wisconsin state laboratory of hygiene cystic fibrosis gary hoffman wisconsin newborn screening...
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WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Cystic Fibrosis
Gary HoffmanWisconsin Newborn Screening
Laboratory
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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 dysfunctiondigestive system reproductive system (males)
respiratory tract
• Clinical manifestationsIrreversible lung damage (infections)
Severe Malnutrition
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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 STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Mutation Methods
• FDA approved kits:
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
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Mutation Testing Summary
• Increase specificity and detection sensitivity
• Minimize false negatives
• Expedite management and treatment
• Identification of carriers
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Maple Syrup Urine Disease
Gary HoffmanWisconsin Newborn Screening
Laboratory
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Maple Syrup Urine Disease
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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)
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Molecular Testing Advantages
• Support of the screening results
• Provide for earlier intervention
• High risk births – immediate testing
• Inexpensive testing
WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
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