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An association study on contrasting cystic fibrosis endophenotypes recognizes KRT8 but not KRT18 as a modifier of cystic fibrosis disease severity and CFTR mediated residual chloride secretion.

Stanke F, Hedtfeld S, Becker T, Tümmler B - BMC Med. Genet. (2011)

Bottom Line: In consistency, the KRT8 haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype.The two opposing KRT8 alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation.As the mild KRT8 allele is associated with CFTR mediated residual chloride secretion among F508del-CFTR homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Hannover Medical School, Hannover, Germany. mekus.frauke@mh-hannover.de

ABSTRACT

Background: F508del-CFTR, the most frequent disease-causing mutation among Caucasian cystic fibrosis (CF) patients, has been characterised as a mutant defective in protein folding, processing and trafficking. We have investigated the two neighbouring cytokeratin genes KRT8 and KRT18 in a candidate gene approach to ask whether variants in KRT8 and/or KRT18 modify the impaired ion conductance known as the CF basic defect, and whether they are associated with correct trafficking of mutant CFTR and disease severity of CF.

Methods: We have selected contrasting F508del-CFTR homozygous patient subpopulations stratified for disease severity, comparing 13 concordant mildly affected sib pairs vs. 12 concordant severely affected sib pairs, or manifestation of the CF basic defect in intestinal epithelium, comparing 22 individuals who exhibit CFTR-mediated residual chloride secretion vs. 14 individuals who do not express any chloride secretion, for an association. The KRT8/KRT18 locus was initially interrogated with one informative microsatellite marker. Subsequently, a low density SNP map with four SNPs in KRT8 and two SNPs in KRT18, each selected for high polymorphism content, was used to localize the association signal.

Results: KRT8, but not KRT18, showed an association with CF disease severity (Pbest=0.00131; Pcorr=0.0185) and CFTR mediated residual chloride secretion (Pbest=0.0004; Pcorr=0.0069). Two major four-marker-haplotypes spanning 13 kb including the entire KRT8 gene accounted for 90% of chromosomes, demonstrating strong linkage disequilibrium at that locus. Absence of chloride secretion was associated with the recessive haplotype 1122 at rs1907671, rs4300473, rs2035878 and rs2035875. The contrasting haplotype 2211 was dominant for the presence of CFTR mediated residual chloride secretion. In consistency, the KRT8 haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype. Analysis of microsatellite allele distributions on the SNP background suggests that the mild KRT8 haplotype 2211 is phylogenetically older than its severe counterpart.

Conclusions: The two opposing KRT8 alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation. As the mild KRT8 allele is associated with CFTR mediated residual chloride secretion among F508del-CFTR homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.

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Mikrosatellite KRT8Sat allele distributions on KRT8 SNP background. Pictograms show the allele distribution of KRT8Sat. The reference sequence for the dinucleotide repeat KRT8Sat is (CA)12(TA)(CA)2(TA)(CA)4 starting at position 15442813 on contig NT_029419. Alleles were calibrated in arbitrary repeat units using an invariant set of controls for all analyses. Six alleles were observed in the entire population, differing in size by one, three, four, seven and eight dinucleotide units in reference to the smallest allele observed. A: Allele distribution at KRT8Sat, observed among 101 CF families with a total of 171 patients. B: Distribution of KRT8Sat alleles on SNP allele background, given for allele 1 at SNP markers (left column) and allele 2 at SNP markers (right column). Alleles at all PCR-RFLP typed SNPs are named for absence (allele 1) or presence (allele 2) of diagnostic restriction site. SNPs rs1907671 (top row), rs4300473 (2nd row), rs2035878 (3rd row) and rs2035875 (bottom row) are shown. See text for details.
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Figure 2: Mikrosatellite KRT8Sat allele distributions on KRT8 SNP background. Pictograms show the allele distribution of KRT8Sat. The reference sequence for the dinucleotide repeat KRT8Sat is (CA)12(TA)(CA)2(TA)(CA)4 starting at position 15442813 on contig NT_029419. Alleles were calibrated in arbitrary repeat units using an invariant set of controls for all analyses. Six alleles were observed in the entire population, differing in size by one, three, four, seven and eight dinucleotide units in reference to the smallest allele observed. A: Allele distribution at KRT8Sat, observed among 101 CF families with a total of 171 patients. B: Distribution of KRT8Sat alleles on SNP allele background, given for allele 1 at SNP markers (left column) and allele 2 at SNP markers (right column). Alleles at all PCR-RFLP typed SNPs are named for absence (allele 1) or presence (allele 2) of diagnostic restriction site. SNPs rs1907671 (top row), rs4300473 (2nd row), rs2035878 (3rd row) and rs2035875 (bottom row) are shown. See text for details.

Mentions: We wanted to know which of the two frequent KRT8 haplotypes is older and have analyzed the four two-marker-haplotypes each composed of one out of the four KRT8 SNPs rs1907671, rs4300473, rs2035878 and rs2035875 in combination with the microsatellite KRT8Sat (Figure 2). Four major alleles, each present on more than 10% of chromosomes and all of them accounting for 98% of chromosomes are observed at the (CA)n-repeat KRT8Sat in the entire patient sample (Figure 2). SNP alleles 2 at rs1907671, 2 at rs4300473, 1 at rs2035878 and 1 at rs2035875 were observed to occur with three major microsatellite alleles that differ by more than one repeat motif in size, showing a broad distribution that has likely been generated by more than one recombination event (Figure 2). In contrast, the opposite SNP alleles 1 at rs1907671, 1 at rs4300473, 2 at rs2035878 and 2 at rs2035875 are observed together with two microsatellite alleles that differ by one repeat motif in size, one presumably having emerged from the other by an indel event in the microsatellite's primary sequence (Figure 2). Under the assumption that the KRT8Sat alleles themselves are functionally equivalent and neutral towards the studied phenotypes, we conclude that the benign KRT8 haplotype 2211 is older than the severe 1122 allele as one slippage event, explaining the KRT8Sat allele distribution on the 1122 background, requires fewer generations than the multiple recombination events that have generated the microsatellite's sequence pattern on 2211 alleles.


An association study on contrasting cystic fibrosis endophenotypes recognizes KRT8 but not KRT18 as a modifier of cystic fibrosis disease severity and CFTR mediated residual chloride secretion.

Stanke F, Hedtfeld S, Becker T, Tümmler B - BMC Med. Genet. (2011)

Mikrosatellite KRT8Sat allele distributions on KRT8 SNP background. Pictograms show the allele distribution of KRT8Sat. The reference sequence for the dinucleotide repeat KRT8Sat is (CA)12(TA)(CA)2(TA)(CA)4 starting at position 15442813 on contig NT_029419. Alleles were calibrated in arbitrary repeat units using an invariant set of controls for all analyses. Six alleles were observed in the entire population, differing in size by one, three, four, seven and eight dinucleotide units in reference to the smallest allele observed. A: Allele distribution at KRT8Sat, observed among 101 CF families with a total of 171 patients. B: Distribution of KRT8Sat alleles on SNP allele background, given for allele 1 at SNP markers (left column) and allele 2 at SNP markers (right column). Alleles at all PCR-RFLP typed SNPs are named for absence (allele 1) or presence (allele 2) of diagnostic restriction site. SNPs rs1907671 (top row), rs4300473 (2nd row), rs2035878 (3rd row) and rs2035875 (bottom row) are shown. See text for details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Mikrosatellite KRT8Sat allele distributions on KRT8 SNP background. Pictograms show the allele distribution of KRT8Sat. The reference sequence for the dinucleotide repeat KRT8Sat is (CA)12(TA)(CA)2(TA)(CA)4 starting at position 15442813 on contig NT_029419. Alleles were calibrated in arbitrary repeat units using an invariant set of controls for all analyses. Six alleles were observed in the entire population, differing in size by one, three, four, seven and eight dinucleotide units in reference to the smallest allele observed. A: Allele distribution at KRT8Sat, observed among 101 CF families with a total of 171 patients. B: Distribution of KRT8Sat alleles on SNP allele background, given for allele 1 at SNP markers (left column) and allele 2 at SNP markers (right column). Alleles at all PCR-RFLP typed SNPs are named for absence (allele 1) or presence (allele 2) of diagnostic restriction site. SNPs rs1907671 (top row), rs4300473 (2nd row), rs2035878 (3rd row) and rs2035875 (bottom row) are shown. See text for details.
Mentions: We wanted to know which of the two frequent KRT8 haplotypes is older and have analyzed the four two-marker-haplotypes each composed of one out of the four KRT8 SNPs rs1907671, rs4300473, rs2035878 and rs2035875 in combination with the microsatellite KRT8Sat (Figure 2). Four major alleles, each present on more than 10% of chromosomes and all of them accounting for 98% of chromosomes are observed at the (CA)n-repeat KRT8Sat in the entire patient sample (Figure 2). SNP alleles 2 at rs1907671, 2 at rs4300473, 1 at rs2035878 and 1 at rs2035875 were observed to occur with three major microsatellite alleles that differ by more than one repeat motif in size, showing a broad distribution that has likely been generated by more than one recombination event (Figure 2). In contrast, the opposite SNP alleles 1 at rs1907671, 1 at rs4300473, 2 at rs2035878 and 2 at rs2035875 are observed together with two microsatellite alleles that differ by one repeat motif in size, one presumably having emerged from the other by an indel event in the microsatellite's primary sequence (Figure 2). Under the assumption that the KRT8Sat alleles themselves are functionally equivalent and neutral towards the studied phenotypes, we conclude that the benign KRT8 haplotype 2211 is older than the severe 1122 allele as one slippage event, explaining the KRT8Sat allele distribution on the 1122 background, requires fewer generations than the multiple recombination events that have generated the microsatellite's sequence pattern on 2211 alleles.

Bottom Line: In consistency, the KRT8 haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype.The two opposing KRT8 alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation.As the mild KRT8 allele is associated with CFTR mediated residual chloride secretion among F508del-CFTR homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Hannover Medical School, Hannover, Germany. mekus.frauke@mh-hannover.de

ABSTRACT

Background: F508del-CFTR, the most frequent disease-causing mutation among Caucasian cystic fibrosis (CF) patients, has been characterised as a mutant defective in protein folding, processing and trafficking. We have investigated the two neighbouring cytokeratin genes KRT8 and KRT18 in a candidate gene approach to ask whether variants in KRT8 and/or KRT18 modify the impaired ion conductance known as the CF basic defect, and whether they are associated with correct trafficking of mutant CFTR and disease severity of CF.

Methods: We have selected contrasting F508del-CFTR homozygous patient subpopulations stratified for disease severity, comparing 13 concordant mildly affected sib pairs vs. 12 concordant severely affected sib pairs, or manifestation of the CF basic defect in intestinal epithelium, comparing 22 individuals who exhibit CFTR-mediated residual chloride secretion vs. 14 individuals who do not express any chloride secretion, for an association. The KRT8/KRT18 locus was initially interrogated with one informative microsatellite marker. Subsequently, a low density SNP map with four SNPs in KRT8 and two SNPs in KRT18, each selected for high polymorphism content, was used to localize the association signal.

Results: KRT8, but not KRT18, showed an association with CF disease severity (Pbest=0.00131; Pcorr=0.0185) and CFTR mediated residual chloride secretion (Pbest=0.0004; Pcorr=0.0069). Two major four-marker-haplotypes spanning 13 kb including the entire KRT8 gene accounted for 90% of chromosomes, demonstrating strong linkage disequilibrium at that locus. Absence of chloride secretion was associated with the recessive haplotype 1122 at rs1907671, rs4300473, rs2035878 and rs2035875. The contrasting haplotype 2211 was dominant for the presence of CFTR mediated residual chloride secretion. In consistency, the KRT8 haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype. Analysis of microsatellite allele distributions on the SNP background suggests that the mild KRT8 haplotype 2211 is phylogenetically older than its severe counterpart.

Conclusions: The two opposing KRT8 alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation. As the mild KRT8 allele is associated with CFTR mediated residual chloride secretion among F508del-CFTR homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.

Show MeSH
Related in: MedlinePlus