Lack of Association between NLGN3, NLGN4, SHANK2 and SHANK3 Gene Variants and Autism Spectrum Disorder in a Chinese Population

<div><p>Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication, absence or delay in language development, and stereotyped or repetitive behaviors. Genetic studies show that neurexin-neuroligin (NRXN-NLGN) pathway genes contribute susceptibility to ASD, which include cell adhesion molecules <i>NLGN3</i>, <i>NLGN4</i> and scaffolding proteins <i>SHANK2</i> and <i>SHANK3</i>. Neuroligin proteins play an important role in synaptic function and trans-synaptic signaling by interacting with presynaptic neurexins. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density. Sequence level mutations and structural variations in these genes have been identified in ASD cases, while few studies were performed in Chinese population. In this study, we examined the copy numbers of four genes <i>NLGN4, NLGN3, SHANK2,</i> and <i>SHANK3</i> in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). We also screened the regulatory region including the promoter region and 5′/3′ untranslated regions (UTR) and the entire coding region of <i>NLGN4</i> in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. DNA copy number calculation in four genes showed no deletion or duplication in our cases. No missense mutations in <i>NLGN4</i> were identified in our cohort. Association analysis of 6 common SNPs in <i>NLGN4</i> did not find significant difference between ASD cases and controls. These findings showed that these genes may not be major disease genes in Chinese ASD cases.</p> </div

Assays of copy number in <i>SHANK2</i> and <i>SHANK3</i> genes.

<p>The copy number states of five segments in ASD patients were shown in five panels. Panel A: exon7 of <i>SHANK2</i>; Panel B: intron16-exon17 of <i>SHANK2</i>. Panel C: exon25 of <i>SHANK2</i>; Panel D: exon6 of <i>SHANK3</i>; Panel E: exon22 of <i>SHANK3</i>. Each column indicates a patient. All ASD cases showed two copy of <i>SHANK2/SHANK3.</i></p

Linkage disequilibrium block of <i>NLGN4</i> gene.

<p>The color of each square from light to dark represents the level of LD from low to high. White: complete recombination; blue: partial linkage; red: complete linkage.</p

The distribution of allele frequencies for 6 SNPs in <i>NLGN4</i> gene.

<p>*P value not corrected for the multiple testing.</p

High-density SNP-array Analysis of Chromosome 22q13 Deletions in Four Patients with ID.

<p>Microdeletions and duplications are represented by red and blue bars respectively. Annotated genes in the overlapping region are shown in the bottom of the figure.</p

Identification and functional analysis of <i>SHANK3</i> mutations.

<p>(A) Pedigree of the proband with a <i>de novo</i> Y1015X mutation and localization of this nonsense variation on the linear protein structure of <i>SHANK3</i>. ANK, ankyrin repeats; SH3, Src homology 3 domain; PDZ, postsynaptic density protein; SAM, sterile α motif domain. (B) Representative photographs demonstrating the anatomic differences in traced neurons transfected with empty vector, <i>SHANK3</i> WT, A921T, or Y1015X vectors, respectively. (C–F) Analysis of <i>SHANK3</i> mutations in primary mouse cortical neuronal cultures. Neurite number (C), nodes (D), tips (E) and total length (F) are quantified in bar histograms along with standard error of the mean for each bar. Asterisks indicate a statistically significant difference (**P<0.001, Post Hoc tests). Compared to control, WT and A921T, the overexpression of Y1015X significantly decreased neurite complexity and length.</p

Basic features of 4 cases with 22q13 deletion.

a<p>N:just one parent of the case was tested with MLPA and did not carry the 22q13 deletion.</p