KLF13, a member of the KFL family of transcription factors, has been identified as a novel cardiac factor that is involved in heart development [8, 18]. Previous research by Lavallee et al. showed that KLF13 is expressed during cardiac morphogenesis in mice and knocking it down induced ventricular hypotrabeculation, atrial septal defects (ASDs), and delayed AV-cushion formation in Xenopus embryos [8]. However, variants of KLF13 in CHD patients have remained unidentified and unelucidated. In the present study, two novels heterozygous KLF13 variants were reported using targeted sequencing in 309 patients with complex CHDs. The variant, c.467G>A (S156N), identified in the patients with TA, VSD, and ASD, had altered protein expression and functionality compared with that of the wild-type protein, without affecting the subcellular localization. The other variant, c.487C>T (P163S), found in a D-TGA patient, did not show any abnormalities in protein expression or subcellular localization. However, this variant had altered transcriptional activities of downstream genes and altered physical interactions with another cardiac factor, TBX5.
Both variants, c.467G>A and c.487C>T, were highly preserved in vertebrates based on multiple sequence-alignment analyses and were located in the NLS1 region. Song et al. showed that a bipartite NLS was identified in KLF13 protein based on sequence searching, and also found that AA 147–168 (NLS1) was not the only NLS region for protein subcellular localization because deletion of this signal did not affect nuclear transport [12]. In our present results, although these two variants were located in the NLS1 region, they were also expressed in the nucleus, as was the wild-type KLF13 protein. However, the locations of these two variants were close to Cys2/His2 zinc-finger motifs that bind to gene promoters and enhancer regions to activate or inhibit transcription [20], suggesting that these two variants might have crucial functions in mediating the expression of downstream target genes. Lavallee et al used BNP promoter and showed that KLF13 could activate the promoter by binding to an evolutionarily conserved CACCC box [8]. Although the subcellular localization of the variant protein did not change, which is In contrast to most previously reported CHDs associated variants [21–22], we found that the S156N variant was a gain-of-function mutation, since it significantly increased the transcriptional activity of BNP. Thus, mutation of this sites maybe may promote DNA binding activity by increasing the protein, which may well explain the up-regulated BNP in this study. Although a gain-of-function was detected in our experiments, the protein that might be less stable in vivo could also result in a loss-of-function phenotype [23–24]. Consistent with this, human heterozygous micro-deletions and duplication of the chromosomal band harboring KLF13 (15q13.3), have been demonstrated to be associated with a wide range of cardiac defects [25]. Unlike the S156N variant, the c.487C>T variant decreased the transcriptional activity of BNP in the present study, but we did not find any disruption of expression or subcellular localization. However, a proline-to-serline substitution maybe result in impaired interactions with DNA, and the location of the P163S variant in the tertiary structure that is near the bound DNA also might lead to a decrease in the DNA-binding activity. Thus, dysregulation of downstream target genes during cardiac morphogenesis may be the reason why this variant caused TGA.
The reduced binding ability of our identified variants to targets may be primarily related to decrease DNA binding. However, structural instability, when bound to cofactors is another possibility [26]. The mechanism of cardiac transcription factors in the developing heart involves cooperative interactions with other conserved factors, and any structural defect is often linked to more than one candidate gene. Also, variable expressivity of the phenotype is often observed where CHDs are linked to a specific gene mutation [27]. Thus, variants of these factors may eliminate any cooperative interactions between these cofactors [27–28]. Previously, we showed that KLF13 and TBX5 exhibit physical and functional interactions, and that a combined heterozygous loss of TBX5 and its associated KLF13 leads to decreased postnatal viability and a higher incidence of septal defects compared to that of Tbx5 heterozygous mice. Additionally, several TBX5 mutations are associated with CHDs, as indicated by impaired functional and physical interactions with KLF13, which confirms that TBX5 is a genetic modifier of KLF13 [10]. In the present study, increased transcription activity of BNP was more pronounced when wild type KLF13 was co-transfected with TBX5, which indicated that KLF13 has functional interactions with TBX5 to activate BNP promoters. In our previous study of the structure-function of the TBX5-KLF13 synergy [10], the N-terminal domain of KLF13 was mainly found to support synergy with TBX5. The two novel variants of KLF13 that were detected in the 309 CHDs patients were in the N-terminal domain of the protein. However, the two variants had different effects on the interactions between TBX5. Significantly enhanced synergistic activity was detected in the S156N variant, while the P163S variant eliminated this synergistic activity, which suggested that the loss of function observed in KLF13 was associated with disruption of physical and functional interactions with cofactors (TBX5), while the gain of function could enhance the interaction with cofactors.