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 × m)(6 × 10 9 )(10 13 ) 2.0 × 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 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 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. 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 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): BCR - ABL t(12;21)TEL - AML1 t(1;19): E 2 A - PBX1 t(4;11): MLL - AF4 Myeloid Leukemia Inv(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 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

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 Number of diseases Nature of disease mutation Nature of disease mutation Technology Cost Number of samples Organisation

Diagnostic bottle necks Number of diseases Number of diseases Nature of disease mutation Nature of disease mutation Technology Cost Number of samples Organisation

Disease Mutations Easy tests : Single - common mutations Difficult tests : Private mutations

Disease Mutations Single mutationsFragile X Sickle Cell Anemia Common mutationsDeafness Hemochromatosis Panel of mutationsCystic Fibrosis Private mutationsBreast Cancer Colorectal cancer

DiseaseGeneMutation Fragile XFMR1Repeat FRAXEFMR2Repeat Friedreich ataxiaFRDARepeat Haw RiverDRPLARepeat Huntington type 1HDRepeat KennedyARRepeat Myotonic dystrophy type 1DMPKRepeat Spinocerebellar ataxiaSCA1,2, 3, 6, 7, 8,10, 12,17Repeat Alpha 1 antitrypsinPI2 common mutations Charcot-Marie-Tooth Type 1APMP221 common mutation Cystic fibrosisCFTRCommon mutations DeafnessGJB21 common mutation Hemochromatosis type1HFE2 common mutations Hereditary neuropathy (HNPP)PMP221 common mutation Sickle cell anemiaHBB1 common mutation Spinal muscular atrophySMN11 common mutation Beta thalassemiaHBB1 exon

BRCA testing BRCA1 : 23 exon, 1863 AA, bp BRCA2 : 28 exon, 3418 AA, bp Total : > bp sequence

Diagnostic bottle necks Number of diseases 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

42 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 DeletionDuplicationPoint % DMD Mutation Types

43 Molecular Diagnostics DNA Diagnostic Systems Sequencing Minisequencing by primer extension DNA polymerase + one of the four labeled dNTPs = sequencing of one nucleotide -> HPLC analysis

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