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Molecular Medicine in Clinical Practice

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Title: Molecular Medicine in Clinical Practice


1
Molecular Medicine in Clinical Practice
  • Dr. Osama . I . Nassif , FRCPC
  • Associate Professor and Consultant Pathologist
  • Department of Pathology, Faculty of Medicine
  • King Abdullaziz University Hospital

2
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

3
Introduction
  • DNA isolation
  • RNA isolation

4
Introduction
  • Molecular Bio-techniques
  • Blotting
  • Southern
  • Northern
  • Western
  • Hybridization
  • PCR, RT-PCR
  • DNA sequencing
  • cDNA cloning
  • Recombinant protein

5
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

6
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.

7
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.

8
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

9
Mutational Analysis
  • Detecting DNA deletion
  • Very small deletions can be detected by PCR (e.g.
    cystic fibrosis)
  • Larger deletion (e.g. a thalassaemia) can be
    detected by Southern blotting
  • The largest deletion (e.g. contiguous gene
    syndrome) can be detected by PFGE or FISH

10
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.

11
Mutational Analysis
  • DNA sequencing
  • Chromosomal analysis
  • Karyotyping
  • FISH

12
Applications of Molecular Bio-techniques in
Medical Genetics
13
Diagnosis of Genetic Diseases
  • Two general methods are used
  • Cytogenetic analysis and
  • Molecular analysis.

14
Diagnosis of Genetic Diseases
  • 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 (gt34 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)

15
Diagnosis of Genetic Diseases
  • 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.

16
Diagnosis of Genetic Diseases
  • 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

17
Diagnosis of Genetic Diseases
  • The advantages of molecular diagnosis of genetic
    disorders
  • 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.
  • 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.

18
Diagnosis of Genetic Diseases
  • 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."

19
Diagnosis of Genetic Diseases
  • 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.
  • 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.

20
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.
21
Diagnosis of Genetic Diseases
  • Allele-specific oligonucleotide hybridization
    "dot blot" test
  • e.g. a1 antitrypsin deficiency
  • Direct gene diagnosis by using PCR and an
    allele-specific oligonucleotide probe.
  • Base change converts a normal a1 antitrypsin
    (allele M) to a mutant (Z) allele.

22
  • 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.

23
Diagnosis of Genetic Diseases
  • 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)

24
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.
25
Diagnosis of Genetic Diseases
  • 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.

26
Diagnosis of Genetic Diseases
  • 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.

27
Diagnosis of Genetic Diseases
  • 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.

28
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.
29
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.
30
Diagnosis of Genetic Diseases
  • 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

31
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
32
Diagnosis of Genetic Diseases
  • 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.

33
Diagnosis of Genetic Diseases
  • 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.

34
Applications of Molecular Bio-techniques in
Medical Oncology
35
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

36
Molecular Diagnosis of Cancer
  • Molecular techniques can be used for
  • Cancer diagnosis
  • Ancillary tools for cancer diagnosis
  • Subclassification of tumors

37
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.

38
Molecular Diagnosis of Lymphoma
Gene Rearrangement
39
Molecular Diagnosis of Lymphoma
Gene Rearrangement
40
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

41
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.

42
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.

43
Molecular Techniques as Ancillary Tools for
Cancer Diagnosis
44
Ancillary Tools for Cancer Diagnosis
Malignancy Translocation Affected Genes
Chronic myeloid leukemia (922)(q34q11) Ab1 9q34 bcr 22q11
Acute leukemias (AML and ALL) (411)(q21q23) AF4 4q21 MLL 11q23
Acute leukemias (AML and ALL) (611)(q27q23) AF6 6q27 MLL 11q23
Burkitt lymphoma (814)(q24q32) c- myc 8q24 IgH 14q32
Mantle cell lymphoma (1114)(q13q32) Cyclin D 11q13 IgH 14q32
Follicular lymphoma (1418)(q32q21) IgH 14q32 bcl-2 18q21
T-cell acute lymphoblastic leukemia (814)(q24q11) c- myc 8q24 TCR-alpha 14q11
T-cell acute lymphoblastic leukemia (1014)(q24q11) Hox 11 10q24 TCR-alpha 14q11
Ewing sarcoma (1122)(q24q12) FL-1 11q24 EWS 22q12
Melanoma of soft parts (1222)(q13q12) ATF-1 12q13 EWS 22q12
45
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.

46
Molecular Biology for Medical Oncology
  • Cancer screening and early detection
  • Evaluation of cancer risk
  • Table of familial cancer

47
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.

48
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.

49
Molecular Biology and Cancer Therapeutics
50
Anticancer Smart bombs
Tyrosine kinase inhibitors
Gleevec
Iressa
Monoclonal antibodies
- CML - GIST
Antiangiogenesis
Herceptin
anti-VEGF thalidomide
Cetuximab Rituximab
Retinoids
COX-2 inhibitors
Celecoxib
All-trans retinoic acid
- Breast Cancer
51
Molecular Biology of CML
52
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53
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.

54
Gleevec (STI571, Imatinib)
55
Gleevec (STI571, Imatinib) Kantarjian et al,
NEJM February 2002
  • 532 patients with late chronic phase CML
  • The treatment with interferon a 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.

56
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.

57
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.

58
Breast Cancer and Her2/neu
Fluorescence in situ hybridization
Immunohistochemistry
59
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.

60
Trastuzumab (Herceptin) for Breast CaSlamon et
al NEJM March 2001
61
Future Direction
62
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.

63
cDNA Microarray
64
Internet Resources
  • Genetics Education Center.htm
  • Genetics Education Centre
  • Molecular Tools of Medicine.htm
  • Molecular Tools of Medicine
  • Talking Glossary of Genetic Terms.htm
  • Talking Glossary of Genetic Terms
  • DNALC Biology Animation Library.htm
  • Animation Library

65
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