Dr Mohammad Hasan Sheikhha, M.D., Ph.D. Shahid Sadoghi Medical University, Yazd, Iran Molecular Genetics Lab

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.

The Central Dogma of Molecular Biology

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.



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



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”.



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) : 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




- Pharmacogenetics


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


Deep venous thrombosis

Cardiovascular disease

Alzheimer disease

Osteoporosis


Deep venous thrombosis

Factor V

Factor II

MTHFR




- Pharmacogenetics


Pharmacogenetic tests• Drug specificity• Drug efficacity - toxicity

Drug specificity

Herceptin : HER2

Tyrosine kinase inhibitors

BCR / ABLKITPDGFR A/BEGFR

Pharmacogenetics

• Warfarin metabolism– Polymorphisms

• VKORC1– Vitamin K epoxide

reductase complex 1

• CYP2C9– Part of cytochrome P450

family

– FDA testing recommended




- Pharmacogenetics


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


• Organisation

Disease MutationsEasy tests : Single - common mutations

Difficult tests : Private mutations

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


• Organisation

Mutation Detection

1. Point mutations, frame shifts :

A. Sequencing

B. WAVE

2. Deletions : MLPA

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

Thank you

Documents

Dr Mohammad Hasan Sheikhha, M.D., Ph.D. Shahid Sadoghi Medical University, Yazd, Iran Molecular Genetics Lab