Download Genetics

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
Introduction
• Sources of DNA in clinical practice:
– Any nucleated cell in the body
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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
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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 Biotechniques 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 premarriage 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 Biotechniques in Medical Genetics
Diagnosis of Genetic Diseases
• Two general methods are used:
– Cytogenetic analysis and
– Molecular analysis.
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 (>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)
Diagnosis of Genetic Diseases
Postnatal chromosome analysis:
• This is performed on peripheral blood lymphocytes.
• Indications are as follows:
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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.
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
Diagnosis of Genetic Diseases
The 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.
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Virtually all cells of the body of an affected individual contain the same DNA, each postzygotic
cell carries the mutant gene.
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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.
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."
Diagnosis of Genetic Diseases
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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:
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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.
Diagnosis of Genetic Diseases
b) Allele-specific oligonucleotide
hybridization "dot blot" test:
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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.
Diagnosis of Genetic Diseases
c) Mutations that affect the length of DNA
(e.g., deletions or expansions) can be
detected by PCR analysis.
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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.
Diagnosis of Genetic Diseases
2. Indirect DNA Diagnosis: Linkage
Analysis
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large number of genetic diseases, including
some that are relatively common, information
about the gene sequence is lacking.
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Therefore, alternative strategies are to track
the mutant gene on the basis of its linkage to
detectable genetic markers.
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.
Diagnosis of Genetic Diseases
a) Restriction Fragment Length Polymorphisms (RFLPs).
– Background:
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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.
Diagnosis of Genetic Diseases
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:
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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
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.
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.
Applications of Molecular Biotechniques in Medical Oncology
Molecular Biology for Medical
Oncology
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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
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 HindIIIdigested 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
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
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 HER2nue 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”
Tyrosine kinase inhibitors
Gleevec
Iressa
Monoclonal antibodies
- CML
Herceptin
Cetuximab
- GIST
Antiangiogenesis
anti-VEGF
thalidomide
Rituximab
Retinoids
All-trans retinoic acid
COX-2 inhibitors
Celecoxib
- 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 BCRABL 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
Immunohistochemistry
Fluorescence in situ hybridization
Trastuzumab (Herceptin) for Breast Ca
Slamon 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 Ca
Slamon 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.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
Thank You