Progress in Genetic Studies of Tourette’s Syndrome

Tourette’s Syndrome (TS) is a complex disorder characterized by repetitive, sudden, and involuntary movements or vocalizations, called tics. Tics usually appear in childhood, and their severity varies over time. In addition to frequent tics, people with TS are at risk for associated problems including attention deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), anxiety, depression, and problems with sleep. TS occurs in most populations and ethnic groups worldwide, and it is more common in males than in females. Previous family and twin studies have shown that the majority of cases of TS are inherited. TS was previously thought to have an autosomal dominant pattern of inheritance. However, several decades of research have shown that this is unlikely the case. Instead TS most likely results from a variety of genetic and environmental factors, not changes in a single gene. In the past decade, there has been a rapid development of innovative genetic technologies and methodologies, as well as significant progresses in genetic studies of psychiatric disorders. In this review, we will briefly summarize previous genetic epidemiological studies of TS and related disorders. We will also review previous genetic studies based on genome-wide linkage analyses and candidate gene association studies to comment on problems of previous methodological and strategic issues. Our main purpose for this review will be to summarize the new genetic discoveries of TS based on novel genetic methods and strategies, such as genome-wide association studies (GWASs), whole exome sequencing (WES) and whole genome sequencing (WGS). We will also compare the new genetic discoveries of TS with other major psychiatric disorders in order to understand the current status of TS genetics and its relationship with other psychiatric disorders.


Clinical Features of Tourette's Syndrome
Tourette's Syndrome (TS) is a complex neuropsychiatric and developmental disorder characterized by repetitive, sudden, involuntary movements and vocalizations, called tics. Vocal tics demonstrated as the involuntary outburst of obscene words, or socially inappropriate and derogatory remarks, called coprolalia, are rare (in about 8% of TS patients) according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) [1]. TS is the most serious form of a spectrum of tic disorders [1], not only due to its complexity and duration of tic symptoms, but also due to its higher comorbidity, impairment of social function and compromised quality of life [2]. In TS,

Genome-Wide Linkage Studies of TS
Due to the autosomal dominant-like inheritance pattern in some large extended pedigrees with TS and comorbidities, earlier stage of genetic research of TS had focused on genome-wide linkage studies (GWLSs) of TS families, which assess the probability in a given pedigree or pedigrees, where the disease and the genetic marker(s) are cosegregating. Several chromosomal regions were reported as potential candidate loci for TS through GWLSs, including chromosome 3p21-p14 [41], 4q34-q35 [42,43], 5q35.2-q35.3 [43], 6p21 [44], 7q31 [45][46][47], 11q23-24 [48][49][50], 13q31.1 [51], 15q21.1-15q21.3 [52], and 17q25 [43,53]. However, despite the earlier excitement and optimism about gene discovery for TS through GWLS approach, and some chromosomal regions have been implicated in some TS families, no gene or causal mutation of major effect has been discovered for the above-mentioned TS loci, except for the SLITRK1 locus on chromosome 13 [51] and the HDC locus on chromosome 15 [52]. The reasons behind these earlier disappointing gene discovery endeavors through GWLSs may be multiplicative, including phenotypic complexity, such as clinical and genetic heterogeneity of TS and its associated comorbidities, limited sample size, as well as limitation of the previous genetic technologies and statistical methodologies, e.g., the low coverage and resolution of the microsatellite marker panels. Since linkage analysis is based on calculation of recombination events in limited number of generations, it is sensitive to misspecified genetic information, such as individual affection status, allele frequency, mode and parameters about inheritance. Nevertheless, limited significant statistical results of TS GWLSs strongly suggest that the underlying genetic architecture of TS and associated comorbidities is more complex than simple Mendelian inheritances.

Candidate Gene Association Studies of TS
After unsuccessful GWLS attempts in TS gene discovery, researchers had turned to direct candidate gene linkage and association studies to identify the potential genetic contribution of TS. Based on progresses in neuroimaging, postmortem, animal models and pharmacological studies, it has been hypothesized that the cortico-striatal-thalamo-cortical (CSTC) pathways may underlay the pathogenesis of TS and related symptomatology; and abnormalities of those neurotransmitters and their signal transmissions could lead to the dysfunction of CSTC neural network and subsequent clinical manifestation of TS. Multiple neurotransmitter systems have been implicated in CSTC pathways [54], including dopaminergic, serotonergic, glutamatergic, gamma-amino butyric acid-(GABAergic), histaminergic, and other neurotransmitters [55][56][57]. Furthermore, a neurotransmitter dysfunction may also cause change in other neurotransmitters due to interaction or self-regulation among them [57].
Compared to genetic linkage analysis, genetic association is when one or more genotypes within a population, i.e., unrelated cases and controls, co-occur with a phenotypic trait more often than would be expected by chance occurrence. In earlier stage of genetic research of TS, many studies have been carried out to test genetic linkage and associations between various neurotransmitters and TS. Table 1 is a summary of candidate gene linkage and association studies of TS. In general, none candidate gene showed positive linkage result. Some association studies of TS showed positive results; however, their effects in TS seemed to be very small; and very often results were non-replicable; sometimes even controversial. As shown in Table 1, most studies were limited by sample sizes, therefore under power according to current standards. Interestingly, since 2005, majority of genetic association studies have achieved positive results, accounting for bigger sample size, more advanced technical methods and selected genes. One pedigree, 85 interviewed in 116 members (NA) [58] 110 trios (French Canadian) [47] 61 TS and 109 parents (Germany) [67] DRD3 (Dopamine receptor D3) 3q13.3 Both family and case-control studies did not find association between DRD3 (Mscl, rs6280) and TS.

Genome-Wide Association Studies of TS and Other Psychiatric Disorders
Facilitated by the advent of SNP array technologies and statistical methodologies, as well as the collective efforts of large scale biobanks for research materials, genome-wide association study (GWAS) has become the main stream of genetic studies of common diseases in the past decade. The current GWAS Catalog contains 3055 publications and reports 39,360 unique SNP-trait associations in nearly 2000 traits or diseases [95]. Psychiatric disorders fall into the complex trait category; and most psychiatric disorders are common and highly heritable. Therefore, GWAS has been the focus of considerable effort in field of psychiatric genetics in the past decade. To date, over 400 GWAS studies have been published in psychiatric disorders like SCZ (104 GWAS analyses) [95][96][97], ASD (23 GWAS analyses) [95,98,99], BPD (85 GWAS analyses) [95,100,101], MDD (major depressive disorder) (55 GWAS analyses) [95,102,103], OCD (5 GWAS analyses) [95,104,105] and ADHD (33 GWAS analyses) [95,106,107]. These research results have markedly increased our understanding and knowledge of the genetic basis of these psychiatric disorders, and have yielded empirical data on genetic architecture and pathways critical to address the long-standing debates in the field of psychiatry. Table 2 shows a summary of GWASs of several main psychiatric disorders. In general, the effects of risk variants from GWASs are small but wide-spread genome-wide; and the majority of GWAS-identified variants fall in noncoding regions of the human genome. However, further annotations indicate that these regions are enriched for active elements for gene expression regulation in relevant cell functions [108,109].
So far, only two GWASs of TS and one replication study have been published to date; one reached a marginal genomewide significance and the other GWAS failed to identify genome-wide significant loci for TS [110][111][112]. These results may have a close relationship with the sample size (1285 cases/4964 controls, and 2498 cases/6277 controls, respectively for each TS GWAS), which were much smaller than other major psychiatric GWASs. To date, there are no significant finding in ADHD and OCD GWASs either, most likely due to limited sample sizes as well. Nevertheless GWASs of ADHD and OCD found some top signals within some interesting candidate genes, such as LMOD2, WASL, ASB15, and SHFM1 genes in ADHD GWASs [113,114]; and PTPRD [104] and FAIM2 [105] genes in OCD GWASs.
One unambiguous conclusion from all GWASs is that complex traits are polygenic; and multiple lines of evidence are consistent with widespread pleiotropy for complex traits; these features are particularly striking in GWASs of psychiatric disorders. Widespread but small effects indicate that these common risk variants are not the main cause of disease phenotype under study, but rather just reflect the minimal peripheral polymorphic effects of the underlying molecular networks in different individuals and populations; and in case of TS GWAS, the subtle functional diversity or polymorphisms of the CSTC network.   [114] D: Discovery, R: Replication, C: Combined; NR: ethnicity not reported; OR: odds ratio; SCZ: schizophrenia; BPD: bipolar disorder; MDD: major depression; ASD: autism spectrum disorder; ADHD: attention-deficit hyperactivity disorder; GWAS: genomewide association study.

Chromosomal Abnormalities and Copy Number Variants (CNVs) of TS
Chromosomal aberrations studies are an important aspect of TS genetics with more significant findings. By single or combined in situ hybridization, chromosomal microarray, cytogenetic and next-generation sequencing, chromosomal aberrations studies have identified several large, rare structural aberrations associated with TS and related phenotypes. Table 3 is a summary of some main candidate loci and genetic findings. Among the most interesting candidate genes, Slit and Trk-like, family member 1 (SLITRK1) gene on 13q31.1, discovered in a TS patient with a de novo inversion [51]. SLITRK1 is a transmembrane protein that regulates neurite outgrowth by phosphorylation-dependent manner; and it has been shown to control neurite outgrowth; and it is expressed in the embryonic and postnatal brain, including the cortex, thalamus, and basal ganglia, which correlate with the neuroanatomical regions most commonly implicated in TS [119][120][121]. Inner mitochondrial membrane protein 2L (IMMP2L) gene on 7q22-q31 is also an interesting candidate gene for TS, disrupted by a breakpoint in 7q31 in TS patients, as well as implicated in autism and speech-language disorders [45,46,122,123]. IMMP2L encodes the inner membrane peptidase subunit 2, a mitochondrial protease involved in cleaving the space-sorting signals of mitochondrial membrane proteins; and defective IMMP2L may lead to disrupted mitochondria function [123]. Mitochondrial dysfunction has been associated with a range of human disorders, including neuropsychiatric disorders [123]. Disruptions of Contactin associated protein-like 2 gene (CNTNAP2) on 7q35-q36 [124,125] and Neuroligin 4 (NLGN4) on Xp22.3 [126] have also been reported to be associated with TS and other neurodevelopmental disorders.  CNV is a special type of structural variation, i.e., a type of duplication or deletion event that affects a considerable number of base pairs at the same chromosomal location [142]. CNVs have been identified as causal genetic variants in other psychiatric disorders [143,144]. It has been reported that de novo CNVs are strongly associated with ASD and SCZ [143][144][145][146][147][148][149]. In ASD, de novo CNV in simplex families is 5 times and 10 times higher than multiplex families and controls, respectively [145]. In SCZ, de novo CNV is 8 times higher in sporadic cases than in controls [148]. It has been estimated that increased large CNV and chromosomal rearrangement may contribute to 5-10% risk of ASD [149]. The other neurodevelopmental conditions, such as bipolar disorder, epilepsy, and intellectual deficiency also share a highly similar large CNV landscape with potential pathogenicity [150,151]. Table 4 summarizes CNV studies in TS. Approximately 1% of TS cases carry one of these CNVs, indicating that rare structural variation contributes significantly to the genetic architecture of TS structural variants, as well as increased global CNV burden that is mainly driven by large, rare, clinically relevant events, contribute significantly to the genetic architecture of TS, in which larger CNVs could create imbalance for more genes during neurodevelopment and lead to more severe outcomes. CNV: copy number variation.

Whole Exome/Genome Sequencing Studies of TS and Other Psychiatric Disorders
Whole exome sequencing (WES) is a fast and cost-effective method by sequencing all coding regions of the entire human genome to detect rare deleterious variants, which disrupt encoded protein function. Whole genome sequencing (WGS) is to sequence the entire genome, including intron, exon, flanking sequence and interval regions. Unlike GWASs that identify common variants (frequency ≥ 5%), WES/WGS are used to identify rare (frequency ≤ 1%) and novel variants genome-wide. WES has achieved great success in Mendelian disorders [157]; but also used as an effective means to identify loci for complex traits or diseases [158]; and these protein-coding regions contain 85% of disease-causative mutations, even if they only cover less than 2% of the entire genome [159][160][161]. Coming after WES, WGS allows to identify private genetic variants, small insertions and deletions (indels), more complex CNVs, and other structural alterations; and most of these variants reside in the~98% noncoding genome largely unexplored by SNP microarray and WES studies. With the application of WES/WGS, some rare mutations including de novo point mutations, gene-disrupting structural mutations have been found in psychiatric disorders [162][163][164][165], in which loss of functional variants (nonsense, splice-site variants), and increased burden of de novo mutations, have been shown to play an important role in etiology of psychiatric disorders [165].  2015-2017 [182][183][184][185] Some major studies and findings of WES and WGS in psychiatric disorders are summarized in Table 5. So far, the application of WES has generated the most significant results in ASD than in any other psychiatric disorders, and multiple large scale studies have identified numerous candidate genes for ASD [166,170,171,174,[186][187][188][189][190][191][192][193][194][195]. Compared to WES, WGS studies are much newer and with fewer number of studies and with smaller sample sizes for current studies. However, with reduced cost and improved methodologies, the application of WGS has been rapidly increased in genetic studies of complex disorders [179][180][181][196][197][198]. WES/WGS studies have further demonstrated that psychiatric disorders like SCZ, BPD, ASD belong to complex traits, rather than monogenetic disorders; and the underlying pathogenic mechanisms and pathways are perplexing. There are fewer studies of WES/WGS in TS. Sundaram et al. performed WES in ten members of one pedigree with seven affected by TS; and identified three novel nonsynonymous variants within MRPL3, DNAJC13 and OFCC1 genes [199]. Willsey et al. performed WES in two TS cohorts (325 trios from the Tourette International Collaborative Genetics cohort; 186 trios from the Tourette Syndrome Association International Consortium on Genetics), and suggested that de novo damaging variants found in approximately 400 genes may contribute to genetic risk in 12% of clinical cases of TS; and they particularly reported 4 likely risk genes for TS, i.e., WWC1 (WW and C2 domain containing 1), CELSR3 (Cadherin EGF LAG seven-pass G-type receptor 3), NIPBL (Nipped-B-like), and FN1 (fibronectin 1) [200]. Eriguchi et al. identified 30 de novo mutations, including four missense mutations (RICTOR, STRIP2, NEK10, and TNRC6A genes) in WES of nine trio families and one quartet family of TS [201]. Sun et al. identified a nonsense mutation through WES in the PNKD gene segregating with TS and Tic disorders in six out of nine members in a three-generation TD multiplex family [202]. These five above-mentioned candidate genes are enriched in CSTC regions, which are implicated TS; and their expression levels vary in different development stages, suggesting that disruptive protein function of those genes might affect neuronal development or activity in these brain structures [201,202]. Compared to ASD, SCZ and MDD, WES/WGS in TS are limited by the small sample sizes [199][200][201][202]. Further studies with enlarged sample size will be helpful to understand the genetic architecture of TS and its overlap with other neuropsychiatric disorders.

Epigenetics and TS
Decades of genetic research have shown that there are no direct correlations between genotypes and phenotypes in psychiatric disorders; and there ought to be missing pieces of information between genomic variations and disease phenotypes, i.e., changes of genomes will only become effective through transcsriptome, epigenome and result in phenotypic outcomes. Epigenetics refers to the ongoing regulation of gene expression, which includes structural modification of chromatin, post-translational modification of histones (i.e., acetylation and methylation), chemical modification of DNA through methylation or hydroxymethylation of cysteins, as well as expression of interfering non-coding RNAs (i.e., miRNAs and long-non-coding RNAs) [203,204]. These epigenetic mechanisms allow reprogramming of the genome upon environmental inputs at specific time-points during development and through lifetime. The epigenetic changes triggered by early life events stand as a valuable hypothesis of the environmentally induced behavioral changes.
Epigenetic regulation has been shown to have an impact on the development of many neuropsychiatric disorders [205,206], and to play a pivotal role in embryonic and adult neurogenesis [207]. Some studies on epigenetic processes in the expression and inheritance of behaviors have helped us to further understand the complexity of brain functions [208]. Abelson et al. identified a noncoding RNA variant (var321) at the binding site for microRNA hsa-miR-189 in two unrelated TS patients on chromosomal 13 [51] (see above). In 2015, Rizzo et al. found that miR-429 was significantly underexpressed in TS patients and suggested as an useful molecular biomarker to aid TS diagnosis in the future [209]. Another study suggested that methylation levels of the DRD2 gene were higher in adults with TS than in sex-, age-matched controls [210]; and methylation of DRD2 was positively correlated with tic severity. However, methylation of DAT was negatively correlated with tic severity in the same study [210]. State et al. reported that there might be relationship between chromosome 18(q21-q22) inversion and epigenetic mechanism in a 12 year old boy with TS and OCD [140]. The results from the first epigenome-wide association study, which investigated DNA methylation in patients with tic disorders, suggested that the top hits of methylation signal were enriched for genes involved in brain-specific and developmental processes [211]. Delgado et al. did not find significant methylation difference in TS patients compared with controls in a region of chromosome 8 for KCNK9 and TRAPPC9 genes [212].

Conclusions
TS is a complex disorder with highly variable phenotypic manifestations and high frequency of comorbidities. It is more common in males than in females. Family and twin studies have shown that genetic factors play an important role in the pathogenesis of TS. Candidate genes studies have shown that multiple genes (DRD2, DRD4, 5-HT2C, SERT) in multiple neural systems, including dopaminergic, serotonergic, histaminergic pathways, might be associated with pathogenesis of TS, but results are not yet convincing enough. In recent years, several new candidate genes, e.g., SLITRK1, IMMP2L, CNTNAP2, NLGN4, have been identified through linkage studies and structural genomic aberrations, in which very rare genetic variants with large effects were found in TS patients and families.
Compared to genetic studies of other psychiatric disorders, TS studies are currently still largely limited by the relatively small sample sizes, because of the complexity and heterogeneity in clinical manifestation. We expect that more rare and common variants with various effects underlying the genetic susceptibility of TS will be discovered through larger-scale international collaborative projects in the near future. Combinational analyses of common variants with rare variants, structural variations and CNVs, plus epigenetic factors; i.e., integrative "multi-omics" together with environmental factor analyses [213,214] will be the future direction. As promising starts, four large collaborative groups with joint effort and multiple resources have been developed for TS genetic research, focusing on existing large well-characterized patient cohorts, which include (1) the Tourette Syndrome Association International Consortium for Genetics (TSAICG) [215]-genomewide association studies for TS; (2) the Tourette international collaborative genetics (TIC genetics) study-whole exome sequencing in families with TS [216], which is funded by the National Institute of Mental Health (NIMH) in the USA; (3) the European multicentre tics in children (EMTICS) study-exploring gene-environment interactions that underlie TS etiology, which is funded by the European Commission under the Seventh Framework Programme, including 17 clinical sites from across Europe [217]; (4) TS-EUROTRAIN-coordinating large-scale studies and training the next generation of experts for TS, supported by the European Commission [218,219]; (5) Of particular interest in this field, the PsychENCODE project has been created and developed by an international consortium to provide an enhanced framework of regulatory genomic elements (promoters, enhancers, silencers and insulators), catalog epigenetic modifications and quantify coding and non-coding RNA and protein expression in tissue and cell-type-specific samples from healthy (neurotypical) control and disease-affected post-mortem human brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. [220]. The Project is currently focusing on three major psychiatric disorders: ASD, BPD and SCZ, for example, 98 of the 108 independently associated loci were found in non-coding region in SCZ GWAS [96,221] suggesting that the PsychENCODE project will be a valuable platform to study the epigenetics of psychiatric disorders in the future, including TS studies, in which some preliminary results indicated that epigenetics might play a role in TS as well [51,140,[209][210][211].
In the near future, GWAS by SNP arrays will most likely be gradually replaced by WGS, with increased power by including rare variants in the genetic analyses. For many years, genotyping technology was the limiting step to genetic discoveries, but now discovery is limited by phenotypic descriptors that could link with genetic data to allow disease stratification, which might be more aligned with treatments; therefore, deep phenotyping aligned with genetic studies will also facilitate new discoveries for disease risk and mechanism.
Although with limited results, epigenetic research has shown promising hope to link the genomic variations with environmental exposures and disease outcomes, particularly for psychiatric disorders and behavioral phenotypes. For TS, further studies with larger sample size could focus on further understanding of the effect of dynamic epigenome on the regulation of developmental genes and behaviors. Additional large-scale studies could also aim to disentangle common versus disorder-specific genomic and epigenomic variations in TS with other psychiatric and behavioral phenotypes. Understanding the mechanism behind the sex differences in TS may also help us better understand the regulation of gene expression in the brain and its implication in TS pathogenesis because TS is a male-biased disease.