Transcript Diagnosis of genetic diseases

Molecular
Genetics Lab
Dr Mohammad Hasan Sheikhha,
M.D., Ph.D.
Shahid Sadoghi Medical University,
Yazd, Iran
• 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.
The Central Dogma of Molecular Biology
Clinical Applications of
Molecular Diagnosis
CA García-Sepúlveda MD PhD
Viral & Human Genomics Laboratory
Facultad 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 disease
23% - Blood Screening
13% - Genetic Testing
7% - Cancer.
Prediction of risk – Oncotype.
Early detection - Fragile X.
Classification of disease – Leukemias.
Therapeutic homming of presumptive target.
Prediction of toxicity & response – Herceptin.
Laboratorio de Genómica Viral & Humana
- Facultad de Medicina Universidad Autónoma de San Luis Potosí
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 Pharmacogenomics
Warfarin 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
Genetic Diagnostics
• Cytogenetic tests
• FISH
• Molecular tests
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
Genetic Identification
- Paternity Testing
- Forensics
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) :
t(12;21)
t(1;19) :
t(4;11) :
BCR - ABL
TEL - AML1
E2A - PBX1
MLL - AF4
Myeloid Leukemia
Inv(16)
t(8;22) :
t(9;22) :
CBF - MYH11
AML - ETO
BCR - ABL
Diagnosis of genetic diseases
- Somatic rearrangements in cancer
- Genetic risk factors
-
Pharmacogenetics
- Mutations in monogenic diseases
Genetic Risk Factors
Monogenic 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
Genetic Risk Factors
Deep venous thrombosis
Cardiovascular disease
Alzheimer disease
Osteoporosis
Genetic Risk Factors
Deep venous thrombosis
Factor V
Factor II
MTHFR
Diagnosis of genetic diseases
- Somatic rearrangements in cancer
- Genetic risk factors
-
Pharmacogenetics
- Mutations in monogenic diseases
Pharmacogenetic tests
• Drug specificity
• Drug efficacity - toxicity
Drug specificity
Herceptin : HER2
Tyrosine kinase inhibitors
BCR / ABL
KIT
PDGFR A/B
EGFR
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 diseases
•
Nature of disease mutation
•
Technology
•
Cost
•
Number of samples
•
Organisation
Diagnostic bottle necks
•
Number of diseases
•
Nature of disease mutation
•
Technology
•
Cost
•
Number of samples
•
Organisation
Disease Mutations
Easy tests : Single - common mutations
Difficult tests : Private mutations
Disease Mutations
Single 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 diseases
•
Nature of disease mutation
•
Technology
•
Cost
•
Number of samples
•
Organisation
Mutation Detection
1. Point mutations, frame shifts :
A. Sequencing
B. WAVE
2. Deletions : MLPA
Molecular Diagnostics
DNA 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
DMD Mutation Types
60
%
40
20
Deletion
Duplication
Point
42
Molecular Diagnostics
DNA Diagnostic Systems
Sequencing
Minisequencing by primer extension
DNA polymerase + one of the four labeled dNTPs
= sequencing of one nucleotide
-> HPLC analysis
43
Thank you