Transcriptome-wide association identifies KLC1 as a regulator of mitophagy in non-syndromic cleft lip with or without palate

Abstract

This study investigated pathogenic genes associated with non-syndromic cleft lip with or without cleft palate (NSCL/P) through transcriptome-wide association studies (TWAS). By integrating expression quantitative trait loci (eQTL) data with genome-wide association study (GWAS) data, we identified key susceptibility genes, including KLC1. Notably, the variant rs12884809 G>A was associated with an increased risk of NSCL/P by enhancing the binding of the transcription factor ELK1 to the KLC1 promoter, thereby activating its expression. This alteration in KLC1 expression subsequently impacted mitophagy, leading to significant changes in cellular behavior and zebrafish morphology. Our findings illuminate the genetic mechanisms underlying NSCL/P and provide valuable insights for future prevention strategies and a deeper understanding of this condition.

FIGURE 1

Genetic associations by both genome‐wide association studies (GWAS) and transcriptome‐wide association studies (TWAS) and allele‐specific effect of rs12884809. (A) Manhattan plot of the cross‐tissue TWAS results for NSCL/P. The red horizontal line indicates genes with nominal significance (p < 1 × 10⁻⁴). (B) GWAS and expression quantitative trait loci (eQTL) associations for KLC1. Left panel: The relationship between GWAS and eQTL is presented as a scatter plot, of which the x‐axis represents GWAS p‐value in −log₁₀ scale for NSCL/P and the y‐axis represents the whole blood eQTL p‐value in −log₁₀ scale. Right panel: The regional association plots present the GWAS association (upper panel) and the eQTL association (lower panel) for rs12884809 in whole blood. (C) Overview of the H3K4me1, H3K27ac, and H3K4me3 chromatin modifications, and DNase cluster distribution in the regions surrounding rs12884809 of the early human craniofacial tissues supported from the UCSC genome browser. Rs12884809 is indicated by the orange vertical line. (D) Histone H3K4me3 enrichment at SNP rs12884809 site in human oral keratinocyte (HOK) and human palatal mesenchymal (HEPM) cells by chromatin immunoprecipitation followed by quantitative PCR (ChIP‐qPCR) analysis. The p values were calculated using a two‐tailed unpaired Student's t‐test. *** indicates p < 0.001. (E) Dual‐luciferase reporter assay of promoter activity affected by rs12884809. Rs12884809 had allelic effects on promoter activity in HOK and HEPM cells, where A allele had higher activity than G allele. The p values were calculated using a two‐tailed unpaired Student's t‐test. * indicates p < 0.05. (F) The ELK1 binding site to the A allele of rs12884809 was predicted using PERFECTOS‐APE.

FIGURE 2

In vivo and in vitro role of KLC1 and its regulation of mitophagy. (A) The klc1a crispant embryos showed a shorter body length, curvature of the spine, craniomaxillofacial deficiency (indicated by red dashed lines) and edema around the heart (indicated by red arrow). (B) Quantitative analysis of body length in 120 hpf zebrafish embryos. (C) Upper panel: Alcian blue staining was used to assess the length of the palatoquadrate in zebrafish embryos at 144 hpf. The klc1a crispant embryos displayed a shorter palatoquadrate length compared to the other groups (indicated by red arrow). Lower panel: Alcian blue staining of the length and width of the ethmoid palate in zebrafish embryos at 144 hpf. The klc1a crispant embryos showed a cleft in the ethmoid palate (indicated by red arrow). (D) The lip morphology of zebrafish. The klc1a crispant embryos exhibited multiple lip deformities and clefts (indicated by red dashed lines), whereas zebrafish in other groups showed intact, oval‐shaped lips. oe, overexpression. (E) Gene‐set analysis on the NSCL/P GWAS data. Blue dotted lines represent statistically significant differences. (F) Western blot validation of mitophagy‐related genes in HOK and HEPM cells with KLC1 knockdown (si) or overexpression (oe). Relative quantification numbers of protein expression are shown below the lanes. (G) Live cell imaging showed co‐localization of lysosomes and mitochondria (Lyso‐Tracker and Mito‐Tracker, indicated by white arrows), and autophagosomes and mitochondria (GFP‐LC3B and Mito‐Tracter, indicated by red arrows) in HEPM cells. (H) Transmission electron microscopy (TEM) images showed mitophagy changes in HEPM cells with or without KLC1 knockdown/overexpression (scale bar = 2 μm and 500 nm). Red arrows indicate autophagosomes involved in the mitophagy process. (I) JC‐1 staining demonstrated changes in mitochondrial membrane potential in HEPM cells. KLC1 knockdown led to (J) increased apoptosis, (K) reduced migration, and (L) proliferation in HEPM cells, all of which were partially rescued by treatment with the mitophagy inhibitor Mdivi‐1. ns, not significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.