Description of the stratification protocol used in this study.

<p>The 4 original sub-phenotypes (denoted by four different colors) were further stratified by removal of families containing affected siblings of another sub-phenotype to yield the Gs level subgroups. These subgroups were further divided according to male only or female-containing pedigrees. Due to intra-family heterogeneity in multiplex cases, some families were included in more than one stratified group. Therefore, the sum of individual and family numbers in subgroups exceeds the numbers listed for the original combined cohort (ALL).</p

Improvement of maximum LOD scores in subgroups with addition of new families.

1<p>Previously reported linked region (see <b>Table S1</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067569#pone.0067569.s001" target="_blank">File S1</a></b> for references).</p

A Novel Stratification Method in Linkage Studies to Address Inter- and Intra-Family Heterogeneity in Autism

<div><p>Most genome linkage scans for autism spectrum disorders (ASDs) have failed to be replicated. Recently, a new ASD phenotypic sub-classification method was developed which employed cluster analyses of severity scores from the Autism Diagnostic Interview-Revised (ADI-R). Here, we performed linkage analysis for each of the four identified ADI-R stratified subgroups. Additional stratification was also applied to reduce intra-family heterogeneity and to investigate the impact of gender. For the purpose of replication, two independent sets of single nucleotide polymorphism markers for 392 families were used in our study. This deep subject stratification protocol resulted in 16 distinct group-specific datasets for linkage analysis. No locus reached significance for the combined non-stratified cohort. However, study-wide significant (P = 0.02) linkage scores were reached for chromosomes 22q11 (LOD = 4.43) and 13q21 (LOD = 4.37) for two subsets representing the most severely language impaired individuals with ASD. Notably, 13q21 has been previously linked to autism with language impairment, and 22q11 has been separately associated with either autism or language disorders. Linkage analysis on chromosome 5p15 for a combination of two stratified female-containing subgroups demonstrated suggestive linkage (LOD = 3.5), which replicates previous linkage result for female-containing pedigrees. A trend was also found for the association of previously reported 5p14-p15 SNPs in the same female-containing cohort. This study demonstrates a novel and effective method to address the heterogeneity in genetic studies of ASD. Moreover, the linkage results for the stratified subgroups provide evidence at the gene scan level for both inter- and intra-family heterogeneity as well as for gender-specific loci.</p></div

Linkage data obtained for four overlapping regions, between two different subgroups.

<p>Calculated LOD scores were improved after combining the two respective subject groups. It further validates the original computed LOD scores and serves as a partial replication of our linkage results.</p>1<p>Previously reported linked region (see <b>Table S1</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067569#pone.0067569.s001" target="_blank">File S1</a></b> for references).</p>a<p>A positive LOD score of 2.94 was obtained when no stratification was applied to 392 families (i.e., ALL). The linkage results shown here for the SNP rs11658900 suggest that the subgroups G3 and G4 are the strongest contributors to the original LOD score in the unstratified cohort (ALL). Therefore, combining G3 and G4 data resulted in an improvement in the LOD score relative to ALL with fewer families (i.e., 249).</p

Heat map of LOD scores.

<p>A graphical representation (heat map) of the LOD score data (cut-off ≥2.0) was generated to visually demonstrate the computed linkage scores for each subgroup in a hierarchy. The heat map compares LOD score patterns for the 16 subgroups. As expected, there were more similarities within each ADI-R group (e.g., G1, G1s, G1M, and G1Fc) than between two different ADI-R groups. Each horizontal band represents a SNP while each column represents a stratified subgroup, with the exception of the first column which represents the combined (ALL) cohort. <b>Table S4</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067569#pone.0067569.s002" target="_blank">File S2</a></b> lists the SNPs and LOD scores contributing to the identified segregation patterns by subgroups (i.e., hot spots). The corresponding genomic positions of the SNPs contributed to the heat map (Y-axis) are listed in <b>Table S4</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067569#pone.0067569.s002" target="_blank">File S2</a></b>. The heat map was generated using MeV software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067569#pone.0067569-Saeed1" target="_blank">[41]</a>.</p

Loci with highest LOD scores for a given subtype.

<p>The highest LOD scores (shown in bold font), were obtained after including additional families (i.e., n2), as described in <b>Table S2</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067569#pone.0067569.s001" target="_blank">File S1</a></b>.</p>1<p>Previously reported linked region (see <b>Table S1</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067569#pone.0067569.s001" target="_blank">File S1</a></b> for references).</p>*<p>According to permutation tests reached a study-wide significant (i.e., p = 0.02, see <b>Table S9A</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067569#pone.0067569.s003" target="_blank">File S3</a></b>); G1 = 87% white, G1s = 98% white.</p>a<p>An example of loci with highest LOD scores for the first level of subgrouping (intra-family heterogeneity included). This is potentially a shared linked region for all affected siblings in a pedigree regardless of concordance status, for a given subtype (i.e., G1).</p>b<p>Loci with highest LOD scores when only group-specific concordant autistic subjects were maintained (intra-family heterogeneity reduced). It is potentially a linked region only for concordant siblings in a given subtype (i.e., G1s and G4s).</p

Runtime and memory usage in Fast using simulated data compared with publicly available stand-alone implementations (denoted Orig).

<p>All runtimes are in minutes and memory usages are in megabytes. ‘Linear/Logistic’ uses genotype data while ‘Summary’ uses summary data. All indicates running Gwis, Bimbam, Vegas, Minsnp, Minsnp-gene and Single-snp simultaneously in Fast.</p>*<p>No permutations.</p

Defining the Contribution of <i>CNTNAP2</i> to Autism Susceptibility

<div><p>Multiple lines of genetic evidence suggest a role for <i>CNTNAP2</i> in autism. To assess its population impact we studied 2148 common single nucleotide polymorphisms (SNPs) using transmission disequilibrium test (TDT) across the entire ~3.3 Mb <i>CNTNAP2</i> locus in 186 (408 trios) multiplex and 323 simplex families with autistic spectrum disorder (ASD). This analysis yielded two SNPs with nominal statistical significance (rs17170073, <i>p</i> = 2.0 x 10^-4; rs2215798, <i>p</i> = 1.6 x 10^-4) that did not survive multiple testing. In a combined analysis of all families, two highly correlated (<i>r</i>² = 0.99) SNPs in intron 14 showed significant association with autism (rs2710093, <i>p</i> = 9.0 x 10^-6; rs2253031, <i>p</i> = 2.5 x 10^-5). To validate these findings and associations at SNPs from previous autism studies (rs7794745, rs2710102 and rs17236239) we genotyped 2051 additional families (572 multiplex and 1479 simplex). None of these variants were significantly associated with ASD after corrections for multiple testing. The analysis of Mendelian errors within each family did not indicate any segregating deletions. Nevertheless, a study of <i>CNTNAP2</i> gene expression in brains of autistic patients and of normal controls, demonstrated altered expression in a subset of patients (<i>p</i> = 1.9 x10^-5). Consequently, this study suggests that although <i>CNTNAP2</i> dysregulation plays a role in some cases, its population contribution to autism susceptibility is limited.</p> </div

A plot of -log₁₀<i>p</i> across <i>CNTNAP2</i> from family-based association test (TDT) results on 323 simplex families.

<p>A plot of -log₁₀<i>p</i> across <i>CNTNAP2</i> from family-based association test (TDT) results on 323 simplex families.</p

Demonstration of independent effects for rs16847548 and rs16856785 on QT interval stratified by ethnicity.

<p>Single SNP indicates a regression model with rs16847548 OR rs16856785, and multi-SNP indicates both SNPs are in the model. A1 refers to the minor allele in the CEU HapMap population. <b>Bold</b> signifies P-values<0.05. Genomic position is given relative to Build35 of the Human Genome. β is the effect size under an additive genetic model. SE = standard error.</p