Variants in the 3' untranslated region of the KCNQ1-encoded Kv7.1 potassium channel modify disease severity in patients with type 1 long QT syndrome in an allele-specific manner

Abstract

Aims: Heterozygous mutations in KCNQ1 cause type 1 long QT syndrome (LQT1), a disease characterized by prolonged heart rate-corrected QT interval (QTc) and life-threatening arrhythmias. It is unknown why disease penetrance and expressivity is so variable between individuals hosting identical mutations. We aimed to study whether this can be explained by single nucleotide polymorphisms (SNPs) in KCNQ1's 3' untranslated region (3'UTR).

Methods and results: This study was performed in 84 LQT1 patients from the Academic Medical Center in Amsterdam and validated in 84 LQT1 patients from the Mayo Clinic in Rochester. All patients were genotyped for SNPs in KCNQ1's 3'UTR, and six SNPs were found. Single nucleotide polymorphisms rs2519184, rs8234, and rs10798 were associated in an allele-specific manner with QTc and symptom occurrence. Patients with the derived SNP variants on their mutated KCNQ1 allele had shorter QTc and fewer symptoms, while the opposite was also true: patients with the derived SNP variants on their normal KCNQ1 allele had significantly longer QTc and more symptoms. Luciferase reporter assays showed that the expression of KCNQ1's 3'UTR with the derived SNP variants was lower than the expression of the 3'UTR with the ancestral SNP variants.

Conclusion: Our data indicate that 3'UTR SNPs potently modify disease severity in LQT1. The allele-specific effects of the SNPs on disease severity and gene expression strongly suggest that they are functional variants that directly alter the expression of the allele on which they reside, and thereby influence the balance between proteins stemming from either the normal or the mutant KCNQ1 allele.

Figure 1

Genetic variation in the 3′ untranslated region of KCNQ1. Single nucleotide polymorphisms (SNPs) found in the study cohorts of the Academic Medical Center Amsterdam (AMC) and the Mayo Clinic (MC). Position of the nucleotide change is starting from the ATG start codon (NCBI build 36, hg18).

Figure 2

Allele-specific effects of single nucleotide polymorphisms (SNPs) rs2519184, rs8234, and rs10798 on QTc. Allele-specific effects of SNPs rs2519184, rs8234, and rs10798 on QTc are displayed in panels (A and B) and (C) for the AMC population, and (D  and E), and (F) for the MC population. Effects of SNP rs2519184 are displayed in (A) and (D). Effects of SNPs rs8234 and rs10798 are displayed in (B) and (E). Allele-specific haplotype analysis of the three SNPs is displayed in (C) and (F). The red line represents mean QTc of all individuals regardless of the specific LQT1-causative mutation and the 3′ untranslated region SNP status. Numbers below genotypes denote group sizes. Data are presented as mean; I bars represent standard errors. N, normal KCNQ1 allele; M, mutant KCNQ1 allele; green box, ancestral SNP variant; yellow box, derived SNP variant.

Figure 3

Allele-specific effects of single nucleotide polymorphisms (SNPs) rs2519184, rs8234, and rs10798 on QTc in three single families. Allele-specific haplotype analysis of SNPs rs2519184, rs8234, and rs10798 with regard to QTc are displayed for the three largest families: one family carrying the KCNQ1-R243C mutation (A), one family with the KCNQ1-I235N mutation (B), and one family carrying the KCNQ1-339delF mutation (C). Numbers below genotypes denote group sizes. Data are presented as mean; I bars represent standard errors. N, normal KCNQ1 allele; M, mutant KCNQ1 allele; green box, ancestral SNP variant; yellow box, derived SNP variant.

Figure 4

Allele-specific effects of single nucleotide polymorphisms (SNPs) rs2519184, rs8234, and rs10798 on symptomatology. Allele-specific effects of SNPs rs2519184, rs8234, and rs10798 with regard to the occurrence of cardiac symptoms [unexplained syncope, documented torsades de pointes ventricular tachycardia/ventricular fibrillation, and/or (aborted) sudden death] are shown in the total combined study population (AMC and MC). Effects of SNP rs2519184 are displayed in (A). Effects of SNPs rs8234 and rs10798 are displayed in (B). Allele-specific haplotype analysis of the three SNPs with regard to the occurrence of symptoms is displayed in (C). Numbers below genotypes denote group sizes. Data are presented as mean; I bars represent standard errors. N, normal KCNQ1 allele; M, mutant KCNQ1 allele; green box, ancestral SNP variant; yellow box, derived SNP variant.

Figure 5

The functional effects of genetic variation in the 3′untranslated region (3′UTR) of KCNQ1 in vitro. (A) Luciferase assays in primary neonatal rat cardiomyocytes transfected with two independent reporter plasmids containing either the ancestral or the derived haplotype of single nucleotide polymorphisms (SNPs) in KCNQ1's 3′UTR. Observed differences in luciferase activity between the ancestral and derived haplotype suggest that expression is inhibited. (B) Luciferase assays in neonatal rat cardiomyocytes transfected with a reporter plasmid containing either the ancestral haplotype of the 3′UTR of KCNQ1 or the same reporter where the SNPs rs2519184, rs8234, and rs10798 were separately changed to the derived allele. Introducing either of the derived SNP variants was sufficient to decrease the luciferase activity. Data are presented as means; I bars denote standard errors. Green box, ancestral SNP variant; yellow box, derived SNP variant.

Figure 6

The postulated mechanism where single nucleotide polymorphisms (SNPs) in the 3′UTR of KCNQ1 modulate the assembly of the K_v7.1 potassium channel in type 1 long QT syndrome. Individuals with LQT1 are heterozygous for the disease-causing mutation in KCNQ1. Four KCNQ1-encoded subunits co-assemble to form one tetrameric channel. Single nucleotide polymorphisms in this region may influence repolarization by altering the balance and composition of the K_v7.1 tetramers derived from expression of the normal allele and the mutant allele. If SNPs exert no effect on expression, the balance between normal and mutant subunits within each channel is equal. However, if the derived variants of the SNPs cause reduced expression, then the balance between normal and mutant subunits within each channel depends on whether the ‘suppressive’ SNP variants reside on the normal allele or the mutant allele. If the ‘suppressive’ SNP variants reside on the normal allele, the number of normal subunits in the channels would decrease. Inversely, if the ‘suppressive’ SNP variants reside on the mutant allele, this would decrease the relative number of mutant subunits and shift the K_v7.1 tetramers to a greater percent of normal allele-derived monomeric subunits.