List of complete mtDNA sequences included in Figure 1.

a<p>Family ID numbers correspond to the numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone-0042242-g001" target="_blank">Figure 1</a>.</p>b<p>This family is originally from Lebanon.</p>c<p>This family is originally from Turkey.</p>d<p>This family is originally from Benin.</p>e<p>Haplogroup classification based on the most updated human mitochondrial phylogeny <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-vanOven1" target="_blank">[35]</a>.</p>f<p>Nucleotide positions and changes refer to rCRS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-Andrews1" target="_blank">[57]</a>. The non-synonymous nucleotide changes at nps 8860 (8860G) and 15326 (15326G), present in all LHON samples, are not included because they are private mutations of the reference sequence. LHON mutations are in bold, while those with a possible synergistic effect are underlined.</p>g<p>Only the entire mitochondrial sequence #6 was already published (Olivieri et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-Olivieri1" target="_blank">[44]</a>).</p

Phylogenetic tree of 16 complete mtDNA sequences from LHON patients.

<p>Rare LHON mutations are shown in bold. The position of rCRS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-Andrews1" target="_blank">[57]</a> is indicated for reading off sequence motifs. Mutations are shown on the branches; they are transitions unless a base is explicitly indicated. The prefix @ designates reversions, while suffixes indicate: transversions (to A, G, C, or T), indels (+, d), gene locus (∼t, tRNA; ∼r, rRNA), synonymous or non-synonymous changes (s or ns) and heteroplasmies (h). Recurrent mutations are underlined. One mtDNA sequence (I; black circle) from a control subject (GenBank accession number FJ190383) was also included to illustrate that sequences 11 and 12 acquired independently the LHON mutation m.14568C>T. The haplogroup affiliation of each haplotype is based on mutational motifs and follows the most updated human phylogeny <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042242#pone.0042242-vanOven1" target="_blank">[35]</a>.</p

RFLP evaluation of heteroplasmy.

<p>Electrophoresis on Metaphor gel of the restriction fragments is shown. Wild type and mutant fragments are indicated with the corresponding size, expressed as base pairs (bps). Non affected negative control individual is indicated as C. DNA samples were extracted from whole blood, except when indicated: whole blood is indicated as B, urinary epithelium is indicated as U, platelet fraction is indicated as P, hairs are indicated as H and skeletal muscle is indicated as M. Molecular weight marker is indicated as MWM.</p

Sequence alignment and conservation analysis of ND1, ND4L and ND6 protein sequences.

<p>Alignments of protein sequences from eukaryotes and mammals, including <i>Homo sapiens,</i> are reported. The upper lines represent the human sequence with the amino acid changes induced by the corresponding LHON mutation. Mutated positions are in bold and indicated by an asterisk. Different shading corresponds to increasing conservation levels: amino acid conservation between 70% and 90% are highlighted in light grey, amino acid conservation between 90% and 99% are highlighted in dark grey, and invariant positions (100% conservation) are highlighted in black. Alignment gaps are indicated by a hyphen (-).</p

Conservation analysis of rare primary LHON mutations.

a<p>The GenBank database was searched on March 2^nd 2012 when a total of 10,304 mtDNA coding regions were deposited.</p

Additional file 1: of Increased Wnt and Notch signaling: a clue to the renal disease in Schimke immuno-osseous dysplasia?

Supplementary Methods, Tables, and Figures. (PDF 45904 kb

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

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

Scatter plots of the intellectual quotient and the Autism Diagnostic Interview-Revised (ADI-R) scores of the patients with ASD screened for <i>SHANK1-3</i> mutations.

<p>Mutations in <i>SHANK1-3</i> are associated with a gradient of severity in cognitive impairment. <i>SHANK1</i> mutations were reported in patients without ID (green dots). <i>SHANK2</i> mutations were reported in patients with mild ID (orange dots). <i>SHANK3</i> mutations were found in patients with moderate to severe deficit (red dots). Black dots correspond to the patients enrolled in the PARIS cohort screened for deleterious <i>SHANK1-3</i> mutations (n = 498). In addition to the PARIS cohort <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Durand1" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Pinto1" target="_blank">[8]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Leblond1" target="_blank">[18]</a>, three patients with a <i>SHANK1</i> deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Sato1" target="_blank">[19]</a> and two patients with a <i>SHANK2</i> deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004580#pgen.1004580-Berkel1" target="_blank">[14]</a> were included in the scatter plot. A high score of the ADI-R is associated with a more severe profile. The threshold of the “Social”, “Verbal”, “Non-Verbal” and “Repetitive Behavior” Scores are 10, 8, 7 and 3, respectively.</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