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.

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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.

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Mutational Analysis

• DNA sequencing
• Chromosomal analysis
• Karyotyping
• FISH

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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.

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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

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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
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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.

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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.

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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.

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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
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Molecular Biology of CML
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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.

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Gleevec (STI571, Imatinib)
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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.

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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.

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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.

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

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

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cDNA Microarray
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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

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Thank You