocol by gradually reducing anxiety and depressive symptoms over the 12-month treatment period. The best responders were ACE-I/D (r=-0.89) > ACE-D/D (r=-0.68) > ACE-I/I (r=-0.08). Depressive symptoms were also similarly improved in all ACE-I/D variants. The best responders were ACE-I/D (r=-0.88) > ACE-D/D (r=-0.55) > ACE-I/I (r=-0.13). Com-paratively, the worst responders among ACE-I/D variants were carriers of the ACE-I/I genotype which were also the poorest responders in anxiety and cognition10,50,52.

The combination of APOE and ACE poly-morphic variants in bigenic clusters yields different anxiety and depression patterns at baseline and after one year of treat-

APOE-CYP2D6 AssociationDistribution of APOE Genotypesin CYP2D6-Related EM, IM, PM and UM Genotypes

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Fig.13 5Distribution and frequency of APOE genotypes in CYP2D6-related extensive (EM), intermediate (IM), poor (PM), and ultra-rapid metabolizers (UM). Accumulation of APOE-4/4 in PMs and UMs.(Adapted from Cacabelos42)

42

Personalized Medicine of Dementia

ment. The most anxiogenic patients at baseline are those with the 23DD, 44ID, and 34II genotypes, and the least anxio-genic patients are those harbouring the 23II, 44DD, and 23ID genotypes. The most depressive clusters at baseline are those harbouring the 23DD, 33ID, and 33II genotypes, with a clear accumulation of APOE-3/3 carriers in these groups, and the least depressive clusters are those rep-resented by carriers of the 23II, 44ID, and 23ID genotypes. All bigenic clusters show a positive anxiolytic and anti-depressive response to the multifactorial treatment, except 44DD carriers who exhibited the worst response10,47,50,52.

APOE influences liver function and CYP2D6-related enzyme activity probably via regulation of hepatic lipid metabolism. It has been observed that APOE may influ-ence liver function and drug metabolism by modifying hepatic steatosis and transam-inase activity. There is a clear correlation between APOE-related TG levels and GOT,

GPT, and GGT activities in AD10,50. Both plasma TG levels and transaminase activ-ity are significantly lower in AD patients harbouring the APOE-4/4 genotype, prob-ably indicating (a) that low TG levels pro-tect against liver steatosis, and (b) that the presence of the APOE-4 allele influences TG levels, liver steatosis, and transaminase activity. Consequently, it is very likely that APOE influences drug metabolism in the liver through different mechanisms, in-cluding interactions with enzymes such as transaminases and/or cytochrome P450-related enzymes encoded in genes of the CYP superfamily10,48,50,52.

When APOE and CYP2D6 genotypes are integrated in bigenic clusters and the APOE+CYP2D6-related therapeutic re-sponse to a combination therapy is analy-sed in AD patients, it becomes clear that the presence of the APOE-4/4 genotype is able to convert pure CYP2D6*1/*1 EMs into full PMs, indicating the existence of a powerful influence of the APOE-4 ho-mozygous genotype on the drug metabo-lising capacity of pure CYP2D6-EMs. In ad-dition, a clear accumulation of APOE-4/4

genotypes is observed among CYP2D6 PMs and UMs42 (Fig.13).

From these studies we can conclude the following: (i) Most studies with acetylcho-linesterase inhibitors indicate that the pres-ence or absence of the APOE-4 allele influ-ences the therapeutic outcome in patients with AD. (ii) Multifactorial treatments combining neuroprotectants, endogenous nucleotides, nootropic agents, vasoactive substances, cholinesterase inhibitors, and NMDA antagonists associated with meta-bolic supplementation on an individual ba-sis adapted to the phenotype of the patient may be useful to improve cognition and slow-down disease progression in AD. (iii) The therapeutic response in AD seems to be genotype-specific under different phar-macogenomic conditions. (iv) In mono-genic-related studies, patients harbouring the APOE-4/4 genotype are the worst re-sponders (Fig.11). (v) APP, PSEN1 (Fig.12) and PSEN2 mutations influence the thera-peutic response in AD (vi) In trigenic-related studies (APOE+PSEN1+PSEN2) the best responders are those patients car-rying the 331222-, 341122-, 341222-, and

Fig.14 5Pharmacogenetic and pharmacogenomic components of the therapeutic process in dementia.(Adapted from Cacabelos42)

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10q24.1-q24.3CYP2C19

10q24CYP2C9

10q24CYP2C8

19q13.2CYP2B6

1913.2CYP2A13

19q13.2CYP2A7

19q13.2CYP2A3/6

2p22-p21CYP1B1

15q22-qterCYP1A2

15q22-q24CYP1A1

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diciembre 2009 43

Pharmacogenomics of Alzheimer’s disease

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441112- genomic clusters. (vii) The worst responders in all genomic clusters are pa-tients with the 441122+ genotype. (viii) The interaction of several AD-related genes seems to be determinant for drug efficacy and safety. (ix) APOE-CYP2D6 interactions might influence the therapeutic response in AD via changes in lipid metabolism and liver function. (x) APOE may also interact with PSEN1, ACE, A2M and other genes to regulate the effect of drugs on cognition and behavioural changes in dementia. (xi) The APOE-4/4 genotype seems to acceler-ate neurodegeneration anticipating the onset of the disease by 5-10 years; and, in general, APOE-4/4 carriers show a faster disease progression and a poorer therapeu-tic response to all available treatments than any other polymorphic variant. (xii) Phar-macogenomic studies using monogenic, bigenic, trigenic, tetragenic or polygenic clusters as a harmonization procedure to reduce genomic heterogeneity in clinical trials are very useful in order to widen the therapeutic scope of limited pharmacologi-cal resources3-15,28,47-52.

Practical Considerations

The great variability in the therapeutic response of AD patients to conventional treatments (<20% effective responders), the heterogeneity of the disease and its complex pathogenesis, the occurrence of neuropsychiatric disorders associated with cognitive deterioration, as well as the pres-ence of other age-related disorders in pa-tients with dementia, seem to suggest that: (a) it is very unlikely that a single drug may be able to halt disease progression after the onset of the disease; (b) multifactorial interventions (as in other complex disor-ders, such as cardiovascular disease, cancer, AIDS, etc) might be an alternative strategy; however, drug-drug interactions in elderly patients who receive over 6 different drugs per day can represent a serious drawback in terms of safety; (c) the co-administration of many different drugs in patients with concomitant pathologies (i.e. coronary disease, hypertension, atherosclerosis, hy-perlipidemia, dementia) may represent an obstacle for an effective pharmacological management of dementia since some drugs effective for a peripheral medical condition can exert a deleterious effect on brain func-tion and brain perfusion with severe effects on cognition, behaviour and psychomotor function; (d) the fact that approximately 50-60% of patients with dementia exhibit

a marked cerebrovascular dysfunction rec-ommends that cerebrovascular protection should not be neglected in the treatment of AD; (e) the co-administration of psy-chotropic drugs should be carried out with extreme care as most psychotropics dete-riorate cognitive function, psychomotor ac-tivity, and cerebrovascular function; (f) the conventional procedures currently used in drug development (i.e. trial-and-error) and serendipity are not cost-effective nowadays; (g) the bimodal fashion of the amyloid-tau hypothesis of AD as a major target for fu-ture drug developments is a focus of con-troversy with unpredictable consequences for the industry and the public; (h) the reluctant attitude of the medical commu-nity to incorporate genomic procedures as diagnostic aids and disease biomarkers is not contributing to accelerating our under-standing of the dementia syndrome and its biological diversity; and (i) the underdevel-oped field of pharmacogenetics and phar-macogenomics is delaying the possibility of optimising our limited therapeutic resourc-es for the treatment of AD42..

The introduction of novel procedures into an integral genomic medicine pro-tocol for CNS disorders is an imperative requirement in drug development and in the clinical practice in order to im-prove diagnostic accuracy and to optimise therapeutics. This kind of protocol should integrate the following components: (i) clinical history, (ii) laboratory tests, (iii) neuropsychological assessment, (iv) car-diovascular evaluation, (v) conventional X-ray technology, (vi) structural neuroim-aging, (vii) functional neuroimaging, (viii) computerized brain electrophysiology, (ix) cerebrovascular evaluation, (x) structural genomics, (xi) functional genomics, (xii) pharmacogenetics, (xiii) pharmacogenom-ics, (ix) nutrigenetics, (x) nutrigenomics, (xi) bioinformatics for data management, and (xii) artificial intelligence procedures for diagnostic assignments and probabilis-tic therapeutic options4 (Table 8). All these procedures, under personalized strategies adapted to the complexity of each case, are essential in order to depict a clinical pro-file based on specific biomarkers correlat-ing with individual genomic profiles.

Our understanding of the pathophysiology of CNS disorders has advanced dramati-cally during the last 30 years, especially in terms of their molecular pathogenesis and genetics. The drug treatment of CNS disor-ders has also made remarkable strides, with the introduction of many new drugs for the

treatment of schizophrenia, depression, anxiety, epilepsy, Parkinson’s disease, and Alzheimer’s disease, among many other quantitatively and qualitatively important neuropsychiatric disorders. Improvement in terms of clinical outcome, however, has fallen short of expectations, with up to one third of the patients continuing to experi-ence clinical relapse or unacceptable med-ication-related side effects in spite of ef-forts to identify optimal treatment regimes with one or more drugs. Potential reasons to explain this historical setback might be that: (a) the molecular pathology of most CNS disorders is still poorly understood; (b) drug targets are inappropriate, not fit-ting into the real etiology of the disease; (c) most treatments are symptomatic, but not anti-pathogenic; (d) the genetic com-ponent of most CNS disorders is poorly defined; and (e) the understanding of ge-nome-drug interactions is very limited4,42.

Assuming that the human genome con-tains about 20,000-30,000 genes, at the present time only 0.31% of commercial drugs have been assigned to correspond-ing genes whose gene products might

The human genome contains

about 20,000-30,000 genes. At the present

time only 0.31% of commercial

drugs have been assigned to

corresponding genes

44

Personalized Medicine of Dementia

be involved in pharmacokinetic and phar-macodynamic activities of a given drug; and only 4% of the human genes have been assigned to a particular drug metabolic pathway. Supposing a theoretical number of 100,000 chemicals in current use world-wide, and assuming that practically all hu-man genes can interact with drugs taken by human beings, each gene in the human genome should be involved in the metabo-lism and/or biopharmacological effect of 30-40 drugs; however, assuming that most xenobiotic substances in contact with our organism can influence genomic function, it might be possible that for 1,000,000 xe-nobiotics in daily contact with humans, an average of 350-500 xenobiotics have to be assigned to each one of the genes poten-tially involved in drug metabolism and/or the processing of xenobiotics. To fulfil this task a single gene has to possess the capac-ity of metabolizing many different xenobi-otic substances and at the same time many different genes have to cooperate in or-chestrated networks in order to metabolise a particular drug or xenobiotic under se-quential biotransformation steps (Fig.14). Numerous chemicals increase the meta-bolic capability of organisms by their abil-ity to activate genes encoding various xe-nochemical-metabolising enzymes, such as CYPs, transferases and transporters. Many natural and artificial substances induce the hepatic CYP subfamilies in humans, and these inductions might lead to clinically important drug-drug interactions. Some of the key cellular receptors which mediate such inductions have been recently identi-fied, including nuclear receptors, such as the constitutive androstane receptor (CAR, NR1I3), the retinoid X receptor (RXR, NR2B1), the pregnane X receptor (PXR, NR1I3), and the vitamin D receptor (VDR, NR1I1) and steroid receptors such as the glucocorticoid receptor (GR, NR3C1)86. There is a wide promiscuity of these recep-tors in the induction of CYPs in response to xenobiotics. Indeed, this adaptive system acts as an effective network where recep-tors share partners, ligands, DNA response elements and target genes, influencing their mutual relative expression86,87.

The optimisation of CNS therapeutics re-quires the establishment of new postulates regarding (a) the costs of medicines, (b) the assessment of protocols for multifacto-rial treatment in chronic disorders, (c) the implementation of novel therapeutics ad-dressing causative factors, and (d) the set-ting-up of pharmacogenetic/pharmacog-enomic strategies for drug development42.

The cost of medicines is a very important issue in many countries due to (i) the growing of the aging population (>5% disability), (ii) neuropsychiatric and de-mented patients (>5% of the population) belonging to an unproductive sector with low income, and (iii) the high cost of health care systems and new health tech-nologies in developed countries. Despite the effort of the pharmaceutical industry to demonstrate the benefits and cost-ef-fectiveness of available drugs, the general impression in the medical community and in some governments is that some psycho-tropics and most anti-dementia drugs pres-ent in the market are not cost-effective2. Conventional drugs for neuropsychiatric disorders are relatively simple compounds with unreasonable prices. Some new prod-ucts are not superior to conventional an-tidepressants, neuroleptics, and anxiolyt-ics. There is an urgent need to assess the costs of new trials with pharmacogenetic and pharmacogenomic strategies, and to implement pharmacogenetic procedures for the prediction of drug-related adverse events. Pharmacogenomics can also help to reduce costs in drug development as well as the number of patients in clinical trials with high risk of toxicity. It has been suggested that the two critical strategies for pipeline genetics must make use of

fewer patients: (i) the early identification of efficacy signals so that they can be ap-plied early in development for targeted therapies, and (ii) identification of safety signals which can subsequently be vali-dated prospectively during development using the least number of patients with ad-verse responses83.

Cost-effectiveness analysis has been the most commonly applied framework for evaluating pharmacogenetics. Pharmacogenetic test-ing is potentially relevant to large popula-tions which incur in high costs. For instance, the most common drugs metabolised by CYP2D6 account for 189 million prescrip-tions and US$12.8 billion annually in expen-ditures in the US, which represent 5-10% of total utilization and expenditures for outpa-tient prescription drugs88. Pharmacogenom-ics offer great potential to improve patients’ health in a cost-effective manner; however, pharmacogenetics/pharmacogenomics will not be applied to all drugs available in the market, and careful evaluations should be made prior to investing resources in R&D of pharmacogenomic-based therapeutics and making reimbursement decisions89.

In performing pharmacogenomic studies in dementia, it is necessary to rethink the therapeutic expectations of novel drugs,

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redesign the protocols for drug clinical trials, and incorporate biological markers as assessable parameters of efficacy and prevention. In addition to the character-ization of genomic profiles, phenotypic profiling of responders and non-respond-ers to conventional drugs is also important (and currently neglected).

An important issue in AD therapeutics is that anti-dementia drugs should be effec-tive in covering the clinical spectrum of de-mentia symptoms represented by memory deficits, behavioural changes, and func-tional decline. It is difficult (or impossible) for a single drug to be able to fulfil these criteria. A potential solution to this prob-lem is the implementation of cost-effective, multifactorial (combination) treatments integrating several drugs, taking into con-sideration that traditional neuroleptics and novel antipsychotics (and many other psychotropics) deteriorate both cognitive and psychomotor functions in the elderly and may also increase the risk of stroke90. Few studies with combination treatments have been reported and most of them are poorly designed. We also have to realize that the vast majority of dementia cases in people older than 75-80% are of a mixed type, in which the cerebrovascular compo-nent associated with neurodegeneration

cannot be therapeutically neglected. In most cases of dementia, the multifactorial (combination) therapy appears to be the most effective strategy10,42,47-52. The combi-nation of several drugs increases the direct costs (e.g. medication) by 5-10%, but in turn, annual global costs are reduced by approximately 18-20% and the average survival rate increases about 30% (from 8 to 12 years post-diagnosis)4,42.

There are major concerns regarding the validity of clinical trials in patients with se-vere dementia. If we assume that AD is a complex disorder where genomic and en-vironmental factors interact to induce the premature death of neurons (which begins 30 years prior to the onset of the disease), it seems clear that future therapeutic strat-egies must be addressed towards the pre-vention of neurodegeneration because when the first symptoms appear thousands of millions of neurons have already died, and under these circumstances the possi-bility of being therapeutically effective is very remote.

Major impact factors associated with drug efficacy and safety include the following: (i) the mechanisms of action of drugs, (ii) drug-specific adverse reactions, (iii) drug-drug interactions, (iv) nutritional factors,

(v) vascular factors, (vi) social factors, and (vii) genomic factors (nutrigenetics, nutrigenomics, pharmacogenetics, phar-macogenomics). Among genomic factors, nutrigenetics/nutrigenomics and pharma-cogenetics/pharmacogenomics account for more than 80% of efficacy-safety out-comes in current therapeutics9,10,50,52.

To achieve a mature discipline of pharma-cogenetics and pharmacogenomics in CNS disorders and dementia it would be conve-nient to accelerate the following processes: (a) to educate physicians and the public on the use of genetic/genomic screening in the daily clinical practice; (b) to standard-ize genetic testing for major categories of drugs; (c) to validate pharmacogenetic and pharmacogenomic procedures accord-ing to drug category and pathology; (d) to regulate ethical, social, and economic is-sues; and (e) to incorporate pharmacoge-netic and pharmacogenomic procedures to both drugs in development and drugs on the market in order to optimise thera-peutics4,9,10,42,47-52.

Future Trends

The globalisation of the present economic crisis will negatively affect future invest-ments in dementia research and drug de-velopment; however, for the first half of the coming decade, after an initial period with some programmes put on standby, it is ex-pected that progress in AD pathogenesis, molecular diagnosis, and therapeutics will evolve favourably. Genome-wide family-based association studies, using single SNPs or haplotypes, will help to identify associa-tions with genome-wide significance30,31,91,92; similarly, genome-wide expression analysis will be useful for the discovery of new drug targets. Some studies will try to elucidate the weight of genome-environment inter-actions in the pathogenesis and clinical course of dementia, and also the emerg-ing role of epigenetics, as well. The valida-tion of protocols for genomic screening will contribute to introducing structural genomics (genotyping, genome-wide analysis), functional genomics (genotype-phenotype correlations), and proteom-ics as diagnostic aids and therapeutic tar-gets93. New initiatives for the prevention of dementia will also emerge94, together with new insights into the role of nutrition and nutrigenomics in brain function and neu-rodegeneration95.

46

Personalized Medicine of Dementia

Priority areas for pharmacogenetic research are to predict serious adverse reactions (ADRs) and to establish variation in efficacy96. Both require-ments are necessary in dementia to cope with efficacy and safety issues associated with both current anti-dementia drugs and new drugs. Since drug response is a complex trait, genome-wide approaches (oligonucleotide microarrays, proteomic profiling) may provide new insights into drug metabolism and drug response. Of paramount importance is the identification of polymorphisms affecting gene regulation and mRNA processing in genes encoding cyto-chrome P450s and other drug-metabolising en-zymes, drug transporters, and drug targets and receptors, with broad implication in pharmaco-genetics since functional polymorphisms which alter gene expression and mRNA processing appear to play a critical role in shaping human phenotypic variability97. It is also most relevant, from a practical point of view, to understand the pharmacogenomics of drug transporters, espe-cially ABCB1 (P-glycoprotein/MDR1) variants, due to the pleiotropic activity of this gene on a large number of drugs98. There are over 170 hu-man solute carrier transporters which transport a variety of substrates, including amino acids, lip-ids, inorganic ions, peptides, saccharides, metals, drugs, toxic xenobiotics, chemical compounds, and proteins99.

In approximately 3-5 years novel data on clinical trials with anti-amyloid vaccines will be delivered and AD immunotherapy will face new vaccine models (active and passive immunization) and new therapeutic challenges regarding the amy-loid burden in AD100. Other expected develop-ments in AD therapeutics include γ-secretase inhibitors, ß-secretase inhibitors, ß-sheet break-ers and chaperone inhibitors, regulators of the

ubiquitin-proteasome system, small molecule activators (non-peptide neurotrophic factors) of the Trk receptors, p38α mitogen-activated pro-tein kinase (MAPK) regulators, ADNP (activity-dependent neuroprotective protein) derivatives (NAP peptides), GSK-3ß modulators, phospho-lipase A2 inhibitors, the medium-chain triglyc-eride AC-1202, inhibitors of insulin-regulated aminopeptidase, amphiphilic pyridinium salts, and some other novel compounds, still in a pre-clinical stage, most of which are intended to be Aß lowering agents. There will be some initia-tives for nanotechnology approaches to crossing the blood-brain barrier and drug delivery to the CNS, as well as for new transdermal and intrana-sal delivery systems.

Another important issue in the pathogenesis and therapeutics of CNS disorders is the role of microRNAs (miRNAs). New inventories of miRNA expression profiles from CNS regions must be reported. These inventories of CNS miRNA profiles will provide an important step toward further elucidation of miRNA function and miRNA-related gene regulatory networks in the mammalian CNS. RNA interference (RNAi) has led in recent years to powerful ap-proaches to silencing targeted genes in a se-quence-specific manner with potential therapeu-tic applications in neurodegenerative diseases. RNAi procedures for gene selective inhibition must improve (a) cytoplasmic delivery of short sdRNA oligonucleotides (siRNA), which mimics an active intermediate of an endogenous RNAi mechanism, and (b) nuclear delivery of gene expression cassettes which express a short hair-pin RNA (shRNA), which mimics the micro in-terfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism. These technologies, complemented by non-viral gene delivery systems and ligand-targeted plasmid-based nanoparticles for RNAi agents, will bring new hopes for the treatment of different com-plex disorders101,102. We need more information about the feasibility of targeting AD genes (e.g. APP-London mutation, APP-Swedish mutation, PS1, APLP1, APLP2, PEN-2, APH-1a, Nicastrin, BACE, MAPT-V337M) with RNAi and making sure that gene silence in CNS disorders does not affect proteomic and/or metabolomic networks which are fundamental for a correct brain func-tion103. Another area of growing interest is the role of adult neurogenesis and stem cells in AD. Stem cell therapy has been suggested as a pos-sible strategy for replacing damaged circuitry and restoring learning and memory abilities in patients with AD; however, there is a long path ahead from the promising investigations which are raising hopes, and the challenges behind translating underlying stem cell biology into an effective therapy for AD104.

Prof. Dr. Ramón Cacabelosrcacabelos@gen-t.es

Some studies will try to elucidate the weight of genome-environment interactions in the pathogenesis and clinical course of dementia

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50

popTosis has been an intensive research area, which involves the study of compounds that trigger or inhibit this mechanism of death. Be-ing an important process implicated in many pathological diseases, including cancer, a num-ber of new natural compounds has been inves-tigated in the attempt to inhibit or trigger this fundamental cellular process making apoptosis amenable to new biopharmaceutical interven-tions. The aim of this study was to investigate the antitumor effect of FR-91, a standardized lysate of microbial cells belonging to the Ba-cillus genus, against SW872 (human adipose cells), SW982 (human synovial sarcoma line),

HL-60 (promyelocytic cells), HS 274.T (breast adenocarcinoma), HS 313.T (lymphoma), H2126 (lung adenocarcinoma), TOV 21G (epi-thelial ovarian cancer), WM 115 (melanoma), and HS 281T (breast adenocarcinoma) human tumor cell lines. We used the MTT (3-(4,5-dy-methylthiazol-2-y1)2,5-diphenyltetrazolium bromide) assay to study the growth inhibition activity of FR-91 and apoptosis. We report the potential apoptogenic activity after 24 hours incubation with 10, 25 and 50 μl/ml of FR-91. Apoptosis, determined by DNA cellular frag-mentation, was observed in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines treated

VRM Lombardi1, E. Martínez2, R. Chacon2, I. Etcheverría1, R. Cacabelos1

1. EBIOTEC, Department of Cellular Immunology , La Coruña, Spain2. Georgian Alternative Medicine, Madrid, Spain

A

Effects of FR-91on human tumor cell lines

diciembre 2009 51

with 25 and 50 μl/ml of FR-91. The highest lev-el of growth inhibition, cytotoxicity assay, was observed in SW872 (55, 78, 87%), SW982 (50, 70, 87%) and TOV-21G (42, 66, 85%), with re-spect to untreated cells, while the results of the expression of genes associated with apoptosis indicated a down-regulation of Bcl-2 in all cell lines. Taken together these results suggest that FR-91 contains small peptides which can con-tribute to the induction of apoptosis. Further investigations will be required to clarify the na-ture of these bioactive constituents and the in vivo effects of FR-91 on tumor-induced in ex-perimental animal models.

Introduction

Dysregulated proliferation appears to be a hall-mark of increased susceptibility to neoplasia. Cancer prevention is generally associated with inhibition, reversion, or retardation of cellular hyperproliferation. Both epidemiological and experimental studies have shown that a variety of food and food components, by exhibiting several biological properties that are able to modulate mammalian immune system, could be used for the prevention of many degenera-tive diseases1,2,3, and would be more economical and less painful, representing a new rational

52

Effects of FR-91 on human tumor cell lines

potentially pathogenic bacteria in the gut is de-pendent on the diet.

Studies in rats have shown that probiotics can inhibit the formation of aberrant crypt foci, thought to be a pre-cancerous lesion in the co-lon. Some of the best results were obtained with a probiotic strain consumed with inulin, a type of fructooligosaccharide. Total aberrant crypt foci, chemically induced, were reduced 74% by the treatment of rats with inulin and B. longum, but only 29 and 21% by B. longum and inulin alone, respectively24. There was a synergistic ef-fect in using both products together. Similar synergy was seen in rats with azoxymethane-in-duced colon cancer in another study. Rats fed Raftilose, a mixture of inulin and oligofructose, or Raftilose with Lactobacilli rhamnosus (LGG) and Bifidobacterium lactis (Bb12) had a signifi-cantly lower number of tumors compared to the control group25. A probiotic mixture, without any prebiotic, given to rats fed azoxymethane reduced colon tumors compared to the control (50% vs 90%), and also reduced the number of tumors per tumor-bearing rat26.

In the present study, different concentrations of FR-91, a standardized lysate of microbial cells belonging to the Bacillus genus, have been used to investigate potential antitumor activity against SW872, SW982, HL-60, HS 274.T, HS 313.T, H2126, TOV 21G, WM 115, and HS 281T human tumor cell lines.

Materials and methods

Cell lines and cell culturesHuman adipose cells (SW872) and human syn-ovial sarcoma line (SW 982) were obtained from the American Type Culture Collection (Rock-ville, MD, U.S.A.). Cells were maintained in L-15 (Leibovitz-15 medium with l-glutamine [PAA,

approach for cancer control. It is well known that dietary flavonoids and isoflavonoids behave as general cell growth inhibitors. One of their biological properties in plants, in fact, is provid-ing resistance to fungal or bacterial growth, and although most flavonoids and isoflavonoids ap-pear nontoxic to humans and animals, they have demonstrated to inhibit proliferation in many kinds of cultured human cancer cell lines4,5,6,7. Antiproliferative effects of quercetin8, taxifolin9, nobiletin10, and tangretin11 at 2-8 μg/ml for 3-7 day on squamous cell carcinoma, meningioma, colon carcinoma, leukaemia, and lung carcino-ma tumor cell lines have been reported.

Studies on mice, rats, and chickens have shown that many animals carry latent oncogenic viruses that usually remain harmless but occasionally become activated to cause the development of tumors or leukaemia12. The activation process can be triggered by several external or internal factors, such as radiation, certain hormones, or chemicals. However, the activation of the latent tumor-inducing viruses can be substantially de-layed, or even prevented, by avoiding or reduc-ing exposure to tumor-inducing factors, and also by the use of natural products13.

The bacteria that reside in the intestinal tract generally have a symbiotic relationship with their host. Beneficial bacteria produce natural antibiotics14 to keep pathogenic bugs in check (preventing diarrhea and infections) and pro-duce some B vitamins in the small intestine where they can be utilized. Beneficial bacteria help a) with food digestion15,16,17 by providing extra enzymes, such as lactase, in the small intes-tine; b) strengthen the immune system18,19 right in the gut where much of the interaction be-tween the outside world and the body goes on; and c) prevent food allergies20,21,22. In addition, they can help to prevent cancer at various stages of development23. These good bacteria can im-prove mineral absorption, maximizing food uti-lization. However, the balance of beneficial and

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MTT reduction assayCell proliferation assay was determined using MTT assay. Cell lines (1 x 105 cells/ml) were in-cubated in 96 well plates with different doses of FR-91 (10, 25 and 50 μl/w) for 24 hours. Ten μl of MTT (10 mg/ml) was added to each well and incubated further at 37ºC for 4 hours. After incubation, MTT-formazan precipitate was dis-solved in 100 μl of DMSO and absorbance was recorded at 570 nm in an automatic plate read-er (BioRAD instrument). Data are presented as percentage of cytotoxicity of treated versus un-treated cells.

DNA ladder assayDNA ladder assay was carried out as per standard method. This method prevents the contamina-tion of entire genomic DNA with fragmented DNA. Briefly, after treatment with FR-91, cells were harvested, washed twice with cold PBS and lysed for 30 min at 4ºC in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2% Triton X-100) using zirconium beads and automatic cell lyser. After centrifugation at 15,000 x g for 20 min, the supernatants was treated with pro-tease inhibitor cocktail and 0.5% SDS for 1 hour at 37ºC. DNA was extracted twice with phenol and precipitated with 150 mM NaCl and two vol-umes of ethanol at -20ºC. DNA precipitate was washed twice with cold 70% ethanol, dissolved in TE buffer and treated for 1 hour with Rnase at 37ºC. Finally, DNA precipitates were stained with propidium iodide, electrophoresed on 2% agarose gel and visualized in an automatic gel documentation system (BioRAD system).

Quantitative estimation of DNA fragmentation using an enzyme-linked immunosorbent assay (ELISA)After trypsin treatment, cells collected by brief centrifugation were plated to a density of 1104 cells per well on flat-bottomed 96-well plates (Co-star) and incubated overnight in complete me-dium at 37°C under a 5% CO2 atmosphere. The next day, the culture medium was exchanged for 100 μl of complete medium with or without the indicated concentration of 10, 25 and 50 μl of FR-91 and incubated for an additional

The Cell Culture Company, Pasching, Austria]), supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA) and sodium bicarbon-ate [1.5 g/liter; Gibco]) and incubated at 37°C and 5% CO2. HL-60 (promyelocytic cells) cell line was obtained from the American Type Cul-ture Collection. Cells were grown in RPMI 1640 medium supplemented with 10% heat inacti-vated FBS, 100 IU penicillin, 100 μg/ml strepto-mycin and 2 mM L-glutamine. HS 274.T (breast adenocarcinoma) cell line was obtained from the American Type Culture Collection. Cells were grown in Dulbecco’s modified Eagle’s me-dium (DMEM) supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. HS 313.T (lymphoma) cell line was obtained from the American Type Culture Collection. Cells were grown in DMEM supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. H2126 hy-potriploid cell line from metastatic site, pleural effusion adenocarcinoma, was obtained from the American Type Culture Collection. Cells were grown in DMEM/F12 supplemented with 5% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin, G5 supplement, and 2 mM L-glutamine. TOV 21G epithelial ovarian cancer cell line was obtained from the American Type Culture Collection. Cells were grown in DMEM supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. WM 115 (melanoma) cell line was obtained from the American Type Culture Collection. Cells were grown in Mini-mal essential Medium Eagle (MEM), supple-mented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. HS 281T (breast adenocarcinoma) cell line was obtained from the American Type Culture Collection. Cells were grown in DMEM supplemented with 10% heat inactivated FBS, 100 IU penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Cell stocks were maintained in liquid nitrogen.

Prior to use in experimental assays, the cells growing in monolayer were released from the culture flask with 0.25% trypsin (PAA), washed twice with fresh medium, and seeded onto 96-well microculture flat plates. Viable cell counts were confirmed prior to each experiment using trypan exclusion.

ChemicalsHEPES-buffered RPMI 1640 medium, DMEM, DMEM/F12, L-15, MEM, FBS, trypsin, G5 sup-plement, and CryoMaxx S were obtained from PAA. Glutamine and antibiotics (penicillin and streptomycin) were purchased from Sigma-Al-drich (Madrid, Spain).

The balance of beneficial and potentially pathogenic

bacteria in the gut is dependent on the diet

54

Effects of FR-91 on human tumor cell lines

merase for 30 cycles of denaturing at 94ºC for 1 min, annealing at 55ºC for 1 min and extension at 72ºC for 2 min. The resulting PCR products were analyzed by 1.5% agarose gel electropho-resis. Sequences for the specific primers used in the PCR were summarized in Table 1.

Statistical analysisAll statistical tests were performed with the use of SPSS (version 11.0; SPSS Inc, Chicago) and a P value <0.05 indicated statistical significance. Data were analyzed by using a two-factor re-peated-measures analysis of variance (ANOVA) followed by a post hoc analysis where relevant (one-factor repeated-measures ANOVA, fol-lowed by Tukey’s tests for a significant effect of dose and paired t tests for a significant effect of FR-91 treatment).

Results

FR-91 growth inhibition in tumor cell linesTo examine the possible antineoplastic effect of FR-91 in tumor cells, we first determined its effects on cell growth by the MTT assay, which measures the metabolically live cells based on their mitochondrial dehydrogenase activity. As shown in Fig 1, FR-91 caused growth inhibition in a dose-dependent manner. The cytotoxicity was not restricted to a specific cell line, since five different cell lines were sensible to the ef-fects of FR-91. The highest level of growth in-hibition was observed in SW872 (55%, 78%, 87%), SW982 (50%, 70%, 87%) and TOV-21G (42%, 66%, 85%), while HL-60 (6%, 33%, 48%) and HS 313.T (5%, 50%, 60%) showed a slight, but significant, cytotoxic effect with respect to untreated cells. At the lowest concentration of FR-91 (10μl/ml) a significant difference be-tween controls and treated cells was observed in SW 872, SW 982, and TOV-21G cell lines. No cytotoxic effects were observed with HS 274.T, H2126, WM 115, and HS281.T tumor cell lines. To determine if the antiproliferative effect was reversible, SW872, SW982, and TOV-21G cells were treated with 25 μl/ml FR-91 or culture me-dium for 48 hours. The medium-treated and FR-91-treated cells were then incubated in complete medium for 0, 6, 24, 48, and 72 hours followed by trypsinization and counting of the cells. After 48 hours of treatment with FR-91, the cell num-ber had decreased by 50% in all cell lines. Once FR-91 was removed, the cell number did not increase but rather showed a small further de-crease, whereas the cell number in the absence of treatment, as expected, increased with time (Fig. 2). These data indicate that the FR-91 effect was not reversible.

24 h. Insome experiments, cells were prein-cubated with z-VAD-fmk (Promega) or the ad-enosine kinase inhibitor 5’-iodotubercidin (IT; Sigma) prior to incubation with FR-91. Thereaf-ter, DNA fragmentation was quantitatively esti-mated in the remaining attached cells using an ELISA (Cell Detection ELISAPLUS, Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s protocol. Absorbance at 405 nm (reference at 492 nm) was measured in each well, and the means±SD were plotted as a function of the concentration of the indicated reagent. All control incubations contained the maximal concentration of DMSO, which was typically <0.1%; DMSO concentrations of up to 1.25% did not induce significant DNA fragmen-tation (data not shown). Each independent ex-periment was carried out in triplicate using all the different tumor cell lines.

RNA isolation and RT-PCRTotal cellular RNA was isolated by lysis in a guani-dinium isothiocyanate buffer followed by single step phenol-chloroform-isoamyl alcohol extrac-tion27. Briefly, cells were harvested and lysed in a solution containing 4M guanidium isothiocya-nate, 25mM sodium citrate (pH 7.0), 0.5% so-dium sarkosine and 0.1M β-mercaptoethanol. Sequentially, 1/10 volume of 2M sodium acetate (pH 4.9), one volume of phenol and 1/5 vol-ume of chloroform-isoamyl alcohol (49:1, v:v) were added to the homogenate. After vigorous shaking for 30 seconds, the solution was centri-fuged at 10,000 x g for 15 minutes at 4ºC. RNA in the aqueous phase was precipitated by the addition of 0.5 ml isopropanol. One μg of total RNA was reverse-transcribed into cDNA by in-cubating with 200 units of reverse transcriptase in 20 μl of reaction buffer containing 0.25 μg of random primers and 0.8 mM dNTPs at 42ºC for one hour. Two μl of the cDNA was used for the PCR reaction as templates. The PCR was per-formed in buffer containing 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 μM of each primer and 5 units Taq DNA poly-

Oligonucleotides used in the RT-PCR

p53 sense: antisense:

5’-AAAACTTACCAAGGCAACTA-3’5’-TGAAATATTCTCCATCGAGT-3’

p21 sense: antisense:

5’CATGTCCGATCCTGGTGATG-3’5’-AGTGCAAGACAGCGACAAGG-3’

Bcl-2 sense: antisense:

5’-TGCACCTGACGCCCTTCAC-3’5’-AGACAGCCAGGAGAAATCAAACAG-3’

Bax sense: antisense:

5’-ACCAAGAAGCTGAGCGAGTGTC-3’5’-ACAAAGATGGTCACGGTCTGCC-3’

Table 1

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FR-91 induction of apoptosis in tumor cell linesNext, we tested wether the administration of FR-91 induced any cytotoxic effects on SW872, SW982, HL-60, HS 274.T, HS 313.T, H2126, TOV 21G, WM 115, and HS 281T human tumor cell lines. When cells were treated with 10, 25, and 50 μl/ml of FR-91 for 24 hours and then ex-amined morphologically by light microscopy, a portion of the cells exhibited condensation (ar-row-head) and cleavage (arrow) of the nuclei, findings that are typical of apoptosis (Fig.3). No such images were observed in control, untreated cells. The following results, expressed as absor-bances (570 nm), clearly show that the treatment with FR-91 indeed induced apoptosis in HL-60 (FR-91 25 μl/ml: 0.96, p<0.001 vs negative, 0.12, and positive, 1.47, controls); FR-91 50 μl/ml: 1.1, p<0.001 vs negative, 0.18, and positive, 1.72, con-trols); HS 313T (FR-91 25 μl/ml: 0.77, p<0.001 vs negative, 0.18, and positive, 1.72, controls; FR-91 50 μl/ml: 0.68, p<0.001 vs negative, 0.18, and positive, 1.72, controls ); SW872 (FR-91 10 μl/ml: 0.58, p<0.001 vs negative, 0.17, and positive, 1.64, controls; FR-91 25 μl/ml: 0.76, p<0.001 vs negative, 0.17, and positive, 1.64, controls; FR-91 50 μl/ml: 1.55, p<0.001 vs negative, 0.17, and positive, 1.64, controls); SW982 (FR-91 10 μl/ml: 0.77, p<0.002 vs negative, 0.24, and positive, 1.8, controls; FR-91 25 μl/ml: 0.99, p<0.001 vs negative, 0.17, and positive, 1.64, controls; FR-91 50 μl/ml: 1.48, p<0.001 vs negative, 0.17, and positive, 1.64, controls); and TOV-21G (FR-91 25 μl/ml: 0.58, p<0.001 vs negative, 0.17, and posi-tive, 1.8, controls; FR-91 50 μl/ml: 1.6, p<0.001 vs negative, 0.17, and positive, 1.8, controls), cell lines (Fig. 4). DNA prepared from HL-60, HS 313T, SW872, SW982 and TOV-21G cells treated with FR-91 for 24 hours showed oligo-nucleosomal ladder fragmentation on agarose gel electrophoresis (data not shown). No signs of apoptosis were observed on HS 274.T (FR-91 10 μl/ml: 0.22, p=0.28 vs negative, 0.2, and posi-tive, 1.65, controls; FR-91 25 μl/ml: 0.24, p=0.08 vs negative, 0.22, and positive, 1.65, controls; FR-91 50 μl/ml: 0.2, p=0.33 vs negative, 0.22, and positive, 1.65, controls), H2126 (FR-91 10 μl/ml: 0.18, p=0.32 vs negative, 0.2, and positive, 1.75, controls; FR-91 25 μl/ml: 0.23, p=0.78 vs negative, 0.2, and positive, 1.75, controls; FR-91 50 μl/ml: 0.22, p=0.52 vs negative, 0.2, and positive, 1.75, controls), WM 115 (FR-91 10 μl/ml: 0.23, p=0.25 vs negative, 0.14, and positive, 1.69, controls; FR-91 25 μl/ml: 0.22, p=0.27 vs negative, 0.14, and positive, 1.69, controls; FR-91 50 μl/ml: 0.24, p=0.07 vs negative, 0.14, and positive, 1.69, controls), and HS 281T (FR-91 10 μl/ml: 0.2, p=0.25 vs negative, 0.24, and positive, 1.81, controls; FR-91 25 μl/ml: 0.22, p=0.27 vs negative, 0.24, and positive, 1.81, controls; FR-91 50 μl/ml: 0.24, p=0.07 vs negative, 0.24, and positive, 1.81, controls) (Fig. 5).

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% o

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itio

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FR-91 25 _l

FR-91 50 _l

5 Figure 1Growth inhibition of SW872, SW982, HL-60, HS 274.T, HS 313.T, H2126, TOV 21G, WM 115, and HS 281T human tumor cell lines. Cells were plated on 96 well plates and exposed to three 10, 25 and 50 μl/ml of FR-91. The growth inhibition is expressed as percentage from cells exposed to culture medium. The mean shown was calculated from five independent experiments performed by triplicate.

5 Figure 2Antiproliferative effects of FR-91 in SW 872, SW 982, and TOV-21G cells. Cells were treated for 48 hours with 25 μl/ml of FR-91 or complete medium. The medium was replaced with only medium, and after various times (0-72 hours) the cells were trypsinized and counted. Results are expressed relative to control (48 hours incubation with medium). All values are mean of triplicate cultures in three independent experiments.

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SW 872 TREATED

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SW982 TREATED

TOV-21G UNTREATED

TOV-21G TREATED

56

Effects of FR-91 on human tumor cell lines

Discussion

In the attempt of understanding and treating diseases, natural components have been dis-covered, by trial and error, and used for thou-sands of years by a significant fraction of the population in many countries or regions of the world. It is estimated that approximately 25% of the drugs prescribed worldwile at present derive from plants and 60% of antitumor/anti-infectious drugs already on the marker or under clinical investigations are of natural origin, and extracts from plants such as Taxol28, curcumin29, phenolic acids30 and flavonoids31 are reported to inhibit tumor growth in many types of can-cer. Tumor growth is generally associated with marked changes in hematopoiesis and immune response, myelosuppression and anemia. The immune system has several potential means to limit or even prevent tumor growth. These in-clude: a) specific T cell-mediated immunity against tumor associated transplantation anti-gens (TATAs) on tumor cells; b) production of antibodies against TATAs and/or other antigen-ic structures associated with the tumor cells; c) natural cell-mediated immunity against tumors, which consists mainly of natural killer (NK) cells and activated macrophages; and d) natural anti-tumor antibodies.

Although there is some evidence for substan-tial anti-tumor effects of specifically induced or natural antibodies, particularly if they are cyto-toxic in the presence of complement or in com-bination with lymphocytes or macrophages that express receptors for the Fc portion of immu-noglobulins (and hence can mediate antibody-dependent cell-mediated cytotoxicity (ADCC), most attention and experimental data have been focused on cell-mediated immunity as the basis for possible resistance of the host against pro-gressive growth of tumor. There has been much interest and controversy for many years over the possible role of each of these immunological mechanisms in prevention of the initial develop-ment of detectable tumors.

Considerations along these lines are encom-passed in the hypothesis of immunological sur-veillance against tumors. An updated version of this hypothesis, which includes the potential in-volvement of NK cells and other aspects of natu-ral immunity as well as specific T-cell mediated immunity has been reported32,33,34.

The most compelling support for this hypoth-esis has come from observations, in clinical situations as well as in experimental animal models, of substantially more frequent tumor development in immunodeficient individuals as compared to individuals with normally function-ing immune system. For example, the frequent

5 Figure 3Morphological examination of FR-91 treated SW 982 cells. SW 982 cells were cultured in the presence or absence of either 25 μl/ml of FR-91 and 1 μl/ml of apoptosis inducer mix (Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, Etoposide), as a positive control of apoptosis induction, for 24 hours as indicated in the materials and methods section. After Giemsa-staining, the morphological appearance of the cells was examined using light microscopy. The black arrows indicate nuclear condensation. Typical apoptotic cells, characterized by cleaved nuclei, are indicated by the white arrows. Magnification 400x.

Down-regulation of Bcl-2 gene expression in FR-91-treated tumor cell linesTo further investigate the molecular mechanisms responsible for the FR-91 induced apoptosis in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines, the gene expression of some apopto-sis-related genes such as p53, p21, Bax and Bcl-2 was analyzed by RT-PCR. In Fig. 6, FR-91 treat-ment caused the down-regulation of Bcl-2 gene expression, while other genes were not affected, which therefore resulted in the relative increase of Bax/Bcl-2 ratio.

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development of lymphoproliferative disease in immunosuppressed organ transplant recipients, patients with infection by the human immuno-deficiency virus (HIV), or in children with con-genital immunodeficiencies points strongly to a

key role of the immune system in preventing this type of cancer35,36. However, it has been very dif-ficult to determine clearly which components of the immune system are most critical for effective immunological surveillance.

p < 0 . 0 0 1

vs CN

00 , 2

0 , 40 , 6

0 , 81

1 , 21 , 4

1 , 61 , 8

N . C . P . C . FR-91 10 _l FR-91 25 _l FR-91 50 _l

e x p 1

e x p 2

e x p 3

e x p 4

e x p 5p < 0 . 0 0 1

vs CN

HL-60

H.S. 313.

0

0 , 5

1

1 , 5

2

2 , 5

C . N . C . P . FR-91 10 _l FR-91 25 _l FR-91 50 _l

e x p 1

e x p 2

e x p 3

e x p 4

e x p 5

p < 0 . 0 0 1

vs CN

p < 0 . 0 0 1

vs CN

SW 872

0

0 , 5

1

1 , 5

2

C . N . C . P . FR-91 10 _l FR-91 25 _l FR-91 50 _l

e x p 1

e x p 2

e x p 3

e x p 4

e x p 5

p < 0 . 0 0 1

vs CN

p < 0 . 0 0 1

vs CNp < 0 . 0 0 1

vs CN

SW 982

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e x p 1

e x p 2

e x p 3

e x p 4

e x p 5

p < 0 . 0 0 1

vs CN

p < 0 . 0 0 1

vs CNp < 0 . 0 0 1

vs CN

TOV-21G

0

0 , 5

1

1 , 5

2

2 , 5

C . N . C . P . FR-91 10 _l FR-91 25 _l FR-91 50 _l

e x p 1

e x p 2

e x p 3

e x p 4

e x p 5p < 0 . 0 0 1

vs CN

p < 0 . 0 0 1

vs CN

3 Figure 4Detection of DNA fragmentation in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines after culturing 24 hours in the presence of the indicated concentrations of FR-91 and 1 μl/ml of apoptosis inducer mix (Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, Etoposide), as a positive control of apoptosis induction. Experiments were repeated five times and the results are expressed as optical density.

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Effects of FR-91 on human tumor cell lines

It remains quite possible that several different arms of the immune system contribute to resis-tance against primary tumor development, with the appearance of overt malignant disease pos-sibly representing failure of more than one line of defense.

Although at least 120 chemical substances use-ful as antineoplastic drugs, and among them Paclitaxel (Taxol) isolated from three species

of the genus Taxus, are still isolated from plants throughout the world, research regarding the role of natural compounds in the treatment of different types of tumors still remains contro-versial. Epidemiological studies assessing dietary intake of natural compounds, although firmly based on biologically plausible hypotheses, have provided support for the association between reduced risk and bioactive intake, in most, but not all, studies published to date. Although the

H.S. 274

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WM 115

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H.S. 281.T

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e x p 1

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e x p 4

e x p 5

Figure 5 4Detection of DNA fragmentation

in HS 274.T, H2126, WM 115, and HS 281T cell lines after culturing

24 hours in the presence of the indicated concentrations of FR-91

and 1 μl/ml of apoptosis inducer mix (Actinomycin D, Camptothecin,

Cycloheximide, Dexamethasone, Etoposide), as a positive control of apoptosis induction. Experiments were repeated five times and the

results are expressed as optical density.

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regulatory proteins but also based on the ratio of them. If the ratio is high, the cells go to apop-tosis40. All cell lines used in the presented study have been induced by a variety of chemical re-agents to undergo apoptosis through different pathways such as p53-dependent pathway or Bcl-2 family-related pathway.

To clarify the molecular mechanism of apoptosis mediated by different concentrations of FR-91, we examined the expression of genes including p53, p21, Bax and Bcl-2 family by RT-PCR. Re-sults indicated that apoptosis occurred in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines treated with 25 μl/ml of FR-91 accompanied by the dose-dependent down-regulation of Bcl-2 gene expression, while others were not signifi-cantly changed (Fig. 6). In the present study, we demonstrated that the relative increase of apop-totic Bax/Bcl-2 ratio correlated well with FR-91-induced apoptosis in different human cell lines. It is possible that FR-91, through an appropriate signal, induces a conformational change in the Bax which moves to the mitochondrial mem-brane where it causes release of mitochondrial cytochrome c into the cytosol.

In conclusion, the use of FR-91 extracts exhib-ited an apoptosis-inducing effect in various hu-man tumor cell lines. Although further studies must be performed to elucidate the mechanisms by which FR-91 induces apoptosis in tumor cell lines, the present data indicate that FR-91 might be a useful chemotherapeutic compound for pa-tients with different types of tumors.

FR-91 extract has demonstrated significant in vitro and in vivo antineoplastic activities against different tumor cell lines, the mechanism of this effect has not been fully examined. In this study, FR-91 demonstrated selective cytotoxicity in vitro for human tumor cells tested (liposarcoma, sar-coma, promyelocitic, ovarian, and lymphoma cancer cell lines) when compared to untreated cells. Moreover, FR-91 showed the greatest cyto-toxicity towards HS 313.T cells that are resistant to chemotherapy. This compound’s ability to effectively kill several types of tumors without significant cytotoxicity to normal cells indicates that this compound may be a potentially chemo-therapeutic agent. The FR-91 induced apopto-sis occurred in a dose-dependent manner and was accompanied by the disruption of the mi-tochondrial transmembrane potential (data not shown) and the activation of caspase-3 and per-haps caspase-8.

The results of this study suggest that FR-91 inhibited proliferation of tumor cells by a mechanism that involves cytotoxicity. The pre-dominant form of cell death is likely apoptosis since evidence of apoptotic cell death was seen initially. However, at longer times of incuba-tions, necrotic cell death was also observed. It is likely that necrotic cell death occurred as a sec-ondary event and is a phenomen seen in vitro due to the lack of white cell phagocytosis. The possibility cannot be excluded, however, that FR-91 causes both forms of cell death and the incidence of these forms of death depends on the concentration that is used and the length of incubation.

Apoptosis is regulated and executed by differ-ent interplay of many genes responsive to vari-ous stimuli. There are two central patways that lead to apoptosis: 1) positive induction by ligand binding to a plasma membrane receptor and 2) negative induction by loss of a suppressor activ-ity. Positive-induction involves ligands related to TNF, while negative induction of apoptosis by loss of a suppressor activity involves the mito-chondria. The study of apoptosis in cancer ther-apy is very important37. It has been proved that occurrence of cancers is due to the loss of con-trol of normal apoptosis and the disturbance of balance between cell apoptosis and cell pro-liferation38. The apoptotis related genes (bcl-2 family) are divided in two categories: apoptotic repressor and apoptotic promoter. Bcl-2 is an important apoptotic repressor, while Bax is one of the most important apoptotic promoters. The protein it encodes can combine with Bcl-2 to form compounds which resist the action of repressing apoptosis. But it has a positive regulatory action39. Recent studies indicate that the regulation of apoptosis by Bcl-2 and Bax is not only based on the level of either of the two

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H.S. 313.T H.S. 313.T +

FR-91

SW 872 SW 872 +

FR-91

SW982 SW982 +

FR-91

TOV-21G TOV-21G +

FR-91

% o

f g

en

e e

xp

ressio

n

p53

p21

Bax

Bcl-2

5 Figure 6FR-91 induced the Bcl-2 down-regulation in HL-60, HS 313T, SW872, SW982 and TOV-21G cell lines. Cells were treated with 25μl of FR-91 as indicated for 24 hours and cells were harvested for RNA isolation and RT-PCR. Positive controls were treated with 1 μl/ml of apoptosis inducer mix (Actinomycin D, Camptothecin, Cycloheximide, Dexamethasone, Etoposide). Experiments were repeated three times and the results are expressed as % of gene expression compared to positive controls.

Dr. Valter R.M. Lombardibiotecnologia@ebotec.com

Effects of FR-91 on human tumor cell lines

60

29. Hussain AR, Ahmed M, Al-Jomah NA, Khan AS, Ma-nogaran P, Sultana M, Abubaker J, Platanias LC, Al-Kuraya KS, Uddin S. Curcumin suppresses constitu-tive activation of nuclear factor-kappa B and requires functional Bax to induce apoptosis in Burkitt’s lym-phoma cell lines. Mol Cancer Ther 2008; 7:3318-29.

30. Seeram NP. Berry fruits for cancer prevention: cu-rrent status and future prospects. J Agric Food Chem 2008; 56:630-5.

31. Raza H, John A. In vitro effects of tea polyphenols on redox metabolism, oxidative stress, and apoptosis in PC12 cells. Ann N Y Acad Sci 2008; 1138:358-65.

32. Varela JC, Imai M, Atkinson C, Ohta R, Rapisardo M, Tomlinson S. Modulation of protective T cell immu-nity by complement inhibitor expression on tumor cells. Cancer Res 2008; 68:6734-42.

33. Bergmann C, Strauss L, Wang Y, Szczepanski MJ, Lang S, Johnson JT, Whiteside TL. T regulatory type 1 cells in squamous cell carcinoma of the head and neck: mechanisms of suppression and expansion in advanced disease. Clin Cancer Res 2008; 14:3706-15.

34. Rethi B, Vivar N, Sammicheli S, Fluur C, Ruffin N, Atlas A, Rajnavolgyi E, Chiodi F. Priming of T cells to Fas-mediated proliferative signals by interleukin-7. Blood 2008; 112:1195-1204.

35. Sullivan RJ, Pantanowitz L, Casper C, Stebbing J, Dezube BJ. HIV/AIDS: epidemiology, pathophysio-logy, and treatment of Kaposi sarcoma-associated herpesvirus disease: Kaposi sarcoma, primary effu-sion lymphoma, and multicentric Castleman disease. Clin Infect Dis 2008; 47:1209-15.

36. Chua I, Quinti I, Grimbacher B. Lymphoma in com-mon variable immunodeficiency: interplay between immune dysregulation, infection and genetics. Curr Opin Hematol 2008; 15:368-74.

37. Johansson M, Persson JL. Cancer therapy: targeting cell cycle regulators. Anticancer Agents Med Chem 2008; 8:723-31.

38. Brantley EC, Nabors LB, Gillespie GY, Choi YH, Pal-mer CA, Harrison K, Roarty K, Benveniste EN. Loss of protein inhibitors of activated STAT-3 expression in glioblastoma multiforme tumors: implications for STAT-3 activation and gene expression. Clin Cancer Res 2008; 14:4694-704.

39. Heath-Engel HM, Chang NC, Shore GC. The en-doplasmic reticulum in apoptosis and autophagy: role of the BCL-2 protein family. Oncogene 2008; 27:6419-33.

40. Reagan-Shaw S, Nihal M, Ahsan H, Mukhtar H, Ahmad N. Combination of vitamin E and selenium causes an induction of apoptosis of human prostate cancer cells by enhancing Bax/Bcl-2 ratio. Prostate 2008; 68:1624-34.

mixtures of probiotics or probiotics and prebiotics in a randomized controlled trial. Am J Clin Nutr 2008; 87:1365-73.

16. Macfarlane GT, Steed H, Macfarlane S. Bacterial me-tabolism and health-related effects of galacto-oligo-saccharides and other prebiotics. J Appl Microbiol 2008; 104:305-44.

17. Blaut M, Clavel T. Metabolic diversity of the intestinal microbiota: implications for health and disease. J Nutr 2007; 137:751S-5S.

18. Isolauri E, Salminen S; Nutrition, Allergy, Mucosal Im-munology, and Intestinal Microbiota (NAMI) Research Group Report. Probiotics: use in allergic disorders: a Nutrition, Allergy, Mucosal Immunology, and Intesti-nal Microbiota (NAMI) Research Group Report. J Clin Gastroenterol 2008; 42:S91-S96.

19. Klein K, Stevens R. The clinical use of probiotics for young children. J Fam Health Care 2008; 18:66-8.

20. Ouwehand AC. Antiallergic effects of probiotics. J Nutr 2007; 137:794S-797S.

21. del Giudice MM, Rocco A, Capristo C. Probiotics in the atopic march: highlights and new insights. Dig Liver Dis 2006; 38:S288-S290.

22. Parvez S, Malik KA, Ah Kang S, Kim HY. Probiotics and their fermented food products are beneficial for health. J Appl Microbiol 2006; 100:1171-85.

23. Milner JA. Nutrition and cancer: essential elements for a roadmap. Cancer Lett 2008; 269:189-98.

24. Rowland IR, Rumney CJ, Coutts JT, Lievense LC. Effect of Bifidobacterium longum and inulin on gut bacterial metabolism and carcinogen-induced aberrant crypt foci in rats. Carcinogenesis 1998; 19:281-5.

25. Femia AP, Luceri C, Dolara P, Giannini A, Biggeri A, Salvadori M, Clune Y, Collins KJ, Paglierani M, Caderni G. Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on azoxymethane-induced colon carcinogenesis in rats. Carcinogenesis 2002; 23:1953-60.

26. Marotta F, Naito Y, Minelli E, Tajiri H, Bertuccelli J, Wu CC, Min CH, Hotten P, Fesce E. Chemopreventive effect of a probiotic preparation on the development of preneoplastic and neoplastic colonic lesions: an experimental study. Hepatogastroenterology 2003; 50:1914-8.

27. Wang JW, Cheng JQ. A simple method for profi-ling miRNA expression. Methods Mol Biol 2008; 414:183-90.

28. Drago-Ferrante R, Santulli A, Di Fiore R, Giuliano M, Calvaruso G, Tesoriere G, Vento R. Low doses of paclitaxel potently induce apoptosis in human reti-noblastoma Y79 cells by up-regulating E2F1. Int J Oncol 2008; 33:677-87.

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6. Yang H, Landis-Piwowar KR, Chen D, Milacic V, Dou QP. Natural compounds with proteasome inhibitory activity for cancer prevention and treatment. Curr Protein Pept Sci 2008; 9:227-39.

7. Yu W, Jia L, Wang P, Lawson KA, Simmons-Mencha-ca M, Park SK, Sun L, Sanders BG, Kline K. In vitro and in vivo evaluation of anticancer actions of natu-ral and synthetic vitamin E forms. Mol Nutr Food Res 2008; 52:447-56.

8. Choi EJ, Bae SM, Ahn WS. Antiproliferative effects of quercetin through cell cycle arrest and apoptosis in human breast cancer MDA-MB-453 cells. Arch Pharm Res 2008; 31:1281-5.

9. Brusselmans K, Vrolix R, Verhoeven G, Swinnen JV.Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid syn-thase activity. J Biol Chem 2005; 280: 5636-45.

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11. Kong D, Aoki S, Sowa Y, Sakai T, Kobayashi M. Sme-nospongine, a sesquiterpene aminoquinone from a marine sponge, induces g1 arrest or apoptosis in different leukemia cells. Mar Drugs 2008; 6: 480-8.

12. Rezacova M, Zaskodova D, Vavrova J, Vokurkova D, Tichy A. Antileukemic activity of the combination of ionizing radiation with valproic acid in promyelocytic leukemia cells HL-60. Neoplasma 2008; 55:519-25.

13. Lollini PL, Nicoletti G, Landuzzi L, De Giovanni C, Nanni P. New target antigens for cancer immunopre-vention. Curr Cancer Drug Targets 2005; 5:221-8.

14. Salvini F, Granieri L, Gemmellaro L, Giovannini M. Probiotics, prebiotics and child health: where are we going? J Int Med Res 2004; 32: 97-108.

15. Chouraqui JP, Grathwohl D, Labaune JM, Hascoet JM, de Montgolfier I, Leclaire M, Giarre M, Steenhout P. Assessment of the safety, tolerance, and protective effect against diarrhea of infant formulas containing

L

62

a farmacogenómica se encuentra en pleno desarrollo. Pero 20 años de investigación han sido suficientes para desmostrar que los méto-dos basados en la farmacogenómica permiten detectar factores hereditarios que causan altera-ciones en el metabolismo de fármacos y en la predisposición a padecer reacciones adversas. Y estos factores hereditarios pueden ser detecta-dos con sencillos análisis genéticos. El resultado se conoce en horas. Uno de los objetivos de la farmacogenómica es ajustar la dosis del fármaco a la capacidad metabólica del paciente.

Los métodos basados en la farmacogenómica muestran su mayor utilidad con fármacos cuyo

metabolismo predominante es llevado a cabo por una enzima polimórfica, cuando las muta-ciones que se analizan son frecuentes en la po-blación de estudio y tienen un claro efecto sobre los parámetros farmacocinéticos y/o la respues-ta clínica a ese fármaco.

Con la mayor parte de los antiinflamatorios no esteroideos (AINEs), se dan las tres situaciones anteriores, y, por este motivo, estos fármacos constituyen un excelente ejemplo de cómo la determinación de mutaciones en determinados genes puede ser de utilidad en la aplicación personalizada de los tratamientos más adecua-dos y seguros para cada paciente según su ge-

Personalizar el Tratamiento

diciembre 2009 63

notipo. Con la mayor parte de AINEs, los genes más relevantes son los que codifican las enzimas citocromos P450 CYP2C8 y CYP2C9. En estos genes existen determinadas mutaciones y, lo que es más importante aún, determinadas com-binaciones de ellas (haplotipos) que se asocian a la aparición de reacciones adversas severas. Ac-tualmente, disponemos de test genéticos funda-mentales en la detección de pacientes con alto riesgo de desarrollar reacciones adversas con estos medicamentos y con cuyo uso se pueden seleccionar, para cada paciente, los AINEs que comporten un menor riesgo de provocar reac-ciones adversas.

La variabilidad interindividual en la respuesta a los fármacos es una importante causa de efec-tos adversos. En muchos casos, esta variabilidad está ligada al polimorfismo de genes que codi-fican las enzimas responsables del metabolismo de dichos fármacos. La mayoría de enzimas que metabolizan fármacos es polimórfica, debido a la presencia de mutaciones en los genes que las codifican. Estas mutaciones, que consisten en la ausencia completa del gen, polimorfismos de un solo nucleótido, aislados o combinados, y duplicaciones génicas, causan ausencia, reduc-ción, alteración o incremento de la actividad enzimática 1, 2.

Prof. Dr. J.A. García-Agúndez Departamento de Farmacología y Psiquiatría, Facultad de Medicina

Universidad de Extremadura, Badajoz, España

64

Farmacogenómica de los AINEs

Las enzimas citocromo P450 2C8 (CYP2C8) y 2C9 (CYP2C9) pertenecen a una de las princi-pales familias de enzimas implicadas en el me-tabolismo de fármacos. Los genes que codifican estas dos enzimas, junto a los que codifican los otros componentes de CYP2C (denominados CYP2C18 y CYP2C19) se agrupan en dos clusters consecutivos en el cromosoma 10 y muestran un alto grado de asociación, de modo que la pre-sencia de mutaciones en uno de los genes sue-le coincidir con mutaciones en otros genes del cluster 7.

La importancia clínica de los polimorfismos de CYP2C8 y CYP2C9 radica en la concurrencia de dos factores: ambas enzimas están implicadas en el metabolismo de numerosos fármacos de uso clínico, algunos de los cuales tienen un margen terapéutico muy estrecho, y, además, un porcen-taje elevado de la población española es porta-dora de mutaciones en los genes que codifican estas enzimas.

Más de 30 millones de personas son tratadas diariamente con antiinflamatorios no esteroideos (AINEs) y cerca del 25% de la población ha experimentado alguna vez en su vida reacciones adversas causadas por AINEs que han requerido tratamiento médico 8, 9. Entre estas reacciones adversas, las que tienen una mayor relevancia clínica, por su severidad y frecuencia, son las relacionadas con las hemorragias digestivas altas (HDA) que, solamente en los estados Unidos causan más de 30.000 hospitalizaciones anuales10. Se define como hemorragia digestiva

Los portadores de mutaciones en genes codifi-cadores de enzimas metabolizadoras de fárma-cos, cuando son tratados con dosis estándar de un fármaco que sea sustrato de la enzima afec-tada, suelen presentar niveles plasmáticos más elevados, cifras de aclaramiento más bajas 3, 4, y un incremento en la frecuencia y severidad de reacciones adversas secundarias al uso de dicho fármaco 5, 6.

La farmacogenómica es un área de la farmacolo-gía que se encuentra en pleno desarrollo y que estudia la contribución de factores genéticos a las diferencias interindividuales en la respuesta a fármacos. Aunque el ámbito de estudio de la far-macogenómica implica también la variabilidad farmacodinámica - por ejemplo, a través del es-tudio de polimorfismos en genes que codifican receptores -, es en el ámbito de la farmacocinéti-ca, y, en particular, en el metabolismo de fárma-cos donde la farmacogenómica ha adquirido un desarrollo pleno y donde se están obteniendo los primeros resultados de utilidad clínica.

La utilidad clínica de la farmacogenética es ya un hecho en el metabolismo de los fármacos

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alta (HDA) la que se origina en una lesión situada por encima del ángulo de Treitz. Es una causa frecuente de consulta en servicios de urgencias y de ingreso hospitalario. En España, presenta una incidencia de entre 85 y 106 casos por 100.000 habitantes/año 11. La mortalidad asociada a la HDA ha permanecido invariable en las últimas décadas 12 , debido, fundamentalmente, al incremento de la edad de los pacientes con HDA y a la coexistencia de enfermedades asociadas 13.

La tasa de mortalidad por HDA secundaria a AI-NEs en España es de más del 5% 11. En estudios realizados en otros países, se ha estimado que uno de cada 1.200 pacientes tratado con AINEs por vía oral durante al menos 2 meses morirá, debido a complicaciones gastroduodenales di-rectamente relacionadas con el uso de AINEs 14.

Además del peligro que pueden suponer las re-acciones adversas a AINEs, las implicaciones eco-nómicas para el sistema sanitario son también muy relevantes. El coste de los efectos adversos gastrointestinales a veces supera el propio coste de los AINEs 15, 16. No sorprende, por lo tanto, el creciente interés de las autoridades sanitarias en la identificación de pacientes con alto riesgo de desarrollar hemorragias digestivas con el uso de AINEs.

En los últimos años se han realizado considera-bles avances en el conocimiento de las enzimas implicadas en el metabolismo de los AINEs y en la identificación de metabolitos de estos fárma-cos. Adicionalmente, se han desarrollado pro-cedimientos para identificar pacientes con una alteración en el metabolismo de estos fármacos determinada genéticamente. Aunque los AINEs constituyen un grupo de fármacos químicamen-te heterogéneo, la mayor parte de ellos compar-te las principales enzimas implicadas en su meta-bolismo, que son CYP2C8 y CYP2C9.

Sin embargo, el papel relativo de estas enzimas difiere entre diferentes AINEs. La Tabla 1 resu-me las principales enzimas implicadas en el me-tabolismo de AINEs. Los polimorfismos en los genes que codifican estas enzimas causan impor-tantes cambios en la farmacocinética de algunos de estos AINEs, de los cuales los más importantes son aquellos cuyo metabolismo predominante es a través de estas enzimas: celecoxib, ibuprofe-no, lornoxicam y piroxicam son metabolizados en más de un 90% por estas enzimas, mientras que aceclofenaco, diclofenaco, flurbiprofeno, indometacina, meloxicam y tenoxicam son me-tabolizados en más de un 50% por CYP2C8 y/o CYP2C9.

Dado que existen numerosas mutaciones en los genes que codifican estas enzimas que pue-

30 millones de personas son tratadas diariamente

con AINEs

Tabla 1: Principales enzimas implicadas en el metabolismo de AINEs

FármacoRelevancia de

CYP2CEnzima

principalEnzima

secundaria

Aceclofenaco parcial CYP2C9 Esterasas plasmáticas

Aspirina secundario UGT1A6 CYP2C9

Celecoxib predominante CYP2C9

Diclofenaco parcial CYP2C9 UGT2B7, y diversos CYPs

Dipirona secundario CYP2C19, CYP2C8 CYPs

Etoricoxib secundario CYP3A4

Flurbiprofeno parcial CYP2C9

Ibuprofeno predominante CYP2C8, CYP2C9

Indometacina parcial CYP2C9 Carboxil esterasas

Ketoprofeno secundario UGTs CYP

Lornoxicam predominante CYP2C9

Meloxicam parcial CYP2C9 CYP3A4

Naproxeno secundario UGT2B7 CYP2C9, CYP1A2

Parecoxib secundario Hidrolisis a valdecoxib

CYP3A4, CYP2C9

Piroxicam predominante CYP2C9

Rofecoxib secundario UGT2B7, UGT2B15 CYP2C9, CYP3A4

Sulindac secundario UGTs CYP2C9

Tenoxicam parcial CYP2C9

Valdecoxib secundario CYP3A4 CYP2C9

Relevancia de las enzimas CYP2C en el metabolismo primario de AINEs. Predominante: más del 90% del fármaco es metabolizado por CYP2C8 o CYP2C9. Parcial: del 50% al 90% del fármaco es metabolizado por CYP2C8 o CYP2C9. Secundario: Menos del 50% del fármaco es metabolizado por CYP2C8 o CYP2C9.

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Farmacogenómica de los AINEs

Tabla 2: Variantes del gen CYP2C8 que causan sustituciones de aminoácidos.

Nombre del aleloCambio de aminoácido

Efecto en la actividad

Identificación del SNP.

CYP2C8*2 I269F Aumento en la Km rs11572103

CYP2C8*3 R139K; K399RDisminución rs11572080;

rs10509681

CYP2C8*4 I264M No concluyente rs1058930

CYP2C8*5 159 Frame shift Ausente Sin designación

CYP2C8*7 R186X Ausente Sin designación

CYP2C8*8 R186G Desconocido Sin designación

CYP2C8*9 K247R Desconocido Sin designación

CYP2C8*10 K383N Desconocido Sin designación

CYP2C8*12 V461deletion Desconocido Sin designación

CYP2C8*13 I223M Desconocido Sin designación

CYP2C8*14 A238P Desconocido Sin designación

Sin designación A82S Desconocido rs17851796

Sin designación I244V Desconocido rs11572102

Sin designación L361F Desconocido rs45438799

En cuanto a CYP2C9, existen numerosas varian-tes que conducen a una baja actividad enzimáti-ca, pero la mayor parte de ellas son muy infre-cuentes. Entre las más frecuentes, se encuentran, para individuos de origen caucásico, CYP2C9*2 y CYP2C9*3. En individuos orientales, son muy infrecuentes y se limitan a CYP2C9*3 y en indivi-duos de origen africano aparecen diversas varian-tes con frecuencias entre el 1% y el 3% que inclu-yen CYP2C9*2, *3, *5, *6 y *11. Además se han demostrado variaciones en las frecuencias entre distintas poblaciones del mismo origen étnico. Por ejemplo, la variante CYP2C9*3 aparece con una frecuencia del 11% en Españoles y menos de la mitad de esta frecuencia en Suecos 7, 17.

Se ha descrito una clara asociación entre la pre-sencia de variantes de CYP2C8 y/o de CYP2C9 y la capacidad de metabolizar diversos AINEs.

La farmacocinética de celecoxib, diclofenaco, flurbiprofeno, ibuprofeno, lornoxicam, pi-roxicam y tenoxicam se ve alterada en portado-res de estas variantes alélicas que, comparados con los no portadores, presentan una farmaco-cinética más lenta y, por lo tanto, pueden pre-sentar efectos adversos con más facilidad que los no portadores.

Recientes estudios llevados a cabo por nuestro grupo – y, posteriormente, confirmados por va-rios grupos independientes 18-23 - indican de for-ma inequívoca que los portadores de variantes de CYP2C8 y/o CYP2C9 presentan un mayor riesgo de desarrollar hemorragias digestivas altas (HDA) asociadas al tratamiento con AINEs. En nuestra población, la frecuencia de portadores de al menos una de estas mutaciones es de más del 40% 3, 4. Estas personas tienen un mayor ries-go de presentar HDA cuando son tratados con AINEs que son sustratos de CYP2C8 y CYP2C9.

Sin embargo, el grupo más llamativo es el de portadores de varias de estas mutaciones simul-táneamente, especialmente si son homocigotos 20-23. Cerca del 10% de la población española pertenece a este grupo que tiene un riesgo glo-bal de desarrollar hemorragias digestivas cuan-do son tratados con AINEs que puede ser 4 veces superior al de la población general, especial-mente cuando concurren variantes de CYP2C8 y CYP2C9 en el mismo paciente.

Por otra parte, el incremento del riesgo en este grupo de portadores de variantes genéticas varía para cada fármaco. Así, fármacos considerados seguros para la mayor parte de la población, como el ibuprofeno, pueden convertirse en fár-macos inseguros para este 10% de personas que son portadores homocigotos. En estos pacien-tes, es más seguro el uso de aspirina o diclofena-co, que son AINEs teóricamente menos seguros

den modificar la actividad metabólica, y, por ende, la farmacocinética de estos AINEs, los portadores de estas mutaciones pueden tener un mayor riesgo de desarrollar reacciones ad-versas cuando sean tratados con estos fármacos.

La Tabla 2 resume las principales mutaciones en el gen CYP2C8, y la Tabla 3 resume las prin-cipales mutaciones en el gen CYP2C9. Por ra-zones de espacio, solamente se han incluido aquellas en las que la variante causa una sustitu-ción de aminoácido. Las variantes CYP2C8*2, *3, *5, *7 y *8 conducen a una alteración de la actividad enzimática. Las más frecuentes son CYP2C8*2 en individuos de origen africano, CYP2C8*3 y CYP2C8*4 en individuos de ori-gen caucásico (europeo) y CYP2C8*5 en indi-viduos de origen oriental. El resto aparece con frecuencias muy bajas.

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Tabla 3: Variantes del gen CYP2C9 que causan sustituciones de aminoazidos.

para la población general que el ibuprofeno. Con el caso del naproxeno, el efecto es aún más llamativo. El naproxeno está considerado como un AINE de riesgo intermedio para el desarrollo de HDA; sin embargo, en este 10% de personas que son portadoras de variantes de CYP2C8 y/o CYP2C9 en homocigosidad, el riesgo de presen-tar HDA con naproxeno se multiplica en un fac-tor de casi 5, lo que convierte a este fármaco en uno de los AINEs con mayor riesgo 20-23.

Por los motivos expuestos, resulta de extraordi-nario interés saber si un paciente pertenece a ese 40% de portadores de mutaciones, y espe-cialmente si pertenece a ese 10% de portadores homocigotos, antes de iniciar un tratamiento con AINEs. Existen varios AINEs alternativos en los que las variantes de CYP2C8 y CYP2C9 tie-nen una escasa relevancia, como la aspirina, el paracetamol, los coxibs etoricoxib, rofecoxib o parecoxib, el ketoprofeno o la dipirona (meta-mizol). En los pacientes con alteraciones en los genes CYP2C8 y/o CYP2C9, deberían utilizarse estos AINEs en lugar de aquellos cuya farmaco-cinética o sus efectos adversos se asocian a las variaciones genéticas de CYP2C8 y CYP2C9.

Una buena predicción del riesgo genético pue-de incrementar la calidad de vida y la seguridad de estos pacientes. Uno de los primeros objetivos de la farmacogenómica es el ajuste de la dosis de acuerdo a la capacidad metabólica del paciente. Este objetivo es especialmente relevante cuando hay pocos tratamientos alternativos, por ejem-plo, con determinados antineoplásicos. Pero, afortunadamente, la farmacología actual dispo-ne de un amplio rango de AINEs que nos permi-te simplemente seleccionar otros tratamientos más seguros para los pacientes con un factor ge-nético de riesgo.

Es previsible, y deseable, que en breve, antes de iniciar un tratamiento con AINEs, los médicos sepamos a qué tipo genético de paciente esta-mos tratando para seleccionar no sólo la dosis más adecuada, sino también el fármaco más seguro para este paciente en concreto. De este modo, la farmacogenómica, con el concurso de otras disciplinas que ahora están en sus inicios, como la toxicogenómica y la metabolómica, nos ayudará a llegar al objetivo de una medicina lo más personalizada, segura y eficaz posible.

Nombre del alelo

Cambio de aminoácido

Efecto en la actividad

Identificación del SNP.

CYP2C9*2 R144C Disminución rs1799853

CYP2C9*3 I359L Disminución rs1057910

CYP2C9*4 I359T Desconocido rs56165452

CYP2C9*5 D360E Disminución rs28371686

CYP2C9*6 273 Frame shift Ausente rs9332131

CYP2C9*7 L19I Desconocido Sin designación

CYP2C9*8 R150H No concluyente rs7900194

CYP2C9*9 H251R Desconocido rs2256871

CYP2C9*10 E272G Desconocido rs9332130

CYP2C9*11 R335W Disminución rs28371685

CYP2C9*12 P489S Disminución rs9332239

CYP2C9*13 L90P Disminución Sin designación

CYP2C9*14 R125H Disminución Sin designación

CYP2C9*15 S162X Ausente Sin designación

CYP2C9*16 T299A Disminución Sin designación

CYP2C9*17 P382S Desconocido Sin designación

CYP2C9*18 I359L; D397A Disminución rs1057910; sin designación

CYP2C9*19 Q454H Desconocido Sin designación

CYP2C9*20 G70R Desconocido Sin designación

CYP2C9*21 P30L Desconocido Sin designación

Nombre del alelo

Cambio de aminoácido

Efecto en la actividad

Identificación del SNP.

CYP2C9*22 N41D Desconocido Sin designación

CYP2C9*23 V76M Desconocido Sin designación

CYP2C9*24 E354K Desconocido Sin designación

CYP2C9*25 118 Frame shift Ausente Sin designación

CYP2C9*26 T130R Disminución Sin designación

CYP2C9*27 R150L Desconocido Sin designación

CYP2C9*28 Q214L Disminución Sin designación

CYP2C9*29 P279T Desconocido Sin designación

CYP2C9*30 A477T Disminución Sin designación

CYP2C9*31 I327T Desconocido rs57505750

CYP2C9*32 V490F Desconocido Sin designación

CYP2C9*33 R132Q Disminución Sin designación

CYP2C9*34 R335Q Desconocido Sin designación

Sin designación I112L Desconocido rs5030781

Sin designación R124Q Desconocido rs12414460

Sin designación R150C Desconocido rs17847037

Sin designación P337L Desconocido rs58368927

Sin designación Y358C Desconocido rs1057909

Sin designación L413P Desconocido rs28371687

Sin designación L447F Desconocido rs59485260

Prof. Dr. J.A. García-Agúndezjagundez@unex.es

Farmacogenómica de los AINEs

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13. Allan R, Dykes P. A study of the factors influencing mortality rates from gastrointestinal haemorr-hage. Q J Med 1976;45(180):533-50.

14. Tramer MR, Moore RA, Reynolds DJ, McQuay HJ. Quantitative estimation of rare adverse events which follow a biological progression: a new model applied to chronic NSAID use. Pain 2000;85(1-2):169-82.

15. Walan A, Wahlqvist P. Pharmacoeconomic aspects of non-steroidal anti-inflammatory drug gastro-pathy. Ital J Gastroenterol Hepatol 1999;31 Suppl 1:S79-88.

16. Rahme E, Joseph L, Kong SX, Watson DJ, LeLorier J. Cost of prescribed NSAID-related gastrointes-tinal adverse events in elderly patients. Br J Clin Pharmacol 2001;52(2):185-92.

17. Garcia-Martin E, Martinez C, Ladero JM, Gamito FJ, Agundez JA. High frequency of mutations related to impaired CYP2C9 metabolism in a Caucasian population. Eur J Clin Pharmacol 2001;57(1):47-9.

18. Pilotto A, Seripa D, Franceschi M, et al. Gene-tic susceptibility to NSAID-related gastro-duodenal bleeding: Role of cytochrome P450 (CYP) 2C9 polymorphisms. Gastroenterology 2007;doi:10.1053/j.gastro.2007.05.025.

19. Vonkeman HE, van de Laar MA, van der Palen J, Brouwers JR, Vermes I. Allele variants of the cyto-chrome P450 2C9 genotype in white subjects from The Netherlands with serious gastroduo-denal ulcers attributable to the use of NSAIDs. Clin Ther 2006;28(10):1670-6.

20. Blanco G, Martinez C, Ladero JM, et al. Interaction of CYP2C8 and CYP2C9 genotypes modifies the risk for nonsteroidal anti-inflammatory drugs-related acute gastrointestinal bleeding. Pharma-cogenet Genomics 2008;18(1):37-43.

21. Agundez JA, Garcia-Martin E, Martinez C. Genetica-lly based impairment in CYP2C8- and CYP2C9-dependent NSAID metabolism as a risk factor for gastrointestinal bleeding: is a combination of pharmacogenomics and metabolomics required to improve personalized medicine? Expert Opin Drug Metab Toxicol 2009;5(6):607-20.

22. Agundez JA, Martinez C, Garcia-Martin E, Ladero JM. Cytochrome P450 CYP2C9 polymorphism and NSAID-related acute gastrointestinal blee-ding. Gastroenterology 2007;133(6):2071-2.

23. Martinez C, Blanco G, Ladero JM, et al. Genetic predisposition to acute gastrointestinal bleeding after NSAIDs use. Br J Pharmacol 2004.

Referencias Bibliográficas

1. Meyer UA. Pharmacogenetics - five decades of therapeutic lessons from genetic diversity. Nat Rev Genet 2004;5(9):669-76.

2. Ingelman-Sundberg M. Pharmacogenetics of cytochrome P450 and its applications in drug the-rapy: the past, present and future. Trends Pharmacol Sci 2004;25(4):193-200.

3. Martinez C, Garcia-Martin E, Blanco G, Gamito FJ, Ladero JM, Agundez JA. The effect of the cyto-chrome P450 CYP2C8 polymorphism on the dis-position of (R)-ibuprofen enantiomer in healthy subjects. Br J Clin Pharmacol 2005;59(1):62-9.

4. Garcia-Martin E, Martinez C, Tabares B, Frias J, Agundez JA. Interindividual variability in ibuprofen pharmacokinetics is related to interaction of cy-tochrome P450 2C8 and 2C9 amino acid polymor-phisms. Clin Pharmacol Ther 2004;76(2):119-27.

5. Martinez C, Blanco G, Ladero JM, et al. Genetic pre-disposition to acute gastrointestinal bleeding after NSAIDs use. Br J Pharmacol 2004;141(2):205-8.

6. Sanderson S, Emery J, Higgins J. CYP2C9 gene va-riants, drug dose, and bleeding risk in warfarin-treated patients: a HuGEnet systematic review and meta-analysis. Genet Med 2005;7(2):97-104.

7. Garcia-Martin E, Martinez C, Ladero JM, Agundez JA. Interethnic and intraethnic variability of CYP2C8 and CYP2C9 polymorphisms in healthy individuals. Mol Diagn Ther 2006;10(1):29-40.

8. Singh G, Triadafilopoulos G. Epidemiology of NSAID induced gastrointestinal complications. J Rheu-matol Suppl 1999;56:18-24.

9. Bloom BS. Direct medical costs of disease and gastrointestinal side effects during treatment for arthritis. Am J Med 1988;84(2A):20-4.

10. Tarone RE, Blot WJ, McLaughlin JK. Nonselecti-ve nonaspirin nonsteroidal anti-inflammatory drugs and gastrointestinal bleeding: relative and absolute risk estimates from recent epidemiolo-gic studies. Am J Ther 2004;11(1):17-25.

11. Lanas A, Perez-Aisa MA, Feu F, et al. A nationwi-de study of mortality associated with hospi-tal admission due to severe gastrointestinal events and those associated with nonsteroidal antiinflammatory drug use. Am J Gastroenterol 2005;100(8):1685-93.

12. Olsen KM. Use of acid-suppression therapy for treatment of non-variceal upper gastrointestinal bleeding. Am J Health Syst Pharm 2005;62(10 Su-ppl 2):S18-23.

HepatoSar® es el primer nutracéutico compuesto por un 57% de una estructura lipoproteica natural de origen marino(S. pilchardus) y un extracto vegetal de Cynara scolymus L. estandarizado al 5%, que aporta propiedades muy beneficiosas sobre la función hepatobiliar y la digestión de las grasas.

70

Genómica de la patología cerebrovascular

l accidenTe cerebrovascular (ACV) se pro-duce por la interacción de múltiples facto-res ambientales y mutaciones en diferentes genes. Estos polimorfismos genéticos deter-minan la susceptibilidad o la resistencia a la enfermedad y la respuesta al tratamiento. Debido a la interacción entre genes y am-biente, las enfermedades complejas se pue-den prevenir a través de la actuación sobre

Elos factores ambientales con un plan de prevención adecuado, siempre y cuando se tenga conocimiento del riesgo genético re-lativo de padecer la enfermedad, lo que se consigue con un buen panel genético de sus-ceptibilidad.

La definición del panel de riesgo genético cerebrovascular pasa por abordar el estudio

Dr. Juan Carlos Carril Departamento de Genómica e Identificación Humana Euroespes Biotecnología, Bergondo, Coruña, España

diciembre 2009 71

de genes implicados en los diferentes eventos que desencadenan el proceso aterogénico: metabolismo lipídico, respuesta inmunitaria y estabilidad de la placa de ateroma.

La determinación de los factores genéticos y sus interacciones con factores ambientales que ocasionan el desarrollo de la enfermedad, tan sólo es la mitad del camino hacia la obtención

de un test genético predictivo de utilidad en la práctica diagnóstica.

La validación del panel genético, la determina-ción de diferencias entre individuos enfermos y controles sanos, así como la elección del mode-lo estadístico adecuado, es la clave para obtener una herramienta predictiva de utilidad real en la práctica médica.

72

Genómica de la patología cerebrovascular

Accidente Cerebrovascular

Un accidente cerebrovascular (ACV), ictus o in-farto cerebral consiste en la alteración perma-nente o transitoria de la función cerebral que aparece como consecuencia de un trastorno cir-culatorio, bien de los vasos cerebrales o bien de alteraciones hemáticas.

La incidencia del accidente cerebrovascular, igual que otras enfermedades, es variable en di-ferentes países y tiene relación con factores ge-néticos, edad de la población y factores ambien-tales asociados1. La incidencia de nuevos casos en España se sitúa alrededor de 156 por 100.000

habitantes, aunque es presumible que estén alre-dedor de los 200 casos anuales2.

Existen muy pocos datos sobre la prevalencia de ictus en España, con frecuencias que oscilan entre el 2.1% en la población mayor de 20 años hasta el 8.5% en la población mayor de 65 años3. La mortalidad por ictus en España oscila entre un 10% y un 34% en las estadísticas hospitala-rias, siendo mucho más elevada en los casos de hemorragia cerebral4.

El accidente cerebrovascular es un episodio neu-rológico agudo, con afectación de las funciones del sistema nervioso central. Según su etiología se suelen clasificar en:

Accidentes isquémicos:También se llaman infartos cerebrales y se deben a la oclusión de alguna de las arterias que irrigan la masa encefálica, generalmente por aterosclerosis.

Podemos distinguir:

a. Accidente isquémico transitorio.- Episodio de déficit focal de la circulación cerebral, de comienzo brusco, con altera-ciones que duran generalmente unos 2-10 minutos pero que pueden persistir hasta las 24 horas.

b. Déficit neurológico isquémico reversible.- La duración del cuadro deficitario es superior a las 24 horas, pero los síntomas y signos clínicos desaparecen de forma total durante las tres semanas siguientes al episodio.

c. Infarto cerebral.- Como consecuencia de la falta de aporte circulatorio a un territorio cerebral se presenta un déficit neurológico, de duración superior a 24 horas. El infarto puede ser silente, pero generalmente da manifestaciones clínicas neurológicas según el territorio afectado.

d. Infarto cerebral de tipo aterotrombótico.- La lesión de la pared del vaso determina una estenosis u oclusión de la luz arterial y se produce una lesión dentro de su territorio de irrigación que puede ser total o parcial, dependiendo de la posible compensación de la circulación colateral.

Accidentes hemorrágicos:También se denominan hemorragias cerebrales o apoplejías y se deben a la ruptura de un vaso sanguíneo encefálico debido a un pico hipertensivo o a un aneurisma congénito.

Tipos de hemorragias cerebrales:

a. Infarto cerebral por embolismo de origen cardíaco.- La lesión de las válvulas cardíacas, del miocardio y/o los trastornos del ritmo cardíaco dan origen a trombos que llegan a las arterias cerebrales.

b. Infarto hemorrágico cerebral.- Sobre la lesión isquémica se produce un fondo hemorrágico por alteración de la barrera hematoencefálica en una zona de reperfusión, generalmente tras la lisis del émbolo.

c. Infarto lacunar.- Es un infarto pequeño, de menos de 15 mm, situado en las áreas profundas del cerebro o del tronco cerebral, que se produce por la oclusión de las ramas perforantes de las arterias cerebrales.

d. Hemorragia intracerebral.- Es una colección hemática dentro del parénquima encefálico debido a la rotura de un vaso encefálico.

diciembre 2009 73

Aterogénesis

La aterosclerosis es un síndrome caracterizado por el depósito de sustancias lipídicas, llamado placa de ateroma, en las paredes de las arterias de mediano y grueso calibre. El término “ateros-clerosis” proviene de los vocablos griegos athero (pasta) y skleros (duro/piedra).

No debe confundirse con arterioesclerosis, ya que esta última se refiere al endurecimiento de las paredes arteriales - arterio de arteria, esclerosis de endurecimiento - y en todo caso, el término arterioesclerosis abarca varias afecciones que llevan al endurecimiento, incluyendo la ateros-clerosis.

La aterosclerosis puede ser considerada como una forma de inflamación crónica resultado de la interacción entre lipoproteínas modifica-das, macrófagos derivados de monocitos, célu-las T, y los elementos celulares normales de la pared arterial. Este proceso inflamatorio puede dar lugar a la formación de lesiones complejas, o placas, que resaltan en el lumen arterial. La ruptura de la placa y la trombosis dan lugar a complicaciones clínicas agudas como el infarto de miocardio y el ictus5.

A mediados del siglo XIX, Virchow6 describió el engrosamiento de la íntima debido al acú-mulo de cristales de colesterol extracelular, así como en el interior de las llamadas “células es-pumosas”, como característica histológica clave en la aterosclerosis. De este modo, formuló la denominada como “Hipótesis de Infiltración”, en la que se establece la aterosclerosis como el resultado de la infiltración de lípidos y células inflamatorias procedentes de la sangre.

En 1972, Ross y Glomset7 establecieron la deno-minada “Hipótesis de Respuesta a la Lesión”, según la cual la pared vascular responde, por disfunción endotelial y proliferación de células vasculares de músculo liso, cuando es dañada mecánicamente por flujo anormal o por otros agentes nocivos como el tabaquismo o las pro-teínas glicoxiladas de pacientes con diabetes.

Basándose en el trabajo sobre los mecanismos de incorporación celular del colesterol de Golds-tein y Brown8 (por el que recibieron el Premio Nobel), Steinberg et al.9 postularon la denomi-nada “Hipótesis del LDL modificado”, según la cual las modificaciones de LDL-colesterol, incluida la oxidación, aumentan su captación por los macrófagos dando lugar a las células es-pumosas.

Fig. 1 5Fases iniciales de la lesión aterosclerótica: Oxidación de LDL, migración celular y

formación de células espumosas.

Fig. 2 5Evolución de la lesión aterosclerótica: Respuesta inmunitaria,

migración de células de músculo liso y formación de la capa fibrosa.

Fig. 3 5Fases tardías de la lesión aterosclerótica: Muerte celular programada, formación de las

“gachas” de lípido extracelular y agregación plaquetaria.

74

Entre los factores de riesgo modificables, rela-cionados con hábitos de vida, podemos destacar el tabaquismo y la vida sedentaria. Las sustan-cias tóxicas que contiene el tabaco, como la ni-cotina, tienen un efecto tóxico directo sobre la pared de las arterias, provocando una respuesta inflamatoria. Otros factores de riesgo no ge-nético serían: la dieta, las infecciones víricas y bacterianas, el ambiente fetal y la polución am-biental.

Evolución de la lesión aterosclerótica

Las lesiones ateroscleróticas comienzan como una línea grasa bajo el endotelio de las grandes arterias. La llegada de macrófagos y colesterol derivado de LDL son los principales eventos celulares que contribuyen a la formación de la línea grasa.

El primer evento de carácter aterogénico consis-te en la oxidación del LDL-colesterol. Los lípi-dos y moléculas de ApoB que forman parte del LDL sufren modificaciones oxidativas mínimas y da lugar al mmLDL, que todavía es reconocido por los receptores de LDL (Fig. 1).

Cuando estas modificaciones se hacen más ex-tensivas, ApoB se fragmenta y los residuos de li-sina se modifican covalentemente con reactivos producto de la fragmentación de lípidos oxida-dos, dando lugar a oxLDL, que ya no tiene capa-cidad para unirse a los receptores de LDL, y es capturado por los receptores limpiadores (“sca-venger receptors”) que expresan en macrófagos y células musculares lisas.

Las células endoteliales, en respuesta al estímu-lo inflamatorio que provoca el oxLDL, generan moléculas de adhesión celular en la superficie y tiene lugar el reclutamiento de monocitos a las regiones propensas a lesiones de las grandes ar-terias y su subsiguiente diferenciación en macró-fagos. Este fenómeno de migración a la íntima, si bien puede cumplir inicialmente una función protectiva eliminando partículas oxLDL proin-flamatorias y citotóxicas y células apoptóticas, finalmente contribuye en el desarrollo de lesio-nes ateroscleróticas.

A continuación se desarrollan las denominadas “células espumosas” o macrófagos “foam cell”, que contienen cantidades masivas de ésteres de colesterol.

La transición desde la relativamente simple lí-nea grasa hasta la lesión más compleja se carac-teriza por la inmigración de células de músculo liso desde la capa media de la pared arterial

Factores de riesgo de enfermedad vascular

Entre los muchos factores de riesgo ambientales y genéticos, los niveles elevados de colesterol en suero son, por sí solos, suficientes para provocar el desarrollo aterosclerótico, incluso en ausen-cia de otros factores de riesgo. El estudio de los mecanismos moleculares que controlan la bio-síntesis de colesterol y los niveles de colesterol en suero condujo al desarrollo de las “estatinas”, una potente clase de drogas que reducen el co-lesterol y que se han demostrado efectivas en la reducción de la mortalidad cardiovascular en pacientes con hipercolesterolemia.

Otro factor de riesgo relevante es la hipertensión arterial, que provoca fuerzas de cizallamiento que rompen el frágil endotelio que recubre la su-perficie interior de las arterias. Por otra parte, se ha descrito que las hormonas masculinas son ate-rogénicas, mientras que los estrógenos protegen de la aterosclerosis, por eso las mujeres se afec-tan después de la menopausia. Este factor, junto con la edad, no depende del estilo de vida.

En definitiva, podemos enumerar los principa-les factores de riesgo genéticos y su grado de he-redabilidad como:

• LDL y VLDL colesterol elevados (40%–60%) (Hipercolesterolemia).

• HDL colesterol bajo (45%–75%).

• Triglicéridos elevados (40%–80%).

• Índice de Masa Corporal incrementado (25%–60%) (Obesidad).

• Presión sistólica elevada (50%–70%) (Hipertensión arterial).

• Presión diastólica elevada (50%–65%).

• Lipoproteína (a) elevada (90%).

• Homocisteína elevada (45%).

• Diabetes mellitus tipo 2 (40%–80%).

• Fibrinógeno elevado (20%–50%).

• Proteína C-reactiva elevada (40%).

• Sexo (Hormonas sexuales).

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Genes y polimorfismos relacionados con enfermedad vascular

Símbolo Gen Locus Polimorfismo dbSNP

ABCA1 ATP-binding cassette, subfamily A, member 1 9q22-q31 T-477C

ABCA1 ATP-binding cassette, subfamily A, member 1 9q22-q31 G1051A (Arg219Lys) rs2230806

ABCA1 ATP-binding cassette, subfamily A, member 1 9q22-q31 A2583G (Ile823Met) rs4149313

ACE Angiotensin I- converting enzyme 17q23 A-240T rs4291

ACE Angiotensin I- converting enzyme 17q23 Intron 16 Alu 287bp I/D

ADRB1 Beta-1-adrenergic receptor 10q24-q26 G1165C (Gly389Arg) rs1801253

ADRB2 Beta-2-adrenergic receptorHA 5q32-q34 A46G (Arg16Gly) rs1042713

ADRB2 Beta-2-adrenergic receptor 5q32-q34 C79G (Gln27Glu) rs1042714

ADRB3 Beta-3-adrenergic receptor 8p12-p11.2 T190C (Trp64Arg) rs4994

AGT Angiotensinogen 1q42-q43 G-6A rs5051

AGT Angiotensinogen 1q42-q43 M235T rs699

AGT Angiotensinogen 1q42-q43 T174M rs4762

AGTR1 Angiotensin receptor 1 3q21-q25 C-535T rs1492078

AGTR1 Angiotensin receptor 1 3q21-q25 A1166C rs5186

AGTR1 Angiotensin receptor 1 3q21-q25 G→A (Ala163Thr) rs12721226

AGTR1 Angiotensin receptor 1 3q21-q25 G→T (Ala244Ser) rs12721225

AGTR1 Angiotensin receptor 1 3q21-q25 A→C (Thr336Pro) rs1801021

AGTR2 Angiotensin II receptor, type 2 Xq22-q23 G1675A rs1403543

AGTR2 Angiotensin II receptor, type 2 Xq22-q23 C3123A rs11091046

ALOX5 Arachidonate 5-lipoxygenase 10q11.2 C3175G rs12762604

ALOX5 Arachidonate 5-lipoxygenase 10q11.2 Sp1 STR

ALOX5 Arachidonate 5-lipoxygenase 10q11.2 G→A (Phe42Leu) rs12762604

ALOX5 Arachidonate 5-lipoxygenase 10q11.2 G→A (Glu254Lys) rs2228065

ANXA5 Annexin A5 4q26-q28 C-1T rs11575945

AP2M1 Adaptor-related protein complex 2, MU-1 subunit 3q28 G62T rs1501299

APOA1 Apolipoprotein A-I 11q23 G-75A rs670

APOA1 Apolipoprotein A-I 11q23 T84C rs5070

APOA5 Apolipoprotein A-V 11q23 T-1131C rs662799

APOB Apolipoprotein B 2p24 C2488T (XbaI)

APOB Apolipoprotein B 2p24 G10708A (Arg3500Gln)

APOB Apolipoprotein B 2p24 C10800T (Arg3531Cys)

APOC3 Apolipoprotein C-III 11q23 C-482T rs2854117

APOC3 Apolipoprotein C-III 11q23 C1100T rs4520

APOC3 Apolipoprotein C-III 11q23 C3175G (S1/S2) rs5128

Tabla 1

Continúa 6

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Genómica de la patología cerebrovascular

Símbolo Gen Locus Polimorfismo dbSNP

APOE Apolipoprotein E 19q13.2 G-219T rs405509

APOE Apolipoprotein E 19q13.2 T3932C (Cys112Arg) rs429358

APOE Apolipoprotein E 19q13.2 C4070T (Arg158Cys) rs7412

CAPN10 Calpain 10 2q37.3 G4852A rs3792267

CCL2 Chemokine, CC motif, ligand 2 17q11.2-q12 A-2518G

CCL5 Chemokine, CC motif, ligand 5 17q11.2-q12 C-28G rs2280788

CCL5 Chemokine, CC motif, ligand 5 17q11.2-q12 G-403A rs2107538

CCL11 Chemokine, CC motif, ligand 11 17q21.1-q21.2 G→A (Ala23Thr) rs3744508

CCND1 Cyclin D1 11q13 G→T (Ala30Ser) rs2220247

CCR2 Chemokine, CC motif, receptor 2 3p21 G190A (Val64Ile) rs1799864

CCR5 Chemokine, CC motif, receptor 5 3p21 G59029A rs1799987

CD14 Monocyte differentiation antigen CD14 5q31.1 C-260T rs2569190

CD36 CD36 antigen 7q11.2 G30294C rs1049673

CD36 CD36 antigen 7q11.2 C12293T (Pro90Ser)

CD40 CD40 antigen 20q12-q13.2 A455T

CETP Cholesteryl ester transfer protein, plasma 16q21 G279A (TaqB1/B2)

CETP Cholesteryl ester transfer protein, plasma 16q21 C-629A rs1800775

CETP Cholesteryl ester transfer protein, plasma 16q21 A1061G (Ile405Val) rs5882

COL1A2 Collagen, type I, alpha-2 7q22.1 G→C (Ala459Pro) rs42524

COL3A1 Collagen, type III, alpha-1 2q31 G2209A (Ala698Thr) rs1800255

COL3A1 Collagen, type III, alpha-1 2q31 A3730G (Ile1205Val) rs2271683

CRP C-reactive protein, pentraxin-related 1q21-q23 C1444T rs1130864

CX3CR1 Chemokine, CX3C motif, receptor 1 3pter-p21 C926T (Thr280Met) rs3732378

CXCL16 Chemokine, CXC motif, ligand 16 17p13 C→T (Ala181Val) rs2277680

ELN Elastin 7q11.2 G1264A (Gly422Ser) rs2071307

EPHX2 Epoxide hydrolase 2, cytosolic 8p21-p12 G→A (Arg287Gln) rs751141

ESR1 Estrogen receptor 1 6q25.1 T-1989G rs2071454

F3 Coagulation factor III 1p22-p21 A-603G rs1361600

F7 Factor VII 13q34 G11496A (Arg353Gln) rs6046

F12 Factor XII 5q33-qter C46T rs17876008

FABP2 Fatty acid-binding protein 2 4q28-q31 G2445A (Ala54Thr) rs1799883

FBN1 Fibrillin 1 15q21.1 T1875C rs25458

Símbolo Gen Locus Polimorfismo dbSNP

FGB Fibrinogen, B beta polypeptide 4q28 G-455A rs1800790

FGB Fibrinogen, B beta polypeptide 4q28 G8059A (Arg448Lys) rs4220

Continuación Tabla 1

Continúa 6

diciembre 2009 77

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Símbolo Gen Locus Polimorfismo dbSNP

HNF4A Hepatocyte nuclear factor 4-alpha 20q12-q13.1 A→G rs2425640

ICAM1 Intercellular adhesion molecule 1 19p13.3-p13.2 G1462A (Glu469Lys) rs5498

IGF2R Insulin-like growth factor II receptor 6q26 A5002G (Arg1619Gly) rs629849

IL1B Interleukin 1-beta 2q14 C-511T rs16944

IL1RN Interleukin 1 Receptor Antagonist 2q14.2 IL1RN*2 VNTR

IL6 Interleukin 6 7p21 G-174C

IL6 Interleukin 6 7p21 G-572C rs1800796

IL10 Interleukin 10 1q31-q32 A-1082G

IL10 Interleukin 10 1q31-q32 T-819C rs1800871

IL10 Interleukin 10 1q31-q32 A-592C rs1800872

INS Insulin 11p15.5 T-23A rs689

INSR Insulin receptor 19p13.2 C7067365A rs2860172

IPF1 Insulin promoter factor 1 13q12.1 -108/3G→4G (S82168)

IRS1 Insulin receptor substrate 1 2q36 A3694G (Ser892Gly) rs1801277

IRS1 Insulin receptor substrate 1 2q36 G3931A (Gly972Arg) rs1801278

ITGA2 Integrin, alpha-2 5q23-q31 A1648G (Lys505Glu) rs10471371

ITGB2 Integrin, beta-2 21q22.3 C1323T rs235326

LDLR Low density lipoprotein receptor 19p13.2 G1184A (Ala370Thr) rs11669576

LMNA Lamin A/C 1q21.2 A→G (Glu2Gly) rs11549669

LPL Lipoprotein lipase 8p22 C1595G (Ser447Stop) rs328

MMP1 Matrix metalloproteinase 1 11q22-q23 -1607/1G→2G rs1799750

MMP1 Matrix metalloproteinase 1 11q22-q23 A-519G

MMP1 Matrix metalloproteinase 1 11q22-q23 T-340C

MMP2 Matrix metalloproteinase 2 16q13 C-1306T rs243865

MMP3 Matrix metalloproteinase 3 11q23 -1171/5A→6A rs3025058

MMP3 Matrix metalloproteinase 3 11q23 indel-1612A

MMP3 Matrix metalloproteinase 3 11q23 A-709G

MMP3 Matrix metalloproteinase 3 11q23 A→G (Lys45Glu) rs679620

MMP3 Matrix metalloproteinase 3 11q23 A→C (His113Pro) rs11606831

MMP9 Matrix metalloproteinase 9 20q11.2-q13.1 C-1562T rs3918242

MMP9 Matrix metalloproteinase 9 20q11.2-q13.1 G855A (Arg279Gln) rs2664538

MMP12 Matrix metalloproteinase 12 11q22.2-q22.3 A-82G rs2276109

MPO Myeloperoxidase 17q23.1 G-463A (NT_035425)

MPO Myeloperoxidase 17q23.1 G-129A (AH002972)

MTHFR 5,10-Methylenetetrahydrofolate reductase 1p36.3 C677T (Ala222Val) rs1801133

Continuación Tabla 1

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Símbolo Gen Locus Polimorfismo dbSNP

NFKB Nuclear Factor Kappa B, subunit 1 4q23-q24 indel-94ATTG

NOS3 Nitric oxide synthase 3 7q36 G37498A

NOS3 Nitric oxide synthase 3 7q36 T-786C rs2070744

NOS3 Nitric oxide synthase 3 7q36 4a/4b

NOS3 Nitric oxide synthase 3 7q36 G894T

NPY Neuropeptide Y 7p15.1 T1128C (L7P)

OLR1 Low density lipoprotein, oxidized, receptor 1 12p13-p12 G501C (Lys167Asn) rs11053646

P2RY12 Purinergic receptor P2Y, G protein-coupled, 12 3q24-q25 T744C NC_000003

PAI1 Plasminogen activator inhibitor 1 7q21.3-q22 -668/4G→5G rs1799768

PAI1 Plasminogen activator inhibitor 1 7q21.3-q22 A→C (His25Pro) rs2227647

PAI1 Plasminogen activator inhibitor 1 7q21.3-q22 G→A (Arg209His) rs2227669

PAI1 Plasminogen activator inhibitor 1 7q21.3-q22 A→G (Tyr243Cys) rs13306846

PAX4 Paired box gene 4 7q32 C567T (Arg121Trp) (AF043978)

PECAM1 Platelet-endothelial cell adhesion molecule 1 17q23 C1454G (Leu125Val) rs668

PECAM1 Platelet-endothelial cell adhesion molecule 1 17q23 G2201A (Gly670Arg) rs1131012

PIK3R1 Phosphatidylinositol 3-kinase, regulatory, 1 5q13 G1020A (Met326Ile) rs3730089

PON1 Paraoxonase 1 7q21.3 G-162A rs705381

PON1 Paraoxonase 1 7q21.3 A532G (Arg160Gly) rs13306698

PON1 Paraoxonase 1 7q21.3 G584A (Gln192Arg) rs662

PON2 Paraoxonase 2 7q21.3 C475G (Ala148Gly) rs11545941

PPARD Peroxisome proliferator-activated receptor-delta 6p21.2-p21.1 T294C rs2016520

PPARG Peroxisome proliferator-activated receptor-gamma 3p25 C-681G rs10865710

PPARG Peroxisome proliferator-activated receptor-gamma 3p25 C34G (Pro12Ala) rs1801282

PPARGC1 Peroxisome proliferator-activated receptor-gamma, coactivator 1 4p15.1 G1564A (Gly482Ser) rs8192678

SAH Hypertension-associated SA, rat, homolog of 16p13.11 A→G (-7 from exon 13) rs13306607

SELE Selectin E 1q23-q25 A561C (Ser128Arg) rs5361

SELP Selectin P 1q23-q25 A37674C (Thr715Pro) rs6136

SELP Selectin P 1q23-q25 G→T (Val640Leu) rs6133

SRA Scavenger Receptor A1 8p22 C877T

SRB1 Scavenger Receptor B1 12q24.31 G2S

SREBF1 Sterol regulatory element-binding transcription factor 1 17p11.2 indel-36G (AX977070)

TCF1 Transcription factor 1 12q24.2 C→T (Ala98Val) rs1800574

TGFB1 Transforming growth factor, beta-1 19q13.1 C-509T rs1800469

TGFBR2 Transforming growth factor-beta receptor, type II 3p22 C1167T (Asn389Asn) rs2228048

THBD Thrombomodulin 20p11.2 C2136T (Ala455Val) rs1042579

Continuación Tabla 1

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pasando la lámina elástica interna hasta la ínti-ma o el espacio subendotelial (Fig. 2).

Las células de músculo liso de la íntima pueden proliferar y captar lipoproteínas modificadas contribuyendo a la formación de células espu-mosas, así como sintetizar proteínas de matriz extracelular para la formación de la capa fibro-sa. Esta fase del desarrollo de la lesión está in-fluenciada por interacciones entre monocitos/macrófagos y células T dando lugar a un amplio rango de respuestas celulares y humorales y a la adquisición de muchas características del estado inflamatorio crónico.

Aunque las lesiones ateroscleróticas avanzadas pueden dar lugar a síntomas isquémicos como resultado del progresivo angostamiento del lu-men del vaso, los eventos cardiovasculares agu-dos que resultan en infarto de miocardio e ictus se achacan generalmente a la ruptura de la pla-ca y trombosis. La ruptura de la placa expone los lípidos de la placa y factores tisulares a los componentes sanguíneos, iniciando la cascada de coagulación, adherencia de plaquetas y trom-bosis (Fig. 3).

El análisis de la aterosclerosis humana sugiere que la evolución de las placas avanzadas puede involucrar ciclos repetitivos de microhemorra-gias y trombosis. Las roturas de la placa asocia-das con infartos de miocardio, generalmente

tienen lugar en las regiones laterales de la placa y ocurren más probablemente en lesiones con capas fibrosas finas, una relativamente elevada concentración de células espumosas en las re-giones laterales, y grandes núcleos necróticos. La apoptosis de macrófagos y células musculares lisas aparece como resultado de interacciones célula-célula y composición de citoquinas en la pared arterial, involucrando la acción de proteí-nas pro- y antiapoptóticas que incluyen recepto-res de muerte celular, proto-oncogenes y genes supresores tumorales.

La liberación de lípidos oxidados e insolubles por parte de las células necróticas contribuye a la formación de las características “gachas” de las lesiones avanzadas. La apoptosis de macrófagos y células de músculo liso pueden no sólo ser

Símbolo Gen Locus Polimorfismo dbSNP

THBS2 Thrombospondin II 6q27 T3949G rs8089

THBS4 Thrombospondin IV 5q13 G1186C (Ala387Pro) rs1866389

THPO Thrombopoietin 3q26.3-q27 A5713G rs6141

TLR4 Toll-like receptor 4 9q32-q33 A896G

TLR4 Toll-like receptor 4 9q32-q33 A2326G (Asp299Gly) rs4986790

TNF Tumor necrosis factor 6p21.3 C-863A rs1800630

TNF Tumor necrosis factor 6p21.3 C-850T rs1799724

TNF Tumor necrosis factor 6p21.3 G-238A rs361525

TNFRSF1A Tumor necrosis factor receptor 1 12p13.2 R92Q

TNFSF4 Tumor necrosis factor ligand superfamily, member 4 1q25 A→G rs3850641

VEGF Vascular endothelial growth factor 6p12 A-2518G

VEGF Vascular endothelial growth factor 6p12 C936T rs3025039

La llegada de macrófagos y colesterol derivado de LDL

son los principales eventos celulares que contribuyen a la

formación de la línea grasa

Continuación Tabla 1

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importantes para determinar la capacidad de las lesiones en sufrir una regresión, sino también pueden influir en la estabilidad de la placa, así como los lípidos del núcleo necrótico pueden incrementar el potencial de trombosis.

Las metaloproteasas de la matriz segregadas por los macrófagos en regiones de ruptura de la pla-ca influyen en la estabilidad de la misma degra-dando proteínas de matriz extracelulares.

Adicionalmente, se observa un acúmulo exten-sivo de fibrina en las lesiones más complejas, y se ha propuesto un descenso de la actividad fi-brinolítica como acelerador de la aterogénesis arterial facilitando la trombosis y la deposición de fibrina durante el desarrollo de las lesiones ateroscleróticas.

La neovascularización es frecuente en lesiones ateroscleróticas asociadas con ruptura de la pla-ca, hemorragias o angina inestable (episodios progresivos de isquemia cardiaca temporal de-bidos a formación de trombos transitorios). La angiogénesis ocurre en asociación con la remo-delación y la activación de proteasas en tejidos circundantes, sugiriendo que la neovasculariza-ción puede contribuir a la inestabilidad y ruptu-ra de la placa.

Genes de susceptibilidad

Aunque la aterosclerosis ha sido objeto de inten-sivas investigaciones epidemiológicas y patofisio-lógicas, son considerables las evidencias de que importantes determinantes de la susceptibilidad a la enfermedad permanecen sin identificar. La historia familiar es un importante factor de ries-go tras eliminar otros factores independientes previamente identificados, implicando factores genéticos adicionales (Tabla 1).

Estudios de raros trastornos mendelianos como la hipercolesterolemia familiar y la enfermedad de Tangier podrían seguir facilitando la identifi-cación de nuevos genes que influyen en el desa-rrollo de la aterosclerosis.

Panel de riesgo genético cerebrovascular

La definición del panel de riesgo genético ce-rebrovascular pasa por abordar el estudio de genes implicados en los diferentes eventos que desencadenan el proceso aterogénico, es decir, metabolismo lipídico (modificación de LDL-co-lesterol), función endotelial, respuesta inmuni-taria (reclutamiento de macrófagos y formación de células espumosas) y estabilidad de la placa de ateroma (trombosis).

Panel de Metabolismo LipídicoEl panel de metabolismo lipídico aborda el estu-dio de genes implicados en la modificación de los niveles colesterol en sus distintas formas y su contribución en el proceso aterogénico como factor de riesgo vascular (Tabla 2).

El polimorfismo C3175G (rs5128), también co-nocido como Sst I, se encuentra en la región 3’UTR del mRNA de APOC3. La variante 3175G (S2) está relacionada con mayor estabilidad y mayores niveles de expresión de ApoCIII, por lo que se relaciona con riesgo incrementado de enfermedad vascular debido a su implicación en el metabolismo de triglicéridos10.

Panel de riego cerebrovascular: Metabolismo Lipídico.

METABOLISMO LIPÍDICO

Símbolo Gen Locus Polimorfismo dbSNP

APOC3 Apolipoprotein C-III 11q23 C3175G (S1/S2) rs5128

APOE Apolipoprotein E 19q13.2 T3932C (Cys112Arg) rs429358

APOE Apolipoprotein E 19q13.2 C4070T (Arg158Cys) rs7412

APOB Apolipoprotein B 2p24C7673T (Thr2488Thr, XbaI)

rs693

CETPCholesteryl ester transfer protein, plasma

16q21 G279A (TaqB1/B2) rs708272

LPL Lipoprotein lipase 8p22 C1595G (Ser447Stop) rs328

Panel de riego cerebrovascular: Función Endotelial e Hipertensión.

FUNCIóN ENDOTELIAL E HIPERTENSIóN

Símbolo Gen Locus Polimorfismo dbSNP

NOS3 Nitric oxide synthase 3 7q36 G894T rs1799983

ACEAngiotensin I- converting enzyme

17q23 C547T rs4332

ACEAngiotensin I- converting enzyme

17q23 Intron 16 Alu 287bp I/D

AGT Angiotensinogen 1q42-q43 Met235Thr rs699

AGT Angiotensinogen 1q42-q43 Thr174Met rs4762

Tabla 2

Tabla 3

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El polimorfismo G894T (rs1799983): E298D, y concretamente la presencia del alelo 894T está asociada a una menor actividad del enzima NOS3, lo que implica un mayor riesgo vascular y una mayor susceptibilidad de padecer patolo-gías cardiovasculares16.

La enzima convertidora de angiotensina (ACE), es una dipeptidil carboxipeptidasa que desem-peña un papel importante en la regulación de la presión arterial y en el balance de elec-trolitos y la presión sanguínea hidrolizando la angiotensina I en angiotensina II, un potente vasopresor, y un péptido estimulante de aldos-terona. La enzima también es capaz de inactivar la bradicinina, un potente vasodilatador. Los polimorfismos a estudiar son C547T (rs4332) y la presencia (inserción, I) o ausencia (delec-ción, D) de una secuencia alu repetitiva de 287 pb en el intrón 16 del gen está asociada a nive-les circulantes de la enzima y a patologías

ApoE interviene en el catabolismo de proteínas ricas en triglicéridos y en la homeostasis del co-lesterol. La presencia del alelo ε4 del gen APOE está ligada a niveles altos de colesterol y de beta-lipoproteínas, así como a la propensión a sufrir enfermedades cardiovasculares11. La presencia del alelo ε2 está ligada a riesgo incrementado de Hiperlipoproteinemia tipo III, niveles altos de colesterol, triglicéridos y beta-VLDL, así como al desarrollo de aterosclerosis e incremento del riesgo vascular12.

Los niveles aumentados de ApoB se asocian di-rectamente con las lipoproteínas aterógenas, VLDL, IDL y LDL. Se sintetiza principalmente en hígado e intestino. El alelo 7673C está asocia-do a menores niveles de triglicéridos, colesterol y colesterol LDL. Sin embargo, individuos por-tadores del alelo 7673T responden mejor a una dieta baja en grasa y colesterol, con una dismi-nución significativamente mayor de sus niveles de LDL y ApoB.

CETP codifica para la proteína de transferencia de ésteres de colesterol, que facilita el inter-cambio de triglicéridos y ésteres de colesterol estimulando la recuperación de colesterol. El polimorfismo G+279A (rs708272) del gen CETP (también denominado TaqIB) está asociado con niveles bajos de colesterol HDL y niveles altos de actividad CETP en plasma (presencia del alelo +279G o B1), que contribuyen a un incremento en el riesgo de enfermedades car-diovasculares.

La lipoproteína lipasa (LPL) desempeña una función clave en el metabolismo lipoproteico, hidrolizando los triglicéridos que forman par-te del VLDL y los quilomicrones, así como eli-minando las lipoproteínas de la circulación13. LPL influye en la interacción de las lipopro-teínas aterogénicas con la superficie de la cé-lula y con los receptores de la pared vascular14. Estudios recientes relacionan el polimorfismo C1421G (rs328) [S447X] (proteína truncada de 446 aminoácidos en lugar de 448) con un menor riesgo de padecer CAD, debido a su re-lación con un aumento de HDL y una disminu-ción de triglicéridos15. Por lo tanto, la variante 447X tiene una mayor actividad enzimática, por lo que debería tener un efecto protector contra el desarrollo de la aterosclerosis y CAD posterior.

Panel de Función Endotelial e HipertensiónLa transición desde la relativamente simple lí-nea grasa hasta la lesión más compleja se carac-teriza por la inmigración de células de músculo liso desde la capa media de la pared arterial pa-sando la lámina elástica interna hasta la íntima o el espacio subendotelial (Tabla 3).

Panel de riego cerebrovascular: Respuesta Inmunitaria.

RESPUESTA INMUNITARIA

Símbolo Gen Locus Polimorfismo dbSNP

IL1B Interleukin 1-beta 2q14 T5887C rs1143634

IL6 Interleukin 6 7p21 G-174C rs1800795

IL6 Interleukin 6 7p21 G-573C rs1800796

IL6R Interleukin 6 receptor 1q21 A1510C rs8192284

TNFATumor necrosis factor alpha

6p21.3 G-308A rs1800629

Panel de riego cerebrovascular: Trombosis.

TROMBOSIS

Símbolo Gen Locus Polimorfismo dbSNP

F2 Coagulation factor II (thrombin) 11p11 G20210A rs1799963

F5Coagulation factor V proaccelerin)

1q23 G1691A rs6025

MTHFR5,10-Methylenetetrahydrofolate reductase

1p36.3 A1298C rs1801131

MTHFR5,10-Methylenetetrahydrofolate reductase

1p36.3 C677T (Ala222Val) rs1801133

Tabla 4

Tabla 5

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cardiovasculares. El alelo D (delección) está asociado a una alta predisposición a desarrollar hipertensión arterial esencial lo que favorece el padecimiento de otras patologías cardiovascu-lares17.

El gen AGT codifica el angiotensinógeno, el cual, mediante la renina, se transforma en an-giotensina I. Los alelos 235T y 174M están aso-ciados con un mayor riesgo de sufrir hiperten-sión arterial esencial18, 19.

Panel de Respuesta InmunitariaActualmente se conoce el papel relevante de la inflamación en el inicio y progresión de la aterosclerosis20 y se sospecha que influye en el desarrollo trombótico activando el proceso de coagulación21 (Tabla 4).

Se han descrito niveles incrementados de mar-cadores de inflamación con enfermedad vas-cular isquémica22, 23. Se postula la influencia de polimorfismos en IL1 en la modulación del pa-trón inflamatorio involucrado en la formación de trombos que pudiera desencadenar procesos arteriales isquémicos24.

La interleuquina 6 (IL-6) es una citoquina pleio-trópica implicada en la regulación de la reacción de fase aguda, la respuesta inmune, y la hema-topoyesis, pudiendo jugar un papel en la me-gacariocitopoyesis y producción plaquetaria. El polimorfismo G-174C (rs1800795) en la región 5’ parece estar asociado con diferencias en los niveles plasmáticos de IL-6 en voluntarios sanos. Fernández-Real et al.25 encontraron que los por-tadores del alelo -174G, que se asocia con mayor secreción de IL-6, tienen niveles incrementados de triglicéridos plasmáticos, VLDL y ácidos gra-

sos libres, así como niveles más bajos de HDL-co-lesterol. Por otra parte, se ha descrito una fuerte asociación entre el genotipo -174CC y el infarto lacunar26. El polimorfismo G-573C (rs1800796) en la región 5’ está significativamente asociado con infarto cerebral aterotrombótico y hemorra-gia intracerebral27.

El factor de necrosis tumoral (TNF-alfa) es una citoquina proinflamatoria secretada predomi-nantemente por monocitos y macrófagos y que afecta al metabolismo lipídico, coagulación, resistencia a insulina y función endotelial. Se han encontrado evidencias in vivo de la impli-cación de TNF-alfa en la hidrólisis de esfingo-mielina, producción de ceramida y apoptosis mediada por ceramida28. El polimorfismo G-308A (rs1800629) se ha relacionado con niveles incrementados de cortisol en saliva y obesidad en individuos homocigotos AA29. También se ha descrito una asociación entre la variante -308G en homocigosis y un riesgo incrementado de pa-decer migraña30, debido probablemente al efec-to de este polimorfismo sobre el flujo sanguíneo cerebral.

Panel de TrombosisAunque las lesiones ateroscleróticas avanzadas pueden dar lugar a síntomas isquémicos como resultado del progresivo angostamiento del lumen del vaso, los eventos vasculares agudos que resultan en infarto de miocardio e ictus se achacan generalmente a la ruptura de la placa y trombosis (Tabla 5).

El Factor II de coagulación o protrombina está implicado en la coagulación sanguínea. Esta pro-teína plasmática es la precursora de la trombina, responsable de la formación del coágulo. El po-

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limorfismo G20210A (rs1799963) se encuentra en el 3% de la población del sur de Europa. Esta alteración está relacionada con un aumento de los niveles plasmáticos de protrombina. Las personas que llevan una copia de esta mutación (alelo 20210A) tienen 6 veces más probabilida-des de sufrir una trombosis31. Las mujeres em-barazadas o tratadas con anticonceptivos tienen un riesgo 16,3 veces mayor de sufrir trombosis si son portadoras de la mutación32.

El Factor V de Leiden es uno de los factores implicados en la coagulación sanguínea. La función del Factor V es inactivada por la Pro-teína C, que constituye uno de los mecanismos anticoagulantes más importantes. La trombina, cuando se une a la trombomodulina en la su-perficie endotelial, activa a la proteína C y ésta a su vez, inactiva a los factores V y VIII. La muta-ción G1691A (rs6025): Arg506Gln en el gen F5, presenta una alta prevalencia en caucasoides, entre un 5 y un 10%. La presencia de la muta-ción 1691A impide la inactivación del factor V por parte de la proteína C, provocando un es-tado de hipercoagulabilidad y un aumento del riesgo trombótico. Algunos estudios sugieren un aumento de 50 a 100 veces en el riesgo de trom-bosis venosa para los portadores en homocigosis del alelo 506Q y de 5 a 10 veces para los porta-dores heterocigotos R506Q33.

La metilentetrahidrofolato reductasa (MTHFR) cataliza la conversión de 5,10-metilentetrahidro-folato a 5-metiltetrahidrofolato, un cosustrato para la remetilación de homocisteína a metioni-na. El polimorfismo C677T (rs1801133): A222V da lugar a una proteína con actividad enzimática reducida y termolabilidad incrementada cuan-do aparece la variante 222V en homocigosis o heterocigosis. Los individuos 677TT presentan niveles en plasma de homocisteína elevados y tienen niveles de riesgo de padecer una enfer-medad cardiovascular prematura hasta tres ve-ces superiores al resto34. Otra mutación también relacionada con una reducción en la actividad enzimática es la A1298C (rs1801131): E429A, aunque este descenso en la actividad no pare-ce estar relacionado con niveles plasmáticos de homocisteína incrementados ni concentracio-nes menores de folato en plasma como ocurre con los 677T homocigotos. Aumentar la ingesta de folato (0,8 mg de ácido fólico) reduce en un 16% el riesgo de cardiopatía isquémica y en un 24% el de accidente cerebrovascular35.

Dr. Juan C. Carrilgeneticaforense@ebiotec.com

Capacidad predictiva del panel genético

En las enfermedades multifactoriales, la heren-cia genética así como los factores ambientales, físicos y de estilo de vida son determinantes en el establecimiento de un perfil de riesgo de enfermedad. La ponderación inadecuada de dichos factores, así como la baja o incompleta penetrancia, provocan resultados de asociacio-nes débiles en los estudios epidemiológicos, con resultados, muchas veces, no reproducibles e in-cluso contradictorios entre estudios.

La pregunta que pretendemos responder es simple, lo complejo es llegar a una respuesta con suficientes garantías: ¿Cuál es el riesgo ob-jetivo de un individuo sano (del que conocemos su perfil genético, sus características físicas y su estilo de vida) de padecer la enfermedad? La respuesta pasa por la elección de las herramien-tas de detección adecuadas (polimorfismos de interés) y del modelo de tratamiento de los datos que mejor se ajuste a la enfermedad en cuestión.

La determinación de los factores genéticos y de las interacciones entre ellos y con los factores ambientales que desencadenan el desarrollo de la enfermedad, tan sólo es la mitad del camino hacia la obtención de un test genético predicti-vo de utilidad en la práctica diagnóstica.

La validación del panel genético, es decir, la de-terminación de que las diferencias entre casos y controles son causales y no espúreas, así como la elección del modelo estadístico adecuado, es la clave para obtener una herramienta predictiva de utilidad real en la práctica médica.

La pregunta que pretendemos responder es simple:

¿Cuál es el riesgo objetivo de un individuo sano de padecer

la enfermedad?

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19. Katsuya T, Koike G, Yee TW et al. Association of angiotensino-gen gene T235 variant with increased risk of coronary heart disease. Lancet 1995; 345: 1600-1603.

20. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420(6917):868-74.

21. Luther T, Mackman N. Tissue factor in the heart. Multiple roles in hemostasis, thrombosis, and inflammation. Trends Cardio-vasc Med. 2001; 11(8):307-12.

22. Danesh J, Appleby P. Persistent infection and vascular disea-se: a systematic review. Expert Opin Investig Drugs. 1998; 7(5):691-713.

23. Rost NS, Wolf PA, Kase CS et al. Plasma concentration of C-reactive protein and risk of ischemic stroke and tran-sient ischemic attack: the Framingham study. Stroke. 2001; 32(11):2575-9.

24. Iacoviello L, Di Castelnuovo A, Gattone M et al. Polymor-phisms of the interleukin-1ß gene affect the risk of myocardial infarction and ischemic stroke at young age and the response of mononuclear cells to stimulation in vitro. 2004. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 222-227.

25. Fernández-Real JM, Broch M, Vendrell J et al. Interleukin-6 gene polymorphism and lipid abnormalities in healthy sub-jects. J Clin Endocrinol Metab. 2000; 85(3):1334-9.

26. Revilla M, Obach V, Cervera A et al. A -174G/C polymorphism of the interleukine-6 gene in patients with lacunar infarction. Neurosci Lett. 2002; 324(1):29-32.

27. Yamada Y, Metoki N, Yoshida H et al. Genetic risk for ischemic and hemorrhagic stroke. Aterosclerosis, thrombosis, and vas-cular biology 2006; 26 (8): 1920-5.

28. Obeid LM, Linardic CM, Karolak LA et al. Programmed cell dea-th induced by ceramide. Science. 1993; 259(5102):1769-71.

29. Rosmond R, Chagnon M, Bouchard C et al. G-308A polymor-phism of the tumor necrosis factor alpha gene promoter and salivary cortisol secretion. J Clin Endocrinol Metab. 2001; 86(5):2178-80.

30. Rainero I, Grimaldi LM, Salani G et al. Association between the tumor necrosis factor-alpha -308 G/A gene polymorphism and migraine. Neurology 2004; 62(1):141-3.

31. De Stefano V, Martinelli I, Mannucci PM et al. The risk of recu-rrent deep venous thrombosis among heterozygous carriers of both factor V Leiden and the G20210A prothrombin mutation. N Engl J Med. 1999; 341(11):801-6.

32. Gerhardt A, Scharf RE, Beckmann MW et al. Prothrombin and factor V mutations in women with a history of thrombosis during pregnancy and the puerperium. N Engl J Med. 2000; 342(6):374-80.

33. Casas JP, Hingorani AD, Bautista LE et al. Meta-analysis of genetic studies in ischemic stroke: thirty-two genes involving approximately 18,000 cases and 58,000 controls. Arch Neu-rol. 2004; 61(11):1652-61.

34. Bethke L, Webb E, Murray A et al. Functional polymorphisms in folate metabolism genes influence the risk of meningio-ma and glioma. Cancer Epidemiol Biomarkers Prev. 2008; 17(5):1195-202.

35. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. Brit Med J 2002; 325: 1202–1207.

Referencias Bibliográficas

1. Bonita R. Epidemiology of Stroke. Lancet 1992; 339: 342-44.

2. Instituto Nacional de Estadística. Salud. España en cifras 2008. Madrid: INE; 2008:18.

3. Hervás-Angulo A, Cabasés-Hita JM, Forcén-Alonso T. Costes del ictus desde la perspectiva social. Enfoque de incidencia retrospectiva con seguimiento a tres años. Rev Neurol 2006; 43: 518-25.

4. Medrano MJ Boix R, Cerrato E et al. Incidencia y prevalencia de cardiopatía isquémica y enfermedad cerebrovascular en España: revisión sistemática de la literatura. Rev Esp Salud Pública 2006; 80: 5-15.

5. Glass CK, Witztum JL. Atherosclerosis. the road ahead. Cell. 2001;104(4): 503-16.

6. Virchow R. Die cellularpathologie in ihrer Begründung auf phy-siologische und pathologische Gewebenlehre. 1860.University of Würzburg.

7. Ross R, Glomset JA. Atherosclerosis and the arterial smooth muscle cell: Proliferation of smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science 1973; 180(93):1332-9.

8. Goldstein JL, Brown MS. The low-density lipoprotein pathway and its relation to atherosclerosis. Ann. Review Biochem. 1977; 46: 897–930.

9. Steinberg D et al. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 1989; 20: 915–924.

10. Paré G, Serre D, Brisson D et al. Genetic analysis of 103 candidate genes for coronary artery disease and associated phenotypes in a founder population reveals a new association between endothelin-1 and high-density lipoprotein choleste-rol. Am J Hum Genet. 2007; 80(4):673-82.

11. Pedro-Botet J, Sentí M, Nogués X et al. Lipoprotein and apolipoprotein profile in men with ischemic stroke. Role of lipoprotein(a), triglyceride-rich lipoproteins, and apolipoprotein E polymorphism. Stroke. 1992; 23(11):1556-62.

12. Breslow JL, Zannis VI, SanGiacomo TR, et al. Studies of fami-lial type III hyperlipoproteinemia using as a genetic marker the apoE phenotype E2/2. J Lipid Res. 1982; 23(8):1224-35.

13. Eckel RH. Lipoprotein lipase. A multifunctional enzyme rele-vant to common metabolic diseases. N Engl J Med. 1989; 320(16):1060-8.

14. Beisiegel U, Weber W, Bengtsson-Olivecrona G. Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc Natl Acad Sci USA. 1991; 88(19):8342-6.

15. Stocks J, Thorn JA, Galton DJ. Lipoprotein lipase genotypes for a common premature termination codon mutation detec-ted by PCR-mediated site-directed mutagenesis and restric-tion digestion. J Lipid Res. 1992; 33(6):853-7.

16. Rankinen T, Rice T, Perusse L, et al. NOS3 Glu298Asp geno-type and blood pressure response to endurance training: the HERITAGE family study. Hypertension 2000; 36:885–9.

17. Slowik A, Turaj W, Dziedzic T et al. DD genotype of ACE gene is a risk factor for intracerebral hemorrhage. Neurology 2004; 63: 359-361.

18. Jeunemaitre X, Soubrier F, Kotelevtsev YV et al. Molecular basis of human hypertension: role of angiotensinogen. Cell 1992; 71: 7-20.

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a fUndación galega para a Sociedade do Coñe-cemento, FSC, se creó con el objetivo de establecer puentes de comunicación y colaboración permanen-te entre la empresa, la universidad y la adminis-tración de Galicia, en la búsqueda de un objetivo común: incrementar la prosperidad de Galicia y sus ciudadanos, a través del incremento de la competiti-vidad del tejido empresarial gallego.

La colaboración triple hélice es un modelo de co-operación esencial en el contexto actual, caracteriza-do por la cada vez mayor importancia estratégica del conocimiento, en sus distintas realizaciones (forma-ción, talento, tecnologías, creación y combinación de conocimiento, etc), el cual tiene su mayor expo-nente en el mundo académico.

El mundo empresarial, verdadero agente creador de riqueza para una sociedad, pone en valor el co-nocimiento a través de la innovación y el emprendi-miento, lo cual faculta a una sociedad la internacio-nalización de la misma. Y la Administración, como elemento dinamizador, que debe encauzar sus polí-ticas de una manera eficiente, alineando los recursos y capacidades de una sociedad para el desarrollo de las necesidades reales de la empresa, así como establecer las pautas para favorecer un crecimiento equilibrado y sostenible en el tiempo de la sociedad en su conjunto.

La estructura de la FSC está diseñada en torno a cuatro Comités de expertos, formados por agen-tes de la triple hélice, con el objetivo de trabajar de forma coordinada en aquellos ámbitos clave que permitan incrementar la competitividad de Galicia. Los comités se centran en: coope-ración, como elemento que facilita alcanzar las sinergias y masa crítica necesaria para competir; innovación, en sentido amplio, como elemento fundamental para impulsar la competitividad; nuevos negocios y emprendedores, como ele-mento para valorizar/comercializar las innova-ciones que nos permitan dar un salto cualitativo

y, en cuarto lugar, la internacionalización, como resultado de los tres elementos anteriores y ex-presión de la competitividad de la empresa.

En este entorno, cada vez más competitivo y glo-balizado, la interdependencia entre entidades y la creación de sinergias han hecho de la cooperación un elemento esencial. La Fundación Galega para a Sociedade do Coñecemento impulsa el modelo de cooperación Cluster, dado que facilita una perspec-tiva más significativa en cuanto al modo en que fun-ciona una economía y cómo se interrelacionan las empresas; asimismo, ofrece la configuración y ex-ternalidades adecuadas para impulsar la innovación y el incremento de la productividad.

Desde la FSC se promueve la figura del Metadis-trito, como punto de encuentro y colaboración de los clusters gallegos para el impulso de la coopera-ción entre los mismos, que propicie el trabajo en red, abierto al conocimiento, la investigación aplicada y la innovación, desarrollando modelos de coopera-ción multisectorial para alcanzar la excelencia com-petitiva, a través de valores y estrategias comunes abiertas al mercado mundial.

La Unión Europea está alentando y promoviendo entre sus miembros (países y regiones) el uso de los clusters y la mejora de la calidad del entorno empresarial como elementos para dinamizar la eco-nomía y marco de referencia para el incremento de la innovación y la productividad y, por ende, la competitividad. Prueba de ello es el nuevo mar-co Europeo de apoyo a los clusters, buscando la excelencia y cooperación entre los clusters de Europa. Asimismo, hay que mencionar que las re-giones/países más desarrollados aplican la política cluster, desde hace algunos años, como elemento central de su política de crecimiento económico (Cataluña, País Vasco, Navarra, Baden-Württen-berg, Ille de France, Dinamarca, Estados Unidos, Reino Unido, etc.).

Entre los objetivos de la Fundación está el propiciar el incremento de la productividad, generar mejores empleos, difundiendo la tecnología y el conocimien-to para, en definitiva, poder alcanzar un desarrollo sostenible de Galicia, a través de la excelencia em-presarial y la innovación, teniendo en cuenta las ca-racterísticas únicas de nuestra comunidad autónoma, sus recursos naturales, capacidades, localización y activos culturales e históricos.

Cooperación Multisectorial para Impulsar el Desarrollo de GaliciaFundación Galega para a Sociedade do Coñecemento

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diciembre 2009 87

Desde la FSC hemos lanzado el Observatorio de Competitividad de Galicia (proyecto financiado por el IGAPE, Caixanova y Caixa Galicia), centro dinámico formado por agentes locales del conoci-miento y entes públicos con el objetivo de potenciar y medir la competitividad de nuestra economía.

En los trabajos de lanzamiento del Observato-rio, se ha realizado un análisis y diagnostico de la situación competitiva de Galicia, con la utili-zación de herramientas avanzadas de benchmar-king internacional. Asimismo, se ha articulado una Visión Galicia 2014 y un plan de acción concreto, utilizando como referencia las expe-riencias de éxito llevadas a cabo en otras eco-nomías avanzadas, pero adaptado a la realidad y circunstancias gallegas, con el fin último del incremento de la prosperidad socio-económica de Galicia. Para determinar la situación compe-titiva de partida, se han utilizado dos herramien-tas avanzadas que permiten la comparación de Galicia con otras localizaciones: Ranking de Competitividad de Galicia y Benchmarking com-petitivo basado en clusters. Dichas herramien-tas han sido facilitadas por las colaboraciones que ha establecido la Fundación Galega para a Sociedade do Coñecemento con dos entidades de reconocido prestigio internacional: Institute for Management Development (IMD) y Monitor Group.

El IMD, con sede en Lausanne (Suiza), es una de las principales escuelas de negocios a nivel mundial que dirige y gestiona la elaboración de uno de los dos rankings de competitividad de referencia a nivel internacional (el “IMD World Competitiveness Yearbook”). Su programa

MBA se clasificó en el 1er puesto mundial en los rankings del Financial Times “Ranking of the Rankings”.

Monitor Group, empresa de consultoría, fun-dada en 1983 por Michael Porter, con amplia experiencia en competitividad regional, dispo-ne de bases de datos únicas en el mundo facili-tando el benchmarking de la situación de Galicia en distintos aspectos. Recientemente, la Unión Europea, a través de la Comisión Europea, está trabajando con Monitor Group para aplicar su metodología basada en clusters a toda Europa.

Felicito a la Fundación EuroEspes por el impor-tante trabajo que llevan a cabo y, en concreto, por la revista Gen-T, que supone un paso más en la difusión del conocimiento científico, agra-deciendo su amable invitación para dar a cono-cer a la sociedad las líneas de trabajo llevadas a cabo por la FSC. Les invito a visitar nuestro sitio web www.fgsc.es en el que podrán seguir de cerca toda la actividad desarrollada por la fundación.

Son objetivos de la FSC incrementar la productividad

y difundir la tecnología y el conocimiento

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José Mª Martín Morenojmartin@fgsc.es

Director general de la Fundación Galega para a Sociedade do Coñecemento

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La III Conferencia Anual se celebró en A Coruña en diciembre de 2008

La III Conferencia Anual EuroEspes, celebrada en A Coruña los días 12 y 13 de diciembre de 2008, reunió a los más prestigiosos especialistas en medicina genómica de todo el mundo. La Fundación EuroEspes organizó, en el marco de la III Conferencia Anual, el Primer Encuentro de la Asociación Mundial de Medicina Genómica.

Ist Meeting of the World Association of Genomic Medicine

www.euroespesannualconference.org

Genomic Medicine and PHARMACOGENOMICS

Future Challengesfor PERSONALIZED MEDICINE

in the European Union

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Ramón Cacabelos, director del Centro de Investi-gación Biomédica EuroEspes, primer grupo genó-mico en Europa, habló de la importancia de la me-dicina genómica para conocer mejor las patologías que afectan al sistema nervioso central.

En su intervención, Ramón Cacabelos explicó que “los mecanismos patogénicos de la mayoría de los desórdenes del sistema nervioso central son poco conocidos aún. Los estudios genéticos realizados en las dos últimas décadas han demostrado que las patologías del Sistema Nervioso Central (SNC) son multifactoriales y poligénicas. De ahí que los recientes avances en medicina genómica puedan contribuir a acelerar nuestro conocimiento de la patogénesis de los desórdenes del SNC, mejoran-do la precisión del diagnóstico con la introduc-ción de nuevos biomarcadores, y personalizando los tratamientos con la incorporación de procedi-mientos farmacogenéticos y farmacogenómicos al desarrollo farmacológico y la práctica clínica”.

John R. Cockcroft, del Instituto de Investigación Cardiaca de la Universidad de Gales, en el Reino Unido, abordó el papel de la Medicina Genómica y de la Farmacogenómica en las patologías cardiacas.

Según este especialista, “desde que las cardiopa-tías son una de las mayores causas de morbilidad y fallecimiento, se han convertido en un objetivo central de la investigación sobre la interacción entre el genoma y los fármacos cardiovascula-res. Sin embargo, aunque la farmacogenómica cardiovascular es un campo prometedor, todavía estamos trabajando en la consecución de un test con verdadera utilidad clínica”.

xperTos como los profesores Urs A. Meyer, Allen D. Roses, Filipo Guglielmo de Braud, Francesco Marotta, Masatoshi Takeda, Christian A. Scerri, Valter Lombardi, John R. Cockcroft y Ramón Cacabelos, entre otros, analizaron y debatieron las posibilidades - y los retos - que ofrece la medicina genómica en el tratamiento de distintas enfermeda-des. Entre ellas, el cáncer, los trastornos neuropsi-quiátricos y las patologías respiratorias y cardiovas-culares.

El nivel científico del encuentro fue altísimo. Las previsiones de la organización del evento (“Medici-na Genómica y Farmacogenómica. Futuros desa-fíos para la Medicina Personalizada en la Unión Europea”) se superaron ampliamente.

Una de las intervenciones más esperadas fue la del profesor de Farmacología del Biocentro de la Uni-versidad Basilea ( Suiza ), Urs A. Meyer. En su ponencia, explicó que la “la farmacogenética per-mitirá mejorar los resultados clínicos a través de un diagnóstico preciso mediante la optimización de la elección del fármaco y de la dosis adecuada para cada paciente. Con ello, se producirán me-nos reacciones adversas y los tratamientos serán más efectivos”.

El profesor Meyer repasó también los desarrollos en genómica más recientes, como el proyecto interna-cional HapMap y también los proyectos 1000 Ge-nomas, Genoma Personal, Atlas del Genoma del Cáncer y las iniciativas de “consumer genomics” que genotipan más de 500.000 SNP por menos de 400 dólares.

La ponencia plenaria de Allen D. Roses causó gran expectación. El profesor de Neurología de la Uni-versidad de Duke, en California (Estados Unidos), explicó que “para que la farmacogenética sea inte-grada en la práctica clínica, el modelo de negocio farmacéutico debe evolucionar. Durante el desa-rrollo de fármacos, se debe aplicar, de forma rigu-rosa, la ciencia, de manera que se recopilen datos significativos que puedan ser empleados para de-mostrar los valores predictivos positivos y negati-vos para las necesidades médicas no cumplidas”.

EUROESPES REÚNE A LOS MEJORES ESPECIALISTAS EN MEDICINA GENÓMICA DEL MUNDO

E5Acto de inauguración III Con-ferencia Anual EuroEspes.El Dr. Cacabelos, en primer término.

La nutrigenómica ayudará a prevenir enfermedades como

la diabetesMarvin Edeas

III Conferencia Anual EuroEspes

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director del Instituto Nacional de Medicina Genómi-ca de México, explicó cómo la medicina genómica se ha convertido en una prioridad para el Gobierno mexicano como un medio para encontrar nuevas es-trategias para abordar patologías comunes: “Dicho compromiso - contó - se ha plasmado con la crea-ción del Instituto Nacional de Medicina Genó-mica (INMEGEN) por el Congreso Mexicano en 2004. Un centro diseñado para desarrollar inves-tigación transnacional centrada en los problemas de salud de la población mexicana, que tiene una estructura genómica ancestral particular, debido a su mezcla de orígenes”.

Masatoshi Takeda, jefe del Departamento de Psi-quiatría y Ciencia del Comportamiento de la Univer-sidad de Osaka, centró su conferencia en la implan-tación de la Medicina Genética en su país, en Japón, y, en especial, en las patologías neurodegenerativas. Su conclusión es clara: “Los estudios genéticos han acelerado dramáticamente la investigación en Alzheimer, y no solo para el conocimiento de la enfermedad, también para el desarrollo del tratamiento y del diagnóstico”.

La ponencia sobre los biomarcadores en oncología corrió a cargo de Filippo Guglielmo de Braud, di-rector de la Unidad de Farmacología Clínica y Nue-vos Fármacos de la Facultad Europea de Oncología de Milán.

“Los biomarcadores genómicos”, explicó, “son la base de la medicina personalizada. Pueden ser utilizados para seleccionar poblaciones de pacientes adecuados para recibir tratamientos adecuados evitando la toxicidad, o seleccionar el fármaco óptimo para poblaciones de células can-cerosas específicas. Sin embargo, la metodología de los ensayos clínicos y del desarrollo farmaco-lógico debe cambiar para que la farmacogenética mejore los resultados del tratamiento, permitien-do la selección de los pacientes más adecuados para beneficiarse del tratamiento propuesto o para excluir aquellos tumores con menos proba-bilidades de ser sensibles al tratamiento. El futu-ro de la oncología pasa por la identificación de los perfiles químico-genéticos de diversos fármacos antitumorales”.

El genetista maltés Christian A. Scerri abordó las oportunidades que una población insular puede ofrecer a la farmacogenética, que, en su opinión, son muchas. Por su parte, Gerardo Jimenez-Sánchez,

Los biomarcadores genómicos son la base de la medicina personalizadaFilippo Guglielmo de Braud

El Prof. Dr. Allen D. Roses (EE.UU.), durante su ponencia6

5Vista del acto de inauguración celebrada en el Palacio de Exposiciones y Congresos de A Coruña (Palexco)

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Para Francesco Marotta, especialista en Biogeronto-logía de la Universidad de Pavia, en Italia, “los ali-mentos funcionales representan una oportunidad emergente y jugarán un papel importante en el futuro. La nutrigenómica no solo promoverá la nu-trición, sino que, además, ayudará a prevenir en-fermedades como la diabetes, la obesidad, las pato-logías neurodegenerativas y el envejecimiento”.

Munir Pirmohamed, del Departamento de Farma-cología de la Universidad de Liverpool, en el Reino Unido, fue el encargado de analizar el papel de la Me-dicina Genómica en las enfermedades metabólicas. Ian P. Hall, de la División de Medicina Molecular

y Terapéutica de la Universidad de Nottingham, re-pasó el valor del abordaje genético en las patologías respiratorias. “Puede -dijo- ser de gran utilidad, bien definiendo nuevos marcadores predictivos de eficacia, o respuestas adversas a fármacos, o bien identificando nuevos objetivos potenciales para el desarrollo farmacológico”.

Valter Lombardi, director del Departamento de In-munología Molecular de EuroEspes Biotecnología, disertó sobre la medicina genómica y farmacogenó-mica en las patologías inmunológicas y las vacu-nas. Eugenio Luigi Dorio, nutricionista y bioquími-co de la Universidad de Nápoles (Italia), abordó los retos de la redoxómica.

El desarrollo de fármacos y la farmacogenómica en Estados Unidos fue otro de los asuntos tratados en la III Conferencia Anual EuroEspes.

Para hablar de este tema, viajó a A Coruña Carmen Vigo, directora de Diagnóstico y Transplante de In-vitrogen (Estados Unidos). Vigo asegura que “uno de los retos más importantes en la definición de los rasgos farmacogenéticos es la necesidad de contar con pacientes bien caracterizados que ha-yan sido uniformemente tratados y sistemática-mente evaluados para hacer posible cuantificar, objetivamente, la respuesta farmacológica”.

Las ponencias sobre Nutrigenómica fueron también muy seguidas por los asistentes a la conferencia.

Marvin Edeas, profesor de medicina preventiva en la Universidad de París XIII, sostiene que “el im-pacto futuro de la nutrigenómica en Europa aún no se comprende bien. Por ello, nuestro reto ac-tual es unir las visiones científica, industrial y po-lítica de la nutrigenómica para discutir el modo de lograr el objetivo último, que es mejorar la salud del consumidor”.

La farmacogenéticapermite mejorar

los resultados clínicosUrs A. Meyer

El Prof. Dr. Urs A. Meyer (Suiza) en plena inter-vención

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Premios Fundación EuroEspesLa III Conferencia Anual EuroEspes y el Primer Encuentro Mundial de Medicina Genómica fi-nalizaron con la entrega de la Primera Edición de los Premios Fundación EuroEspes.

Se entregaron tres galardones.

Uno de los premios correspondió al Cuerpo Na-cional de Policía por la “creación del Laborato-rio Territorial de Biología y ADN de la Jefatura Superior de Policía en Galicia”.

Recogió el premio Luis García Mañá, jefe supe-rior de policía en Galicia.

El presidente de Laboratorios PARGGON de Guadalajara ( México ), Rafael López Martínez, recibió el premio Fundación EuroEspes por su “apuesta empresarial por el desarrollo de la bio-tecnología aplicada a la salud, la nutracéutica médica y la farmacogenómica”.

Otro de los galardones recayó en Eulalia Puig Colominas, presidenta de Genomax Iberplus.

El patronato de la Fundación EuroEspes la pre-mió por ser “la primera compañía española de-dicada a la difusión del conocimiento genómico y a la implementación de instrumentos para el diagnóstico genómico y la personalización del tratamiento basado en la farmacogenómica”.

El director general de Terapias Avanzadas y Transplantes del Ministerio de Sanidad y Consu-mo, Augusto Silva González, asistió a la entrega de premios.

Para Amalio Telenti, del Instituto de Microbiología de Lausanne, en Suiza, las “grandes diferencias en la susceptibilidad a patógenos como la tubercu-losis, la malaria o el VIH y en la progresión de la enfermedad que muestran los humanos hacen necesario analizar la patogénesis de dichas enfer-medades”.

El cofundador y director ejecutivo de la empresa alemana FarmacoGenómica GmbH, Stefan Prause, fue el último de los ponentes de la III Conferencia.

Su conclusión es que la “implementación de la farmacogenética en la medicina diaria es difícil por el consumo de tiempo y lo costoso de las tec-nologías, que aún requieren equipamientos muy sofisticados. Los test farmacogenéticos aún están reservados a los laboratorios centralizados. Sin embargo, nosotros ya disponemos de un test far-macogenético que se dirige a ese vacío diagnósti-co con un método rápido y coste efectivo que uti-liza equipamiento poco costoso y que es adecuado para introducir la farmacogenética en la práctica clínica diaria y en las aplicaciones diagnósticas rutinarias”.

Para el director de la III Conferencia Anual EuroEspes, Ramón Cacabelos, la conclusión es clara: Seguir trabajando en la divulgación e im-plantación de la Medicina Genómica. “Esta con-ferencia”, recordó, “es sólo un pequeño ejemplo de hacia dónde debe ir en el futuro la medicina genómica”.

Durante la III Conferencia, la nueva Asociación Mundial de Medicina Genómica (WAGEM) ce-lebró su reunión inaugural. Los principales ob-jetivos de esta asociación es educar y concienciar sobre los beneficios de la medicina geómica a la población general y al colectivo sanitario en particular.

5Luis García Mañá, Jefe Superior de Policía de Galicia, recoge el galardón otorgado por la Fundación EuroEspes en presencia del Dr. Augusto Silva, Director General de Terapias Avanzadas y Transplantes del Ministerio de Sanidad. Entrega el Presidente de la Fundación, el Dr. Cacabelos.

5El Dr. Valter Lombardi, di-rector del departamento de inmunología celular de Ebiotec, durante su ponencia.

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Noticias EuroEspes

Ebiotec comercializará productos nutracéuticos en PortugalLa distribución de nutracéuticos en Portu-gal es posible gracias al acuerdo alcanzado entre Ebiotec y la distribuidora de pro-ductos farmacéuticos IA FARMA, con sede en Lisboa. El acuerdo de colaboración fue firmado por el presidente del Grupo EuroEspes, Ramón Cacabelos, y el director general de IA FARMA, Joao Alves.

Para el doctor Ramón Cacabelos, la firma de este acuerdo “significa la entrada en un estado de la Península Ibérica con el que formamos una unidad geográfíca común con hábitos muy semejantes que nos lle-van a compartir dietas y enfermedades se-mejantes”.

Conferencia en el Club Tomás de Mercado La Junta Directiva del Club Tomás de Mercado invitó al Dr. Ramón Cacabelos, Presidente del Grupo EuroEspes, a un en-cuentro en torno a una cena coloquio en la que debatió sobre “la importancia de la medicina predictiva en la salud de la em-presa”.

El Club Tomás de Mercado tiene la sede en Barcelona y allí fueron invitados mi-nistros de diferentes gobiernos así como empresarios de importantes sociedades de origen multinacional. El número de socios está en 60, y su objetivo es que el debate que se produzca entre los asistentes sirva para incrementar su nivel de conocimien-to en las materias de actualidad que se pro-graman.

Psiquiatras japoneses se interesan por la Farmacogenética Una docena de psiquiatras japoneses, per-tenecientes a su asociación nacional que engloba a 1.214 hospitales, visitaron las instalaciones de EuroEspes para conocer los avances en la medicina genómica y en especial mostraron un gran interés por la tarjeta farmacogenética que ha desarrolla-do la citada institución médica gallega que dirige el doctor Ramón Cacabelos.

Yasuo Okuyama, jefe de la delegación, co-mentó que “el conocimiento de la gené-tica del paciente”, es algo “fundamental” para “acertar con la medicación correcta”, y además darle “la dosis adecuada”. “La tarjeta farmacogenética viene a marcar el camino a seguir en un futuro próximo”, apostilló.

Ebiotec lanza HepatoSar Nutracéutico,que ayuda a la digestión de las grasas y favorece la función hepatobiliarEste es un nutracéutico con lipoproteínas naturales de origen marino que se obtie-nen mediante procesos biotecnológicos no desnaturalizantes que preservan todas las propiedades biológicas de la especie mari-na original, S. pilchardus (E-SAR-94010®), un pescado azul de la familia de la sardina y extracto de alcachofa (cynara scolymus) estandarizado al 5%.

Las excelentes propiedades de este pesca-do azul, junto con las del extracto de alca-chofa, hacen que, HepatoSar Nutracéuti-co, ayude al funcionamiento fisiológico de nuestro organismo.

diciembre 2009 95

EuroEspes crea una unidad para tratar los problemas de la visión asociados a trastornos neurológicos (UNO)El nuevo departamento se denomina Uni-dad de Neuro-oftalmología (UNO). La Neuro-oftalmología es una rama de la of-talmología que se dedica al estudio anato-mofisiológico y patológico así como de la estrecha relación que existe entre el ojo y el sistema nervioso central.

En esta Unidad de Neuro-oftalmología se pueden tratar casos de visión doble, dismi-nución del campo visual, pupilas con dis-tinto tamaño, molestias visuales, disminu-ción inexplicada de la visión, problemas del nervio óptico o molestias visuales.

Con la puesta en funcionamiento de este servicio, EuroEspes se convierte en el pri-mer centro de España con un programa in-tegral que vincula la estrecha relación que existe entre la visión y el cerebro: el 70% del cerebro participa, de alguna manera, en la función visual.

Directivos de la Fundación Sociedade Galega para O Coñecemento visitan el Grupo EuroEspesJosé María Martín Moreno, Director Ge-neral y Luis González, Secretario General de la Fundación Sociedade Galega para O Coñecemento realizaron una visita al Gru-po EuroEspes. Era la primera toma de con-tacto entre ambas organizaciones y en ella se realizó un amplio recorrido por Ebiotec donde escucharon y vieron los diferentes proyectos y estudios en los que los inves-tigadores de EuroEspes Biotecnología se encuentran trabajando en la actualidad. En el transcurso de la visita mostraron in-terés por los procesos de liofilización, la genética y por la nutracéutica así como por el proyecto Ebiosea, que les fue explicado detalladamente.

En el Centro de Investigación Biomédica EuroEspes (CIBE) fueron recibidos por el Dr. Cacabelos, quien les introdujo en la filosofía del Grupo y mostró los últimos avances tecnológicos para la detección y diagnóstico de enfermedades neurodege-nerativas del sistema nervioso central pu-diendo comprobar el alto grado de innova-ción e investigación que se está realizando en la actualidad.

El Presidente del Grupo EuroEspes participa en el VI Congreso de Directivos CEDE El Dr. Ramón Cacabelos, Presidente del Grupo EuroEspes, participó en el progra-ma del VI Congreso Directivos CEDE, los días 28 y 29 de octubre, en Pamplona, que se celebró bajo el lema “Liderando en posi-tivo”. Su intervención tuvo lugar dentro de la sesión temática “Biotecnología”.

En este evento, al que asistieron destaca-dos profesionales de la gestión empresa-rial de nuestro país, EuroEspes participó como empresa colaboradora del mismo, con el fin de difundir la importancia de la predicción y la prevención en “la salud del cerebro del directivo”. El acto de clau-sura fue presidido por S.A.R. el Príncipe de Asturias.

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Noticias EuroEspes

Anfaco Cecopesca, Fundación EuroEspes y Ebiotec organizaron una jornada de biotecnología aplicada La biotecnología aplicada a la nutrición humana en la industria de conservación de productos de la pesca y la acuicultura es el título de la Jornada que el pasado día 24 de junio tuvo lugar en las instalaciones de ANFACO CECOPESCA dirigida a los representantes del sector conservero ga-llego y a empresas de biotecnología. Con el enunciado que da titulo a la Jornada el Dr. Ramón Cacabelos, presidente del Gru-po EuroEspes expuso que la biotecnología aplicada a la salud es un poderoso instru-mento para la innovación tecnológica e industrial en el sector de la nutrición hu-mana y la medicina.

Desde la perspectiva de la investigación indicó las líneas de trabajo en las que se mueve el Grupo EuroEspes y, especialmen-te, Ebiotec. Participaron como represen-tantes de Ebiotec y con sendas ponencias, el Dr. Valter Lombardi con el trabajo “Ali-mentos funcionales y productos nutracéu-ticos: una estrategia de futuro en nutrición humana”, por su parte Ramón Alejo, Di-rector Técnico de Producción de Ebiotec habló de “Liofilización industrial: nuevas tecnologías para la conservación y preser-vación de propiedades de los productos de la pesca”.

La jornada fue inaugurada por Juan Ma-nuel Vieites Baptista de Sousa, secretario general de ANFACO y director general de CECOPESCA.

NOTA ACLARATORIA

Sr. Director de la revista “Gen-T. The Euroespes Journal”:

En relación con el artículo titulado “Sobre la ética

de los análisis de ADN”, firmado por mí, Juan Car-

los Carril, y publicado en la sección “Tribuna Abier-

ta” de la revista “Gen-T The EuroEspes Journal”,

número 2, y en respuesta al acto de conciliación

entre la Universidad de Santiago de Compostela

(USC) y el que suscribe celebrado en Bergondo

el 27 de junio de 2008, y en interés de una mejor

comprensión de lo expuesto en el citado artículo,

solicito a la dirección de la revista la publicación de

la siguiente nota aclaratoria:

En el artículo mencionado, se hace referencia al

Instituto de Medicina Legal de Santiago de Compos-

tela (en adelante IML), únicamente como ejemplo

de laboratorio serio y riguroso en sus actuaciones, y

no como sospechoso de ningún tipo de actuación

irregular: “(…) participan en rigurosos controles de

calidad, cumplen normas internacionales de buenas

prácticas de laboratorio y están avalados por socieda-

des científicas internacionales como la Sociedad In-

ternacional de Genética Forense (ISFG). El Institu-

to de Medicina Legal de Santiago de Compostela es

uno de estos laboratorios serios, pero no el único.”

El autor no pretende afirmar, ni de la lectura del

mencionado artículo se puede deducir, que exista

demora por parte del IML en la realización de las

pruebas solicitadas por los Juzgados y Tribunales. El

autor alerta de la necesidad de que no tengan lugar

demoras más allá de las estrictamente derivadas de

la complejidad de los análisis realizados.

En ningún caso se afirma - y ni tan siquiera se insi-

núa - que se prioricen las pruebas privadas en detri-

mento de las solicitadas por los órganos judiciales.

No existe ni una sola frase en toda la redacción

del artículo que insinúe que los responsables o los

trabajadores, o la propia Universidad, se estén lu-

crando con la realización en el IML de pruebas de

paternidad privadas.

En ningún caso se hace referencia a las tarifas vi-

gentes en el IML ya que no es sobre este laboratorio

que el autor está dando la voz de alarma.

Por todo lo anteriormente expuesto, el autor en-

tiende que el artículo aquí mencionado no difama

ni calumnia ni al Instituto de Medicina Legal, ni a

sus trabajadores, ni a la Universidad de Santiago de

Compostela, además de considerar fruto de una in-

terpretación errónea la asunción de cualquier tipo

de ataque o falsedad contra los antes mencionados.

Es por ello que el autor considera oportuna la publi-

cación de esta nota aclaratoria que, en esencia, consi-

dera que se corresponde con lo expuesto en el escri-

to propuesto por la USC en el acto de conciliación.

Fdo. Juan Carlos CarrilBergondo, 10 de septiembre de 2008

Máster en Biotecnología de la Salud y Programa de Doctorado en Medicina GenómicaLa Universidad Camilo José Cela de Ma-drid crea, en colaboración con el Grupo EuroEspes, el Máster Universitario en Biotecnología de la Salud. Se impartirá a partir del curso académico 2010/11, y será dirigido por el profesor Ramón Cacabelos, presidente del Centro de Investigación Biomédica EuroEspes (CIBE) y Director de la Cátedra EuroEspes de Biotecnología y Genómica.

La Agencia Estatal de Evaluación de la Ca-lidad y la Acreditación (ANECA) ha infor-mado favorablemente el Plan de Estudios presentado por la Universidad Camilo José Cela.

La Comisión de Emisión de Informes de Máster reconoce que la Universidad Camilo José Cela, en su informe, “ aporta diferentes evidencias” que ponen de manifiesto el “in-terés y la relevancia académica y científica” del Máster en Biotecnología de la Salud.

Así mismo el Ministerio de Educación, a tra-vés del Consejo de Universidades, aprobó la creación del Doctorado en Medicina Genó-mica de la Universidad Camilo José Cela.

Ramón Cacabelos, será el responsable del Programa de Doctorado.

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