In this study, to identify novel candidate genes for NDDs, we performed exome or targeted sequencing on DNAs of 558 Japanese NDD patients with a rather predominant focus on those associated with trigonocephaly, and identified rare de novo, hemizygous, homozygous, and compound heterozygous variants in genes including functionally related MSX2 and PJA1. Functional analyses of these variants in vitro and in vivo further supported that these are genes for NDDs. This evidence concerns the gene PJA1 and trigonocephaly.