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

Mitochondrial defects in the cerebral cortex of <i>Psen</i> cDKO mice.

<p>(<i>A</i>) (<i>Top</i>) Electron micrographs of mitochondria from the neocortex at 2 months and 6 months of age. Red arrows point to mitochondria typical of those quantified in (<i>A–C</i>). Scale bar, 1 µm. (<i>Bottom</i>) Quantification of mitochondria per 100 µm² in neocortex. The density of <i>Psen</i> cDKO mitochondria was comparable to controls at 2 months (control: 62.5±4.1 vs. <i>Psen</i> cDKO: 68.4±4.1; p>0.05) but reduced by 6 months (control: 61.7±4.8 vs. <i>Psen</i> cDKO: 44.2±4.6; p<0.001), revealing age-dependent loss of mitochondria in <i>Psen</i> cDKO (n = 4 mice per genotype (2 mo) and 3 mice per genotype (6 mo)). (<i>B,C</i>) Analysis of size distribution of cortical mitochondria determined that <i>Psen</i> cDKO has no change in mitochondrial morphology at 2 months (<i>B</i>), but a sizeable reduction in small mitochondria by 6 months of age (<i>C</i>) (0.01–0.1 µm² bin: control, 40.0±4.4 vs. <i>Psen</i> cDKO, 28.6±4.3 (p<0.001); 0.11–0.2 µm² bin: control, 15.5±2.3 vs. <i>Psen</i> cDKO, 7.3±1.6 (p<0.00001)). (<i>D</i>) (<i>Top</i>) High-magnification electron micrographs of cortical mitochondria from mice at 6 months of age. Both control (<i>left</i>) and <i>Psen</i> cDKO samples (<i>middle</i>) have normal mitochondria with clearly visible cristae and outer membrane structures; however, <i>Psen</i> cDKO mice also have a slight increase in the number of swollen mitochondria (<i>right</i>). Scalebar, 0.4 µm (all 3 images). (<i>Bottom</i>) Quantification of large (area >0.3 µm²) mitochondria from mice age 2 months and 6 months. The percentage of large <i>Psen</i> cDKO cortical mitochondria relative to the total number was normal at 2 months of age (control: 0.028±0.017 vs. <i>Psen</i> cDKO: 0.024±0.009; p>0.05), but at 6 months of age, the percentage of large <i>Psen</i> cDKO mitochondria is twice the number observed in controls (control: 0.048±0.018 vs. <i>Psen</i> cDKO: 0.098±0.035; p<0.05), implicating a dysregulation of mitochondrial morphology in the absence of PS function. (n = 4 at 2 months and 3 at 6 months). All data are presented as the mean ± s.e.m.</p

More dramatic increases in apoptosis in the lateral cortex of <i>Psen</i> cDKO mice.

<p>Black vertical lines superimposed on an image of a Nissl-stained coronal brain section depict the relative positions of sagittal brain sections spaced 300 µm apart that were analyzed for either Fluoro-Jade B+ or TUNEL+ cells. The average (± s.e.m.) of the three medial-most sections (M) was compared to the average of the three lateral-most sections (L). Scale bar: 1 mm. For each timepoint, 4 mice per genotype (10 sections per mouse) were analyzed. (<i>A, B</i>) Quantification of numbers of Fluoro-Jade B+ cells in sagittal brain sections from mice at 2 months (<i>A</i>) or 4 months (<i>B</i>). At 2 months of age, increases in degenerating neurons are more pronounced in the lateral sections (control: 1.7±0.7; cDKO: 34.1±3.1; p<0.01) than in the medial sections (control: 2.5±0.3; cDKO: 3.4±0.3; p>0.05). By 4 months of age, the number of degenerating neurons increases significantly in medial sections of cDKO mice, compared to controls (control: 2.6±0.3; cDKO: 14.3±1.1; p<0.01). Furthermore, the number of degenerating neurons in lateral sections of cDKO mice is much greater than in medial sections (control: 1.4±0.1 vs. <i>Psen</i> cDKO: 32.1±3.0; p<0.02). *, p<0.05; **, p<0.01; ***, p<0.001. (<i>C, D</i>) Quantification of numbers of TUNEL+ cells in sagittal brain sections from mice age 2 months (<i>C</i>) or 4 months (<i>D</i>). (<i>C</i>) No significant increase was observed in TUNEL+ cells in the <i>Psen</i> cDKO medial cortex at 2 months (control: 3.0±0.3 vs. <i>Psen</i> cDKO: 4.9±0.5; p = 0.05), in contrast to a large increase in lateral <i>Psen</i> cDKO cortex (control: 3.4±0.4 vs. <i>Psen</i> cDKO: 54.8±8.0; p<0.05). (<i>E</i>) At 4 months, lateral <i>Psen</i> cDKO cortex showed a significant increases in TUNEL+ cells (control: 2.3±0.5 vs. <i>Psen</i> cDKO: 32.9±4.1; p<0.05), while medial <i>Psen</i> cDKO cortex showed only a trend toward increased apoptosis (control: 1.3±0.3 vs. <i>Psen</i> cDKO: 11.9±2.3; p = 0.05).</p

Quantitative analysis of neurodegeneration and cell death in <i>Psen</i> cDKO Cx^†^‡.

†<p>Values for Fluoro-Jade B and TUNEL represent percentage of positive cells based on stereological estimates of total cell number. Note that although <i>Psen1</i> gene inactivation, measured by <i>Psen1</i> mRNA levels, occurs by 3–4 weeks, death is not increased at this timepoint. Since Psen1 protein levels persist and gradually diminish between P22 and 6 weeks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010195#pone.0010195-Yu2" target="_blank">[14]</a>, the delay in onset of death is likely due to the continued presence of Psen1 protein subsequent to gene inactivation.</p>‡<p>All calculations of total % death are based on stereological estimates of cortical cell number at 2 months of age (4.8×10⁶ cells/hemisphere; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010195#pone.0010195-Saura1" target="_blank">[10]</a> and at 4 months of age (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010195#pone-0010195-g001" target="_blank">Fig. 1</a>) combined with an estimate of 5 mm width of one hemisphere. Raw data (# dead cells/20 µm-thick sagittal section) was converted to a % by the following calculations: 1) the fraction of a hemisphere represented by one 20-µm thick section (1 section =  20 µm/5000 µm, or 1/250^th of a hemisphere), 2) the average number of cortical cells per 20 µm -thick section (4,800,000 cells/hemisphere (2 mo) or 1,500,000 cells/hemisphere (4 mo) ×1/250^th of a hemisphere  = 19,200 cells/20 µm section), and 3) the percentage of dying cells per section (average number of dead cells per 20 µm section/19,200 total cells).</p><p>#actual value = −9.6% (p = 0.09; n = 4 pairs); <i>ND</i>, not determined. *<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010195#pone.0010195-Saura1" target="_blank">[10]</a></p

Apoptotic neuronal death in the cerebral cortex of <i>Psen</i> cDKO mice.

<p>(<i>A</i>) Left: Confocal images of TUNEL-stained cells in the neocortex of control and cDKO mice at low magnification (20×). Right: High magnification (100×) images of individual TUNEL+ cells. Scale bar: 10 µm. (<i>B</i>) Quantification of TUNEL+ cells at the ages of 6 weeks, 2 and 4 months. At 6 weeks, similar low numbers of TUNEL+ cells are present in the neocortex between control and <i>Psen</i> cDKO mice. In contrast, dramatic increases in TUNEL+ cells are observed in the cDKO neocortex at 2 months (7.4-fold increase) and 4 months (15.2-fold increase)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). (<i>C</i>) Quantification of cells positive for activated caspase-9 or caspase-3. The number of active caspase-9+ cells is significantly increased in the neocortex of cDKO mice at 2 and 4 months of age (2m: p<0.005; 4m: p<0.001). The number of active caspase-3+ cells is not significantly different in the cDKO neocortex at 2 months (p>0.05), but is significantly increased in the cDKO neocortex at 4 months (1.7-fold increase; p<0.01)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). Data are presented as the mean ± s.e.m.</p

Increases in degenerating cortical neurons at 2 months followed by significant loss of cortical neurons and volume at 4 months in <i>Psen</i> cDKO mice.

<p>(<i>A</i>) Nissl-stained images of coronal sections from age-matched control (<i>left</i>) and <i>Psen</i> cDKO mutant (<i>right</i>) brains from 2 to 22 months of age are shown. Black horizontal bars delineate neocortical layers. At 2 months, no detectable difference is found in size or shape of the <i>Psen</i> cDKO brain relative to control. However, subsequent ages reveal a gradual decrease in cortical thickness in <i>Psen</i> cDKO mice. Scale bar: 1 mm. (<i>B</i>) Stereological measurement of cortical volume from control (open bars) and <i>Psen</i> cDKO mutant (black bars) brains at 2 and 4 months. Values are presented per hemisphere. At 2 months of age, the cortical volume is similar between control and cDKO mice (p>0.05). At 4 months, the cortical volume in cDKO mice is significantly smaller (21.7% reduction; p<0.001). NS, not significant; ***, p<0.001; n = 4 mice per genotype. (<i>C</i>) Stereological quantification of neuron number in the neocortex. At 4 months, an 8.7% decrease in neuron number (1.15×10⁶) is present in <i>Psen</i> cDKO mice, relative to controls (1.05×10⁶; **p<0.01)(n = 3 control mice +4 cDKO mice). (<i>D</i>) Quantification of degenerative neurons. <i>Top</i>: Fluoro-Jade B labels degenerating neurons in brain sections, and high magnification (100×) confocal images of single cells stained with Fluoro-Jade B. Scale bar: 10 µm. <i>Bottom</i>: Increased numbers of Fluoro-Jade-positive neurons are detected at 2 months in the <i>Psen</i> cDKO cortex (7.6-fold increase; p<0.0001) relative to control, with even greater numbers of dying neurons present by 4 months of age (9.0-fold increase; p<0.0001)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). Data are presented as the mean ± s.e.m.</p

Neurodegeneration and increased adult neurogenesis in the hippocampus of <i>Psen</i> cDKO mice.

<p>(<i>A</i>) Images of Nissl-stained brain sections at 2 and 22 months of age. At 2 months, the neocortex (NCx), hippocampal areas CA1 and CA3, and dentate gyrus (DG) are indistinguishable between <i>Psen</i> cDKO and age-matched controls. By 22 months, extensive loss of white and grey matter is evident in the neocortex of <i>Psen</i> cDKO mice. In the hippocampus, loss of white matter in cDKO mice coincides with the increase in neurons in the dentate gyrus. Scale bar: 1 mm. (<i>B</i>) Stereological quantification of hippocampal volumes. The volume of the <i>Psen</i> cDKO hippocampus is normal at 2 months (control: 4.85×10⁵; cDKO: 4.54×10⁵; p>0.05) and 4 months (control: 4.78×10⁵; cDKO: 4.61×10⁵; p>0.05). By 16–23 months, a 15.5% reduction in hippocampal volume is observed (control: 4.76×10⁵; cDKO: 4.02×10⁵; *p<0.05). NS: not significant; n = 4 mice per genotype (2 mo, 4 mo); n = 6 mice per genotype (16–23 mo). (<i>C</i>) Stereological quantification of the number of cells within the hippocampal dentate gyrus (DG). Cell number in the DG is comparable between cDKO and control mice at 2 months (control: 2.94×10⁵; cDKO: 3.21×10⁵; p>0.05) and 6 months (control: 2.80×10⁵; cDKO: 3.15×10⁵; p>0.3). In contrast, by 19 months, <i>Psen</i> cDKO mice have significantly greater numbers of cells in the DG (control: 3.18×10⁵; cDKO: 4.51×10⁵; **p<0.01)(n = 4 mice per genotype per age). (<i>D</i>) Quantification of Fluoro-Jade B+ degenerating neurons. At 2 months, similar numbers of Fluoro-Jade B+ cells are present in control and cDKO mice (p>0.05), in contrast to a 2.3-fold increase in <i>Psen</i> cDKO mice by 4 months of age (*p<0.05)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). (<i>E</i>) Quantification of apoptotic neurons. Increases in TUNEL+ cells are present in the <i>Psen</i> cDKO hippocampus at 2 months (*p<0.05) and 4 months (***p<0.001), whereas the number of TUNEL+ cells is similar in control and cDKO mice at 6 weeks (p> 0.05)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). (<i>F</i>) More active caspase-9+ cells are present in the <i>Psen</i> cDKO hippocampus at 2 months (p<0.05) and 4 months (p<0.05)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). (<i>G</i>) The number of cells positive for active caspase-3+ in the hippocampus is increased in cDKO mice at 2 months (p<0.05), but not significantly increased at 4 months (p>0.05)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). Data are presented as the mean ± s.e.m.</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

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