De Novo Sequence and Copy Number Variants Are Strongly Associated with Tourette Disorder and Implicate Cell Polarity in Pathogenesis

Abstract

We previously established the contribution of de novo damaging sequence variants to Tourette disorder (TD) through whole-exome sequencing of 511 trios. Here, we sequence an additional 291 TD trios and analyze the combined set of 802 trios. We observe an overrepresentation of de novo damaging variants in simplex, but not multiplex, families; we identify a high-confidence TD risk gene, CELSR3 (cadherin EGF LAG seven-pass G-type receptor 3); we find that the genes mutated in TD patients are enriched for those related to cell polarity, suggesting a common pathway underlying pathobiology; and we confirm a statistically significant excess of de novo copy number variants in TD. Finally, we identify significant overlap of de novo sequence variants between TD and obsessive-compulsive disorder and de novo copy number variants between TD and autism spectrum disorder, consistent with shared genetic risk.

Keywords: TIC Genetics; Tourette disorder; cell polarity; copy number variants; de novo variants; gene discovery; microarray genotyping; multiplex; simplex; whole exome sequencing.

Figure 1.. Study Overview

Our group previously generated and analyzed WES data from 511 TD trios, generated by the TIC Genetics (325 trios) and TAAICG (186 trios) consortia (Willsey et al., 2017). In this study, we expand the number of trios with WES data by 291 (92 from TIC Genetics, 18 from UTC, and 181 from TSGENESEE). We leverage recurrent de novo variants occurring within the same gene in unrelated individuals to identify a high-confidence gene, CELSR3. Next, we identify de novo CNVs from the WES data and significantly associate these variants with TD. Third, we replicate the association of de novo CNVs by analysis of microarray data from 399 partially overlapping TIC Genetics trios. Finally, based on the rate of de novo variants, we assess the genomic architecture of TD. CNVs, copy number variants; SSC, Simons Simplex Collection; TAAICG, Tourette Association of America International Consortium for Genetics; TD, Tourette disorder; TIC Genetics, Tourette International Collaborative Genetics consortium; TSGENESEE, Tourette Syndrome Genetics Southern and Eastern Europe Initiative; UTC, Uppsala Tourette Cohort. See Figure S1 for an overview of quality control and sample filtering and Table S1 for sample metrics.

Figure 2.. Combined Burden Analysis Identifies Differences in De Novo Rate in Simplex versus Multiplex Families

We defined a consensus region, consisting of a set of intervals with high-quality coverage across all samples. We then estimated the de novo mutation rates per base pair in this consensus region (STAR Methods). We converted the mutation rate per base pair to an expected rate per child (proband or control) by multiplying the mutation rate per base pair by the size of the total RefSeq hg19 “coding” region (33,828,798 bp). (A) De novo variants are overrepresented in simplex TD trios only. LGD variants are significantly increased in simplex TD probands compared to SSC controls (RR 1.93; p = 0.0028; one-sided rate ratio test). Mis3 variants also trend toward enrichment (RR 1.18; p = 0.08). Therefore, de novo damaging variants as a group are overrepresented in simplex TD (RR 1.29; p = 0.0061). In contrast, de novo variants in any category are not significantly increased in multiplex TD families, though de novo damaging variants trend in that direction (RR 1.16; p = 0.26). Additionally, the rate of de novo LGD variants may be higher in simplex versus multiplex trios though the difference does not reach statistical significance (RR 1.73; p = 0.20). (B) Restricting the analysis to de novo variants in mutation-intolerant genes (missense Z score ≥ 3.891 or pLI ≥ 0.9; Lek et al., 2016) reveals much larger effect sizes, particularly in simplex families. Comparing simplex to multiplex trios reveals significant differences for de novo nonsynonymous variants (RR 3.91; p = 0.023) and for de novo missense variants (RR 5.15; p = 0.047), but not for de novo LGD variants only (RR 2.66; p = 0.28; STAR Methods). Damaging, LGD + Mis3; LGD, likely gene disrupting (insertion of premature stop codon, disruption of canonical splice site, and frameshift insertion-deletion variant); Mis, missense; Mis3, probably damaging missense variants (PolyPhen2 [HDIV] score ≥ 0.957; Adzhubei et al., 2010, 2013); Nonsyn, nonsynonymous; RR, rate ratio; Syn, synonymous. Error bars in (A) and (B) represent the 95% confidence interval (CI). When necessary, we truncated the lower bound of the CI to 0. See Figures S2, S4, and S5 and Table S3.

Figure 3.. De Novo CNV Burden Analysis

We called de novo CNVs from WES data and array data with CoNIFER (Krumm et al., 2012) and PennCNV (Wang et al., 2007), respectively. We utilized different methods for normalization to make the results comparable across different samples sets. For the WES data (A), we normalized the de novo CNV rate by the number of discontinuous capture array intervals in each cohort (Figure S3A). For the microarray data (B), we restricted de novo CNV calling to a set of SNPs shared across all arrays and further removed any outlier SNPs based on the LRR (Figure S3B; see STAR Methods for details). We compared each group with SSC sibling controls using a Wilcoxon rank-sum test in R. We also used the SSC probands as positive controls to validate our de novo calling pipelines. We used all de novo calls (confirmed and unconfirmed) in the burden analysis. Both the WES data (A) and array data (B) demonstrate that de novo CNVs are significantly increased in TD compared to SSC controls and that de novo CNVs occur at approximately the same rate in TD and in ASD. Error bars in (A) and (B) represent the 95% confidence interval (CI). When necessary, we truncated the lower bound of the CI to 0. See also Tables S3, S4, and S5.