Abstract
SCNN1B encodes the beta subunit of the epithelial sodium channel ENaC. Previously, we reported an association between SNP markers of SCNN1B gene and disease severity in cystic fibrosis-affected sibling pairs. We hypothesized that factors interacting with the SCNN1B genomic sequence are responsible for intrapair discordance. Concordant and discordant pairs differed at six SCNN1B markers (Praw = 0.0075, Pcorr = 0.0397 corrected for multiple testing). To identify the factors binding to these six SCNN1B SNPs, we performed an electrophoretic mobility shift assay and captured the DNA–protein complexes. Based on protein mass spectrometry data, the epithelial splicing regulatory protein ESRP2 was identified when using SCNN1B-derived probes and the ESRP2-SCNN1B interaction was independently confirmed by coimmunoprecipitation assays. We observed an alternative SCNN1B transcript and demonstrated in 16HBE14o− cells that levels of this transcript are decreased upon ESRP2 silencing by siRNA. Furthermore, we confirmed that mildly and severely affected siblings have different ESPR2 genetic backgrounds and that ESRP2 markers are linked to the response of CF patients’ nasal epithelium to amiloride, indicating ENaC involvement (Pbest = 0.0131, Pcorr = 0.068 for multiple testing). Our findings demonstrate that sibling pairs clinically discordant for CF can be used to identify meaningful DNA regulatory elements and interacting factors.
Highlights
Genetic variation in humans contributes significantly to phenotypic variation
We previously reported that intrapair discordance for cystic fibrosis (CF) disease severity is associated with three intragenic markers spanning SCNN1B from codon 3 to codon 293
We found a significant difference in two-marker-haplotype distributions for two adjacent genomic fragments defined by markers rs152730–rs152745 and rs152745–rs152740 (Praw = 0.0075 and Praw = 0.00869, respectively; corrected for multiple testing of all informative markers at the SCNN1G/SCNN1B-locus Pcorr = 0.0397, Fig. 1)
Summary
Genetic variation in humans contributes significantly to phenotypic variation. The question of which single nucleotide polymorphism (SNP) determines disease outcome and/or severity, has been addressed in more than 2000 genome-wide association studies (GWAS)[1]. Mapping of the association signal determining intrapair discordance among F508del-CFTR homozygous CF sibling pairs, identification of an alternative SCNN1B transcript and position of SNPs analyzed by EMSA-PSeq. When discordant sibling pairs were compared to concordant sibling pairs, one SCNN1B haplotype defined by SNPs rs238547–rs152730–rs250563 occurred more frequently among discordant than among concordant siblings[11] We concluded that this signal cannot be fully explained by a variant observed within SCNN1B because discordant siblings have a dissimilar phenotype by d efinition[12], and yet these siblings share an SCNN1B intragenic haplotype[11]. The genetic variation of the interaction partner can determine the phenotype causing intrapair discordance in affected sibling pairs
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