KLF15 Loss-of-Function Mutation Underlying Atrial Fibrillation as well as Ventricular Arrhythmias and Cardiomyopathy

Abstract

Atrial fibrillation (AF) represents the most common type of clinical cardiac arrhythmia and substantially increases the risks of cerebral stroke, heart failure and death. Accumulating evidence has convincingly demonstrated the strong genetic basis of AF, and an increasing number of pathogenic variations in over 50 genes have been causally linked to AF. Nevertheless, AF is of pronounced genetic heterogeneity, and the genetic determinants underpinning AF in most patients remain obscure. In the current investigation, a Chinese pedigree with AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy was recruited. Whole exome sequencing and bioinformatic analysis of the available family members were conducted, and a novel heterozygous variation in the KLF15 gene (encoding Krüppel-like factor 15, a transcription factor critical for cardiac electrophysiology and structural remodeling), NM_014079.4: c.685A>T; p.(Lys229*), was identified. The variation was verified by Sanger sequencing and segregated with autosomal dominant AF in the family with complete penetrance. The variation was absent from 300 unrelated healthy subjects used as controls. In functional assays using a dual-luciferase assay system, mutant KLF15 showed neither transcriptional activation of the KChIP2 promoter nor transcriptional inhibition of the CTGF promoter, alone or in the presence of TGFB1, a key player in the pathogenesis of arrhythmias and cardiomyopathies. The findings indicate KLF15 as a new causative gene responsible for AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy, and they provide novel insight into the molecular mechanisms underlying cardiac arrhythmias and hypertrophic cardiomyopathy.

Keywords: KLF15; arrhythmia; atrial fibrillation; cardiomyopathy; genetics; transcription factor.

Figure 1

A novel KLF15 mutation responsible for familial atrial fibrillation. (A) Pedigree structure for the Chinese family with atrial fibrillation. The Chinese family was arbitrarily designated as family 1. Family members are identified by Roman-Arabic numerals. “+” indicates an individual carrying the heterozygous mutation; “–”, an individual with no mutation. (B) Sequence chromatograms showing the heterozygous KLF15 mutation and its wild-type control. An arrow symbol points to the heterozygous nucleotides of T/A in the proband (mutant) or the homozygous nucleotides of A/A in an unaffected individual (wild type). A rectangle marks 3 nucleotides comprising a codon of KLF15. (C) Schematic diagrams of the structural domains of human KLF15 proteins displaying the mutation contributing to atrial fibrillation. COOH, carboxyl-terminus; NH2, amino-terminus; TAD, transcriptional activation domain; ZF, zinc finger.

Figure 2

Functional loss of KLF15 caused by the mutation. In cultured HeLa cells, activation of the KChIP2 promoter-driven luciferase by wild-type (WT) or Lys229*-mutant KLF15 (Mutant), alone or in combination, showed that the mutant had no transcriptional activity. Experiments were done in triplicate, and the results are expressed as mean values ± SD. ^## and ^# indicate p < 0.001 and p < 0.01, respectively, compared with the same amount of homozygous WT.

Figure 3

Diminished inhibitory effect of KLF15 on CTGF resulting from the mutation. In cultivated NIH 3T3 cells, activation of the CTGF promoter-driven luciferase by wild-type (WT) or Lys229*-mutant KLF15 (Mutant), in the absence or presence of TGFB1, showed that the inhibitory effect of the mutant on CTGF was significantly decreased. Experiments were carried out in triplicate, and the data were given as mean values ± SD. ^## and ^# indicate p < 0.05 and p < 0.01, respectively, in comparison with their wild-type counterparts.