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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
