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Overview Molecular Newborn Screening

Overview Molecular Newborn Screening. Michele Caggana, Sc.D. Director, Newborn Screening Program New York State Department of Health Wadsworth Center. Wadsworth Center. ORNL. Human Genome Project. Proposed by Victor McKusick in 1968 DOE and NIH, 15 years, 30 billion dollars

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Overview Molecular Newborn Screening

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  1. Overview Molecular Newborn Screening Michele Caggana, Sc.D. Director, Newborn Screening Program New York State Department of Health Wadsworth Center

  2. Wadsworth Center ORNL

  3. Human Genome Project • Proposed by Victor McKusick in 1968 • DOE and NIH, 15 years, 30 billion dollars • James Watson original head then Francis Collins • International effort • ELSI budget Wadsworth Center

  4. Human Genome Project Five Main Objectives: Generate genetic and physical maps Develop new DNA technologies Accurately sequence the human genome Develop informatics Sequence model organisms Wadsworth Center

  5. Human Genome Project • Accurate Sequence Data: • 3,000,000,000 bases; haploid • Rough draft / 90%, summer 2000, 2/01 • ”finished” • Highly accurate (1 error in 100,000 bases) no gaps or ambiguities by 2003 • First chromosome 22 reported 12/99 • chromosome 21 reported 5/00 • Projected finish 2003, original 2005 Wadsworth Center

  6. Wadsworth Center

  7. Venter & Collins Private vs. Public 1000 Genomes Project Wadsworth Center

  8. Genetics is the study of heredity. It examines a small number of genes, and how they are inherited in families with disease. Genomics is the study of the entire DNA instruction set. 23 pairs of chromosomes 3.1 billion bases “We are 99.9% identical” --Bill Clinton

  9. Linkage: Uses recombination Requires multiple families with disease Requires the investigator to choose a model of inheritance http://genome.wellcome.ac.uk/assets/GEN10000725.jpg http://img.medscape.com/fullsize/migrated/448/388/sp448388.fig1.gif http://www.istockphoto.com/file_thumbview_approve/3450718/2/istockphoto_3450718-needle-in-a-haystack.jpg

  10. Association Studies Non-related, subject to bias

  11. SNPS : ~10 million total • Chromosome 1 AACACGCCA.…TTCGGGGTC….AGTCGACCG…. • Chromosome 2 AACACGCCA….TTCGAGGTC….AGTCAACCG…. • Chromosome 3 AACATGCCA.…TTCGGGGTC….AGTCAACCG…. • Chromosome 4 AACACGCCA.…TTCGGGGTC….AGTCGACCG.... • B. HAPLOTYPES • Haplotype 1 C T C A AA G T A C G G T TC A G G C A • Haplotype 2 T T G A T T G C G C A A C A G T A A T A • Haplotype 3 C CC G A T C T G T G A T A C T G G T G • Haplotype 4 T C G A T T C C G C G G T T C A G A C A • C. Tag SNPs: far fewer A / G C / T C / G From: http://www.dadamo.com/wiki/hapmap.jpg

  12. Factors Affecting Risk and Strengths of Association and NBS • Was the disease defined accurately? • Was the relatedness of the population described? • Could genotyping errors affect results? • Is the test population the same as the reported population, • i.e. ancestry? (population stratification) • Was there ascertainment / selection bias • Was environmental exposure variability considered?

  13. Distinction Between Mutations and SNPs Mutations: Changes in the DNA, which are ‘rare’; can be private; newer SNPs/Polymorphisms: Changes in the DNA occurring at a higher frequency, usually greater than 1%; may start as mutations and reach a higher frequency; older changes. Both are inherited and can be used to track DNA changes cSNPs are in the coding region synonymous: no change to the amino acid (silent) non-synonymous: change to the amino acid Non-coding SNPs: promoter, splice sites, stability, other regulatory changes

  14. Improvement of the Literature Collect data, clinical followup http://www.miragebookmark.ch/images/astronomy-library-utrecht.jpg

  15. TYPES OF MUTATIONS • Normal • CCG GGA AGC AAU • Pro Gly Ser Asn • Missense • CCG GCA AGC AAU • Pro Val Ser Asn • Nonsense • CCG UGA AGC AAU • Pro STOP • Frameshift (insertion) • CCG AGG AAG CAA • Pro Arg Lys Gln • Frameshift (deletion) • CCG GAA GCA AUG • Pro Glu Asp Met • Trinucleotide • CAG CAGCAGCAG • GlnGlnGlnGln Mutations can be helpful – camouflage; selection Mutations can be silent –markers, forensics, mapping, population studies Mutations can be harmful – sickle cell, PKU, CF and other diseases Wadsworth Center

  16. Genetic Disorders • Caused by mutations in genes or chromosomes • Mutations may occur on: -- An autosome (autosomal) -- A sex chromosome (X-linked or Y-linked) -- Multiple genes • Disease expression may be impacted by environmental factors

  17. 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 Most “single gene disorders” are probably influenced by multiple genes / DNA

  18. Classes of Single Gene Disorders • Autosomal Dominant • One copy of a mutated allele results in affected individual • aka: AA or Aa • Heterozygous and homozygous individuals are affected • e.g. achondroplasia, Huntington disease • Autosomal Recessive • Both copies of the gene must be mutated to be affected • aka: aa • Only homozygous individuals are affected. • e.g. Sickle cell anemia, cystic fibrosis, galactosemia

  19. Classes of Single Gene Disorders • X-linked Recessive -- Males affected if X chromosome is mutated -- Females affected only if both X chromosomes are mutated; e.g. Duchenne muscular dystrophy & hemophilia • X-linked Dominant -- Individuals with 1 mutant copy of X chromosome are affected; e.g. Rett syndrome • Y-linked -- Individuals with a mutated Y chromosome are affected -- Rare

  20. Complex / Multifactorial Disorders • Associated with small effects of multiple genes • May be strongly impacted by environmental factors (e.g. lifestyle) • Familial clustering -- No clear-cut pattern of inheritance -- Difficult to determine risk • Heart disease, diabetes, obesity, cancer

  21. 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 • Identification of disease-causing mutations for: • Diagnosis • Predictive testing • Carrier detection • Prenatal screening • Preimplantation testing • Pharmacogenetics

  22. Availability of Genetic Tests GeneTESTS: Availability of Genetic Tests As of 5/1/2012, http://www.ncbi.nlm.nih.gov/sites/GeneTests/

  23. Availability of Genetic Tests As of 5/1/2012, http://www.ncbi.nlm.nih.gov/sites/GeneTests/ site is being revised

  24. Firm Brings Gene Tests to Masses (NYT) Published: January 28, 2010 REDWOOD CITY, Calif. — The new movie “Extraordinary Measures” is based on the true story of a father who starts a company to develop a treatment for the rare genetic disease threatening to kill two of his children before they turn 10…… What condition??

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

  26. History of NBS Molecular Testing • 2005 -- Wisconsin – MSUD (p.Y438N) • 2006 -- New York – Krabbe disease (3 polymorphisms & 5 mutations; full gene DNA sequence analysis) • 2008 -- Wisconsin – SCID – TREC analysis • 1st use of molecular test as a primary full population screen • 2010 -- 36 NBSPs in US use molecular testing for CF

  27. 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

  28. Things for Programs to Consider 2nd tier v. 1st tier • Which tests will have a molecular component? • DNA extraction methods; (cost/labor) • Degree of automation; vendors and contracts • Manipulation (single tube? 96-well? 384-well?) • # Instruments, data collection, interpretation • Staff training (lab and follow-up)

  29. NBS Molecular Tests in US • Primary screen -- SCID • Second-tier screen -- Hemoglobinopathies -- Galactosemia -- Cystic fibrosis -- MCAD and other FAOs -- Phenylketonuria -- Krabbe disease -- Maple syrup urine disease

  30. Does Molecular Testing Add Value?? OR • Increase in sensitivity of a primary test, effect on specificity? • Identification of carriers; teaching moments • Predictions regarding phenotype • Clinicians’ perception, diagnostic tool

  31. 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)

  32. When / Why Use a Molecular Test? • When the primary analyte is transient -- The primary analyte is present for only a limited time after birth and analysis of a second specimen could result in a false negative. (e.g. VLCAD / CPT2) • To speed diagnosis in order to avoid serious medical consequences -- GALT enzyme activity is decreased by heat & humidity, increase in false positive screens -- Genotyping helps sort out the true positives for faster diagnosis.

  33. 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 p.Y438N serves as primary screen for MSUD for Mennonites. -- CPT1a in Alaskan Inuit & Hutterite populations

  34. When / Why Use a Molecular Test? • When diagnostic testing is slow and/or invasive -- Traditional confirmatory testing for VLCAD & CPT1a involves skin biopsy (invasive to collect and slow to grow) • When no other test exists for the analyte • SCID, SMA

  35. Things for Programs to Consider By Contract • Which tests will have a molecular component? • Specimen transport • Screening or confirmatory? • Timing and prioritization for contract lab • Systems integration • Follow-up integration

  36. Things for Programs to Consider In-House 1 • Volume / quality of specimens • Cost ($$$) per sample • “Simple test” mentality • Public health infrastructure • -- Equipment • -- Space • ELSI • Have test, no Tx

  37. Things for Programs to Consider In-House 2 • Capacity – Throughput -- Automation? • IVD v. ASR / LDT; • Expertise / Interpretation • Methods / Manipulation – single tube? 96-well or 384-well plates • Control Materials • Integration into Program / LIMs / Follow-up / TAT

  38. 5’ 3’ 5’ 3’ Starting DNA Template 3’ 5’ 3’ 5’ Separate strands (denature) Forward primer 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ Make copies (extend primers) Reverse primer Add primers (anneal) DNA Amplification with the Polymerase Chain Reaction (PCR) www.nist.gov

  39. Original DNA target region Thermal cycle Thermal cycle Thermal cycle PCR Copies DNA Exponentially through Multiple Thermal Cycles In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created www.nist.gov

  40. Thermal Cyclers (PCR machines) Wadsworth Center Compliments of Colleen Stevens

  41. Illumina Technology BeadArray 5 million SNP markers http://www.nature.com/nmeth/journal/v2/n12/images/nmeth1205-989-I2.jpg

  42. Gel Electrophoresis Cathode D E C R E A S I N G S I Z E Large Small Anode

  43. Restriction Fragment Length Polymorphism (RFLP) ctcctgaggag ctcctgtggag t t Dde I c c c t CTNAG GANTC Wadsworth Center

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