Strategies to identify clinically relevant interaction partners of cystic fibrosis modifying genes: analysis of SCNN1B

Abstract

The course of the monogenic disease cystic fibrosis is influenced by non-genetic factors and by CF modifier genes. During the last years, several CF modifying genes were identified and the clinically relevant variants were mapped by the base in the patient population of the European CF Twin and Sibling study. The identified genetic variants largely are located within the non-coding sequence of the human genome, thus implying that they do not change the amino acid sequence of the corresponding modifier gene but instead reflect an essential regulatory mechanism. During the last years, we have used complementary technologies to describe the molecular mechanism that governs the expression of the cystic fibrosis modifier gene SCNN1B. To identify the causative variants, 3 microsatellites and 45 SNPs were genotyped on 101 families with a total of 171 F508del-CFTR homozygous CF patients. Resequencing of two 8000 bp fragments for which discordant and concordant sibling pairs carry contrasting genetic information has revealed six possible causative SNPs. Bioinformatic predictions and subsequent testing of the predicted interaction partners as candidate genes have so far confirmed one transcription factor as a CF modifying gene. Using both alleles of the six identified SCNN1B SNPs in a comparative electrophoretic mobility shift assay and subsequent protein sequencing of the excised bands has revealed previously unknown novel DNA-protein interactions that are likely to mediate SCNN1B expression. Continuous support by the Fritz-Thyssen-Stiftung is gratefully acknowledged.