Rare mutations and hypermethylation of the ARC gene associated with schizophrenia

Abstract

Activity-regulated cytoskeleton-associated protein (ARC), which interacts with the N-methyl-d-aspartate receptor (NMDAR) complex, is a critical effector molecule downstream of multiple neuronal signaling pathways. Dysregulation of the ARC/NMDAR complex can disrupt learning, memory, and normal brain functions. This study examined the role of ARC in susceptibility to schizophrenia. We used a resequencing strategy to identify the variants of ARC in 1078 subjects, including patients with schizophrenia and normal controls. We identified 16 known SNPs and 27 rare mutations. SNP-based analysis showed no association of ARC with schizophrenia. In addition, the rare mutations did not increase the burden in patients compared with controls. However, one patient-specific allele in the putative ARC promoter region and seven patient-specific mutants in ARC exon regions significantly reduced the reporter gene activity compared with ARC wild-type. Methylation of a putative ARC promoter attenuated reporter activity in vitro, suggesting that ARC expression is regulated by DNA methylation. Pyrosequencing revealed eight hypermethylated CpG sites in the putative ARC promoter region in 64 schizophrenic patients compared with 63 controls. Taken together, our results suggest that both rare variants and epigenetic regulation of ARC contribute to the pathogenesis of schizophrenia in some patients.

Introduction

Schizophrenia is a chronic debilitating mental disorder that affects approximately 1% of the population worldwide. It is characterized by thought abnormalities, hallucinations, delusions, and bizarre behaviors (Freedman, 2003). Twin studies have shown that schizophrenia is a complex trait with genetic and environmental influences (Sullivan et al., 2003). Schizophrenia is a neurodevelopmental disorder with aberrant synaptogenesis and synaptic connectivity (McGlashan and Hoffman, 2000, Stephan et al., 2009).

Abnormal glutamatergic neurotransmission and intracellular signal transduction via NMDARs contributes to the pathophysiology of schizophrenia (Lau and Zukin, 2007, Snyder and Gao, 2013). Rare de novo copy number variants (CNV) were identified in 662 schizophrenia proband-parent trios. These mutations spanned genes encoding members of the discs large family of membrane-associated guanylate kinases that are components of the postsynaptic density (Kirov et al., 2012). Furthermore, defects in NMDAR postsynaptic signaling complexes, which are known to be important in synaptic plasticity and cognition, contribute to the pathogenesis of schizophrenia (Kirov et al., 2012). Recently, two exome sequencing-based studies implicated mutations in genes encoding NMDAR-related and ARC-associated proteins in schizophrenia (Fromer et al., 2014, Purcell et al., 2014). ARC, (also known as Arg3.1), is an immediate early gene that encodes activity-regulated cytoskeleton-associated protein (ARC). ARC is enriched in neuronal dendrites and is implicated in synaptic, experience-dependent plasticity, learning, and memory (Chia and Otto, 2013, Guzowski et al., 2000, Link et al., 1995, Lyford et al., 1995, Plath et al., 2006, Ploski et al., 2008, Vazdarjanova et al., 2002). ARC dysregulation contributes to various mental disorders such as fragile X syndrome (Park et al., 2008) and Alzheimer's disease (Landgren et al., 2012, Palop et al., 2005). Prompted by these findings, we investigated whether ARC is involved in the pathophysiology of schizophrenia.

The genetic basis of complex disorders can be attributed to common variants with small effect and rare variants with high penetrance (Manolio et al., 2009, Schork et al., 2009). High throughput genotyping detects single nucleotide polymorphisms (SNPs) that confer small effects on the pathophysiology of schizophrenia, whereas chromosome microarray and sequencing technologies can detect rare CNVs in genomic DNA that confer large effects (Grozeva et al., 2010, Kenny et al., 2014, Tarabeux et al., 2011, Walsh et al., 2008). Schizophrenia is a complex disorder involving epigenetic factors and has a non-Mendelian inheritance pattern (Burmeister et al., 2008, Diwadkar et al., 2014). Recently, the epigenetic influences on schizophrenia have attracted a lot of attention (Diwadkar et al., 2014, Gavin and Floreani, 2014, Wockner et al., 2014). Thus, we investigated whether ARC is subjected to epigenetic regulation in schizophrenia.

To address these issues, we systemically searched for genetic variants in the putative promoter and exons of ARC in schizophrenia patients and non-psychotic control subjects from Taiwan using a resequencing strategy. We assessed the functional impact of rare mutations identified in this study by in silico analysis. We performed a reporter gene activity assay to assess the potential regulatory impact of mutations located in the putative promoter region on the protein expression. We analyzed the protein function of the identified rare ARC exonic mutants using immunoblotting and immunocytochemistry analysis. Lastly, we conducted an in vitro DNA methylation reaction and pyrosequencing assay to compare DNA methylation in schizophrenic patients and control subjects.