Target Capture/Next-Generation Sequencing for Nonsyndromic Cleft Lip and Palate in the Japanese Population

The pathogenesis of nonsyndromic cleft lip with or without cleft palate (NSCL ± P) and nonsyndromic cleft palate only (NSCP) may be associated with genetic factors. Although some predisposing genes/loci have been reported, their attributable risk is too small to be clinically meaningful. To clarify the genetic causes and mechanisms of NSCL±P or NSCP, we conducted mutation analysis of target genes using a next-generation sequencing (NGS) approach.

The target genes, IRF6, WNT5A, WNT9B, TP63, MSX1, TFAP2A, PAX9, DLX3, DLX4, and MN1, were selected based on previous reports of potential associations with the development of NSCL±P or NSCP from genome-wide association studies and candidate gene analyses. Mutation analysis was conducted using NGS on 74 Japanese trios (patient and parents) and 18 Japanese patients only families.

We detected single-nucleotide variants (SNVs) for 7 genes: IRF6, DLX4, WNT5A, TFAP2A, WNT9B, TP63, and PAX9. The SNVs found on IRF6 and DLX4 were missense mutations, whereas those identified on WNT5A, TFAP2A, WNT9B, TP63, and PAX9 were rare variants in the noncoding region; no de novo mutation was identified in the trio samples. The amino acid change on DLX4 was detected within the highly conserved homeodomain and was predicted to have a deleterious impact on the protein function by in silico analysis.

The DLX4 missense mutation c.359C>T (Pro120Leu) was found in 1 Japanese patient with NSCL±P and was located in the homeodomain region. This mutation likely plays a role in the development of NSCL±P in the Japanese population.