Dec 28, 2015
Dr Mohammad Hasan Sheikhha, M.D., Ph.D.
Shahid Sadoghi Medical University, Yazd, Iran
Molecular Genetics Lab
• If all the DNA in ten human nuclei were enlarged to a full-sized continuous ladder, it could reach more than 30 million miles, or from the earth to the planet Mars:
• (length of 1 bp)(number of bp per cell)(number of cells in the body)
• (0.34 × 10-9 m)(6 × 109)(1013)
• 2.0 × 1013 meters
• That is the equivalent of nearly 70 trips from the earth to the sun and back.
CA García-Sepúlveda MD PhD
Clinical Applications of Molecular Diagnosis
Viral & Human Genomics LaboratoryFacultad de Medicina, Universidad Autónoma de San Luis Potosí
Laboratorio de Genómica Viral & Humana - Facultad de Medicina - Universidad Autónoma de San Luis Potosí Mexico
Molecular Diagnostics Industry
$5.5 Billion industry
$8 Billion by 2010
40 million annual test volumes in the U.S.
Projected to be 1/3 of all diagnostic testing
Laboratorio de Genómica Viral & Humana - Facultad de Medicina -
Universidad Autónoma de San Luis Potosí
Industry Test Volumes & Applications
55% - Infectious disease23% - Blood Screening13% - Genetic Testing 7% - Cancer.
Laboratorio de Genómica Viral & Humana - Facultad de Medicina -
Universidad Autónoma de San Luis Potosí
Prediction of risk – Oncotype.Early detection - Fragile X.Classification of disease – Leukemias. Therapeutic homming of presumptive target.Prediction of toxicity & response – Herceptin.
Breast Cancer and Targeted Therapy
211,000 women diagnosed with breast cancer and 40,000 deaths per year (US 2005 estimate).
Herceptin (trastuzumab) chemotherapy approved by the FDA in 1998.
Risk of congestive heart failure.
Herceptin could benefit women who over-expressed a protein – HER2/Neu.
Molecular diagnostic tests reveal who could and will not benefit from Herceptin.
Herceptin benefit test Cost $500 USD
Herceptin Tx costs $25,000 – $80,000
Getting the “right” women on Herceptin
Laboratorio de Genómica Viral & Humana - Facultad de Medicina -
Universidad Autónoma de San Luis Potosí
Coumarin PharmacogenomicsWarfarin is an oral anticoagulant that inhibits vitamin K reductase.
Discovered 60 years ago and currently one of the most prescribed drugs in the world.
Used to prevent thromboembolisms due to atrial fibrilation, recurring miocardial strokes, Deep vein thrombosis, Pulmonary thromboembolism and that due to valve replacements.
Between 1 and 7% of treated patients will suffer lethal hemorrhagic complications (very tight therapeutical safety index).
CYP2C9 & VKORC1 polimorphisms define metabolic rates and might explain between 10 and 25% of interindividual therapeutic response variations.
“The FDA highlights the opportunity for healthcare providers to use genetic tests (CYP2C9 & VKORC1) to improve their initial estimate of what is a reasonable warfarin dose for individual patients”.
Laboratorio de Genómica Viral & Humana - Facultad de Medicina -
Universidad Autónoma de San Luis Potosí
Molecular Diagnostics
- Diagnosis of infectious diseases
- Genetic identification
- Diagnosis of genetic diseases
Infectious Diseases• MRSA
• VRE
• Group A Strep
• Group B Strep
• TB
• HIV
• HCV
• CMV
• Flu
• Stop me when you’re bored…
Why use a molecular test to diagnose an infectious disease?
• Need an accurate and timely diagnosis– Important for initiating the proper treatment
– Important for preventing the spread of a contagious disease
Leading uses for genetics tests
• Nonculturable agents– Human papilloma virus– Hepatitis B virus
• Fastidious, slow-growing agents– Mycobacterium tuberculosis– Legionella pneumophilia
• Highly infectious agents that are dangerous to culture– Francisella tularensis– Brucella species– Coccidioidis immitis
Leading uses for genetics tests• In situ detection of infectious agents
– Helicobacter pylori– Toxoplasma gondii
• Agents present in low numbers– HIV in antibody negative patients– CMV in transplanted organs
• Organisms present in small volume specimens– Intra-ocular fluid– Forensic samples
Leading uses for genetics tests• Differentiation of antigenically similar agents
– May be important for detecting specific virus genotypes associated with human cancers (Papilloma viruses)
• Antiviral drug susceptibility testing– May be important in helping to decide anti-viral therapy to
use in HIV infections
• Non-viable organisms– Organisms tied up in immune complexes
Leading uses for genetics tests
• Molecular epidemiology– To identify point sources for hospital and
community-based outbreaks
– To predict virulence
• Culture confirmation
Diagnosis of genetic diseases
- Somatic rearrangements in cancer
- Genetic risk factors
- Pharmacogenetics
- Mutations in monogenic diseases
Rearrangements in Cancer Cells
Chromosomal breaks produce fusion genes
These cause leukemias and lymphomas
Diagnosis determines treatment and prognosis
Rearrangements in Cancer Cells
Lymphocytic Leukemia
t(9;22) : BCR - ABL
t(12;21) TEL - AML1
t(1;19) : E2A - PBX1
t(4;11) : MLL - AF4
Myeloid LeukemiaInv(16) CBF - MYH11
t(8;22) : AML - ETO
t(9;22) : BCR - ABL
Diagnosis of genetic diseases
- Somatic rearrangements in cancer
- Genetic risk factors
- Pharmacogenetics
- Mutations in monogenic diseases
Genetic Risk FactorsMonogenic diseases are caused by a
deleterious mutation in a single gene:
Disease-causing mutations
Multifactorial diseases are caused by a
combination of variations in multiple genes:
Genetic Risk Factors
Diagnosis of genetic diseases
- Somatic rearrangements in cancer
- Genetic risk factors
- Pharmacogenetics
- Mutations in monogenic diseases
Pharmacogenetics
• Warfarin metabolism– Polymorphisms
• VKORC1– Vitamin K epoxide
reductase complex 1
• CYP2C9– Part of cytochrome P450
family
– FDA testing recommended
Diagnosis of genetic diseases
- Somatic rearrangements in cancer
- Genetic risk factors
- Pharmacogenetics
- Mutations in monogenic diseases
Diagnostic bottle necks• Number of diseasesNumber of diseases
• Nature of disease mutationNature of disease mutation
• Technology
• Cost
• Number of samples
• Organisation
Diagnostic bottle necks
• Number of diseasesNumber of diseases
• Nature of disease mutationNature of disease mutation
• Technology
• Cost
• Number of samples
• Organisation
Disease MutationsSingle mutations Fragile X
Sickle Cell Anemia
Common mutations Deafness Hemochromatosis
Panel of mutations Cystic Fibrosis
Private mutations Breast Cancer
Colorectal cancer
Disease Gene Mutation
Fragile X FMR1 Repeat
FRAXE FMR2 Repeat
Friedreich ataxia FRDA Repeat
Haw River DRPLA Repeat
Huntington type 1 HD Repeat
Kennedy AR Repeat
Myotonic dystrophy type 1 DMPK Repeat
Spinocerebellar ataxia SCA1,2, 3, 6, 7, 8,10, 12,17 Repeat
Alpha 1 antitrypsin PI 2 common mutations
Charcot-Marie-Tooth Type 1A PMP22 1 common mutation
Cystic fibrosis CFTR Common mutations
Deafness GJB2 1 common mutation
Hemochromatosis type1 HFE 2 common mutations
Hereditary neuropathy (HNPP) PMP22 1 common mutation
Sickle cell anemia HBB 1 common mutation
Spinal muscular atrophy SMN1 1 common mutation
Beta thalassemia HBB 1 exon
BRCA testing
BRCA1 : 23 exon, 1863 AA, 6.200 bp
BRCA2 : 28 exon, 3418 AA, 10.300 bp
Total : > 17.000 bp sequence
Diagnostic bottle necks
• Number of diseasesNumber of diseases
• Nature of disease mutation
• Technology
• Cost
• Number of samples
• Organisation
42
Molecular DiagnosticsDNA Diagnostic Systems
Example: Diagnostic for Duchenne Muscular Dystrophy (DMD)
• X-linked and affect mainly males an estimated 1 in 3500 boys worldwide
• DMD encodes a large structural protein: dystrophin• strengthen muscle cells by anchoring elements of the internal
cytoskeleton to the surface membrane• Mutated dystrophin leads to ”implosion” of muscle cells
60
40
20
DeletionDuplication Point
%
DMD Mutation Types
43
Molecular DiagnosticsDNA Diagnostic Systems
SequencingMinisequencing by primer extension
DNA polymerase + one of the four labeled dNTPs = sequencing of one nucleotide
-> HPLC analysis