A novel KLF13 mutation underlying congenital patent ductus arteriosus and ventricular septal defect, as well as bicuspid aortic valve

Abstract

Recently, mutations in the Kruppel-like factor 13 (KLF13) gene encoding a Kruppel-like transcription factor have been reported to cause congenital heart disease (CHD). However, due to pronounced genetic heterogeneity, the mutational spectrum of KLF13 in other cohorts of cases suffering from distinct types of CHD remain to be ascertained. In the present investigation, by Sanger sequencing of KLF13 in 316 unrelated cases affected by different forms of CHD, a new mutation in heterozygous status, NM_015995.3: c.430G>T; p.(Glu144*), was detected in an index patient affected with patent ductus arteriosus (PDA) and ventricular septal defect (VSD), as well as bicuspid aortic valve (BAV), with a mutation frequency of ~0.32%. Genetic investigation of the available family members of the proband demonstrated that the truncating mutation co-segregated with CHD. The nonsense mutation was not observed in 400 unrelated volunteers without CHD who were enrolled as control subjects. Quantitative biological measurements with dual luciferase reporters revealed that Glu144*-mutant KLF13 did not transactivate the downstream genes vascular endothelial growth factor A and natriuretic peptide A. In addition, the mutation abrogated the synergistic transcriptional activation between KLF13 and T-box transcription factor 5, a well-established CHD-causing gene. In conclusion, the present study indicates that genetically defective KLF13 contributes to familial PDA and VSD, as well as BAV, which expands the phenotypic spectrum linked to KLF13, and reveals a novel molecular pathogenesis of the disease, providing a new molecular target for the early prophylaxis and individualized treatment of CHD.

Keywords: KLF13; biochemical assay; congenital heart defect; molecular genetics; transgene.

Figure 1

A new KLF13 mutation contributing to congenital heart disease. (A) Sequence electropherograms illustrating the KLF13 mutation in the heterozygous status (mutant) and its wild-type base in the homozygous status (wild-type). The arrow indicates the nucleotides where the mutation occurs. (B) Schematic representations exhibiting the structural domains of KLF13. (C) Pedigree structure of the family inflicted with congenital heart disease (+ represents a carrier of the KLF13 mutation and-represents a non-carrier). TAD, transcriptional activation domain; TID, transcriptional inhibitory domain; NLS, nuclear location signal; Zn, zinc finger.

Figure 2

Diminished transcriptional activity of KLF13 caused by the mutation. In cultivated NIH3T3 cells expressing various recombinant plasmids, activation of NPPA-luc by wild-type or Glu144*-mutant KLF13, singly or together, revealed that Glu144*-mutant KLF13 failed to transactivate its downstream target gene NPPA. For each eukaryotic expression plasmid, cellular transient transfection experiments were repeated three times in triplicates. ^*P=0.00056 and ^**P=0.00604 compared with wild-type KLF13. NPPA-luc, human natriuretic peptide precursor A luciferase; KLF13, Kruppel-like factor 13.

Figure 3

Synergistic transcriptional activation between KLF13 and TBX5 disrupted by the mutation. In cultivated NIH3T3 cells, the synergistic transactivation of the promoter of VEGFA by KLF13 and TBX5 was abrogated by the Glu144* mutation. ^*P=0.00058 and ^**P=0.00008 compared with to respective wild-type counterparts. VEGFA, human vascular endothelial growth factor A luciferase; TBX5, T-box transcription factor 5; KLF13, Kruppel-like factor 13.