Analysis of the SNP rs3747333 and rs3747334 in NLGN4X gene in autism spectrum disorder: a meta-analysis

Autism spectrum disorder (ASDs; MIM 209850) is a heterogeneous group of neurodevelopmental diseases defined by significant language delays, social, and communication deficits, and highly repetitive and stereotypic interest and/or behaviors [1]. The global prevalence of autism has risen to 6.2% [2]. Autism patients in China are estimated in the range of 0.6–1.8 million according to the WHO’s report. According to the latest report released by Autistica, people with ASD die on average 18 years before the general population [3]. ASD is a result of complex gene–environment interactions, with strong and clear genetic influences [4]. Although the etiologic and clinical heterogeneity are universally recognized, in practice, many studies still fail to take this into account. As of December, 2015, genetic studies have pointed to 791 potential candidate genes in ASD (https://​gene.​sfari.​org/​autdb/​HG_​Home.​do), making it difficult to find common underlying pathogenic mechanisms.

Neuroligin 4X (NLGN4X) is a member of neuroligins expressed in the postsynaptic neurons and mediate transsynaptic signaling by interacting their ligand, neurexins [5]. Several studies identified significant variants in the NLGN4X gene that were replicated in two or more independent samples and were associated with specific phenotypes of ASD [6‐8]. However, other studies found no variants in the NLGN4X gene in autistic patients in their cohorts [9‐11]. To date, many common single-nucleotide polymorphisms (SNPs) of NLGN4X such as rs72413786, rs2290487, rs7049300, rs3747333, rs3747334, have been studied in extensive research on ASD. Of these, the SNP rs3747333 and rs3747334 have received more favorable observations concerning on the ASD risk [11‐16]. A case–control study of Chinese descendants found that the common SNPs (rs3747333 and rs3747334) in NLGN4X gene significantly associated with risk for autism [16], a previous work showed that seven boys were carriers of mutations at three SNPs including the synonymous SNP rs7049300, the non-synonymous SNP rs3747333 (L593) and the synonymous SNP rs3747334 (L593), but the co-occurrence of rs3747333C>T leading to a substitution of leucine to phenylalanine and rs3747334C>G entails a double substitution of CTC to TTG in codon 593 and thus protect the amino acid leucine [11]. However, a work from German showed that the polymorphisms rs 3747333 and rs 3747334 did not cause any amino acid substitutions in the total of the eight detected carriers [11]. Those inconsistencies could be attributed to the phenotypic heterogeneity of ASD and to relatively small sample sizes.

Given the increasing attention directed to the inconsistencies results, to further clarify the inconclusive association between the two SNPs and ASD, we conducted a meta-analysis with available data published up to Sep. 21th, 2017.

A systematic literature search was conducted to collect studies relevant for the current meta-analysis. First, we searched three online databases (PubMed, Web of Science, Cochrane Library) for articles published in English on associations between the rs3747333, rs3747334 in NLGN4X and ASD up to Sep. 21th, 2017. Combinations of terms identifying the gene (rs3747333, rs3747334, NLGN4X, X-linked Neuroligin 4 gene) and terms identifying the phenotype of interest (autism, autism spectrum disorder, Asperger, childhood disintegrative disorder, pervasive developmental disorder) were entered as Medical Subject Heading terms and text words. After identifying a set of publications returned from each search, we collated the results and eliminated duplicates. We then reviewed the titles, abstracts, keywords and texts of all studies to exclude studies that were clearly irrelevant. Hand-searching was carried out to determine other potential eligible studies through scanning the references cited in the retrieved articles. Finally, we applied criteria to the set of studies to determine their inclusion or exclusion in the present meta-analysis. If the two reviewers (Hongli Sun and Ying Yang) disagreed, all the authors critically evaluated the studies to judge of the inclusion or exclusion of a certain study.

All the eligible articles meet the inclusion criteria:

The two authors independently extracted the following data: (1) first author, (2) year of publication, (3) location, (4) predominant ethnicity of participants, (5) study design, (6) number of participants, (7) diagnostic criteria. When the study did not report sufficient data to calculate effect size, we contacted corresponding authors via e-mail to request additional information. If the contact with the author was not successful, we could not include the study in the meta-analysis.

The association of the rs3747333, rs3747334 in NLGN4X and ASD was assessed by odds ratio (OR) with 95% confidence interval (CI). Subgroup (according to study design) analysis was evaluated by study type. Publication bias was considered present when P-value was less than 0.05. Sensitivity analysis was also carried out to evaluate the stability of the meta-analysis. Statistical heterogeneity among studies was evaluated by the Chi-square-based Q-test and the I² index. I² index < 50% and/or P-value > 0.10 for Q-test indicated a lack of heterogeneity among the studies. The fixed-effect model (the Mantel–Haenszel method) was utilized to calculate the pooled ORs when the heterogeneity was not significant among the studies. Otherwise, the random-effects model (the DerSimonian and Laird method) was employed. Publication bias was evaluated by the funnel plot. All statistical analyses were carried out with the STATA 12.0 (STATA Corp, College Station, Texas).