CFTR GENOTYPING BY PEPTIDE MASS-SIGNATURE GENOTYPING

Pediatrix Screening, Inc., Bridgeville PA

Investigators

Abstract

Cystic Fibrosis (CF) is the most common, lethal, autosomal recessive disorder. It affects 1 in 2500 births and has a carrier frequency of 1 in 25. CF results from mutations in the cystic fibrosis membrane conductance regulator gene (CFTR). The current paradigm of CF screening and diagnosis is being challenged because the traditional battery of assays - immune reactive trypsinogen, Delta F508 analysis, and sweat chloride testing - are proving to be less reliable than previously thought. CFTR genotyping is playing an increasing role in CF diagnosis. CFTR genotyping services are expensive and typically assay for fewer than 10% of known mutations. Peptide MassSignature Genotyping is an inexpensive, high throughput method for CFTR gentyping. PSMG analysis entails 1. Amplification of genomic DNA encompassing the exons and intron/exon boundaries, 2. Cloning the amplicons in-frame with an epitope tag, 3. Expression of the cloned fragment, 4. Affinity purification through the epitope tag, 5. Analysis of peptide mass by MALDI-TOF, and 6. Computational deconvolution of peptide mass data to determine genetic mutations. The exquisite sensitivity of MALDI-TOF allows for multiplex and parallel analysis. The feasibility of CFTR genotyping by PMSG will be demonstrated using exons 10 and 11 as model systems. PROPOSED COMMERCIAL APPLICATIONS: Essentially all CF centers have CFTR analysis performed on their patients. Offering comprehensive CFTR genotyping using PMSG at approx. 1/3 the cost of services currently available has significant commercial potential. Establishing a CFTR genotyping service will set a precedent to expand PMSG services to other disorders where genotype analysis is a valuable tool.