Exome sequencing in schizophrenic patients with high levels of homozygosity identifies novel and extremely rare mutations in the GABA/glutamatergic pathways

Abstract

Inbreeding is a known risk factor for recessive Mendelian diseases and previous studies have suggested that it could also play a role in complex disorders, such as psychiatric diseases. Recent inbreeding results in the presence of long runs of homozygosity (ROHs) along the genome, which are also defined as autozygosity regions. Genetic variants in these regions have two alleles that are identical by descent, thus increasing the odds of bearing rare recessive deleterious mutations due to a homozygous state. A recent study showed a suggestive enrichment of long ROHs in schizophrenic patients, suggesting that recent inbreeding could play a role in the disease. To better understand the impact of autozygosity on schizophrenia risk, we selected, from a cohort of 180 Italian patients, seven subjects with extremely high numbers of large ROHs that were likely due to recent inbreeding and characterized the mutational landscape within their ROHs using Whole Exome Sequencing and, gene set enrichment analysis. We identified a significant overlap (17%; empirical p-value = 0.0171) between genes inside ROHs affected by low frequency functional homozygous variants (107 genes) and the group of most promising candidate genes mutated in schizophrenia. Moreover, in four patients, we identified novel and extremely rare damaging mutations in the genes involved in neurodevelopment (MEGF8) and in GABA/glutamatergic synaptic transmission (GAD1, FMN1, ANO2). These results provide insights into the contribution of rare recessive mutations and inbreeding as risk factors for schizophrenia. ROHs that are likely due to recent inbreeding harbor a combination of predisposing low-frequency variants and extremely rare variants that have a high impact on pivotal biological pathways implicated in the disease. In addition, this study confirms that focusing on patients with high levels of homozygosity could be a useful prioritization strategy for discovering new high-impact mutations in genetically complex disorders.

A novel homozygous mutation in GAD1 gene described in a schizophrenic patient impairs activity and dimerization of GAD67 enzyme

“…Even if a significant role of rare recessive deleterious variants did not emerge in the SCZ 19 – 21 , an enrichment of long Runs of Homozygosity (ROHs) has been found in SCZ cases 22 , 23 , suggesting that large autozygosity regions due to inbreeding could play a role in the disease. These last results are in line with our previous study 24 pointing to the presence of rare SCZ risk variants, in a homozygous state, in subjects bearing long ROHs that are likely due to recent inbreeding.…”

“…Previously we identified a schizophrenic patient that was homozygous for a novel mutation in GAD1 gene ( GAD1 , c.391 A > G). Since this mutation was predicted damaging by bioinformatics tools and fall in a gene whose expression has been described altered in some SCZ patients, we hypothesized it could be a risk variant for SCZ with a recessive effect 24 . Direct sequencing in the two healthy sisters of that patient revealed that both sisters were heterozygotes for the c.391 A > G mutation (Fig.…”

“…Indeed, whole exome sequencing of SCZ patients with high levels of autozygosity, (that is with more than 22 Mb of their genome included in ROHs > 4 Mb, compared to a median value of 8.7 Mb) 24 , allowed us to identify some ultra-rare mutations in a homozygous state. These mutations, mapping in ROHs and affecting genes of the glutamatergic and GABAergic pathways, could be considered good candidate SCZ risk variants 24 . In particular, one of these variants, mapping at position chr2:171,687,546 (hg19 assembly) was a novel missense mutation (c.391 A > G) affecting the Glutamic Acid Decarboxylase I ( GAD1 ) (NM_000817) gene.…”

Exploratory Analysis of Rare and Novel Variants in Mexican Patients Diagnosed with Schizophrenia and Dementia

“…We designed synthetic probes for NGS, targeting genes associated with dementia, SCZ, and several pharmacogenetic targets. The selection of genes was based on a literature search for published works reporting an effect of common variations or rare variants in SCZ, dementia or drug response to different antipsychotics or antidementia drugs 6,7,[26][27][28][29][30][31][32][33][34][35][36] ; a list of the captured genes is reported in Supplementary Table 1. Gene capture was performed using the Haloplex target enrichment system (Agilent Technologies, USA) with 1.51Mb with 40754 amplicons.…”

Global Knockdown of Glutamate Decarboxylase 67 Elicits Emotional and Auditory Abnormalities in Mice

“…Alternative splicing of GAD1 and the epigenetic state may play roles in brain development and the risk of schizophrenia [12]. Recent studies with whole exome sequencing of schizophrenic patients identified missense mutation mapping at the GAD1 gene, which caused a reduction in GAD67 enzymatic activity by ~30% due to impaired homodimerization [13,14]. Therefore, the functional impairment of GAD67 associated with genetic mutations is involved in the neurobiological mechanisms of several psychiatric disorders.…”