FilmArray High Resolution Melting Detection of Mutations Causing Cystic Fibrosis

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is an autosomal recessive, life-shortening disease that affects ~30,000 patients in the U.S., while there are estimated 10 million symptomless carriers in the U.S. (www.cff.org). Approximately one in 2,500-3,300 newborns in the U.S. have CF, however newborn screening is mandated in only 39 states. The gene mutated is the cystic fibrosis transmembrane conductance regulator (CFTR), which encodes a channel that regulates the flow of Cl- ions. Genetic t ests are performed for newborns, suspected CF patients and for prospective parents. The American College of Medical Genetics (ACMG) currently recommends screening for 23 mutations which account for 88% of the CF mutations in non-Hispanic Caucasians. For pa tients who are negative for these mutations, or show only one copy of the screened mutations, sequencing is recommended. However sequencing is costly as the CFTR gene is nearly 190 kb in length, the coding region is ~4,400 bps and there are over 1,000 addi tional mutations associated with disease. The work proposed here will demonstrate the feasibility of producing a fully integrated and automated diagnostic system that will streamline CF testing. Idaho Technology Inc. (ITI) has developed the FilmArray, an a utomated system that performs all of the steps necessary to identify viral and bacterial pathogens in human samples. The FilmArray performs sample preparation, nucleic acid isolation, real time polymerase chain reaction (PCR) and data analysis within one h our. Our goal is to enhance the FilmArray so it can ultimately be used to (1) genotype, or identify the 23 ACMG recommended prevalent mutations and 6 common benign polymorphisms in the CFTR gene, and (2) scan, or screen the entire CFTR coding regions for o ther rare mutations associated with disease. In this Phase I SBIR proposal, the goals are to produce assays and introduce engineering solutions for genotyping and scanning in the FilmArray. The main engineering goal (Aim 1) is better temperature uniformity across the array, where 120 individual real time PCR assays are performed, and genotyping and scanning analysis will be performed. A subset of diverse genotyping and scanning assays are proposed (Aims 2 and 3) to initiate assay development and to monitor the instrument's performance. The Phase II goal of this project is to produce and commercialize a fully integrated, automated FilmArray system that will genotype and scan for CFTR genetic variants in a more comprehensive, error free, rapid and lower cost m anner compared to currently available methods. This turn-key mutation screening system can be adapted to screen other critical disease-causing genes for predisposition or therapeutic treatment. PUBLIC HEALTH RELEVANCE: Over 1,000 recessive mutations in the large cystic fibrosis transmembrane conductance regulator gene lead to cystic fibrosis. Idaho Technology proposes to develop a fully integrated and automated diagnostic system called the FilmArray that will make CFTR genetic testing more comprehensive , rapid and affordable.