wisconsin state laboratory of hygiene cystic fibrosis gary hoffman wisconsin newborn screening...

17
WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

Upload: meghan-cunningham

Post on 02-Jan-2016

219 views

Category:

Documents


2 download

TRANSCRIPT

Page 1: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE

Cystic Fibrosis

Gary HoffmanWisconsin Newborn Screening

Laboratory

Page 2: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin 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

Page 3: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 4: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 5: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 6: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 7: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 8: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 9: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 10: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 11: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE

Maple Syrup Urine Disease

Gary HoffmanWisconsin Newborn Screening

Laboratory

Page 12: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin 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

Page 13: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE

Maple Syrup Urine Disease

Page 14: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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)

Page 15: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 16: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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

Page 17: WISCONSIN STATE LABORATORY OF HYGIENE Cystic Fibrosis Gary Hoffman Wisconsin Newborn Screening Laboratory

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