Identification of a New Susceptibility Gene ALX4 in Non-syndromic Orofacial Cleft

Abstract Objectives Muscle is one of the important tissues that constitute the structure of lip and palate and its development might play some role in the formation of non-syndromic orofacial cleft (NSOC), a congenital orofacial malformation. This study was aimed to identify new susceptibility genes in NSOC. Materials and Methods We collected 18 candidate genes involved in the process of muscle and performed a two-stage case-control association analysis in western Han Chinese population. Then we extracted RNA from the lip tissue of NSCLO patients to perform RNA sequencing and confirmed the up-regulation of differentially expressed gene and microRNA with Real-time quantitative PCR. Bioinformatic analysis and dual luciferase reporter assay were sed to explore the transcriptional regulation of microRNA. Results ALX4 was most significant in the discovery phase and replication analysis further confirmed its association with NSOC subtypes. RNA sequencing found that ALX4 and has-miR-455-3p were both differentially up-regulated in NSCLO patients. Bioinformatics analysis and validation study proved that ALX4 is transcriptionally regulated by has-miR-455-3p as its target gene. Conclusion This study identified that ALX4 was a new susceptibility gene for NSOC in western Han Chinese population. The up-regulation of ALX4 transcription level in NSCLO patients might be regulated by has-miR-455-3p.