Identification of Four Novel Synonymous Substitutions in the X-Linked Genes Neuroligin 3 and Neuroligin 4X in Japanese Patients with Autistic Spectrum Disorder

Mutations in the X-linked genes neuroligin 3 (NLGN3) and neuroligin 4X (NLGN4X) were first implicated in the pathogenesis of X-linked autism in Swedish families. However, reports of mutations in these genes in autism spectrum disorder (ASD) patients from various ethnic backgrounds present conflicting results regarding the etiology of ASD, possibly because of genetic heterogeneity and/or differences in their ethnic background. Additional mutation screening study on another ethnic background could help to clarify the relevance of the genes to ASD. We scanned the entire coding regions of NLGN3 and NLGN4X in 62 Japanese patients with ASD by polymerase chain reaction-high-resolution melting curve and direct sequencing analyses. Four synonymous substitutions, one in NLGN3 and three in NLGN4X, were identified in four of the 62 patients. These substitutions were not present in 278 control X-chromosomes from unrelated Japanese individuals and were not registered in the database of Single Nucleotide Polymorphisms build 132 or in the Japanese Single Nucleotide Polymorphisms database, indicating that they were novel and specific to ASD. Though further analysis is necessary to determine the physiological and clinical importance of such substitutions, the possibility of the relevance of both synonymous and nonsynonymous substitutions with the etiology of ASD should be considered. Introduction Neuroligin 3 (NLGN3) and Neuroligin 4X (NLGN4X) are members of neuroligins expressed in the postsynaptic neurons and mediate transsynaptic signaling by interacting their ligand, neurexins [1]. Mutations in the X-linked genes NLGN3 and NLGN4X (GenBank accession numbers NM_181303.1 and NM_020742.2, resp.) were first reported as being involved in X-linked autistic spectrum disorder (ASD; MIM#300425？and MIM#300495) in Swedish families [2]. Some reports have also indicated that NLGN3 and NLGN4X are responsible for ASD. Mutations in these genes including 5？missense mutations [2–5], two small in-del mutations, which lead to a premature stop codon in the transcript [2, 6], an exon skipping mutation [7], and a large deletion [8], have been identified in ASD patients (Table 1). In vitro experiments using NLGN3 and NLGN4X proteins carrying amino acid changes that were identified in ASD patients indicated that the gene mutations could cause ASD by a loss-of-function mechanism [5, 9]. Neuroligin 3？？ p.R451C knock-in mice and neuroligin 4X-deficient mice exhibited autism-related behaviors [10, 11]. In addition, recent mutation screening studies revealed that the p.K378R substitution in the