1. Gary Hoffman Wisconsin Newborn Screening Laboratory
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 reproductive system (males)
respiratory tract
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. Randomized Control Trial (RCT)
Wisconsin Experience
Randomized Control Trial (RCT)
 1985 – 1990
Immunoreactive trypsinogen
IRT cutoff – 99.8%tile
False negative rate to high 
Incorporated 2nd 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
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

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.  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

8. 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

9. 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

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
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

14. 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)

15.  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

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