Exon resequencing of H3K9 methyltransferase complex genes, EHMT1, EHTM2 and WIZ, in Japanese autism subjects

Abstract

Background: Histone H3 methylation at lysine 9 (H3K9) is a conserved epigenetic signal, mediating heterochromatin formation by trimethylation, and transcriptional silencing by dimethylation. Defective GLP (Ehmt1) and G9a (Ehmt2) histone lysine methyltransferases, involved in mono and dimethylation of H3K9, confer autistic phenotypes and behavioral abnormalities in animal models. Moreover, EHMT1 loss of function results in Kleefstra syndrome, characterized by severe intellectual disability, developmental delays and psychiatric disorders. We examined the possible role of histone methyltransferases in the etiology of autism spectrum disorders (ASD) and suggest that rare functional variants in these genes that regulate H3K9 methylation may be associated with ASD.

Methods: Since G9a-GLP-Wiz forms a heteromeric methyltransferase complex, all the protein-coding regions and exon/intron boundaries of EHMT1, EHMT2 and WIZ were sequenced in Japanese ASD subjects. The detected variants were prioritized based on novelty and functionality. The expression levels of these genes were tested in blood cells and postmortem brain samples from ASD and control subjects. Expression of EHMT1 and EHMT2 isoforms were determined by digital PCR.

Results: We identified six nonsynonymous variants: three in EHMT1, two in EHMT2 and one in WIZ. Two variants, the EHMT1 ankyrin repeat domain (Lys968Arg) and EHMT2 SET domain (Thr961Ile) variants were present exclusively in cases, but showed no statistically significant association with ASD. The EHMT2 transcript expression was significantly elevated in the peripheral blood cells of ASD when compared with control samples; but not for EHMT1 and WIZ. Gene expression levels of EHMT1, EHMT2 and WIZ in Brodmann area (BA) 9, BA21, BA40 and the dorsal raphe nucleus (DoRN) regions from postmortem brain samples showed no significant changes between ASD and control subjects. Nor did expression levels of EHMT1 and EHMT2 isoforms in the prefrontal cortex differ significantly between ASD and control groups.

Conclusions: We identified two novel rare missense variants in the EHMT1 and EHMT2 genes of ASD patients. We surmise that these variants alone may not be sufficient to exert a significant effect on ASD pathogenesis. The elevated expression of EHMT2 in the peripheral blood cells may support the notion of a restrictive chromatin state in ASD, similar to schizophrenia.

Keywords: Autism; G9a; GLP; H3K9; Histone methyltransferase; Rare variant; Wiz.

Figure 1

Genomic structures of EHMT1 , EHMT2 and WIZ genes screened in Japanese autism spectrum disorder (ASD) subjects, and identified missense variants. Black boxes denote coding exons and white boxes denote non-coding exons.

Figure 2

Pedigree structures of autism spectrum disorder (ASD) families harboring novel missense variants in EHMT1, EHMT2 and WIZ . With the exception of Lys968Arg, none of the novel variants were de novo. For the Lys968Arg variant, genotype information of the father was not available.

Figure 3

Expression analysis of (A) EHMT1, (B) EHMT2 and (C) WIZ in lymphocyte samples from a subset of autism spectrum disorder (ASD) cases and control (CNT) subjects who were resequenced for the candidate genes.

Figure 4

Gene expression analysis of (A) EHMT1, (B) EHMT2 and (C) WIZ in Brodmann area (BA) 09, BA21, BA40 and DoRN (dorsal raphe nucleus) of autism spectrum disorder (ASD) cases and controls (CNT). (D) Isoform-specific expression analysis of EHMT1 (variant 1: NM_024757.4 and variant 2: NM_001145527.1) and EHMT2 (isoform a: NM_006709.3 and isoform b: NM_025256.5) in the prefrontal cortex (BA09) of ASD cases and controls.