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Overview of Newborn Screening Molecular Assays Susan M Tanksley, PhD June 28, 2011 Outline Introduction to molecular testing for genetic diseases Brief history of molecular testing in NBS When & why to use a molecular test Availability of NBS molecular tests in different states Potential future applications Genetic Variation in Humans Human genome is 99.9% identical across all people ~3 million nucleotide differences between 2 random individuals Mutation = Any change in the DNA sequence Mutations are the source of differences between individuals Mutations can be.... Helpful – Adaptability Neutral – ‘silent’ or polymorphic Color patterns for camouflage Disease resistance Useful as genetic markers Identification, Forensics, Paternity Gene mapping Population studies Harmful - Disease causing Sickle cell anemia Phenylketonuria (PKU) Cystic fibrosis Genetic Disorders Caused by various types of mutations in genes or chromosomes Mutations may occur on An autosome (autosomal) A sex chromosome (X-linked or Y-linked) Multiple associated genes Disease expression may be impacted by environmental factors Single Gene Disorders Caused by mutations in one gene Generally follow Mendelian inheritance patterns Dominant vs. Recessive Expression may be impacted by genomic imprinting or penetrance Includes most inborn errors of metabolism Classes of Single Gene Disorders Autosomal Dominant One copy of a mutated allele results in affected individual aka: AA or Aa Heterozygotes and Homozygous Dominant Individuals are affected. e.g. Achondroplasia, Huntington’s Disease Autosomal Recessive Both alleles of the gene must be mutated to be affected aka: aa Only Homozygous Recessive individuals are affected. e.g. Sickle Cell Anemia, cystic fibrosis, galactosemia Classes of Single Gene Disorders X-linked Recessive X-linked Dominant Males affected if X chromosome is defective Females affected only if both X chromosomes are defective e.g. Duchenne muscular dystrophy & Hemophilia Individuals with 1 defective copy of X chromosome are affected e.g. Rett syndrome Y-linked Individuals with a defective Y chromosome are affected Rare Complex/Multifactorial Disorders Associated with the effects of multiple genes May be strongly impacted by environmental factors (e.g. lifestyle) Often cluster in families No clear-cut pattern of inheritance Difficult to determine risk e.g. heart disease, diabetes, obesity, cancer Molecular Testing for Genetic Diseases Enabled by gene mapping to identify location of genes on chromosomes AND ability to differentiate between harmful and neutral mutations Goal – identification of disease-causing mutations for: Diagnosis – e.g. MCADD Predictive testing – e.g. Huntington’s Disease, BRCA1 Carrier detection – e.g. Cystic Fibrosis Prenatal screening – e.g. Trisomy 21 Preimplantation testing – e.g. Sickle Cell Anemia Pharmacogenetics – e.g. PKU Availability of Genetic Tests GeneTESTS: Availability of Genetic Tests 599 Laboratories offering in-house molecular genetic testing, specialized cytogenetic testing, and biochemical testing for inherited disorders 2334 Diseases 2072 Clinical Labs 262 Research Labs As of 6/22/2011 Obstacles to Introduction of Genomic Methods in Newborn Screening Volume/quality of specimen Throughput (turn around time) Cost ($$$) per sample “Simple test” mentality Public health infrastructure Equipment Space Trained personnel Have test, no treatment History of Molecular Testing in Newborn Screening 1994 1998 Wisconsin – CFTR mutation analysis for DF508 Washington – hemoglobin confirmatory testing (Hb S, C, E by RFLP) New England – 2 GALT mutations (Q & N) by RFLP 1999 New England – MCADD (985A>G) by RFLP History of NBS Molecular Testing 2005 2006 Wisconsin – MSUD (Y438N) New York – Krabbe (3 polymorphisms & 5 mutations) 2008 Wisconsin – SCID – TREC analysis 1st use of molecular test as a primary full population screen 2010 36 NBS programs in US use molecular testing for CF Uses of Molecular Tests in NBS Primary Screening Test TREC analysis for detection of SCID Second-Tier Test DNA test results provide supplemental information to assist with diagnosis Often provided in separate report b-globin and GALT mutation analysis Genotypic information is required for interpretation of the screen result Cystic fibrosis mutation analysis When/Why Use a Molecular Test? To increase sensitivity without compromising specificity Lower IRT cutoff to avoid missing CF cases To increase specificity of a complex assay Allow differentiation of hemoglobinpathies & thalassemias (e.g. Hb S/b-thalassemia) Distinguish between patient & donor phenotypes when patient was transfused When/Why Use a Molecular Test? When the primary analyte is transient The primary analyte is present in the body for only a limited time (e.g. VLCADD) Analysis of a recollected specimen could result in a false negative. To speed diagnosis in order to avoid serious medical consequences GALT enzyme activity is decreased by heat & humidity, thus increasing false positive screens Genotyping helps sort out the true positives for faster diagnosis. When/Why Use a Molecular Test? When there are significant founder mutations in a population Due to high frequency (1 in 176 live births) of MSUD in Mennonite population in WI, mutation analysis for Y438N serves as primary screen for MSUD for Mennonites. CPT1a in Alaskan Innuit & Hutterite populations When/Why Use a Molecular Test? When diagnostic testing is slow and/or invasive Traditional confirmatory testing for VLCADD & CPT1a involves skin biopsy (invasive to collect and slow to grow) When no other test exists for the analyte SCID, SMA NBS Molecular Tests Available in US Primary Screen - SCID Second-tier Hemoglobinopathies Galactosemia Cystic Fibrosis MCAD and other FAOs PKU and other aminoacidopathies Krabbe Potential Future Applications of Molecular Testing in NBS Genome-wide association studies Susceptibility Testing (heart disease, cancer, obesity, diabetes) Pharmacogenetics and NBS Drugs in clinical trials to treat specific CF causing mutations (VX-770/G551D and VX-890/ DF508) Ataluren (formerly PTC124) is an investigational drug that reads through nonsense or STOP mutations Conclusions NBS Molecular testing began in 1994 with second tier CF DNA testing for DF508 mutation Molecular tests are useful in NBS to: Increase sensitivity or specificity of a primary assay Allow follow-up testing when the primary analyte is transient Aid diagnostic process for disorders with serious consequences or invasive follow-up tests Screen founder populations with greater disease risk Detect disorders for which no biochemical test exists Wide availability of NBS molecular tests in the US With expanding technologies, applications of NBS molecular testing will continue to grow