Cystic Fibrosis. Gary Hoffman Wisconsin Newborn Screening Laboratory WISCONSIN STATE LABORATORY OF HYGIENE

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1 Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

2 Cystic Fibrosis Review Most common lethal genetic autosomal recessive disorder Caucasians 1:3,000 African Americans 1:20,000 Hispanics 1:7,500 Asians 1:30,000 Multi-Organ dysfunction digestive system respiratory tract reproductive system (males) Clinical manifestations Irreversible lung damage (infections) Severe Malnutrition

3 Cystic Fibrosis Review (Cont) Life expectancy 50% die before age 32 Significant morbidity at any age Gene Transmembrane Conductance Regulator (CFTR) On long arm of chromosome 7 lack of salt and water transfer across cell membranes 1,500+ mutations in the CFTR Confirmatory diagnosis Positive sweat chloride measurements

4 Wisconsin Experience Randomized Control Trial (RCT) Immunoreactive trypsinogen IRT cutoff 99.8%tile False negative rate to high Incorporated 2 nd tier DNA F508del mutation Polyacrylamide gel with silver staining IRT cutoff 98.5%tile Significantly reduced false negatives

5 Wisconsin Experience (cont) Routine screening July 1994 Two tiered algorithm 1 st tier Immunoreactive trypsinogen 2 nd tier Mutation analysis for F508del Initial IRT cutoff for mutation testing 94 th %tile Changes Increased percentile to 96 th Changed methodology Roche linear array 23 mutations Hologic (TWT) invader

6 Mutation Analysis Issues Problem Specimens No product formation Insufficient DNA Excessive hemolysis - inhibits PCR Transfusions Delay in reporting results 5 to 8 days depending upon PCR testing schedule

7 Mutation Methods Targeted mutation analysis (most common) Allele specific primers for wild type and mutant bind and amplify small regions around mutation site mutations can be substitutions, insertions, deletions

8 FDA approved kits: Mutation Methods Luminex (xtag) Multiple Allele-Specific Primer Extension (ASPE) beads that generate signals when separated by laser flow cytometry. 39, 60, and 71 mutation panel Program for only ACMG 23 panel Open architecture Hologic InPlex Flurorescence Resonance Energy Transfer (FRET) signal is generated with cleavase ACMG 23 Panel limited hands on time cartridge based

9 Selection of CF mutations Over 1500 unknown mutations Technology can identify about 100 mutations Population demographics Match panel with genotypes mutations will identify >95% of the cases Minimum Core ACMG recommended 23 Disease causing mutations (R117H?) Panels do not need to cover 100% of the disease causing alleles

10 Mutation Testing Summary Increase specificity and detection sensitivity Minimize false negatives Expedite management and treatment Identification of carriers

11 Maple Syrup Urine Disease Gary Hoffman Wisconsin Newborn Screening Laboratory

12 Overview of MSUD Aminoacidopathy of leucine metabolism Primary screening analytes Leucine/Isoleucine & Valine Primary methodology Tandem Mass Spectrometry Prevalence Overall population 1:250,000 Mennonite 1:400 Clinical symptoms seizures, coma, vomiting, sweet smelling urine, death, developmental delay Treatment: low protein diet Recommended treatment age: 72 hours from birth

13 Maple Syrup Urine Disease

14 Gene Selection of Mutations Branched chain keto acid deydrogenase E1, alpha polypeptide (BCKDHA) Long arm of chromosome unknown mutations Disrupt BCKD enzyme from breakdown leucine, valine, and isoleucine Mennonite populations Y438N (Tyr438Asn)

15 Wisconsin Method Tetra-primer ARMS-PCR Two flanking primers; 2 internal primers Two annealing temps Concentrates the DNA fragments Amplicons separated on agarose gel Homozygote one band Heterozygote two bands

16 Molecular Testing Advantages Support of the screening results Provide for earlier intervention High risk births immediate testing Inexpensive testing

17 MSUD SUCCESS STORY Couple known carrier of Y438N mutation Specimen collected at one hour of age Transported to NBS lab within 2 hours Assay setup immediately Result Y438N homozygote (8 hours of age) Dietary treatment at 12 hours of age Outcome One hospitalization in the first week of life Family compliant with therapy At 4 years - no mental or physical issues