By using PCR-HRM analysis we identified an intronic and four exonic substitutions only in 62
Japanese patients with ASD, two out of 62
patients (3.2%) in NLGN3
and three out of 62
patients (4.8%) in NLGN4X
. The exonic substitutions comprised one synonymous substitution in NLGN3
and three synonymous substitutions in NLGN4X
(). The PCR-HRM analysis could detect 90% of the sequence variations with 100% accuracy [19
]; therefore; we were able to identify almost all the changes in NLGN3
in these patients.
In this study, we analyzed genomic DNAs from EBV-transformed cells in patients with ASD. The source of genomic DNA, especially extracted from EBV-transformed cells, should be considerable in each experiment, because there is a possibility that unexpected substitutions occur during the transformation [21
]. Analyses in control genomic DNA showed that there was no sequence alteration or substitution bias in the exons of NLGN3
between genomic DNA isolated from blood and EBV-transformed cells in this study ().
Our mutation screening study in Japanese ASD patients failed to detect novel nonsynonymous mutations and seven known nonsynonymous mutations that were identified in previous studies [2
] as well as a study in a Chinese Han population [12
]. Considering the low frequency of nonsynonymous substitutions in these genes seen in previous reports [15
], a larger number of ASD patients should be sampled. Nevertheless, our results suggest that nonsynonymous substitutions in NLGN3
may account for only a small proportion of Japanese patients with ASD. In addition to ASD, there are some reports indicating that NLGN3
are also relevant to Asperger syndrome [2
], X-linked mental retardation [6
], and Tourette syndrome [8
]. Considering the function of NLGN3
], additional mutation screening studies in such neurobehavioral disorders should be needed in near future.
Experimental evidence is increasing that synonymous substitutions could affect the protein function through transcription or translation impairment [23
]. While the clinical and physiological importance of the four synonymous substitutions and an intronic substitution are not clear at this moment, Mutation Taster software (http://www.mutationtaster.org/
] evaluated that the substitutions might affect protein structure by altering splice site. According to previous reports of mutation screening in NLGN3
, two synonymous mutations, p.Y74Y in NLGN3
and p.A558A in NLGN4X
(), were also observed in ASD patients but not in healthy controls [13
]. A recent study in a Chinese Han population has indicated that a common intronic variation in NLGN3
may influence the susceptibility of males to ASD [12
]. Although further analysis is necessary to demonstrate the biological effects of synonymous and intronic substitutions on ASD, these substitutions as well as nonsynonymous substitutions should be taken into account.