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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.

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Recombination ratio and linkage disequilibrium observed in 762 F508del homozygous samples (1,524 chromosomes) across a 506 kb re-sequencing region surrounding CFTR. Top: recombination ratio plotted by genomic location. A recombination event occurring within intron 15 of CFTR is indicated by the red arrow. Below is an intronic and exonic map of known RefSeq genes, and re-sequencing study variants with minor allele frequency (MAF)>1% (hg19 coordinates). Bottom: LD heat map of variants with MAF>2% in the F508del population. Dashed red lines indicate projection of variants from their genomic positions to the heat-map of r2 values below. Within the heatmap, black boxes indicate an r2 value of 1 or complete LD, while white boxes indicate an r2 of 0 or linkage equilibrium. Three proposed LD blocks are outlined (triangles). The first extends from the start of the sequencing capture to intron 3 of WNT2. LD block 1 then extends from the WNT2 locus to intron 15 of CFTR. Finally, LD block 2 extends from intron 15 of CFTR to the end of the sequencing capture (mid-CTTNBP2). LD blocks 0 and 2 likely extend far beyond the capture design. CFTR, the protein defective in cystic fibrosis; LD, linkage disequilibrium.
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fig3: Recombination ratio and linkage disequilibrium observed in 762 F508del homozygous samples (1,524 chromosomes) across a 506 kb re-sequencing region surrounding CFTR. Top: recombination ratio plotted by genomic location. A recombination event occurring within intron 15 of CFTR is indicated by the red arrow. Below is an intronic and exonic map of known RefSeq genes, and re-sequencing study variants with minor allele frequency (MAF)>1% (hg19 coordinates). Bottom: LD heat map of variants with MAF>2% in the F508del population. Dashed red lines indicate projection of variants from their genomic positions to the heat-map of r2 values below. Within the heatmap, black boxes indicate an r2 value of 1 or complete LD, while white boxes indicate an r2 of 0 or linkage equilibrium. Three proposed LD blocks are outlined (triangles). The first extends from the start of the sequencing capture to intron 3 of WNT2. LD block 1 then extends from the WNT2 locus to intron 15 of CFTR. Finally, LD block 2 extends from intron 15 of CFTR to the end of the sequencing capture (mid-CTTNBP2). LD blocks 0 and 2 likely extend far beyond the capture design. CFTR, the protein defective in cystic fibrosis; LD, linkage disequilibrium.

Mentions: In addition to association testing, we also sought to systematically determine the genetic architecture at this locus in the F508del homozygous population. Haplotypes are combinations of variants that tend to be inherited together (in cis). Their borders are often delineated by meiotic recombination events. Derivation of haplotypes is useful for establishing the degree of genetic diversity in a locus, associating functional variants with background variation, and inferring ancestral origins of disease-causing variants. To assemble haplotypes, single-nucleotide variants with MAF>1% within the 510 kb region surrounding CFTR were phased using SHAPEIT2.24 Samples bearing non-F508del chromosomes were used to deduce the locations of alternative recombination events and additional diversity of the CFTR locus. LD among variants with MAF>2% in 762 F508del homozygotes revealed three primary regions of high LD, two of which encompassed CFTR (Figure 3). A recombination event was observed within intron 15 of CFTR, resulting in an alternative haplotype in LD block 2. This recombination event is not present in the 206 non-F508del chromosomes that were sequenced using the same capture design. The intron 15 recombination event in F508del homozygotes is unique to this population, and is not apparent in HapMap populations (which contain a diversity of CFTR haplotypes only ~5% of which is the F508del ancestral haplotype), where a distinct recombination event within intron 22 is observed.25


Deep resequencing of CFTR in 762 F508del homozygotes reveals clusters of non-coding variants associated with cystic fibrosis disease traits
Recombination ratio and linkage disequilibrium observed in 762 F508del homozygous samples (1,524 chromosomes) across a 506 kb re-sequencing region surrounding CFTR. Top: recombination ratio plotted by genomic location. A recombination event occurring within intron 15 of CFTR is indicated by the red arrow. Below is an intronic and exonic map of known RefSeq genes, and re-sequencing study variants with minor allele frequency (MAF)>1% (hg19 coordinates). Bottom: LD heat map of variants with MAF>2% in the F508del population. Dashed red lines indicate projection of variants from their genomic positions to the heat-map of r2 values below. Within the heatmap, black boxes indicate an r2 value of 1 or complete LD, while white boxes indicate an r2 of 0 or linkage equilibrium. Three proposed LD blocks are outlined (triangles). The first extends from the start of the sequencing capture to intron 3 of WNT2. LD block 1 then extends from the WNT2 locus to intron 15 of CFTR. Finally, LD block 2 extends from intron 15 of CFTR to the end of the sequencing capture (mid-CTTNBP2). LD blocks 0 and 2 likely extend far beyond the capture design. CFTR, the protein defective in cystic fibrosis; LD, linkage disequilibrium.
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Related In: Results  -  Collection

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fig3: Recombination ratio and linkage disequilibrium observed in 762 F508del homozygous samples (1,524 chromosomes) across a 506 kb re-sequencing region surrounding CFTR. Top: recombination ratio plotted by genomic location. A recombination event occurring within intron 15 of CFTR is indicated by the red arrow. Below is an intronic and exonic map of known RefSeq genes, and re-sequencing study variants with minor allele frequency (MAF)>1% (hg19 coordinates). Bottom: LD heat map of variants with MAF>2% in the F508del population. Dashed red lines indicate projection of variants from their genomic positions to the heat-map of r2 values below. Within the heatmap, black boxes indicate an r2 value of 1 or complete LD, while white boxes indicate an r2 of 0 or linkage equilibrium. Three proposed LD blocks are outlined (triangles). The first extends from the start of the sequencing capture to intron 3 of WNT2. LD block 1 then extends from the WNT2 locus to intron 15 of CFTR. Finally, LD block 2 extends from intron 15 of CFTR to the end of the sequencing capture (mid-CTTNBP2). LD blocks 0 and 2 likely extend far beyond the capture design. CFTR, the protein defective in cystic fibrosis; LD, linkage disequilibrium.
Mentions: In addition to association testing, we also sought to systematically determine the genetic architecture at this locus in the F508del homozygous population. Haplotypes are combinations of variants that tend to be inherited together (in cis). Their borders are often delineated by meiotic recombination events. Derivation of haplotypes is useful for establishing the degree of genetic diversity in a locus, associating functional variants with background variation, and inferring ancestral origins of disease-causing variants. To assemble haplotypes, single-nucleotide variants with MAF>1% within the 510 kb region surrounding CFTR were phased using SHAPEIT2.24 Samples bearing non-F508del chromosomes were used to deduce the locations of alternative recombination events and additional diversity of the CFTR locus. LD among variants with MAF>2% in 762 F508del homozygotes revealed three primary regions of high LD, two of which encompassed CFTR (Figure 3). A recombination event was observed within intron 15 of CFTR, resulting in an alternative haplotype in LD block 2. This recombination event is not present in the 206 non-F508del chromosomes that were sequenced using the same capture design. The intron 15 recombination event in F508del homozygotes is unique to this population, and is not apparent in HapMap populations (which contain a diversity of CFTR haplotypes only ~5% of which is the F508del ancestral haplotype), where a distinct recombination event within intron 22 is observed.25

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