Identification of TOX and SLC38A4 as new candidate susceptible genes for non-syndromic cleft lip with or without palate

This study aimed to identify differentially expressed genes (DEGs) associated with non-syndromic cleft lip with or without palate (NSCL/P) and explore their genetic variants related to NSCL/P susceptibility. Microarray datasets GSE42589 and GSE85748 which respectively contained dental pulp stem cells (DPSC) and orbicularis oris muscle mesenchymal stem cells (OOMMS) from NSCL/P cases and controls were used for screening overlapping DEGs of NSCL/P. A case-control study (504 NSCL/P cases and 455 controls) was performed to investigate the associations between single nucleotide polymorphisms (SNPs) in DEGs and NSCL/P susceptibility. Expression quantitative trait loci (eQTL) analysis was conducted from the Genotype-Tissue Expression (GTEx) database. Functions of genes were explored in human embryonic palatal mesenchymal (HEPM) and human oral keratinocytes (HOK) cells, as well as zebrafish models. Co-expression and pathway enrichment analysis were performed based on mouse craniofacial structures data from FaceBase. Three up-regulated genes ( SLC38A4 , TOX , RASGRP3 ) and one down-regulated gene ( MEDAG ) were differentially expressed between NSCL/P and controls. Two SNPs in TOX (rs7838449: P = 1.77 × 10 − 2 ; rs2008398: P = 1.30 × 10 − 2 ) and four SNPs in SLC38A4 (rs12316767: P = 1.15 × 10 − 2 ; rs12582305: P = 1.73 × 10 − 2 ; rs4554975: P = 2.69 × 10 − 2 ; rs11183619: P = 4.00 × 10 − 2 ) were nominally associated with NSCL/P susceptibility. The eQTL analysis suggested that rs7838449 regulated the expression of TOX in cultured fibroblasts, and rs4554975 and rs11183619 were associated with the expression of SLC38A4 in skin and cultured fibroblasts. Overexpression of SLC38A4 and TOX enhanced proliferation and migration but inhibited apoptosis of HEPM and HOK cells; decreased the length of palatoquadrate bone and shortened the length and width of ethmoid plate in zebrafish model. TOX and SLC38A4 were involved in development-related pathways such as PI3K-Akt, Notch, MAPK signaling pathway. Our study identified SLC38A4 and TOX as candidate susceptible genes for NSCL/P.