Extensive Sequencing of the CFTR gene: lessons learned from the first 157 patient samples

Abstract

Cystic fibrosis (CF) is one of the most common monogenic diseases affecting Caucasians and has an incidence of approximately 1:3,300 births. Currently recommended screening panels for mutations in the responsible gene (CF transmembrane regulator gene, CFTR) do not detect all disease-associated mutations. Our laboratory offers extensive sequencing of the CFTR (ABCC7) gene (including the promoter, all exons and splice junction sites, and regions of selected introns) as a clinical test to detect mutations which are not found with conventional screening. The objective of this report is to summarize the findings of extensive CFTR sequencing from our first 157 consecutive patient samples. In most patients with classic CF symptoms (18/24, 75%), extensive CFTR sequencing confirmed the diagnosis by finding two disease-associated mutations. In contrast, only 5 of 75 (7%) patients with atypical CF had been identified with two CFTR mutations. A diagnosis of CF was confirmed in 10 of 17 (58%) newborns with either positive sweat chloride readings or positive immunoreactive trypsinogen (IRT) screen results. We ascertained ten novel sequence variants that are potentially disease-associated: two deletions (c.1641AG>T, c.2949_2853delTACTC), seven missense mutations (p.S158T, p.G451V, p.K481E, p.C491S, p.H949L, p.T1036N, p.F1099L), and one complex allele ([p.356_A357del; p.358I]). We ascertained three other apparently novel complex alleles. Finally, several patients were found to carry partial CFTR gene deletions. In summary, extensive CFTR gene sequencing can detect rare mutations which are not found with other screening and diagnostic tests, and can thus establish a definitive diagnosis in symptomatic patients with previously negative results. This enables carrier detection and prenatal diagnosis in additional family members.

Comprehensive and Rapid Genotyping of Mutations and Haplotypes in Congenital Bilateral Absence of the Vas Deferens and Other Cystic Fibrosis Transmembrane Conductance Regulator-Related Disorders

“…All PCR primers had to be redesigned because of the presence of the M13 linker sequence. In another assay, 27 the CFTR gene was amplified in 30 amplicons with external primers and then sequenced using internal primers in 96-well plates. Redesigning all PCR primers was necessary, and three amplification conditions had to be used because of different annealing temperatures of the primers.…”

ABC transporters, neural stem cells and neurogenesis – a different perspective

“…For instance, the most common fatal autosomal recessive genetic disease affecting Caucasian populations, npg cystic fibrosis, is caused by mutations in the ABCC7 transporter (or CFTR), which is a regulated ion channel [118]. Most of the naturally occurring mutations in the ABCC7 gene either induce an alteration in protein biosynthesis or lead to a defective channel function [60,64,119,120]. Approximately 80% of all mutations found with the ABC transporters are located within the NBDs.…”

“…Moreover, in molecular terms, such functional pleiomorphism is related to the wide range of compounds that ABC family members can transport. Thus, the genotype/phenotype diversity in individuals with mutated ABC transporters may primarily reflect the functional complexity of these large multi-span polypeptides [60,64,119,120].…”

Characterization of a Recurrent Novel Large Duplication in the Cystic Fibrosis Transmembrane Conductance Regulator Gene

“…Using an in-house-developed semiquantitative fluorescent polymerase chain reaction (PCR) assay, we discovered 10 cases of large exonic deletions in patients with CF for whom DNA sequencing failed to identify a second mutated allele. 2,3 Others using similar methods identified large DNA rearrangements in the CFTR gene as well. 4,5 We also identified a deletion in a CBAVD patient.…”