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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

Burden testing of common and rare variants associating with sweat Cl− levels. All variants within each 5 kb window, moved across the entire region in increments of 1,250 bp, were tested for a combined association with sweat chloride levels (mM) via SKAT-O test. The x axis denotes chr7 hg19 bp position, y axis is −log10 of the regional P-value. Association values were plotted at the center of each 5 kb window. Top (red): rare variants with minor allele frequency (MAF)<1% only. Middle (blue): common variants with MAF>1% only. Bottom (purple): combined test of common and rare variants with variants weighted inversely proportional to their frequency. Vertical shaded boxes: regions of significant association in the combined analysis (α=0.01). Genome browser style tracks: Top, packed view of known CFTR regulatory regions of interest and TAD as previously reported (see Supplementary File). Middle, view of genes with exonic/intronic structure. Bottom, CCCTC-binding factor (CTFC) binding signals in two cell types, and mammalian conservation as assayed by genomic evolutionary rate profiling (GERP) (horizontal bar indicating a GERP score of 4). CFTR, the protein defective in cystic fibrosis; TAD, topologically associated domain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121184&req=5

fig1: Burden testing of common and rare variants associating with sweat Cl− levels. All variants within each 5 kb window, moved across the entire region in increments of 1,250 bp, were tested for a combined association with sweat chloride levels (mM) via SKAT-O test. The x axis denotes chr7 hg19 bp position, y axis is −log10 of the regional P-value. Association values were plotted at the center of each 5 kb window. Top (red): rare variants with minor allele frequency (MAF)<1% only. Middle (blue): common variants with MAF>1% only. Bottom (purple): combined test of common and rare variants with variants weighted inversely proportional to their frequency. Vertical shaded boxes: regions of significant association in the combined analysis (α=0.01). Genome browser style tracks: Top, packed view of known CFTR regulatory regions of interest and TAD as previously reported (see Supplementary File). Middle, view of genes with exonic/intronic structure. Bottom, CCCTC-binding factor (CTFC) binding signals in two cell types, and mammalian conservation as assayed by genomic evolutionary rate profiling (GERP) (horizontal bar indicating a GERP score of 4). CFTR, the protein defective in cystic fibrosis; TAD, topologically associated domain.

Mentions: A region-based burden assay was performed to identify groupings of variants that are associated with trait variation. Variants were tested for association with sweat chloride or lung function in groups defined by a series of overlapping 5 kb windows (offset in 1,250 bp increments), with each window generating a test P-value. The P-value for each window was Bonferroni corrected for multiple testing based on the total number of unique windows assayed (see Methods). Regions of significance (hg19 coordinates, study-wide P<0.01) were highlighted for the combined test of common and rare variants associated with either sweat chloride concentration (Figure 1) and/or lung function (Figure 2). Regions that coincided with known regulatory and boundary elements are discussed below (Supplementary Table 4).13,21,22


Deep resequencing of CFTR in 762 F508del homozygotes reveals clusters of non-coding variants associated with cystic fibrosis disease traits
Burden testing of common and rare variants associating with sweat Cl− levels. All variants within each 5 kb window, moved across the entire region in increments of 1,250 bp, were tested for a combined association with sweat chloride levels (mM) via SKAT-O test. The x axis denotes chr7 hg19 bp position, y axis is −log10 of the regional P-value. Association values were plotted at the center of each 5 kb window. Top (red): rare variants with minor allele frequency (MAF)<1% only. Middle (blue): common variants with MAF>1% only. Bottom (purple): combined test of common and rare variants with variants weighted inversely proportional to their frequency. Vertical shaded boxes: regions of significant association in the combined analysis (α=0.01). Genome browser style tracks: Top, packed view of known CFTR regulatory regions of interest and TAD as previously reported (see Supplementary File). Middle, view of genes with exonic/intronic structure. Bottom, CCCTC-binding factor (CTFC) binding signals in two cell types, and mammalian conservation as assayed by genomic evolutionary rate profiling (GERP) (horizontal bar indicating a GERP score of 4). CFTR, the protein defective in cystic fibrosis; TAD, topologically associated domain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121184&req=5

fig1: Burden testing of common and rare variants associating with sweat Cl− levels. All variants within each 5 kb window, moved across the entire region in increments of 1,250 bp, were tested for a combined association with sweat chloride levels (mM) via SKAT-O test. The x axis denotes chr7 hg19 bp position, y axis is −log10 of the regional P-value. Association values were plotted at the center of each 5 kb window. Top (red): rare variants with minor allele frequency (MAF)<1% only. Middle (blue): common variants with MAF>1% only. Bottom (purple): combined test of common and rare variants with variants weighted inversely proportional to their frequency. Vertical shaded boxes: regions of significant association in the combined analysis (α=0.01). Genome browser style tracks: Top, packed view of known CFTR regulatory regions of interest and TAD as previously reported (see Supplementary File). Middle, view of genes with exonic/intronic structure. Bottom, CCCTC-binding factor (CTFC) binding signals in two cell types, and mammalian conservation as assayed by genomic evolutionary rate profiling (GERP) (horizontal bar indicating a GERP score of 4). CFTR, the protein defective in cystic fibrosis; TAD, topologically associated domain.
Mentions: A region-based burden assay was performed to identify groupings of variants that are associated with trait variation. Variants were tested for association with sweat chloride or lung function in groups defined by a series of overlapping 5 kb windows (offset in 1,250 bp increments), with each window generating a test P-value. The P-value for each window was Bonferroni corrected for multiple testing based on the total number of unique windows assayed (see Methods). Regions of significance (hg19 coordinates, study-wide P<0.01) were highlighted for the combined test of common and rare variants associated with either sweat chloride concentration (Figure 1) and/or lung function (Figure 2). Regions that coincided with known regulatory and boundary elements are discussed below (Supplementary Table 4).13,21,22

View Article: PubMed Central - PubMed

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&thinsp;kb encompassing CFTR and extending 10&thinsp;kb 5&prime; and 5&thinsp;kb 3&prime; of the gene was sequenced in 486 F508del homozygotes selected from the extremes of sweat chloride concentration. In phase 2, a 510&thinsp;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 (&alpha;=0.01), five of which overlapped known regulatory elements or chromatin interactions. Notably, the &minus;80&thinsp;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.

No MeSH data available.


Related in: MedlinePlus