Association Between Variants Within Aristaless-Like Homeobox 4 and Non-syndromic Orofacial Cleft in Western Han Chinese

Objective To investigate Aristaless-like homeobox 4 ( ALX4), a paired-like homeodomain transcription factor essential for craniofacial morphogenesis, as a susceptibility gene for non-syndromic orofacial cleft (NSOC) and to explore its potential regulatory mechanisms in the Han Chinese population. Design A two-stage case-control genetic association study complemented by exploratory RNA sequencing (RNA-seq) and functional validation. Setting Tertiary medical center for orofacial cleft treatment in western China. Patients, Participants Discovery phase: 2512 NSOC patients and 2255 controls. Replication phase: 2724 NSOC patients and 1263 controls. RNA-seq: 6 patients with non-syndromic cleft lip only (NSCLO) and 2 lip trauma controls. Real-time quantitative PCR (RT-qPCR) validation: 5 NSCLO patients and 5 controls. Interventions : Genotyping of 76 tag single-nucleotide polymorphisms (SNPs) within the ALX4 gene region using the SNPscan method, with three SNPs selected for independent replication. Exploratory RNA-seq of lip tissues, RT-qPCR validation, and dual-luciferase reporter assays. Main Outcome Measure(s) Allelic and genotypic associations between ALX4 variants and NSOC subtypes; differential expression of ALX4 and hsa-miR-455-3p in NSCLO tissues; functional validation of microRNA-target interactions. Results Multiple ALX4 SNPs showed significant associations with NSOC subtypes after Bonferroni correction ( P < 6.58 × 10 −4 ). rs3861063 demonstrated consistent protective effects for microform cleft lip across both phases. ALX4 and hsa-miR-455-3p were upregulated in NSCLO tissues, confirmed by RT-qPCR. Dual-luciferase assays confirmed that hsa-miR-455-3p directly targets the ALX4 3'UTR (untranslated region), though paradoxical co-upregulation suggests complex regulatory mechanisms. Conclusions ALX4 is a novel NSOC susceptibility gene in Han Chinese, with subtype-specific genetic associations and a complex regulatory interaction involving hsa-miR-455-3p, expanding our understanding of NSOC genetic architecture.