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

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.

To follow up our phase 1 study (Willsey et al., 2017), we conducted WES on 291 new “phase 2” TD trios (802 total trios across phase 1 and 2). We also analyzed 582 new phase 2 control trios from the Simons Simplex Collection (SSC) (1,184 total control trios across phase 1 and 2). After quality control, we trimmed to 777 TD trios and 1,153 SSC trios for de novo sequence variant calling

Cohort characteristics as well as sequencing metrics are summarized per cohort and by phase. 95% confidence intervals are displayed as ±, where relevant.

We leveraged GATK to conduct alignment, quality control, and variant calling (DePristo et al., 2011, McKenna et al., 2010, Van der Auwera et al., 2013). We conducted joint genotyping across the entire set of phase 1 and phase 2 TD trios, as well as the entire set of control trios, in order to reduce batch effects. We further modified our previous de novo calling pipeline (Willsey et al., 2017) to utilize the GATK genotype refinement workflow. We defined likely gene disrupting (LGD) variants as insertion of a premature stop codon, disruption of a canonical splice site, or a frameshift insertion or deletion, and probably damaging missense 3 (Mis 3) variants include missense variants with a PolyPhen2 (HDIV) score ≥ 0.957 (Adzhubei et al., 2010, Adzhubei et al., 2013). We refer to the set of LGD and Mis3 variants as “damaging”.
We detected 309 de novo coding variants from phase 2 samples (1.09 variants per sample). Applying the new pipeline to the phase 1 samples, we detected a total of 466 de novo coding variants (0.94 variants per sample). The number of de novo variants per individual followed a Poisson distribution , and our new pipeline achieved a 95.9% validation rate across phase 1 and 2 TD samples. See STAR Methods for more details. We did not validate the de novo variants in control samples, and therefore, we conducted all burden analyses using all de novo variants identified in TD and control trios. However, for gene discovery, we considered validated de novo variants only. WES coverage varied across cohorts and phases because of the different capture arrays and sequencing protocols used and was positively correlated with the number of de novo variants observed per individual (STAR Methods). To account for these differences, we compared mutation rates, instead of the number of de novo variants observed per individual, to normalize for the number of bases with sufficient joint coverage for de novo calling (Willsey et al., 2017). To further reduce biases, we estimated mutation rates within a high-confidence region with high joint coverage across all cohorts (consensus region; Table 1; STAR Methods). We then compared the rate between TD probands and SSC siblings with a one-sided rate ratio test, as previously described (Willsey et al., 2017). We also confirmed that the overall rate of coding de novo sequence variants does not differ between phase 1 and phase 2 TD trios (rate ratio [RR] 1.03; p = 0.81; two-sided rate ratio test).

We excluded any de novo variants located outside of the consensus regions and then calculated the mutation rate per base pair and 95% CI using t test in R. See also Figures S4 and S5. Comorbid, probands with TD and ADHD/OCD; damaging, LGD + Mis3; in frame, indel causing in-frame deletion or insertion (loss or gain of amino acids); intolerant LGD, de novo LGD variants occurring in genes with pLI greater than 0.9; intolerant Mis, de novo missense variants occurring in genes with missense Z score greater than 3.891; intolerant Nonsyn, intolerant Mis + intolerant LGD; LGD, likely gene disrupting (insertion of premature stop codon, disruption of canonical splice site, and insertion-deletion frameshift); LGD FS, insertion-deletion variant causing frameshift; LGD SNV, point mutation causing insertion of premature stop codon and disruption of canonical splice site; Nonsyn, nonsynonymous; simplex, parents unaffected with TD; Syn, synonymous; unknown, phenotypic data unavailable for parents.

Follow NEWS.am Medicine on Facebook and Twitter