Alpha diversity indices for TcGP63I amplicon diversity derived from pairs of congenital Chagas disease cases.

<p>Diversity indices were derived from STs defined at 99% sequence similarity. Bar plot and associated <i>x</i>-axis on the right hand side shows the Shannon diversity index calculated in Mothur [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003458#pntd.0003458.ref034" target="_blank">34</a>], with error bars defining upper and lower 95% confidence intervals.</p

Principal coordinates analysis of sequence diversity between chronic Chagas Disease patient TcGP63I antigenic repertoires.

<p>Genetic distances are based on a weighted unifrac metric. Plot A shows diversity comparisons among Go-as asymptomatic (asympt) and symptomatic (sympt) clinical cases, as well as one acute case. Plot B shows Goias cases with symptoms categorised as acute, card (cardiopathy), card + mega (cardiopathy as well as megacolon and / or megaesophagous), mega (megacolon and / or megaesophagous) or asympt (asymptomatic). Plot C shows comparisons among Cochabamba clinical cases (not including congenital cases) classified as either asymptomatic (asympt) and symptomatic (sympt). The dashed circle on plot C indicates samples unambiguously defined as TcI at the ND5 locus. Pairs of sequential isolates from the same patient are labelled x and y respectively.</p

Yang and Neilson estimates for positive selection within and among abundant 97% STs identified in this study.

<p>^a Numbers in brackets represent the number of 99% STs define within each cluster from which estimates were generated.</p><p>^b P values are give for Fisher’s exact tests for deviation from the neutral expectation of Ka/Ks = 0.</p><p>Yang and Neilson estimates for positive selection within and among abundant 97% STs identified in this study.</p

Bar plot showing sequence type identity and abundance defined at 97% similarity for the ND5 locus across all samples.

<p>A—Goias cohort chronic/intermediate cases; B—Cochabamba chronic/intermediate cases; C—Cochabamba congenital cases. Y axes show log transformed abundance (read counts). X axes show clustered bars for individual samples. Sequence type identities are given in the legend. Stars denote congenital pair from Goias. Labels x (6416 / 6452), y (6401 / 6536) and z (6379 / 6445) sample pairs from the same patient at different time points (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003458#pntd.0003458.t001" target="_blank">Table 1</a>).</p

Samples provenance and symptoms.

<p>^a Samples from Goias congenital case</p><p>^x Samples from the same patient taken >12 months apart</p><p>^y Samples from the same patient taken < 6 months apart</p><p>^z Samples taken from the same patient >12 months apart</p><p>Samples provenance and symptoms.</p

Genetic alterations identified in the control subject SWE_Q56_508.

<p>A. <i>SHANK2</i> splice mutation (IVS22+1G>T) detected in a Swedish female control, SWE_Q56_508. The mutation altered the donor splicing site of exon 22 and led to a premature stop in all <i>SHANK2</i> isoforms except for the <i>AF1411901</i> isoform, where it altered the protein sequence (G263V). B. CNVs in the same individual altering <i>LOC339822</i>, <i>SNTG2</i>, <i>PXDN</i> and <i>MYT1L</i>. The two close duplications span 264 kb and 245 kb on chromosome 2 and altered <i>LOC339822</i> and <i>SNTG2</i>, and <i>PXDN</i> and <i>MYT1L</i>, respectively. Dots show the B allele frequency (BAF; in green), Log R ratio (LRR; in red), and QuantiSNP score (in blue). Lower panel: all CNVs listed in the Database of Genomic Variants (DGV) are represented: loss (in red), gain (in blue), gain or loss (in brown). H, homer binding site; D, dynamin binding site; C, cortactin binding site.</p

Prevalence and meta-analysis of coding-sequence variant studies in ASD.

<p>A. The prevalence and the confidence interval from a set of single coding-sequence variant studies, and the pooled prevalence and the confidence interval of the meta-analysis. The prevalence is indicated by circles in red, pink, purple and black for “ASD all” (all ASD patients), “ASD IQ<70” (patients with ID; IQ<70), “ASD IQ>70” (patients with normal IQ), and “CTRL” (controls), respectively. Three categories are used to study the prevalence of coding-sequence variants in ASD and controls: all or “A” (all mutation), Damaging or “D” (damaging missense mutation; score obtained from polyphen-2), and Truncating or “T” (mutation altering SHANK protein). The plotted circles are proportional to the corresponding sample size. B. Meta-analysis of coding-sequence variant studies altering <i>SHANK</i> genes. For each study, the Odds ratio and confidence interval is given. Each meta-analysis is calculated using inverse variance method for fixed (IV-FEM) and random effects (IV-REM). The statistics measuring heterogeneity (Q, I² and Tau²) are indicated. The number under the scatter plot correspond to independent studies: 1 = “This study”, 2 = “ Sato et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Sato1" target="_blank">[19]</a>”, 3 = “Berkel et al. (2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Berkel1" target="_blank">[14]</a>”, 4 = “Leblond et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Leblond1" target="_blank">[18]</a>”, 5 = “Boccuto et al. (2012) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Boccuto1" target="_blank">[17]</a>”, and 6 = “[This Study and Durand et al. 2007 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Durand1" target="_blank">[6]</a>]”, 7 = “[Gauthier et al. (2009–2010) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Gauthier1" target="_blank">[16]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Gauthier2" target="_blank">[47]</a>]”, 8 = “Moessner et al. (2007) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Moessner1" target="_blank">[13]</a>”, 9 = “Schaff et al. (2011) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Schaaf1" target="_blank">[35]</a>”. IV, Inverse Variance; FEM, Fixed Effect Method; REM, Random Effect Method; OR, Odds Ratio; CI, Confidence Interval; IQ, Intellectual Quotient; CNV, Copy Number Variant.</p

Characterization of the functional impact of <i>SHANK2</i> mutations in cultured neuronal cells.

<p>A. The colocalization of <i>ProSAP1A/SHANK2</i>-EGFP (postsynaptic marker) and Bassoon (presynaptic marker) indicated that the mutations did not disturb the formation of SHANK2 clusters at excitatory synapses along the dendrites. B. The quantification of synapse density was performed on 20 transfected hippocampal neurons per construct from at least three independent experiments. The majority of the <i>ProSAP1A</i> variants affecting a conserved amino acid among SHANK proteins reduced significantly the synaptic density compared with the variants that affect amino acid non conserved among SHANK proteins (Mann-Whitney U-test: n_WT = 20, n_mut = 20; U_S557N = 82.5, p_S557N = 0.001; U_R569H = 124, p_R569H = 0.04; U_L629P = 149, p_L629P = 0.17; U_V717F = 114, p_V717F = 0.02; U_A729T = 73, p_A729T = 0.000; U_K780Q = 154, p_K780Q = 0.221; U_R818H = 108, p_R818H = 0.012; U_A822T = 154.5, p_A822T = 0.224; U_V823M = 129, p_V823M = 0.056; U_Y967C = 134, p_Y967C = 0.076; U_G1170R = 78, p_G1170R = 0.001; U_R1290W = 142, p_R1290W = 0.121; U_Q1308R = 162, p_Q1308R = 0.314; U_D1535N = 97, p_D1535N = 0.005; U_P1586L = 137, p_P1586L = 0.910; U_L1722P = 79, p_L1722P = 0.001, *p<0.05, **p<0.01, ***p<0.001). <b>C.</b> Effect of the variants on synaptic density. The y-axis represents −log P compared to WT (P obtained with Mann-Whitney test). After Bonferroni correction for 16 tests, only P values<0.003 were considered as significant. Variants represented in red were specific to ASD, in orange were shared by ASD and controls, and in green were specific to the controls. Open circles and filled circles represent non conserved and conserved amino acids, respectively. Prim, primary; second, secondary.</p

<i>SHANK</i> variants in patients with ASD and controls.

<p>Coding-sequence variants identified only in patients with ASD (upper panel), shared by patients and controls (lower panel and underlined), and present only in controls (lower panel). Truncating variants are indicated in red. The variants predicted as deleterious or benign are indicated in orange and green, respectively. Coding-sequence variants with a proven <i>in vitro</i> functional impact are indicated with black stars. Conserved domains are represented in color: SPN (yellow), Ankyrin (red), SH3 (orange), PDZ (blue) and SAM (green).</p

Characterization of CNVs in three patients carrying a <i>de novo</i> deletion of <i>SHANK2</i>.

<p>Paternally or maternally inherited CNVs are indicated by squares and circles, respectively. <i>De novo</i> CNVs are indicated by stars. Deletions and duplications are indicated in red and blue, respectively. CNVs hitting exons or only introns are filled with grey and white, respectively. Squares and circles within star represent <i>de novo</i> CNV of paternal or maternal origin; circles within squares represent CNV inherited by father or mother. ABCC6, ATP-binding cassette, sub-family C, member 6 pseudogene 2; ADAM, ADAM metallopeptidase; AMY1, amylase (salivary); AMY2A, amylase (pancreatic); ARHGAP11B, Rho GTPase activating protein 11B; CAMSAP1L1, calmodulin regulated spectrin-associated protein 1-like 1; CHRNA7, cholinergic receptor, nicotinic, alpha 7; CNTN4, contactin 4; CTNNA3, catenin (cadherin-associated protein), alpha 3; CYFIP1, cytoplasmic FMR1 interacting protein 1; DUSP22, dual specificity phosphatase 22; GALM, galactose mutarotase; GCNT2, glucosaminyl (N-acetyl) transferase 2; GOLGA, golgi autoantigen, golgin subfamily a; GSTT1, glutathione S-transferase theta 1; HLA-DRB, major histocompatibility complex, class II, DR beta; LAMA4, laminin, alpha 4; NIPA, non imprinted in Prader-Willi/Angelman syndrome; NLGN1, neuroligin 1; NME7, non-metastatic cells 7; OR, olfactory receptor; PCDHA, protocadherin alpha; RFPL4B, ret finger protein-like 4B; RHD, Rh blood group, D antigen; SFMBT1, Scm-like with four mbt domains 1; SHANK2, SH3 and multiple ankyrin repeat domains 2; SMC2, structural maintenance of chromosomes 2; TNS3, tensin 3; TUBGCP5, tubulin, gamma complex associated protein 5; UGT2B17, UDP glucuronosyltransferase 2 family, polypeptide B17.</p