the prevention of thalassemia - cold spring...

The Prevention of Thalassemia

Antonio Cao1,3 and Yuet Wai Kan2

1Dipartimento di Scienze Biomediche e Biotecnologie, Universita degli Studi di Cagliari,Via Jenner snc 09121 Cagliari, Sardinia, Italy

2Department of Medicine, University of California, San Francisco, California 94143-0793

Correspondence: [email protected]

The thalassemias are among the most common inherited diseases worldwide, affectingindividuals originating from the Mediterranean area, Middle East, Transcaucasia, CentralAsia, Indian subcontinent, and Southeast Asia. As the diseases require long-term care, pre-vention of the homozygous state constitutes a major armament in the management. Thisarticle discusses the major prevention programs that are set up in many countries in Europe,Asia, and Australia, often drawing from the experience in Sardinia. These comprehensiveprograms involve carrier detections, molecular diagnostics, genetic counseling, and prenataldiagnosis. Variability of clinical severity can be attributable to interactions with a-thalasse-mia and mutations that increase fetal productions. Special methods taht are currently quiteexpensive and not widely applicable are preimplantation and preconception diagnosis. Therecent successful studies of fetal DNA in maternal plasma may allow future prenatal diag-nosis that is noninvasive for the fetus.

The thalassemias are a group of autosomalrecessive disorders caused by reduction or

absent production of one or more of the glo-bin chains that make up the hemoglobin (Hb)tetramers. According to the type of globin chaininvolved, two main types, i.e., thea- andb-thal-assemias can be distinguished. In addition, com-plex thalassemias resulting from defective pro-duction of two to four different globin chains(db-, gdb-, and 1gdb-thalassemia) are recog-nized (Weatherall and Clegg 2001). The clinicaltypes of thalassemias that are targets of preven-tion are b-thalassemia major resulting fromhomozygosity for b-thalassemia and Hb Bart’s

fetal hydrops syndrome caused by deletion ordysfunction of all four a-globin genes (Weath-erall and Clegg 2001).

EPIDEMIOLOGY AND GLOBAL BURDENOF THALASSEMIA DISORDERS

Thalassemias are among the commonest auto-somal recessive disorders worldwide (Modelland Darlison 2008; Weatherall 2010a,b) andare prevalent in populations in the Mediter-ranean area, the Middle East, Transcaucasus,Central Asia, the Indian subcontinent, and theFar East. However, they are also quite common

3Dr. Kan, the editors, and publisher deeply regret that Professor Antonio Cao, a remarkable contributor to the study and alleviation of

thalassemia, passed away on June 21, 2012.

Editors: David Weatherall, Alan N. Schechter, and David G. Nathan

Additional Perspectives on Hemoglobin and Its Diseases available at www.perspectivesinmedicine.org

Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a011775

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in populations of African heritage. Because ofpopulation migration, nowadays thalassemiasare also common in Northern Europe, NorthCentral and South America, and Australia.

Worldwide 56,000 conceptions have a ma-jor thalassemia disorder of which approximate-ly 30,000 are affected by b-thalassemia majorand 3500 succumb perinatally from the hydropsfetalis syndrome. Most (or many) of these pa-tients are born in developing and low-incomecountries where they create an enormous healthburden. Together with sickle cell anemia, it hasalso been estimated that, worldwide, 9 millioncarriers become pregnant annually and 1.33million pregnancies are at risk for a thalassemiamajor condition (Modell and Darlison 2008;Weatherall 2010b).

POPULATION CONTROLOF THALASSEMIAS: PROGRAMSFOR PREVENTION

In the late 1970s pilot population programsdirected to prevent b-thalassemia major by car-rier screening, counseling, and prenatal diagno-sis started in several at-risk populations in theMediterranean area [Cyprus, Sardinia, severalregions of Continental Italy (Delta Po area, Sic-ily), and Greece] (Angastiniotis and Hadjimi-nas 1981; Cao et al. 1996; Loukopoulos 1996).At present, several countries have set up compre-hensive national prevention programs, whichinclude public awareness and education, carrierscreening, and counseling, as well as informa-tion on prenatal diagnosis and preimplanta-tion diagnosis. These countries are Italy, Greece,Cyprus, UK, France, Iran, Thailand, Australia,Singapore, Taiwan, Hong Kong, and Cuba (AEleftheiou and M Angastionitis, pers. comm.).Partial programs including antenatal screen-ing according to ethnic origin are available inseveral countries in Northern Europe (Nether-lands, Belgium, and Germany) (Modell et al.2007). In many developing countries, prenataldiagnosis service has also been introduced (Can-atan et al. 2006; Chern et al. 2006; Weatherall2010b).

In most programs, carrier screening andcounseling are being performed on a voluntary

basis. In Cyprus, however, the Orthodox churchrequires a certificate proving that screening forb-thalassemia has been performed before mar-riage, but allows the final decision on marriageand a reproduction option to be left to thecouple (Angastiniotis and Hadjiminas 1981; Ka-lokairinou 2007). When Cyprus became a mem-ber of the European Union in 2004, the require-ment for this certification became unnecessaryfor civil weddings (Kolnagou and Kontoghi-orghes 2009). In a number of Muslim countriesincluding Lebanon, Iran, Saudi Arabia, Tunisia,United Arab Emirates, Bahrain, Qatar, and Ga-za Strip (Samavat and Modell 2004; Inati et al.2006), the national premarital programs aremandatory and aimed at limiting carrier mar-riage. In most of these countries, nowadays, in-terruption of the pregnancy is permitted for se-vere fetal disorders but only within the fourthmonth of gestation. In China, prenatal screen-ing was compulsory to obtain marriage permis-sion by the marriage law office (Li 2009). Be-cause of worldwide criticism, the requirementfor a certificate of premarital screening was with-drawn.

One of the obstacles for introducing pro-spective screening in Continental Europe is thelack of awareness among policymakers, healthprofessionals, and groups at risk, of hemoglo-bin disorders and the failure to recognize thathemoglobin disorders nowadays present a sig-nificant health problem (Modell et al. 2007). Indeveloping countries, it has been suggested thatthe ideal place where these preventive measurescould be organized is through existing healthcare services. Intensive education of health pro-fessionals as well as the population at large inthe field of preventive genetics is needed (re-viewed in Alwan and Modell 2003) and majorhealth organizations and funding agencies mustsupport these initiatives (Weatherall 2010b).

In a-thalassemia, screening is recommend-ed only to detect couples at risk for hydropsfetalis syndrome because severe toxemia com-plications (hypertension, preeclampsia) in themother may occur (Chui 2005). So far, screen-ing has been mostly retrospective. But prospec-tive screening has recently been introduced inseveral South Asian at-risk countries such as

A. Cao and Y.W. Kan

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Hong Kong, Southern China, Thailand, Taiwan,Malaysia, Singapore, Maldives, and Sri Lanka(Premawardhena et al. 2004; A Eleftheiou andM Angastionitis, pers. comm.). Because of themild phenotype, prevention of HbH disease isnot justified and should be discouraged.

PROGRAMS FOR CONTROL OFb-THALASSEMIA: EDUCATION

The successful programs in the Mediterraneanarea are all characterized by intensive educa-tion directed toward the health personnel andto the population at large. Periodic meetings areheld among physicians and especially pediatri-cians and obstetricians, family planning work-ers, nurses, and social workers, to discuss theclinical characteristics, the natural history, theprinciples of genetic counseling, and the meth-odologies for preventing the birth of affectedchildren (Fig. 1). Population education makesuse of mass media, posters, and informationalbooklets, which are left at various key sites suchas family planning clinics, marriage registries,and counseling rooms. The essential messagesshould include the clinical manifestation, natu-ral history, available therapies, and life expec-

tancy for the homozygous state of thalassemiamajor. It is important to emphasize that the car-rier state can be easily identified at the thalasse-mia centers and is not associated with stigma-tization, whereas couples who are both carriershave several reproductive options, includingprenatal diagnosis. Contact with parents’ andpatients’ associations should be regularly main-tained to update them on the content of themessages to avoid potential misinformation.Furthermore, parents’ associations can play aninvaluable role as an influential group by rais-ing economical support for all aspects of thal-assemia management and providing psycho-logical assistance to parents. Furthermore, inseveral countries, an annual thalassemia meet-ing or thalassemia day is organized and a spe-cific news magazine is regularly issued.

Formal education on thalassemias was alsointroduced in the educational curriculum at theend of secondary school (Cao et al. 2008; Kalo-kairinou 2008; Amato et al. 2009). A criticalevaluation of the information channels by whichthe population was informed, was performedin Sardinia and showed that a large majority ofthe target population (70%) was informed viaphysicians (family doctors, obstetricians, and

• Organizationreference centerand satellite units

• Educationalcampaigns

• Meetings • Consultation withparents’associations

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• Education atschool

• Voluntarism

• Doctors

• Leaders

• Populationat large

• Thalassemiaweeks• Retrospective

versusprospectives• Adequatefacilities

Figure 1. Strategy for prevention of b-thalassemias in Mediterranean at-risk populations.


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genetic counselors). These programs were ini-tially directed to couples with a previous af-fected child (retrospective diagnosis). Althoughvery useful for individual families, retrospectivediagnosis has a limited effect on the controlof homozygous b-thalassemia at the popula-tion level. Nowadays, the target populationsfor screenings are couples before marriage, atpreconception, and early following marriage(prospective diagnosis). Both in Cyprus and inItaly, the percentage of young unmarried cou-ples requesting screening is increasing, indicat-ing a continuous improvement of the knowledgeon thalassemia. In all these programs, carrierscreening has been performed on a voluntarybasis. Information counseling preceded testingbut written informed consent, which should beperformed according to accepted principles, isusually not requested.

Another critical prerequisite for a screeningprogram is the organization of adequate facili-ties to meet the demand both for screening andprenatal diagnosis. In all the successful pro-grams, a large majority of the adults counseledare already fully informed about thalassemia andthe methodology for its prevention, and are ca-pable of making an informed decision aboutreproduction. Furthermore, as seen later, theseprograms have resulted in a consistent decline inthe birth rate of homozygous b-thalassemia.

In Sardinia, a hub and spokes network witha main center and several satellite units was in-strumental in achieving such success. A similarorganization has been set up in Cyprus. In Sar-dinia, the distribution of the population in smallvillages facilitates the spread of informationand extends the counseling and testing to fam-ilies of carriers and parents. The introductionof teaching on thalassemia in secondary schoolhas also played a critical role in enforcing theknowledge of thalassemia in the family mem-bers. Potential economic barriers were prevent-ed by incorporating the screening, counseling,and prenatal diagnosis into the network of theNational Health Service. The experience gath-ered in these preventive programs indicates thatpopulation education should be modulated ac-cording to the cultural and religious character-istics of the target population.

CARRIER DETECTION

Heterozygotes forb-thalassemias, both of theb0

or the bþ variety are characterized hematologi-cally by high red blood cell count, microcytosis,hypochromia, increased HbA2, and unbalancedglobin chain synthesis. Several procedures havebeen proposed for b-thalassemia carrier screen-ing (Cao et al. 1998). The methodology mostoften used in the preliminary selection of indi-viduals at risk of being carriers is the determi-nation of the mean corpuscular volume (MCV)and mean corpuscular Hb concentration (MCH)(Fig. 2). An alternative for this approach pro-posed for the South Asian population (espe-cially for subjects living in rural areas) is theone-tube fragility test (OF) associated with thedichlorophenol indophenol (DCIP) dye test todetect the presence of HbE, which is very com-mon in this region and when interacting withb-thalassemia leads to b-thalassemia major orintermedia (Fucharoen et al. 2004). Those indi-viduals resulting positive at this screening testare further investigated by quantitative determi-nation of HbA2, which is the most importantfeature for identifying heterozygous b-thalas-semia (Weatherall and Clegg 2001). However,both MCV–MCH determination and especial-ly the one-tube fragility test, may miss those b-thalassemia heterozygotes who coinherited a-thalassemia (Melis et al. 1983; Rosatelli et al.1984). In carriers of b0-thalassemia, the a-thal-assemia determinants giving rise to this effectare the deletion of two a-globin genes or one ofthe nondeletion mutations affecting the majora2-globin gene. These carriers may be still easilyidentified because they have the typical increaseof HbA2 levels. Another cause for normal orborderline MCV–MCH values in carriers of b-thalassemia is a mild b-thalassemia mutation,including HbE, Hb Knossos, and Hb Malay(Weatherall and Clegg 2001). A high frequencyof both a- and b-thalassemia is common inmany of the populations at risk such as thoseliving in the Mediterranean area, and the Mid-dle and Far East. In Sardinia, for instance, be-cause of the high frequency of a-thalassemia,13.5% of b-thalassemia carriers have normal–borderline MCV–MCH values. The major

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hematologic characteristic for identifying theb-thalassemia carrier state is the increase of HbA2

levels (3.8%–6% in our laboratory). However, avariable but substantial number (in the differ-ent populations at risk) of b-thalassemia het-erozygotes may have a normal level of HbA2

(normal HbA2 b-thalassemia). The first mech-anism determining this phenotype is the pres-ence of heterozygosity for a b-thalassemia mu-tation associated with a significant residualoutput of b-globin chains from the affected lo-cus (mild b-thalassemia), such as the commonMediterranean bþ-thalassemia IVS nt 6 muta-tions (HBB c. 92 þ 6 T! C) or some promot-er mutation, i.e., the 292 C ! T (HBB c. 142 C! T). However, normal HbA2 b-thalassemiamay also result from coinheritance with b-thal-assemia, of d-thalassemia mutations in cis or in

trans, which determine a decrease in d-chainproduction, thereby resulting in normalizationof HbA2 (normal HbA2 b-thalassemia type 2)(Kattamis et al. 1979; Moi et al. 1988). Doubleheterozygotes for b- and d-thalassemia may bediagnosed by family studies, globin chain syn-thesis analysis, and/or a-, d-, and b-gene anal-ysis. Many different mutations have been so fardefined. d-Thalassemia is a relatively commongenetic condition (Loudianos et al. 1992; Moiet al. 1992). The presence of a d-chain variantmay also be associated with normal HbA2 levels(Waye et al. 2003; Phylipsen et al. 2010; So et al.2011). This condition may be recognized fromthe presence of an additional band in the elec-trophoretic pattern or a peak on the chromato-gram in the region where HbA2 also migrates. Inb-thalassemia carriers, the sum of normal plus

>78

>27

AA2

AA2 > 3.5%

0.1%–7%

NormalDNA analysisfor common β-point mutations

ARMSRDB

Characterizationof undefined

mutationsDGGESSCPdHPLC

Sequencing

DNA analysisfor β-genedeletion

mutationsGAP-PCR

S. blotMLPA

Hb S Hb CHb D

Hb EHb O Arab

Hb Lepore

Others

Furtherinvestigations

required

δ + β Thal traitδβ Thal trait

Normal HbA2 β-Thalcarrier

β-Thal

carrier

DNA analysisfor α-globinmutationsGAP-PCR

MLPAS. blot

PCR + RE

HbH inclusions

α-globin geneanalysis

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<78 MCV f1 <78 Hb variants

Hb electrophoresisHPLC

Mass spectroscopy

<27

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Normal iron

Hematological and DNA studies

MCH pg

Hbs

<27

α-Thal

Figure 2. Flow chart for b-thalassemia carrier screening applied in Sardinia. Thal, thalassemia; S. blot, Southernblotting.



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variant HbA2 is, however, increased. NormalHbA2 b-thalassemia heterozygotes should bedifferentiated from a-thalassemia carriers whoshow the same hematological features. The rare1gdb-,gdb-, and db-thalassemias are also char-acterized by microcytosis and normal HbA2

levels. db-Thalassemia may be easily definedby a marked increase of HbF. Associated irondeficiency may result in decreased levels ofHbA2. This effect, however, is only observed inthe presence of severe anemia (Galanello et al.1981). Iron studies can easily exclude this asso-ciation.

Finally, a critical problem in b-thalassemiacarrier screening is the identification of silentb-thalassemia or the triple-quadruple a-gene ar-rangement, which by interacting with typicalb-thalassemia may result in the clinical featuresof mild or severeb-thalassemia (thalassemia in-termedia or major) (Galanello et al. 1983; Theinet al. 1984; Kulozik et al. 1987; Ristaldi et al.1990; Harteveld et al. 2008; Sollaino et al.2009; Faa et al. 2010). Silentb-thalassemia (nor-mal HbA2 b-thalassemia type 1) shows normalhematological features and HbA2 level and maybe identified solely by globin chain synthesisanalysis or b-globin gene analysis (Kattamiset al. 1979; Gonzalez-Redondo et al. 1989; Gal-anello et al. 1994). The triple or quadruple a-globin gene arrangement, which also displaysthe phenotype of normal HbA2 b-thalassemiatype 1, may show a slight unbalance ofa/b þ g-globin chain synthesis but may also have a ba-lanced synthesis (Harteveld et al. 2008; Faa et al.2010). The presence of these atypical carriers isgenerally suspected in families that already hada child affected by mild b-thalassemia, despitehaving one apparently normal parent, the otherbeing a typical b-thalassemia carrier.

The finding of individuals with borderlineHbA2 (level between 3.3% and 3.8% in our lab-oratory) and normal MCV and MCH createsa relevant problem in the process of thalasse-mia carrier identification. Known determinantscausing this phenotype are the triple a-globingene arrangement, some b-promoter mutation(HBB c. 151 C! T), and rare HBB or HBDgene variant (Table 1) (Galanello et al. 1983,1994; Gasperini et al. 1993). Recently, we per-

formed in the Sardinian population molecularanalysis of theKLF1gene(which codes forazinc-finger transcriptional factor) in a large groupof individuals with borderline HbA2. KLF1 isable to activate in the perinatal period the b-globin gene and at the same time the BCL11Agene (Borg et al. 2010; Zhou et al. 2010), which iscapable in turn to suppress the g-chain produc-tion (Borg et al. 2010; Zhou et al. 2010). KLF1gene mutation has been associated with manydifferent phenotypes including hereditary per-sistenceof fetal hemoglobin (HPFH), congenitaldyserythropoietic anemia, and the (Lu) bloodgroup phenotype (Singleton et al. 2008; Arnaudet al. 2010; Borg et al. 2010; Satta et al. 2011).

In our study, we show that a consistent pro-portion of individuals (35%) with borderlineHbA2 have pathogenic mutation in KLF1, re-sulting in the activation of the HbD gene andthereby in increased HbA2 levels (Perseu et al.2011). This finding may facilitate the carrierdetection and counseling thereby contributingto the success of the program.

In our opinion, a carrier detection proce-dure should be designed to avoid missing anycouple at risk. For this reason we have includedin the first group of examination, in addition todetermination of MCV–MCH, the quantitativeevaluation of HbA2, which may be obtained byelectrophoresis or by high-pressure liquid chro-matography (HPLC). HPLC has the addition-al advantage to quantitate also HbF and to de-tect clinical relevant Hb variants including HbKnossos (a mild b-thalassemia allele), HbS,HbC, HbD Punjab, HbO Arab, and HbE, all ofwhich may interact with b-thalassemia hetero-zygosity leading to thalassemia major or inter-media or sickle cell b-thalassemia. By this ap-proach only silent b-thalassemia or the triple orquadruple a-globin gene arrangement may es-cape detection.

Table 1. Causes of borderline HbA2 levels

Some promoter mutation of the b-globin geneSome silent b-thalassemiaSome HBB or HBD variantsTriple or quadruple a-globin arrangementKLF1 mutations

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The critical issue in the screening processis the differential diagnosis of individuals withthe phenotype of normal HbA2 b-thalassemiatype 2 (low MCV, low MCH, normal, or bor-derline HbA2), which may result from iron de-ficiency, a-thalassemia, gdb-thalassemia, dou-ble heterozygosity for b- and d-thalassemia ormild b-thalassemia. After exclusion of iron de-ficiency by erythrocyte ZnPP and transferrinsaturation determination, we differentiate thedifferent thalassemia genotypes bya-/b-globinchain synthesis or a-, d-, and b-gene analysis.

A hematological phenotype characterizedby low MCV and MCH, normal or reducedHbA2, and high HbF levels indicate the presenceof db-thalassemia. db-Thalassemia may be con-fused with HPFH. The differentiation is basedon globin gene synthesis analysis orb-gene clus-ter studies; in addition, HPFH is characterizedby lack of microcytosis and hypochromia.

MOLECULAR DIAGNOSIS

Molecular diagnosis of a-, b-, and d-thalasse-mia when necessary may be performed by one ofthe PCR-based available procedures. b-Thalas-semias display a marked heterogeneity at themolecular level. So far, .200 different muta-tions have been described. A complete updatedlist is available at the Globin Gene Server Website—http://globin.cse.psu.edu. The large ma-jority of these mutations are point mutationor the addition or deletion of one or a fewnucleotides causing a frameshift. More rarely,b-thalassemias are produced by globin genedeletion. Despite this heterogeneity a limitednumber of molecular defects are present ineach population at risk. This information maybe useful for designing the appropriate primersor probes for each at-risk population (Fig. 3).Nowadays, known mutations are detected bya PCR-based procedure among which the mostcommonly used are commerciallyavailable RDB(reverse oligonucleotide hybridization) analysis(Saiki et al. 1989) and allele-specific amplifica-tion (amplification refractory mutation system[ARMS]) (Newton et al. 1989). Both methods,especially the RDB, can screen in one step up to20–30 different mutations. Methodologies cur-

rently used for identification of unknown mu-tations by scanning the entire gene were dena-turing gradient gel electrophoresis (DDGE)(Myers et al. 1985; Cai and Kan 1990; Rosatelliet al. 1992), denaturing high-pressure liquidchromatography (dHPLC) (Sanger et al. 1977),and single-strand conformation polymorphism(SSCP) analysis (Orita et al. 1989). When mu-tation is not detected by these procedures,polyacrylamide gel electrophoresis of PCR-am-plified product (gapPCR) may lead to the detec-tion of a small deletion of the b-globin gene,which may be suspected by finding unusuallyhigh HbA2. The presence of rare large deletions,including deletion of LCR, is finally investigatedby multiple ligation-dependent probe amplifi-cation (MLPA). These methods are now usuallyreplaced by DNA sequence analysis.

Detection of the most frequent mutations/deletions of the a-globin gene, as for the b-glo-bin gene detection, is also available using thePCR–RDB-based kits. Known deletions maybe detected by PCR-based methods using prim-ers flanking the deletion breakpoint. Primerpanels targeted to the most common deletionfound in the area of the geographic origin ofthe proband can be used (Galanello et al. 1998;Chong et al. 2000; Old et al. 2001). MLPA (Har-teveld et al. 2005) or quantitative real-time PCRassay may also be used to detect less commonmutations or novel deletion (Fallah et al. 2010).a-Thalassemia mental retardation 16 (ATR16),a contiguous gene deletion, may also be detect-ed by MLPA or array comparative genome hy-bridization (CGH) (Gibson et al. 2008).

PREDICTION OF A MILD PHENOTYPE

The severity of homozygous b-thalassemia de-pends on the extent of the imbalance betweena-globin and non-a-globin (including b- andg-chains) chains. Any conditions able to reducethis unbalance results in a mild clinical pheno-type referred to as thalassemia intermedia. Thefirst molecular mechanism is related to severityof the b-thalassemia mutation present in eachcase (Higgs et al. 2011). Mild bþ-thalassemia(which also includes HbE) and silent mutationsare associated with adequate residual output of



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the b-globin gene from the affected b-globinlocus and thereby usually result in attenuatedphenotypes in the homozygosity or compoundheterozygous state. In contrast, compound het-erozygotes for a mild bþ-thalassemia or silentmutation and a severe mutation produce a var-iable phenotype ranging from thalassemia ma-jor to thalassemia intermedia. Therefore, thepresence of this genotype does not consent toconsistently predict a mild phenotype.

The second recognized mechanism forproducing a mild phenotype is the coinheritancewith homozygous b-thalassemia of a-thalas-semia, which, by reducing the output of a-chains, decreases the a-/non-a-chain imbal-ance (Wainscoat et al. 1983; Galanello et al.1989). A consistent ameliorating effect is ob-served when the a-thalassemia determinantconsists of the deletion of two a-globin struc-tural genes or of an inactivating mutation of themajor a2-globin gene (Wainscoat et al. 1983;Galanello et al. 1989; Thein 2008). However, be-

cause this effect is not constantly seen, the coin-heritance of a-thalassemia cannot be recom-mended to predict the phenotype.

Finally, the coinheritance of genetic deter-minants able to sustain a continuous high pro-duction of g-globin chains in adult life, mayameliorate homozygous b-thalassemia by re-ducing the a-/non-a-chain imbalance.

Genetic conditions associated with in-creased HbF in adult life and therefore able toameriolate the phenotype of b-thalassemia arelisted in Table 2. Recent studies using genome-wide association studies (GWAS) have identifiedtwo quantitative trait loci (QTL) (BCL11A onchromosome 2p16 and HBS1L-MYB intergenicregion most likely involving MYB on chromo-some 6q23) that account for �20%–30% ofthe common variation in HbF levels in healthyadults as well as in persons with b-thalasse-mia and sickle cell disease (Uda et al. 2008; Theinand Menzel 2009). In Sardinians, the alleles ofBCL11A associated with high HbF were found

Codon 39 C-T

Mediterranean region

France

SpainItaly Turkey

China

Greece

IVSI-1 G-A

IVSII-1 G-A

Codon 8-AA

IVSI-6 T-C

IVSII-745 C-G

Frameshift 6-A

IVSI-110 G-A

Codon 6-A

Others

CyprusTunisia

India Thailand

Indonesia

–28

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Asian region

Sar

dini

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ly

Codon 17

IVSI-1 G-T

IVSII-654

–619 bp

Codon 15

Codons 41/42

IVSI-5 G-T

Others

Algeria

Figure 3. Population distribution of common b-thalassemia mutations.

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more frequently in patients with thalassemiaintermedia as compared to those affected bythalassemia major, indicating that those loci ex-ert, by increasing HbF, an ameliorating effect onhomozygous b0-thalassemia (Uda et al. 2008;Galanello et al. 2009). These results were con-firmed in patients with sickle cell anemia fromthe United States and Brazil (Lettre et al. 2008)and b-thalassemias in France (Badens et al.2011). The two loci, in addition to coinheriteda-thalassemia, act in additive fashion, account-ing for �75% of the variation in clinical severityin Sardinian b0-thalassemias (Galanello et al.2009). Genotyping at these loci in prospectiveparents may have a practical utility as a prog-nostic indicator for the severity of b-thalasse-mia and sickle cell anemia, thereby improvingthe genetic counseling. It is likely that manyother HbF-associated QTL exist such as thatrecently defined on chromosome 13 (Sankaranand Nathan 2010).

GENETIC COUNSELING

Limited information is available in the literatureconcerning the approach for counseling by thal-assemia centers. A working party several yearsago discussed the problem and presented somerecommendations (WHO 1994). I would liketherefore to concentrate on our own experiencein Sardinia. According to international rules,

counseling has been performed in a nondirec-tive way and was based on a private interview. Apediatrician trained in genetics is responsiblefor counseling the couples identified to be atrisk, whereas a trained nurse deals with individ-uals not yet married. The information providedis aimed at giving an informed basis on which tomake a decision about reproduction. The dis-cussion includes various options available suchas birth control, mate selection, adoption, fetaltesting including prenatal diagnosis and preim-plantation diagnosis, or artificial inseminationby normal donors as well as the details of thenatural history of the disease. When the geno-types indicate the possible development of amild phenotype, we also prudently discuss thispoint, emphasizing that the development of thisphenotype should be considered a probabilityand not a certainty.

I strongly believe that the counseling ac-cording to international guidelines should bebased on truth, complete honesty, and frank-ness. Other experts, before the recent discoveryon HbF QTL, were of the opinion that this phe-notype prediction should not be discussed(Weatherall and Clegg 2001).

In cases with a previous nonaffected child,we propose HLA typing on fetal DNA, to checkwhether the sibling could be HLA identical andtherefore a suitable bone marrow donor (Oro-fino et al. 2003). This information could be use-ful for deciding on noninterruption of the preg-nancy in the case of an affected fetus. In Sardinia,as well in other Mediterranean countries (Cy-prus, Continental Italy, and Greece) and fromrecently published papers also in Muslim coun-tries such as Iran, the option of prenatal diag-nosis is followed by the majority of the couplescounseled. Last, we inform the counselee aboutthe risk for their relatives and recommend in-forming them about the result. This inductivescreening is a very efficient way to multiply theefficacy of the screening as reported also inPakistani populations (Ahmed et al. 2002).

PRENATAL DIAGNOSIS

The possibility of prenatal diagnosis of inheritedhemoglobinopathies was first suggested by the

Table 2. Genetic conditions with increased HbF inadult life

Linked to the b-clusterHPFHdb-ThalassemiasDeletions of the 50 region of the HBB promoterPoint mutation of the Gg (HBG2) (2158 C ! T Gg

mutation)Point mutation of the Ag (HBG1) (2196 G! TAg

mutation)QTL 11q (HS3 locus); locus downstream from HBG1

(Galarneau et al. 2010)

Unlinked to the b-clusterBCL11AHBSA1L-MYB regionOthers (chromosome 13)

HPFH, hereditary persistence of fetal hemoglobin.



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discovery that b-globin chain synthesis in cordblood of b-thalassemia heterozygotes was sig-nificantly lower than normal (Kan and Nathan1968) and that adult hemoglobin synthesiscould be detected in fetuses at midtrimester(Hollenberg et al. 1971). Later on, the bS-chainwas detected in the blood of fetuses (Kan et al.1972). Fetal blood sampling by fetoscopy orplacentocentesis was then set up (Hobbins andMahoney 1974; Kan et al. 1974) and paved theway to introduce prenatal diagnosis by globinchain synthesis on fetal blood (Kan et al. 1975).These techniques were used for 5 years. The firstmolecular diagnosis was performed for the di-agnosis of a-thalassemia by the technique ofmolecular hybridization (Kan et al. 1976). Fora short period diagnosis of b-thalassemia byDNA analysis was obtained indirectly by analy-sis of DNA polymorphisms linked to the b-glo-bin gene (Kan et al. 1980) or directly by muta-tion-specific oligonucleotide hybridization onelectrophoretically separated DNA fragments(Pirastu et al. 1983).

Nowadays, thalassemias are detected direct-ly by the analysis of amplified DNA from fetaltrophoblast or amniotic fluid cells. Fetal DNA isobtained transabdominally by chorionic villussampling. This technique has been chosen bymany centers because of the advantage of beingused within the first trimester and because it isassociated with a low risk (�1%) of fetal mor-tality. Fetal DNA is analyzed with one of themethods described for the detection of knownmutations in the process of carrier identifica-tion. Prenatal diagnosis by DNA analysis is todayavailable in many at-risk populations, includingthose from Hong Kong, Taiwan, China, and In-dia, in addition to those from the Mediterraneanarea, Northern Europe, and Northern and SouthAmerica (Eleftheiou and Angastionitis 2011).

Misdiagnosis may occur for several reasons:i.e., failure to amplify the DNA fragment, mis-paternity, maternal contamination, and sampleexchange. To limit the possibility of misdiagno-sis, we analyze the fetal DNA with two differenttechniques (ARMS and RDB) and carry outDNA polymorphism analysis to detect mater-nal contamination and mispaternity. The PCR-based technique of prenatal diagnosis turned

out to be very reliable. In our center we had nomisdiagnoses in �6000 cases performed so far.Similar results have been reported by others(Angastiniotis et al. 1995).

Fetal anemia may be diagnosed at mid-tri-mester gestation by quantitation of middle ce-rebral artery peak systolic velocity. This tech-nique has been applied successfully to prenatalidentification of Hb Bart’s fetal hydrops syn-drome (Lam and Tang 2002; Srisupundit et al.2009). However, false negatives have been re-corded using this technology. Mutation analysisofa-globin genes may be used to detect deletionand nondeletion a-thalassemia.

PREIMPLANTATION ANDPRECONCEPTIONAL GENETIC DIAGNOSIS

The progress in molecular genetics to analyze thegenotype of a single cell, together with advancesin assisted reproduction techniques, has pavedthe way for developing preimplantation and pre-conceptional diagnosis (Handyside et al. 1990;Monk and Holding 1990). These techniques arenowadays widely available, including countrieswhereb-thalassemia is prevalent such as Cyprus(Kulievet al. 2011), Continental Greece(Zachakiet al. 2011), and China (Jiao et al. 2003; Xu et al.2009). Preimplantation genetic diagnosis is per-formed either by biopsy of one to two blasto-meres in eight-cell embryos after in vitro fertil-ization (by intracytoplasmatic sperm injection)or by biopsy of trophectoderm cells from blas-tocyst (Kuliev et al. 1998; Kokkali et al. 2007).

Preconceptional diagnosis is based on theanalysis of the first polar body of unfertilizedeggs followed by analysis of the second polarbodies after fertilization, which is performedto avoid misdiagnosis resulting from recombi-nation during the first meiosis (Verlinsky et al.1990). Diagnosis was obtained by multiple nest-ed PCR analysis to detect the mutations as well aspolymorphic alleles at the b-globin cluster (Ku-liev et al. 2011; Zachaki et al. 2011). HLA typ-ing of the embryo to select a nonaffected fetusHLA compatible with a previous affected siblingwas recently proposed (Kuliev et al. 2011).

The biopsy of blastomeres from an eight-cellembryo is nowadays the most frequently used

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procedure worldwide. This approach mini-mizes the risk of misdiagnosis that could resultfrom PCR failure, contamination, and allelicdropout that is a definite risk in single-cell anal-ysis. In a large series, misdiagnosis was rare andthe pregnancy rate high (32%) (Kuliev et al.2011). The motivations for couples followingcounseling to opt for preimplantation geneticdiagnosis (PGD) are problems of infertility, anunsuccessful reproductive history owing to re-peated terminations of affected fetuses, moralor religious attitude against pregnancy termina-tion, and the desire to have a nonaffected fetusHLA compatible with a previously homozy-gously affected sibling for future bone marrowtransplantation. Genetic counseling for couplesconsidering PGD should include a discussionon the risk associated with in vitro fertilizationand embryo biopsy, the risk of failure and/ormisdiagnosis, and the need of subsequent pre-natal diagnosis for confirming the results. Atpresent, however, the technical demand forthese procedures, the difficulty in organizingthe service, and the high cost preclude its usefor monitoring worldwide pregnancies at risk.

PRENATAL DIAGNOSIS BY NONINVASIVEPROCEDURES

Several attempts have been made to make fetaldiagnosis by analysis of fetal cells in maternal

circulation (Simpson and Elias 1993; Adinolfi1995; Cheung et al. 1996; Bianchi et al. 1997).However, the complexity of the cell isolationprocess and the lack of reproducibility precludethe use of this approach in clinical practice.Likewise, many studies have been performedto make the diagnosis of b-thalassemia or a-thalassemia by analysis of fetal DNA in maternalplasma (Lo et al. 1997; Lun et al. 2008). Thesenoninvasive diagnostic methods, while gradu-ally being introduced for chromosomal defects,require more refinement for globin and othersingle gene disorders. It should, however, bementioned that a diagnosis of b-thalassemiawas recently realized by the Lo group withnext-generation sequencing (paired and mas-sively, parallel sequencing) of the fetal and ma-ternal DNA genomes present in maternal plas-ma (Lo et al. 2010).

OUTCOME OF THE PROGRAMS

The primary effect of the programs for prevent-ing b-thalassemia in the Mediterranean pop-ulations has been the acquisition by the couplesat risk of the appropriate knowledge to makeinformed decisions on the reproductive optionsavailable to them. This awareness of the geneticrisk and methods of prevention led to a markedacceptance of fetal testing leading to a consistentdecline in the birth rate of thalassemia major in

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77 79 81 83 85 87 89 91 93

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Figure 4. Decline of birth rate of thalassemia major in Sardinia.



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these populations. In Sardinia, for instance, thenumber of thalassemia major children born peryear predicted on the basis of the carrier rate,assuming a random mating, shows a reductionfrom 1:250 live births to 1:1660 in 2009 with aneffective prevention of 85% of the cases (Fig. 4).Similar results have been reported in Cyprus(Angastiniotis and Modell 1998). The dissolu-tion of the marriage or the decision not to havechildren is less commonly followed, except inMuslim countries, where the breakup of mar-riages seems to occur more often.

Despite well-organized prevention pro-grams, reasons for the birth of affected childrenare negative attitudes against fetal testing fol-lowing counseling and decision to continuethe pregnancy despite the documented presenceof an affected fetus for religious or ethical rea-sons. Less common motivations are misinfor-mation, lack of information, mispaternity, andmistakes in fetal testing. Although research onthis topic is not available, it seems that the pro-grams had no adverse effects.

FUTURE PROSPECTIVES

Education on thalassemias may be implement-ed by using informatic channels. The continu-ous reduction in the cost of DNA analysis maylead to its application as the initial step in thescreening process. Using microchip technologytargeted to detect all the known a- and b-thal-assemias as well as DNA polymorphisms is use-ful to predict the phenotype, thereby making itpossible to skip the preliminary hematologicalanalysis.

Technical advances in the methodologies offetal DNA or fetal cells analysis in maternal plas-ma may allow in the future the introduction ofthis important noninvasive technique in clinicalpractice.

Finally, the most important challenge forthe future is the organization of national pre-ventive programs in populations in which thal-assemias are prevalent such as the Middle East,the Indian subcontinent, and the Far East,where several factors previously discussed pre-clude at the present time the realization of suchprograms.

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2013; doi: 10.1101/cshperspect.a011775Cold Spring Harb Perspect Med Antonio Cao and Yuet Wai Kan The Prevention of Thalassemia

Subject Collection Hemoglobin and Its Diseases

The Natural History of Sickle Cell DiseaseGraham R. Serjeant Hemoglobin Synthesis

Transcriptional Mechanisms Underlying

Nathaniel J. Pope, et al.Koichi R. Katsumura, Andrew W. DeVilbiss,

Current Management of Sickle Cell Anemia

WarePatrick T. McGann, Alecia C. Nero and Russell E. Disease

Iron Deficiency Anemia: A Common and Curable

Jeffery L. Miller

TherapiesTargetedNew Disease Models Leading the Way to

Cell-Free Hemoglobin and Its Scavenger Proteins:

Dominik J. Schaer and Paul W. Buehler

Management of the ThalassemiasNancy F. Olivieri and Gary M. Brittenham

-ThalassemiaαClinical Manifestations of Elliott P. Vichinsky

-ThalassemiaβThe Molecular Basis of Swee Lay Thein

Erythroid Heme Biosynthesis and Its DisordersHarry A. Dailey and Peter N. Meissner

Erythropoiesis: Development and DifferentiationElaine Dzierzak and Sjaak Philipsen

Clinical CorrelatesHemoglobin Variants: Biochemical Properties and

Gell, et al.Christopher S. Thom, Claire F. Dickson, David A.

ErythropoietinH. Franklin Bunn

The Prevention of ThalassemiaAntonio Cao and Yuet Wai Kan

Classification of the Disorders of HemoglobinBernard G. Forget and H. Franklin Bunn

The Switch from Fetal to Adult HemoglobinVijay G. Sankaran and Stuart H. Orkin

-ThalassemiaαThe Molecular Basis of Douglas R. Higgs

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

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