Identification and analysis of KLF13 variants in patients with congenital heart disease

Li W, Li B, Li T, Zhang E, Wang Q, Chen S, Sun K.

BMC Med Genet. 2020 Apr 15;21(1):78. doi: 10.1186/s12881-020-01009-x.

PMID: 32293321 Free PMC Article

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Background: The protein Kruppel-like factor 13 (KLF13) is a member of the KLF family and has been identified as a cardiac transcription factor that is involved in heart development. However, the relationship between KLF13 variants and CHDs in humans remains largely unknown. The present study aimed to screen the KLF13 variants in CHD patients and genetically analyze the functions of these variants.

Methods: KLF13 variants were sequenced in a cohort of 309 CHD patients and population-matched healthy controls (n = 200) using targeted sequencing. To investigate the effect of variants on the functional properties of the KLF13 protein, the expression and subcellular localization of the protein, as well as the transcriptional activities of downstream genes and physical interactions with other transcription factors, were assessed.

Results: Two heterozygous variants, c.487C > T (P163S) and c.467G > A (S156N), were identified in two out of 309 CHD patients with tricuspid valve atresia and transposition of the great arteries, respectively. No variants were found among healthy controls. The variant c.467G > A (S156N) had increased protein expression and enhanced functionality compared with the wild type, without affecting the subcellular localization. The other variant, c.487C > T (P163S), did not show any abnormalities in protein expression or subcellular localization; however, it inhibited the transcriptional activities of downstream target genes and physically interacted with TBX5, another cardiac transcription factor.

Conclusion: Our results show that the S156N and P163S variants may affect the transcriptional function of KLF13 and physical interaction with TBX5. These results identified KLF13 as a potential genetic risk factor for congenital heart disease.


Fig. 1 Sequence chromatograms of KLF13 missense variants in patients and controls. a and c: Chromatograms of normal controls. b and d: Chromatograms of the three heterozygous variants. Arrows show heterozygous nucleotide changes; e: Location of the KLF13 variants (highlighted in bold). NLS, nuclear-localization signals; Zn, zinc finger domain. f: Alignments of KLF13 protein among different species indicated that S156N and P163S were conserved among vertebrates


Fig. 2 Characterization of KLF13 variants. a: Immunocytochemical staining to detect the cellular localization of KLF13 variants transfected in 293 T cells. The green color shows the Alexa 488 conjugated secondary antibodies against the anti-myc antibody. The nuclei were visualized with DAPI. b: Western blotting showing the expression of the KLF13 variants. c: The transcriptional activities of KLF13 variants on BNP promoter. Transfections were carried out using the human B-type natriuretic peptide (BNP) promoter and increasing dosages (25, 50, 100, 200, and 300 ng) of expression vectors. Each dosage was conducted in duplicate and the experiment was conducted twice



Fig. 3 KLF13 variants have effects on genetic interaction with TBX5. a: Luciferase activity in NIH3T3 cells cotransfected with wild-type KLF13 or mutant-expression constructs and the BNP promoter, showing that the P163S mutant could decrease the synergistic activation of the BNP promoter, while the S156N mutant increased the synergistic activation of BNP.b: Coimmunoprecipitations were carried out using 293 T cells expressing Myc-KLF13 and TBX5 proteins in combination. The top panel shows Western blots of the nuclear extracts used, with the indicated antibodies. The bottom panel is a Western blot of the anti-TBX5 immunoprecipitates. (*p < 0.05, Statistical significant vs WT, # p < 0.05, Statistical significant vs WT co-transfected with TBX5)