A large French family including members affected by nonspecific X-linked mental retardation, with or without autism or pervasive developmental disorder in affected male patients, has been found to have a 2-base-pair deletion in the Neuroligin 4 gene (NLGN4) located at Xp22.33. This mutation leads to a premature stop codon in the middle of the sequence of the normal protein and is thought to suppress the transmembrane domain and sequences important for the dimerization of neuroligins that are required for proper cell-cell interaction through binding to beta-neurexins. As the neuroligins are mostly enriched at excitatory synapses, these results suggest that a defect in synaptogenesis may lead to deficits in cognitive development and communication processes. The fact that the deletion was present in both autistic and nonautistic mentally retarded males suggests that the NLGN4 gene is not only involved in autism, as previously described, but also in mental retardation, indicating that some types of autistic disorder and mental retardation may have common genetic origins.

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4−/− mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.