molecular medicine in clinical practice

Molecular Medicine in Molecular Medicine in Clinical Practice Clinical Practice

Dr. Osama . I . Nassif , FRCPCAssociate Professor and Consultant PathologistDepartment of Pathology, Faculty of Medicine

King Abdullaziz University Hospital

Introduction

• Sources of DNA in clinical practice:

– Any nucleated cell in the body

• Blood• Tumor sample (tissue or aspirate)• Body discharge• Hair root, semen, or body fluid• Chorionic villi and amnionic fluid• Mouth wash

Introduction

• DNA isolation

• RNA isolation

Introduction

• Molecular Bio-techniques– Blotting

• Southern• Northern• Western

– Hybridization– PCR, RT-PCR– DNA sequencing– cDNA cloning– Recombinant protein

Introduction• Molecular Bio-techniques has many applications in

several fields of clinical practice including:

– Medical genetics

– Fetal and neonatal medicine

– Medical microbiology

– Infectious diseases

– Medical oncology

– Hematology

– Anatomical pathology and tumor diagnosis

– Therapeutics

– Forensic pathology

Applications of Molecular Bio-techniques in Medical Genetics

• Analysis and characterization of genes abnormalities leading to disease.

• Understanding genetic diseases pathogenesis

• Detection of gene mutation (mutational analysis)

• Study of genetic diseases pattern of inheritance

• Diagnosis and screening of genetic diseases

• Prenatal diagnosis

• Identification of diseases carrier to help in genetic and pre-marriage counseling.

Medical Genetics

• Four major categories of genetic disorders:– (1) disorders related to mutant genes of large effect.

most of these follow the classic Mendelian patterns of inheritance, they are also referred to as Mendelian disorders.

– (2) diseases with multifactorial (polygenic) inheritance. These are influenced by both genetic and environmental factors

– (3) chromosomal disorders, includes diseases that result from genomic or chromosomal mutations

– (4) single-gene disorders with nonclassic patterns of inheritance.

Mutational Analysis• It means the identification of changes in DNA which produce

disease or dysfunction.

• Several methods can be used to detect gene mutation including PCR, southern blotting, Pulsed-Field Gel Electrophoresis (PFGE): , FISH, cytogenetic, DNA sequencing.

• Factors that determine the type of methods to be used include:– Nature and size of mutation– Mutation knowledge– The frequency of mutation in the population of interest (hot spot

mutation)– Size of the gene of interest– Nature of the available sample for testing

Mutational Analysis

• Detecting DNA deletion:– Very small deletions can be detected by PCR

(e.g. cystic fibrosis)

– Larger deletion (e.g. α thalassaemia) can be detected by Southern blotting

– The largest deletion (e.g. contiguous gene syndrome) can be detected by PFGE or FISH

Mutational Analysis

• Detecting point mutation:– These occur more frequently than deletion

– They are more difficult to identify because they are small, and heterogeneous.

– PCR is the most useful technique in detecting these mutation if they are known in family of interest.

Mutational Analysis

• DNA sequencing

• Chromosomal analysis– Karyotyping– FISH

Applications of Molecular Bio-techniques in Medical Genetics

Diagnosis of Genetic Diseases

• Two general methods are used: – Cytogenetic analysis and

– Molecular analysis.


Prenatal chromosome analysis:

• This should be offered to all patients who are at risk of cytogenetically abnormal progeny.

• It can be performed on cells obtained by amniocentesis, on chorionic villus biopsy, or on umbilical cord blood.

• indications are the following: – Advanced maternal age (>34 years) because of greater risk of trisomies

– A parent who is a carrier of a balanced reciprocal translocation, robertsonian translocation,

– A previous child with a chromosomal abnormality

– A parent who is a carrier of an X-linked genetic disorder (to determine fetal sex)


Postnatal chromosome analysis:• This is performed on peripheral blood lymphocytes.

• Indications are as follows: – Multiple congenital anomalies.

– Unexplained mental retardation or developmental delay.

– Suspected aneuploidy (e.g., features of Down syndrome).

– Suspected unbalanced autosome (e.g., Prader-Willi syndrome).

– Suspected sex chromosomal abnormality (e.g., Turner syndrome).

– Suspected fragile X syndrome.

– Infertility (to rule out sex chromosomal abnormality).

– Multiple spontaneous abortions.


• Many genetic diseases are caused by subtle changes in individual genes that cannot be detected by karyotyping.

• Traditionally the diagnosis of single-gene disorders has depended on the identification of abnormal gene products (e.g., mutant hemoglobin or enzymes) or their clinical effects, such as anemia or mental retardation (e.g., phenylketonuria).

• Now it is possible to identify mutations at the level of DNA and offer gene diagnosis for several mendelian disorders.

• Examples of inherited diseases that can be detected by PCR

Diagnosis of Genetic DiseasesThe advantages of molecular diagnosis of genetic disorders:

1. It is remarkably sensitive. • The amount of DNA required for diagnosis by molecular hybridization techniques

can be readily obtained from 100,000 cells. • The use of PCR allows several million-fold amplification of DNA or RNA, making

it possible to use as few as 100 cells or 1 cell for analysis. • Tiny amounts of whole blood or even dried blood can supply sufficient DNA for

PCR amplification.

2. DNA-based tests are not dependent on a gene product that may be produced only in certain specialized cells (e.g., brain) or expression of a gene that may occur late in life.

• Virtually all cells of the body of an affected individual contain the same DNA, each postzygotic cell carries the mutant gene.

• These two features have profound implications for the prenatal diagnosis of genetic diseases because a sufficient number of cells can be obtained from a few millilitres of amniotic fluid or from a biopsy of chorionic villus that can be performed as early as the first trimester.


• There are two approaches to the diagnosis of single-gene diseases by DNA based technology: – Direct detection of mutations and

– Indirect detection based on linkage of the disease gene with a harmless "marker gene."


1. Direct Gene Diagnosis:“diagnostic biopsy of the human genome”– Direct gene diagnosis is possible only if the mutant gene and its

normal counterpart have been identified and cloned and their nucleotide sequences are known.

a) One technique relies on:– some mutations alter or destroy certain restriction sites on DNA– e.g.: detecting the mutation of gene encoding factor V. This

protein is involved in the coagulation pathway, and a mutation affecting the factor V gene is the most common cause of inherited predisposition to thrombosis.

Direct gene diagnosis: detection of coagulation factor V mutation by PCR. Base substitution in an exon destroys one of the two Mnl1 restriction sites. The mutant allele therefore gives rise to two, rather than three, fragments by PCR analysis.


b) Allele-specific oligonucleotide hybridization "dot blot" test:

– e.g.: α1 antitrypsin deficiency

– Direct gene diagnosis by using PCR and an allele-specific oligonucleotide probe.

– Base change converts a normal α1 antitrypsin (allele M) to a mutant (Z) allele.

–Two synthetic oligonucleotide probes, one corresponding in sequence to the normal allele (M probe) and the other corresponding to the mutant allele (Z probe), are lined up against normal and mutant genes

– The PCR products from normal individuals, those heterozygous for the Z allele or homozygous for the Z allele, are applied to filter papers in duplicate, and each spot is hybridized with radiolabeled M or Z probe. A dark spot indicates that the probe is bound to the DNA.


c) Mutations that affect the length of DNA (e.g., deletions or expansions) can be detected by PCR analysis.

– e.g.: the fragile X syndrome (associated with trinucleotide repeats)

With PCR, the differences in the size of CGG repeat between normal and premutation gives rise to products of different sizes and mobility. With a full mutation, the region between the primers is too large to be amplified by conventional PCR. In Southern blot analysis the DNA is cut by enzymes that flank the CGG repeat region, and is then probed with a complementary DNA that binds to the affected part of the gene. A single small band is seen in normal males, a higher-molecular-weight band in males with premutation, and a very large (usually diffuse) band in those with the full mutation.


2. Indirect DNA Diagnosis: Linkage Analysis

– large number of genetic diseases, including some that are relatively common, information about the gene sequence is lacking.

– Therefore, alternative strategies are to track the mutant gene on the basis of its linkage to detectable genetic markers.


• Principle:– to determine whether a given fetus or family member

has inherited the same relevant chromosomal region(s) as a previously affected family member.

– the success of such a strategy depends on the ability to distinguish the chromosome that carries the mutation from its normal homologous counterpart.

– This is accomplished by finding naturally occurring variations or polymorphisms in DNA sequences.


a) Restriction Fragment Length Polymorphisms (RFLPs). – Background:

• examination of DNA from any two persons reveals variations in the DNA sequences.

• Most of these variations occur in noncoding regions of the DNA and are hence phenotypically silent.

• these single base pair changes may abolish or create recognition sites for restriction enzymes, thereby altering the length of DNA fragments produced after digestion with certain restriction enzymes.

• Using appropriate DNA probes that hybridize with sequences in the vicinity of the polymorphic sites, it is possible to detect the DNA fragments of different lengths by Southern blot analysis.

• RFLP refers to variation in fragment length between individuals that results from DNA sequence polymorphisms.

RFLP: This technique is to distinguish family members who have inherited both normal chromosomes from those who are heterozygous or homozygous for the mutant gene.

RFLP analysis for the presence of the sickle-cell locus. Genomic DNA is isolated and digested with the restriction enzyme MstII. One MstII site is lost at the sickle-cell locus. The DNA is then Southern blotted and analyzed with a b-globin-specific probe

corresponding to sequences at the 5'-end of the gene.


b) Length polymorphisms: • Background:

– Human DNA contains short repetitive sequences of noncoding DNA.

– the number of repeats affecting such sequences varies greatly between different individuals, the resulting length polymorphisms are quite useful for linkage analysis.

– These polymorphisms are often subdivided on the basis of their length into:

• Microsatellite repeats (usually less than 1 kb and are characterized by a repeat size of 2 to 6 base pairs).

• Minisatellite repeats (these are larger 1 to 3 kb and the repeat is usually 15 to 70 base pairs)

– These stretches of DNA can be used quite effectively to distinguish different chromosomes

allele C is linked to a mutation responsible for autosomal dominant polycystic kidney disease (PKD). Application of this to detect progeny carrying the disease gene is illustrated in one hypothetical pedigree


• Limitations of linkage studies: – For diagnosis, several relevant family members must be

available for testing.

– Key family members must be heterozygous for the polymorphism

– Normal exchange of chromosomal material between homologous chromosomes (recombination) during gametogenesis may lead to "separation" of the mutant gene from the polymorphism pattern with which it had been previously coinherited. This may lead to an erroneous genetic prediction in a subsequent pregnancy.


• Molecular diagnosis by linkage analysis has been useful in the antenatal or presymptomatic diagnosis of disorders such as Huntington disease, cystic fibrosis, and adult polycystic kidney disease.

• In general, when a disease gene is identified and cloned, direct gene diagnosis becomes the method of choice.

• If the disease is caused by several different mutations in a given gene direct gene diagnosis is not possible, and linkage analysis remains the preferred method.

Applications of Molecular Bio-techniques in Medical Oncology

Molecular Biology for Medical Oncology

• Diagnosis

• Cancer screening and early detection

• Evaluation of cancer risk

• Treatment

• Follow up and detection of residual tumor

• Prognosis

• Research and cancer pathogenesis

Molecular Diagnosis of Cancer

• Molecular techniques can be used for:

– Cancer diagnosis

– Ancillary tools for cancer diagnosis

– Subclassification of tumors

Molecular Diagnosis of Cancer

• The gold standard test for cancer diagnosis of almost all tumors is tissue diagnosis.

• PCR and/or Southern blot can be used in diagnosing B and T cell lymphomas.

• PCR-based detection of T-cell receptor or immunoglobulin genes rearrangement allow distinction between monoclonal (neoplastic) and polyclonal (reactive) proliferations.

Molecular Diagnosis of Lymphoma

Gene Rearrangement

Molecular Diagnosis of Lymphoma

• The normal circulating lymphocytes are polyclonal.

• Because of the multiplicity of the gene rearrangement involved, the changes will not be detected at DNA level for polyclonal population.

• The presence of a monoclonal population will usually mean there is a hematological or immunological disorder involving these cells.

• Gene rearrangement indicates a clonal population

• DNA mapping patterns are able to detect monoclonal population in B or T lymphocytes because the same gene rearrangement is now present in large number of cells

Molecular Diagnosis of Lymphoma• TCR-beta gene rearrangements of the DNAs extracted from cells.

• The BamHI-, EcoRI-, and HindIII-digested DNA were hybridized to a probe specific for the joint region of TCR-beta gene.

• Lanes P denote DNAs from this patient and Lanes N from lymphocytes of normal control.

• Arrows denoted rearranged bands and bar, germline bands.

Molecular Techniques as Ancillary Tools for Cancer Diagnosis

• RT-PCR, FISH, or cytogentics can be used to detect certain translocation or gene amplification that specific for some cancer.

• These findings can be used as ancillary tool to help in soft tissue and hematological diagnosis.

Molecular Techniques as Ancillary Tools for Cancer Diagnosis

Malignancy Translocation Affected Genes

Chronic myeloid leukemia (9;22)(q34;q11) Ab1 9q34 bcr 22q11

Acute leukemias (AML and ALL) (4;11)(q21;q23) AF4 4q21 MLL 11q23

(6;11)(q27;q23) AF6 6q27 MLL 11q23

Burkitt lymphoma (8;14)(q24;q32) c- myc 8q24 IgH 14q32

Mantle cell lymphoma (11;14)(q13;q32) Cyclin D 11q13 IgH 14q32

Follicular lymphoma (14;18)(q32;q21) IgH 14q32 bcl-2 18q21

T-cell acute lymphoblastic leukemia (8;14)(q24;q11) c- myc 8q24 TCR-alpha 14q11

(10;14)(q24;q11) Hox 11 10q24 TCR-alpha 14q11

Ewing sarcoma (11;22)(q24;q12) FL-1 11q24 EWS 22q12

Melanoma of soft parts (12;22)(q13;q12) ATF-1 12q13 EWS 22q12

Ancillary Tools for Cancer Diagnosis

Subclassification of Tumors

• Acute myelobalstic leukemia can be classified based on Cytogenetic findings.

• Molecular techniques can help in subclassifications of non-Hodgkin's lymphomas, and pediatric sarcoma.

Molecular Biology for Medical Oncology

• Cancer screening and early detection

• Evaluation of cancer risk

– Table of familial cancer

Follow up and detection of residual tumor

• Detection of BCR-ABL by PCR gives a measure of minimal residual leukemia in patients treated for CML.

Evaluation of Prognosis and Response to Treatment

• FISH or PCR can be used to detect amplification of HER2-nue in breast cancer patient.

• PCR or cytogenetics can be used to detect amplification of C-myc in neuroblastoma patient.

Molecular Biology and Cancer Therapeutics

Anticancer “Smart bombs”

Antiangiogenesis

Tyrosine kinase inhibitors

Iressa

Cetuximab

Rituximab

anti-VEGF

thalidomide

Retinoids COX-2 inhibitors

All-trans retinoic acid Celecoxib

Monoclonal antibodies

Herceptin

Gleevec

- CML

- GIST

- Breast Cancer

Molecular Biology of CML

Gleevec (STI571, Imatinib)Tyrosine Kinase Inhibitor

• in 1993, various compounds tested for ability to block BCR-ABL protein

• STI571 shown to inhibit growth of BCR-ABL expressing cells

• Gleevec: a tyrosine kinase inhibitor with specific activity against BCR-ABL fusion proteins.

Gleevec (STI571, Imatinib)

Gleevec (STI571, Imatinib) Kantarjian et al, NEJM February 2002

• 532 patients with late chronic phase CML:– The treatment with interferon α had failed.– Treated with 400mg of oral Imatinib daily– Evaluated for cytogenetic and hematologic

responses.– Time to progression, survival, and drug toxic

effects were evaluated.

Gleevec (STI571, Imatinib) Kantarjian et al, NEJM February 2002

• 95% of patients had complete hematologic responses.

• 60% had major cytogenetic responses.• After median follow-up of 18 months:

– No progression to accelerated phase in 89%.

– No progression to blast crises in 95%.

• Non hematologic toxic effects were infrequent, and hematologic toxic effects were manageable.

Breast Cancer and Her2/neu

• HER-2/neu (C-erbB-2) is a proto-oncogene, localized to chromosome 17q.

• It encodes a transmembrane tyrosine kinase growth factor receptor.

• Amplification of the HER-2/neu gene or overexpression of the HER-2/neu protein has been identified in 10- 34% of breast cancers.

• Amplification and/or overexpression of HER-2/neu are associated with poor outcome in breast cancer.

Breast Cancer and Her2/neu

Fluorescence in situ hybridizationImmunohistochemistry

Trastuzumab (Herceptin) for Breast CaSlamon et al NEJM March 2001

• Herceptin is a recombinant monoclonal antibody against HER-2/neu.

• In this study efficacy and safety of Herceptin in women with HER2-overexpressing metastatic breast cancer were evaluated.

• Randomly assigned 234 patients to receive standard chemotherapy alone and 235 patients to receive standard chemotherapy plus trastuzumab.

Trastuzumab (Herceptin) for Breast CaSlamon et al NEJM March 2001

Future Direction

The Post-Genome Era

• Associate a specific tumor type with a specific gene expression profile

• Define molecular lesions characteristic of any given cancer

• Inhibit specific deregulated pathways in cancer cells with minimal effect on normal cell function

• Synergistic with other modalities.

cDNA Microarray

Internet Resources

• Genetics Education Center.htmGenetics Education Centre

• Molecular Tools of Medicine.htmMolecular Tools of Medicine

• Talking Glossary of Genetic Terms.htmTalking Glossary of Genetic Terms

• DNALC Biology Animation Library.htmAnimation Library

Thank You

molecular medicine in clinical practice

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