244Functional variants in the 3'Untranslated Region of KCNQ1 strongly modify disease severity in type 1 long QT syndrome in an allele-specific manner by creating binding sites for miR-378

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Type 1 long QT syndrome (LQT1) is a channelopathy caused by mutations in KCNQ1 and characterized by prolonged heart rate-corrected QT interval (QTc) and life-threatening arrhythmias. It is unknown why disease penetrance is so variable between individuals hosting identical causative mutations. We hypothesize that SNPs in the 3'UTR of KCNQ1 modify disease severity by creation of novel miRNA binding sites, resulting in repression of translation of that particular SNP-containing allele.

We included 168 heterozygous KCNQ1-mutation carriers and sequenced the last exon and the 3‘UTR of KCNQ1. We identified three SNPs in the 3'UTR (rs2519184, rs8234, and rs10798) significantly associated with QTc duration. LQT1-patients with the minor alleles of these SNPs on their mutant allele have markedly shorter QTc and fewer symptoms. In contrast, when patients have the minor alleles of these SNPs on their normal allele, they have significantly longer QTc and more symptoms.

To investigate the functional role of the minor alleles of these three SNPs, we generated two reporter constructs containing respectively the major alleles of all three SNPs and the minor alleles. Using these constructs we show that there is a significant 25% repression of translation of the luciferase gene by the minor alleles containing 3'UTR of KCNQ1 in H10-cells and cultured cardiomyocytes. Furthermore we show the allelic-imbalance between the 2 haplotypes by allele-specific Q-PCR on myocardial tissue.

By in silico analysis we determined whether the three SNPs were expected to create target sites for miRNAs and identified miR-378 as the possibly responsible miRNA, because the minor alleles of two of the three SNPs create binding sites for this miRNA. MiR-378 is abundantly expressed in the heart. Overexpression of miR-378 in cos7-cells showed the ability of this miRNA to bind to the minor haplotype and knockdown of this miRNA in cultured cardiomyocytes normalized the luciferase signal of the minor haplotype reporter.

In conclusion, we show that SNPs in the 3'UTR of KCNQ1 suppress translation by creating two functional binding sites for miR-378. In KCNQ1-mutation carriers, these functional SNPs explain a part of the incomplete penetrance and variable expressivity of the mutation associated with LQT1 by creating allelec imbalance.

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