Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism

Abstract

Many studies have supported a genetic etiology for autism. Here we report mutations in two X-linked genes encoding neuroligins NLGN3 and NLGN4 in siblings with autism-spectrum disorders. These mutations affect cell-adhesion molecules localized at the synapse and suggest that a defect of synaptogenesis may predispose to autism.

Fig. 1

Expression of NLGNs in the human brain. Specific RT-PCRs were performed on total RNA from different brain regions using primers in exon 2 and 5 in order to analyse the different alternative spliced transcripts. To distinguish between NLGN4 and NLGN4Y mRNA, RT–PCRs were digested by NcoI. Using specific forward primers in exon 1a or exon 1b and reverse primers in exon 2, two alternative NLGN4 promoters were identified substituting the first exon 1a for 1b. Size of NLGN4 and NLGN4Y PCR differs by 193 bp. Exon 1b is specific to NLGN4 and contains an internal alternative donor splice site. The ages of the two males and the two females studied were 74, 42, 55, and 36 years old with a post-mortem delay of 10, 21, 24, and 2 h, respectively. f: frontal cortex, tc: temporal cortex, o: occipital cortex, h: hippocampus, t: thalamus; c: cerebellum. Normal control human brains were obtained at autopsy under guidelines approved by the ethics committee. DNA amplification and RT-PCRs were performed as described. Alignment and sequences of all NLGN proteins and isoforms are available from the authors.

Fig. 2

Mutation screening of NLGN3 and NLGN4 in autistic subjects. a, Identification of the frameshift NLGN4 mutation in two affected individuals with autism and Asperger syndrome. Pedigree structure and the abnormal SSCP conformer are shown. Using NLGN4 specific primers, sequence of the PCR product revealed a one-base-pair (T) insertion in the mother and her two affected sons, absent in the father and the unaffected son. This insertion occurs in exon 5 of NLGN4 and causes a frameshift that leads to the premature termination of NLGN4 which thus lacks 421 amino acids (51% of the protein) including the transmembrane domain. b, Identification of the NLGN3 mutation. The family comprises a son with autism and his younger brother with AS. The R451C mutation is localised in the esterase domain of the protein and modifies a highly conserved arginine residue present in neuroligins and acetylcholine esterase (ACHE). Affected individuals are indicated by filled symbols (black for autism and grey for AS). Squares are males, circles are females, and a line through a symbol indicates that the person is deceased. SSCP analysis was performed with a GenePhor apparatus (Pharmacia-Biotech). We carried out direct sequencing with BigDye terminator cycle Sequencing Kit (Applied Biosystems) on an ABI 3100 Automated Sequencer.