High-Throughput Sequencing of mGluR Signaling Pathway Genes Reveals Enrichment of Rare Variants in Autism

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism

The mGluR pathway coupling synaptic activity to synaptic protein synthesis.

<p>The diagram illustrates components and interactions in the mGluR pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-KelleherRJ1" target="_blank">[4]</a>. Genes encoding proteins highlighted in yellow were sequenced in this study. Activation of postsynaptic group 1 mGluRs (mGluR1, mGluR5) stimulates protein synthesis by signaling through the Ras/ERK and PI3K/mTOR pathways. Group 1 mGluR function is modulated by interaction with Homer1, which interacts in turn with Shank3 and links mGluRs to the network of postsynaptic density-localized proteins. FMRP regulates synaptic protein synthesis by binding to target mRNAs and repressing their translation. Arc regulates mGluR-dependent synaptic plasticity, and its levels are regulated by FMRP-dependent translation and Ube3a-dependent degradation. The activity of the mGluR pathway is regulated by several pathway components responsible for syndromic ASDs (indicated by asterisks), including NF1 (neurofibromatosis type 1), Ras/ERK cascade members (cardiofaciocutaneous/Noonan syndromes), PTEN (ASD with microcephaly), TSC1 and TSC2 (tuberous sclerosis complex), FMRP (fragile X mental retardation syndrome), and Ube3a (Angelman's syndrome). Mutations in Shank3, Nrxn1, Nlgn3, and Nlgn4 cause rare non-syndromic ASDs, and structural variants in SynGAP1 and DLGAP2/SAPAP2 have been associated with autism (indicated by asterisks) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-Pinto1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-Durand1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-Jamain1" target="_blank">[43]</a>.</p

Autism-specific <i>HOMER1</i> variants affect conserved residues or microRNA binding sites and co-segregate with autism.

<p>(A) Multiple sequence alignments are shown for three segments of the Homer1 protein that contain missense substitutions caused by autism-specific SNVs identified in this study. The amino acid residues altered by these substitutions (highlighted in red) are highly conserved across mammalian and/or vertebrate evolution. (B) The autism-specific <i>HOMER1</i> c.1080C>T variant is predicted to alter multiple microRNA-binding sites in the <i>HOMER1</i> 3′ UTR. The sequence of the <i>HOMER1</i> 3′ UTR is shown at top (the c.1080 position 15 nucleotides distal to the translation stop codon highlighted in red), together with a cluster of microRNA binding sites predicted by the miRanda and Microcosm applications (miRanda target prediction based on ≥ 6-mer seed complementarity and mirSVR score ≤0.1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-John1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-GriffithsJones1" target="_blank">[33]</a>. Predicted pairing between specific microRNAs and the <i>HOMER1</i> 3′ UTR that would be altered by the c. 1080 C>T variant is shown at bottom. (C) Co-segregation with autism was analyzed for the rare, potentially deleterious <i>HOMER1</i> missense variants uniquely identified in AGRE probands by genotyping available parents and siblings. Filled symbols indicate a diagnosis of autism or ASD; unfilled symbols indicate reportedly unaffected individuals. Genotypes are shown for each individual, with “+” designating the wild-type allele and “SNV” designating the indicated variant allele.</p

Rare, potentially deleterious variants identified in mGluR pathway genes in autism cases.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone-0035003-t002" target="_blank">Table 2</a> summarizes the 58 rare, potentially deleterious SNVs that were identified in mGluR pathway genes only in autism cases. For each variant, the nucleotide substitution is shown, and the corresponding amino acid substitution is indicated parenthetically.</p

SNV detection and classification.

<p>(A) The flow diagram depicts the experimental strategy for SNV discovery and confirmation. For the AGRE and control cohorts, orthogonal multiplexing was performed to prepare two distinct sets of sample pools (15 pools of 20 samples each, or 20 pools of 15 samples each). Following enrichment of exonic target regions for all 18 mGluR pathway genes, SNVs were identified and confirmed by deep resequencing of orthogonal pools on two independent NGS platforms (Illumina GAII and the Helicos HeliScope). SNVs concordantly detected on both platforms were then analyzed as shown in panel B. (B) The flow diagram depicts the procedure used to classify the presumptive functional effects of identified variants. SNVs concordantly detected on both NGS platforms were classified as common or rare using a minor allele frequency (maf) threshold of 1%. Common SNVs, rare SNVs occurring in both autism and control populations, and rare synonymous (silent) SNVs were considered likely to be benign. Rare SNVs in intronic sequences flanking exons that did not affect conserved splice donor or acceptor sites or in 5′ untranslated regions were classified as not benign but of unknown significance. Rare SNVs causing missense substitutions or occurring in mRNA 3′ untranslated regions (and therefore possibly affecting mRNA stability or translation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-Chen1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035003#pone.0035003-Conne1" target="_blank">[29]</a>) were considered possibly deleterious, and rare SNVs causing nonsense mutations or affecting conserved splice donor or acceptor sequences were considered probably deleterious. These latter two categories of SNVs were together considered potentially deleterious.</p

Enrichment of rare functional variants in mGluR pathway genes in autism cases detected by high-throughput sequencing.

<p>SNVs in mGluR pathway genes were identified in pools of AGRE or control samples and classified based on allele frequency and predicted functional impact. The values shown represent the numbers of distinct variants identified. A significant excess of rare, potentially deleterious variants in the AGRE group relative to the control group was observed for the <i>HOMER1</i>, <i>SHANK3</i>, <i>TSC1</i>, and <i>TSC2</i> genes (highlighted in bold).</p

Evaluation of a 27-gene inherited cancer panel across 630 consecutive patients referred for testing in a clinical diagnostic laboratory

Abstract Background Extensive clinical and genetic heterogeneity of inherited cancers has allowed multi-gene panel testing to become an efficient means for identification of patients with an inherited predisposition to a broad spectrum of syndromic and nonsyndromic forms of cancer. This study reports our experience with a 27-gene inherited cancer panel on a cohort of 630 consecutive individuals referred for testing at our laboratory with the following objectives: 1. Determine the rates for positive cases and those with variants of uncertain clinical significance (VUS) relative to data published in the recent literature, 2. Examine heterogeneity among the constituent genes on the panel, and 3. Review test uptake in the cohort relative to other reports describing outcomes for expanded panel testing. Methods Clinical and genomic data were reviewed on 630 individuals tested on a panel of 27 genes selected on the basis of high (≥ 40%) or moderate to low (≤ 40%) lifetime risk of hereditary cancer. These patients were not enriched for adherence to the National Comprehensive Cancer Network (NCCN) criteria for Hereditary Breast and Ovarian Cancer (HBOC) or Lynch Syndrome (LS) and constitute a referral laboratory cohort. Results Sixty-five individuals with variants classified as pathogenic or likely pathogenic across 14 genes were identified for an overall positive rate of 10.3%. Although a family history of cancer constituted a major reason for referral, accounting for 84% of our cohort, excluding patients with a known familial variant did not have a significant impact on the observed positive rate (9% vs 10.3%). More than half (58%) of the pathogenic or likely pathogenic variants were observed in high or moderate to low risk genes on the panel, while only 42% occurred in classic HBOC or LS-associated genes. Conclusion These results provide the actual percentage of family or personal history of cancer that can be attributed to pathogenic or likely pathogenic variants in one or more of the genes on our panel and corroborate the utility of multi-gene panels over sequential testing to identify individuals with an inherited predisposition to cancer