Gary Hoffman Wisconsin Newborn Screening Laboratory Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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 reproductive system (males) respiratory tract Clinical manifestations Irreversible lung damage (infections) Severe Malnutrition

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

Randomized Control Trial (RCT) Wisconsin Experience Randomized Control Trial (RCT)  1985 – 1990 Immunoreactive trypsinogen IRT cutoff – 99.8%tile False negative rate to high  1991 - 1994 Incorporated 2nd tier DNA F508del mutation Polyacrylamide gel with silver staining IRT cutoff – 98.5%tile Significantly reduced false negatives

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

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

 Allele specific primers for wild type and mutant 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

Mutation Methods  Luminex (xTAG) FDA approved kits: 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

Selection of CF mutations Over 1500 unknown mutations Technology can identify about 100 mutations Population demographics Match panel with genotypes 23 – 40 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

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

Maple Syrup Urine Disease Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Maple Syrup Urine Disease Leucine, isoleucine, valine are amino acids which come from the protein you eat Purpose of their degradation is to make energy via the TCA cycle MSUD causes block in degradation Increased concentrations of leucine, isoleucine, and valine But most importantly in the increased leucine

Selection of Mutations Gene  Branched chain keto acid deydrogenase E1, alpha polypeptide (BCKDHA)  Long arm of chromosome 19 40 unknown mutations  Disrupt BCKD enzyme from breakdown leucine, valine, and isoleucine Mennonite populations  Y438N (Tyr438Asn)

 Two flanking primers; 2 internal primers 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

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

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