Disruption of the ASTN2/TRIM32 locus at 9q33.1 is a risk factor in males for autism spectrum disorders, ADHD and other neurodevelopmental phenotypes

Abstract

Rare copy number variants (CNVs) disrupting ASTN2 or both ASTN2 and TRIM32 have been reported at 9q33.1 by genome-wide studies in a few individuals with neurodevelopmental disorders (NDDs). The vertebrate-specific astrotactins, ASTN2 and its paralog ASTN1, have key roles in glial-guided neuronal migration during brain development. To determine the prevalence of astrotactin mutations and delineate their associated phenotypic spectrum, we screened ASTN2/TRIM32 and ASTN1 (1q25.2) for exonic CNVs in clinical microarray data from 89 985 individuals across 10 sites, including 64 114 NDD subjects. In this clinical dataset, we identified 46 deletions and 12 duplications affecting ASTN2. Deletions of ASTN1 were much rarer. Deletions near the 3' terminus of ASTN2, which would disrupt all transcript isoforms (a subset of these deletions also included TRIM32), were significantly enriched in the NDD subjects (P = 0.002) compared with 44 085 population-based controls. Frequent phenotypes observed in individuals with such deletions include autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), speech delay, anxiety and obsessive compulsive disorder (OCD). The 3'-terminal ASTN2 deletions were significantly enriched compared with controls in males with NDDs, but not in females. Upon quantifying ASTN2 human brain RNA, we observed shorter isoforms expressed from an alternative transcription start site of recent evolutionary origin near the 3' end. Spatiotemporal expression profiling in the human brain revealed consistently high ASTN1 expression while ASTN2 expression peaked in the early embryonic neocortex and postnatal cerebellar cortex. Our findings shed new light on the role of the astrotactins in psychopathology and their interplay in human neurodevelopment.

Figure 1.

Exonic deletions found at the ASTN2/TRIM32 locus in clinical and control cohorts. Exonic deletions identified in 46 of 89 985 cases and 19 of 44 085 controls are depicted. Filled red bars represent deletions detected in individuals with NDD phenotypes. Empty red bars denote deletions in cases without known NDD phenotypes (from available clinical information) and in controls. Shaded gray region denotes the critical region defined by deletions that disrupt multiple isoforms of ASTN2. Numbers adjacent to the bars are the randomized sample identifiers of individuals with the deletions and correlate with information in Table 2, Supplementary Material, Tables S1 and S3. Gender information was not available for the two control individuals marked with * at the bottom of the figure. Dashed purple lines intersect deletions that overlap exons shared by multiple ASTN2 isoforms and dashed green lines intersect those affecting only the long isoform. Dashed vertical black line intersects deletions that overlap an exon of TRIM32. Genomic locations and coordinates are based on hg18 (NCBI36). Information about genes and transcript isoforms was obtained from the RefSeq database. The three transcript isoforms of ASTN2 possessing different numbers of exons are depicted including the long isoform (NM_014010) and two shorter isoforms (NM_198186 and NM_001184735). The three other shorter isoforms of the gene (NM_198187, NM_198188 and NM_001184734) have the same number and location of exons as NM_198186 but differ slightly in the length of their first and terminal exons and UTRs.

Figure 2.

Exonic CNVs found at the ASTN1 locus in clinical and control cohorts. Red and blue bars represent deletions and duplications, respectively, that overlap ASTN1. Empty bars denote CNVs in cases without known NDD phenotypes (from available clinical information) and in controls. Dashed black lines outline the common region of overlap shared among the three CNVs detected in the clinical case dataset. Genomic locations and coordinates are based on hg18 (NCBI36). Information about genes and transcript isoforms was obtained from the RefSeq database.

Figure 3.

Relative expression of ASTN2 transcript isoforms and exon conservation. (A) Schematic presentation of the ASTN2 transcript isoforms. Red arrows denote location of the primers used for the RT–PCR and/or qRT–PCR assays (Supplementary Material, Table S10). The ‘*’ symbol denotes exons with variable length in different isoforms. (B) Conservation profile of ASTN2 and TRIM32 exons across vertebrates, placental mammals and primates. (C) The expression of ASTN2 transcript isoforms (long, shorter and all) in eight different human brain regions. ACTB was used as a control gene. (D) Quantification of ASTN2 isoform expression levels in triplicate from adult brain and fetal brain RNA samples by qRT–PCR (standard curve method). The expression was normalized using ACTB as a housekeeping gene, and the expression ratio is relative to the expression from all ASTN2 isoforms (mean ratio ± standard deviation). The results were replicated using GAPDH as a housekeeping gene.

Figure 4.

Expression profiles of ASTN2 and ASTN1 across human brain development. The gene level expression profiles of (A) ASTN2 and (B) ASTN1 across developmental time points in nine regions of the human brain; amygdala (AMY), cerebellar cortex (CBC), diencephalon (DIE), frontal cortex (FC), hippocampus (HIP), occipital cortex (OC), parietal cortex (PC), temporal cortex (TC) and ventral forebrain (VF).