Figure 1.
The mGluR pathway coupling synaptic activity to synaptic protein synthesis.
The diagram illustrates components and interactions in the mGluR pathway [4]. 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) [7], [23], [43].
Figure 2.
SNV detection and classification.
(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 [28], [29]) 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.
Table 1.
Enrichment of rare functional variants in mGluR pathway genes in autism cases detected by high-throughput sequencing.
Table 2.
Rare, potentially deleterious variants identified in mGluR pathway genes in autism cases.
Figure 3.
Autism-specific HOMER1 variants affect conserved residues or microRNA binding sites and co-segregate with autism.
(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 HOMER1 c.1080C>T variant is predicted to alter multiple microRNA-binding sites in the HOMER1 3′ UTR. The sequence of the HOMER1 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) [32], [33]. Predicted pairing between specific microRNAs and the HOMER1 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 HOMER1 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.