Overview of Newborn Screening Molecular Assays

1. Overview of Newborn Screening Molecular Assays Susan M Tanksley, PhD June 28, 2011

2. 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

3. 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

4. Mutations can be.... • Helpful – Adaptability • Color patterns for camouflage • Disease resistance • Neutral – ‘silent’ or polymorphic • Useful as genetic markers • Identification, Forensics, Paternity • Gene mapping • Population studies • Harmful - Disease causing • Sickle cell anemia • Phenylketonuria (PKU) • Cystic fibrosis

5. 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

6. 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

7. 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

8. Classes of Single Gene Disorders • X-linked Recessive • Males affected if X chromosome is defective • Females affected only if both X chromosomes are defective • e.g. Duchenne muscular dystrophy & Hemophilia • X-linked Dominant • 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

9. 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

10. 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

11. Availability of Genetic Tests GeneTESTS: Availability of Genetic Tests As of 6/22/2011

12. 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

13. History of Molecular Testing in Newborn Screening • 1994 • Wisconsin – CFTR mutation analysis for DF508 • Washington – hemoglobin confirmatory testing (Hb S, C, E by RFLP) • 1998 • New England – 2 GALT mutations (Q & N) by RFLP • 1999 • New England – MCADD (985A>G) by RFLP

14. History of NBS Molecular Testing • 2005 • Wisconsin – MSUD (Y438N) • 2006 • 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

15. 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

16. 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

17. 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.

18. 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

19. 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

20. NBS Molecular Tests Available in US • Primary Screen - SCID • Second-tier • Hemoglobinopathies • Galactosemia • Cystic Fibrosis • MCAD and other FAOs • PKU and other aminoacidopathies • Krabbe

21. 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

22. 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