Deep resequencing of CFTR in 762 F508del homozygotes reveals clusters of non-coding variants associated with cystic fibrosis disease traits

Abstract

Extensive phenotypic variability is commonly observed in individuals with Mendelian disorders, even among those with identical genotypes in the disease-causing gene. To determine whether variants within and surrounding CFTR contribute to phenotypic variability in cystic fibrosis (CF), we performed deep sequencing of CFTR in 762 patients homozygous for the common CF-causing variant, F508del. In phase 1, ~200 kb encompassing CFTR and extending 10 kb 5′ and 5 kb 3′ of the gene was sequenced in 486 F508del homozygotes selected from the extremes of sweat chloride concentration. In phase 2, a 510 kb region, which included the entire topologically associated domain of CFTR, was sequenced in 276 F508del homozygotes drawn from extremes of lung function. An additional 163 individuals who carried F508del and a different CF-causing variant were sequenced to inform haplotype construction. Region-based burden testing of both common and rare variants revealed seven regions of significance (α=0.01), five of which overlapped known regulatory elements or chromatin interactions. Notably, the −80 kb locus known to interact with the CFTR promoter was associated with variation in both CF traits. Haplotype analysis revealed a single rare recombination event (1.9% frequency) in intron 15 of CFTR bearing the F508del variant. Otherwise, the majority of F508del chromosomes were markedly similar, consistent with a single origin of the F508del allele. Together, these high-resolution variant analyses of the CFTR locus suggest a role for non-coding regulatory motifs in trait variation among individuals carrying the common CF allele.

Cisvariants identified in F508del complex alleles modulate CFTR channel rescue by small molecules

“…We referenced p.Phe508del complex alleles in which the variation in cis is within the coding region and indicated their frequency by combined results found in the literature (Diana et al., ; Fichou et al., ; Vecchio‐Pagan et al., ), the CFTR‐France Database (Bareil et al., ; Claustres et al., ). We also analyzed 100 exon 12 and 153 exon 19 of p.Phe508del containing chromosomes in the French population (Table ).…”

“…We referenced p.Phe508del complex alleles in which the variation in cis is within the coding region and indicated their frequency by combined results found in the literature (Diana et al, 2016;Fichou et al, 2008;Vecchio-Pagan et al, 2016), the CFTR-France Database ( of which were identified in discrete cases. For the five complex alleles found more than once, frequencies ranged from 0.3% up to 6.4%.…”

“…Therefore, these variants are not routinely searched for diagnostic purposes and their frequency is probably underestimated. Nonetheless, their frequency appears to be low (Vecchio‐Pagan et al., ), even if regional cohorts presented higher numbers, for example, the c.[713C > T;1521_1523delCTT] (p.[Ala238Val;Phe508del]) in the South of Italy (Diana et al., ) or c.[1521_1523delCTT;3080T > C] (p.[Phe508del;Ile1027Thr]) in Brittany, France (Fichou et al., ).…”

CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rareCFTRvariants

“…Several complex alleles (e.g., p.[Arg74Trp;Val201Met;Asp1270Asn], p.[Phe508del;Ile1027Thr], or p.[Ser1251Asn;Phe508Cys] are frequent and well known. However, rare associations are also reported in CFTR ‐France, for example, a dozen variants are described in cis of p.Phe508del, some of them not found in the recent article about resequencing of p.Phe508del homozygotes (Vecchio‐Pagán et al., ).…”

Pitfalls in the interpretation of CFTR variants in the context of incidental findings

“…Some benign variants were misclassified as CF‐causing because they were observed in patients with CF but in the context of complex alleles with CF‐causing variants, such as c.3080C>T (p.Ile1027T) reported in cis with c.1521_153del (p.Phe508del; Baatallah et al, ). Notably, a total of 28 such variants have been described in complex alleles in CFTR ‐France, and more than 20 variants have been linked to c.1521_1523del (p.Phe508del; Vecchio‐Pagán et al, ). As other illustrations of complex alleles, among the 27 “frequent” variants (Group 1), c.220C>T (p.Arg74Trp), c.601G>A (p.Val201Met) and c.3808G>A (p.Asp1270Asn) have been described in cis in CFTR ‐France in numerous patients with CFTR‐RD, as well as c.1727G>C (p.Gly576Ala) and c.2002C>T (p.Arg668Cys; El‐Seedy et al, ).…”