Evidence for genotype-dependent activation of <i>CYFIP1</i> by FOXP2.

<p>(A) rs4778298 (orange) associated with variation across the surface area of the left supramarginal gyrus is 2.2 kb upstream of <i>CYFIP1</i>^<i>short</i>. Seven linked variants (r²>0.9), and an expression probe specific for <i>CYFIP1</i>^<i>short</i> (Affymetrix 3583676), are shown. (B) A significant positive association between levels of <i>FOXP2</i> and <i>CYFIP1</i> is seen in human brain (r² = 0.36; p = 2.5x10^-5). (C) rs66903469-T is predicted to bind FOXP2 with 11.8-fold greater affinity than the alternate C allele. MA0593.1 corresponds to the position weight matrix for FOXP2 defined in JASPAR. (D) rs66903469-T (r² with rs4778298-C of 0.93), is strongly associated with increased levels of <i>CYFIP1</i>^<i>short</i> (p = 2.9x10^-5).</p

ASD scores are significantly positively correlated with total grey matter volume.

<p>The volumes of grey and white matter are normalised for total intracranial volume. A regression line is plotted where the Pearson’s product-moment correlation is significant with p<0.05. ASD symptom score refers to an aggregate score from the four Children’s Social and Behavioural Questionnaire (CSBQ) subscales, (1) social interest, (2) social understanding, (3) stereotypy and (4) resistance to change. Abbreviations: con, Control; unaf sib, Unaffected Siblings, adhd, Attention Deficit Hyperactivity Disorder; ASD, autism spectrum disorder.</p

The ADHD microbiome contains significantly increased levels of predicted cyclohexadienyl dehydratase (CDT; KEGG Ortholog K01713; EC:4.2.1.51), responsible for phenylalanine synthesis (Fig B in S1 Appendix).

<p>This analysis is based on functional predictions deriving from 16S profiles of the microbiome, as performed by PICRUSt [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183509#pone.0183509.ref033" target="_blank">33</a>]. Box plots represent the relative abundance of predicted CDT, with 5–95% percentile whiskers (dots represent outliers). The significance was tested with a non-parametric MWU (* p = 0.038), Bonferroni-corrected for 15 K numbers identified.</p

Individual regions of interest (ROIs) drive associations observed in the discovery cohort.

<p>(A) Pairwise analyses of the top signal for each of volume, surface area, and thickness from the discovery cohort determined that aggregate effects were attributable to individual ROIs (and see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158036#pone.0158036.s006" target="_blank">S5 Table</a>). Post-hoc permutation testing, accounting for all 52 tag-SNPs and ROIs examined, found a significant association between rs4778298 genotype and the surface area of the left supramarginal gyrus (lh.SMG) in Caucasian subjects (p_{permutation corrected} = 7.5x10^-3). (B) Linear effects at the CC and lh.SMG were observed for rs12102024 and rs4778298, respectively, consistent with an effect of allele dosage. rh.ACC and CC correspond to right anterior cingulate cortex and corpus callosum, respectively.</p

Final, generalised mixed-effect model showing fixed effects for ASD score modelled against white matter residual and grey matter volumes.

<p>Formula: </p><p></p><p></p><p>“∼” means modelled against, and “(1| factor)” means that a factor is included as a random effect.</p><p>A generalised mixed-effect model is run using normalised volumes of grey and white matter as explanatory variables together with age as a random effect. ASD score is set as the response variable. The final model is derived following an iterative model selection procedure that involves comparing successive models using Akaike’s Information Criterion (see Methods for detailed description of model selection procedure). ASD score refers to an aggregate score from the four Children’s Social and Behavioural Questionnaire (CSBQ) subscales, (1) social interest, (2) social understanding, (3) stereotypy and (4) resistance to change.</p><p>Abbreviations: WM, normalised WM volume; GM, normalised GM volume; US, unaffected siblings; WM:GM, WM by GM interaction; US: Age, unaffected sibling by Age interaction; ADHD:Age, ADHD by Age interaction.</p

Descriptive characteristics of the study samples.

<p>Descriptive characteristics of the study samples.</p

Linear association between common variant genotype at <i>CYFIP1</i> and surface area across the left supramarginal gyrus (lh.SMG) in an independent cohort.

<p>(A) An association between rs4778298 genotype and lh.SMG surface area was observed (p_nominal = 3.2x10^-2) in a large independent population (n = 1276 for rs12102024 and rs1051288; n = 2621 for rs4778298). Similar to what we observed in our discovery cohort, this effect became stronger on analysis of Caucasian subjects alone and survived correction for multiple comparisons (p_nominal = 1.3x10^-2; p_{bonferroni corrected} = 4.9x10^-2). (B) As in the discovery cohort, a linear relationship between genotype and lh.SMG surface area was observed. Notably, however, directionality was the reverse of what was seen previously. rh.ACC and CC correspond to right anterior cingulate cortex and corpus callosum, respectively.</p

ASD score contour graph for the interaction between WM, GM and ASD scores.

<p>The best mixed effects model was converted into a function in R which allowed ASD scores to be extrapolated for a range of GM and WM volumes (see Methods for full description). The best mixed effects model included GM and WM residual terms (see Methods). Volumes are normalised for total intracranial volume. ASD score refers to an aggregate score from the four Children’s Social and Behavioural Questionnaire (CSBQ) subscales, (1) social interest, (2) social understanding, (3) stereotypy and (4) resistance to change.</p

Common variant genotypes at 15q11.2 arssociated with differences in regional cortical volume, thickness, and surface area in a discovery cohort.

<p>(A) Aggregate -log p-values for single nucleotide polymorphisms (SNPs) are plotted against genomic position. P-values reflect the relationships between genotype in the discovery cohort and volume (left), surface area (middle), or thickness (right) across all regions of interest (ROIs). Significant associations (p_{permutation corrected}<0.05) appear above the horizontal red line. Linkage disequilibrium of top hits (diamonds) to typed variants is specified, and estimated recombination rates plotted (blue lines). r² in the legend indicates the correlation between genotype at each tag-SNP (circles) and genotype at the top hit (diamond). (B) Top aggregate p-values and their SNP descriptions for each trait examined in the full cohort and the Caucasian subset are summarized.</p

ASD scores decrease significantly with age within the ADHD groups.

<p>A regression line is plotted where the Pearson’s product-moment correlation is significant with p<0.05. ASD score refers to an aggregate score from the four Children’s Social and Behavioural Questionnaire (CSBQ) subscales, (1) social interest, (2) social understanding, (3) stereotypy and (4) resistance to change. Abbreviations: con, Control; unaf sib, Unaffected Siblings, ADHD, Attention-deficit/hyperactivity disorder; ASD, autism spectrum disorder.</p