Gilles de la Tourette syndrome (GTS) is neuropsychiatric disorder in which motor and vocal tics occur. They are usually accompanied by psychiatric comorbidities such as attention-deficit hyperactivity disorder (ADHD), obsessive–compulsive disorder (OCD), depression or self-injury. Segregation analysis in GTS-risk families suggests a complex, polygenic mode of inheritance involving various loci (Mcmahon et al., 1996). Susceptibility genes associated with GTS include: Slit and Trk-like1 (SLITRK1), L-histidine decarboxylase (HDC), mitochondrial inner membrane protease subunit 2 (IMMP2L), neuroligin 4, X-linked (NLGN4X), contactin-associated protein 2 (CNTNAP2) (for details consult review by Paschou et. al (Paschou et al., 2014), cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) (Willsey et al., 2017; Wang et al., 2018), WW and C2 domain containing 1 (WWC1), Nipped-B-like (NIPBL), fibronectin 1 (FN1) (Willsey et al., 2017), arylacetamide deacetylase (AADAC) (Yuan et al., 2018) and others.

Abnormal dopaminergic neurotransmission in the cortex and in the basal ganglia interacting with other neuronal pathways, including endocannabinoid system (ECS), seems to be crucial in GTS pathophysiology (Martino and Leckman, 2013). Endocannabinoids mediate two pathways in the basal ganglia: neurons of the direct pathway expressing dynorphin and D1 dopamine receptor, as well as the indirect pathway neurons expressing enkephalin and D2 receptor (Steiner and Gerfen, 1998; Mechoulam and Parker, 2013). There are many further indications for an involvement of ECS in GTS (Sandyk, 1988; Leckman et al., 1988; Haber and Wolfer, 1992; Muller-Vahl, 1998; Muller-Vahl, 2002; Müller-Vahl et al., 2002; Hasan et al., 2010; Brunnauer et al., 2011; Mechoulam and Parker et al., 2013; Trainor et al., 2016; Jakubovski and Müller-Vahl, 2017; Kanaan et al., 2017). Some post-mortem studies support cannabinoid dysregulation in GTS, as decreased immunoreactivity with dynorphin A [1–17] has been detected in the striatal area of GTS patients (Haber and Wolfer, 1992). Furthermore, increased levels of dynorphin A [1–8] were found in the cerebrospinal fluid of GTS patients which correlated with OCD severity (Leckman et al., 1988). The ECS involvement in GTS is also indicated by a neuroimaging study using single photon emission computed tomography (SPECT) with [¹²³I]AM281 (Berding et al., 2004). The clinical efficacy of cannabis-based medicines (CBM) also supports the involvement of ECS (Müller-Vahl et al., 2002; Müller-Vahl et al., 2003). Additionally, a number of retrospective reports on self-medication with CBM (Hemming and Yellowlees, 1993; Muller-Vahl, 2002; Brunnauer et al., 2011; Jakubovski and Müller-Vahl, 2017) as well as case reports (Muller-Vahl, 2002; Hasan et al., 2010; Brunnauer et al., 2011; Trainor et al., 2016; Jakubovski and Müller-Vahl, 2017; Kanaan et al., 2017) suggest effectiveness of CBM in GTS.

The two main cannabinoid receptors are CB1 and CB2, cannabinoid receptors type 1 and 2. CB1 receptor is mostly present in the central nervous system, with a high density in basal ganglia (Pertwee, 2006), the brain region implicated in movement control, while CB2 receptor is mainly present in the immune system and in hematopoietic cells (Russo, 2013) where it modulates cytokine release (Suárez-Pinilla et al., 2015). Variants of the CNR1 gene encoding CB1 receptor have been associated with various psychiatric and neurological diseases (Gadzicki et al., 1999; Gadzicki et al., 2004; Domschke et al., 2008; Evans et al., 2016; Ruiz-Contreras et al., 2017; Yao et al., 2018) (see Table 1 ). Gadzicki et al. (1999) were the first to investigate polymorphisms in a coding exon of the CNR1 gene finding the 1359(G/A) polymorphism in German GTS patients. Posteriorly they focused on CNR1 variants in a larger group of GTS patients (Janik et al., 2014). However, they failed to find a correlation between those variants and GTS.

Despite that negative outcome we decided to revise the question of a possible role of CB1 receptor in GTS. We focused on the CB1 receptor as it is mainly expressed in the brain and mediates inhibition of dopaminergic, glutaminergic, and GABAergic neurotransmission in CNS (Haughey et al., 2008). We hypothesized that ECS dysregulation in GTS could be related to some minor variants of the CNR1 gene and therefore tested four such variants as possible risk factors of GTS in Polish GTS patients.

The genotyping success rate was 100% and the consensus rate (on the basis of 10% duplicates) was 100% for DNA isolated from both leukocytes and buccal cells. The quality of genotyping was the same regardless of the biological source and method used for DNA isolation. The genotype frequencies of all four SNPs were in accordance with the Hardy–Weinberg equilibrium in the control group (p>0.9 for rs806379, p>0.3 for rs2023239, p>0.6 for rs2180619 and p>0.6 for rs1049353). In the patient group the genotype frequencies of three SNPs were in accordance with the Hardy–Weinberg equilibrium (p>0.2 for rs2023239, p>0.1 for rs2180619 and p>0.2 for rs1049353) while the distribution of the rs806379 genotype deviated slightly from the Hardy-Weinberg equilibrium (p=0.047) due to a deficit of heterozygotes.

A significant association with the GTS was found for the rs2023239 variant. The MAF of CNR1 rs2023239 was significantly higher in GTS patients compared to control group. When the TT genotype was used as a reference, genotypes CC+CT (dominant model, p = 0.001) and CT alone (p = 0.0009) were significantly associated with a higher risk of GTS. This association remained significant after Bonferroni correction for multiple tests (Bonferroni-corrected significance level for four SNPs and six genetic models is 0.05/(4*6)=0.002). There were no statistically significant associations between the rs2180619, rs806379 rs1049353 variants and the GTS ( Table 2 ). Multivariate logistic regression analysis adjusted for gender and age showed that the presence of CNR1 rs2023239 allele C (genotypes CC+CT, dominant model) is an independent factor associated with a higher risk of GTS ( Table 3 ).

Genotypic and allelic association analysis of all four CNR1 polymorphisms with most common co-morbid disorders in patients with GTS are shown in Table 4 . The only significant association was found for rs806379. The minor allele T of this SNP was more frequent in GTS patients with ADHD than in the patients without ADHD (OR 1.45, 95% CI: 1.01–2.08, p=0.045). No associations were found between the CNR1 genotype and age of tic onset (p>0.4), total YGTSS or YGTSS Total Tic Score (p>0.15), family history of tics (p>0.15) or family history of OCB/OCD (p>0.2).

So far the only comprehensive study investigating genetic variation in the CNR1 gene in GTS (Janik et al., 2014) was aimed to localize pathogenic mutations in CNR1 and failed to show differences with healthy controls. Our study is the first to examine common SNP variants of CNR1 gene in a group of GTS patients. A significant association was found both in the allelic and genotypic analysis between the risk of GTS and the rs2023239 polymorphism. Multivariate analysis proved that CC+CT genotypes are gender-independent factors associated with a higher GTS risk. Our study shows that GTS is associated with rs2023239 polymorphism along with various others neuropsychiatric disorders, such as the cannabis dependence (Benzinou et al., 2008), eating disorders (Yu et al., 2013), schizophrenia (Wiskerke et al., 2012), impulsivity (Juhasz et al., 2009), depression (Ruiz-Contreras et al., 2017), and migraine (Ketcherside et al., 2017). We can only speculate on the molecular mechanism underlying the role of this variant in the pathogenesis of GTS. rs2023239 allele C was found to be associated with increased CB1 receptor density (Janik et al., 2015) which could modulate neurotransmission. It is unlikely that this intronic variant directly affects the splicing, as it is too distant the adjacent exon (122bp). Human Splicing Finder predicts that the minor allele C does not create any new potential splice sites or new potential branch points compared to the common T allele. Additionally, this variant could affect gene expression either directly or through a linkage to functional variant(s) located in regulatory regions.

Our earlier association studies in Polish patients with GTS have revealed a relationship between some SNPs of BTBD9, ADORA1 and ADORA2A genes and co-morbid psychiatric disorders (Muller-Vahl et al., 1999; Martiny, 2017). Therefore, we hypothesized that CNR1 variants could also contribute to the disease complexity predisposing the patients to psychiatric comorbidities as all the CNR1 polymorphisms studied here had been found to be associated with various psychiatric disorders ( Table 1 ). However, both genotypic and allelic association analyses of all the CNR1variants examined, including rs2023239 associated with a higher risk of GTS, failed to demonstrate a significant association with any psychiatric co-morbidity. The only statistically significant association found, that of co-morbid ADHD with rs806379, should probably be regarded as a false positive because of its marginal significance (0.05>p>0.04) in multiple statistical tests performed (4 SNPs x 4 disorders x 6 models).

A number of open as well as randomized controlled studies and case reports support the premise that the use of CBM improves not only tics, but also ADHD (Abi-Jaoude et al., 2017), OCB/OCD (Hasan et al., 2010; Denys et al., 2013), depression (Yoon et al., 2007) and anxiety (Jakubovski and Müller-Vahl, 2017). Our study does not confirm that this beneficial effect of CMB on co-morbid disorders in GTS could be related to CNR1 SNPs as we did not find any association of the CNR1 variants with co-morbidity. Additionally, we did not find any correlation of CNR1 variants with any clinical data, such as age of tic onset, tic severity measured with YGTSS or familial history of tics and OCD/OCB. It can therefore be speculated that while rs2023239 contributes to the pathogenesis of tics per se, other genetic and environmental factors contribute to further clinical features of GTS.

The present results on the CNR1 variants could also be interpreted in relation to an earlier SNP study by our group, in which we found association of ADORA1 and ADORA2A variants with a higher risk of GTS (Muller-Vahl et al., 1999). In this context we note that abnormality of the dopaminergic system has been confirmed to be one of the primary causes of GTS (Singer et al., 1982; Sandyk and Bamford, 1987; Ferré et al., 2009; Ferré et al., 2010; Filbey et al., 2010), both adenosine and endocannabinoids act as modulators of dopamine neurotransmission in the striatum (Müller et al., 2007) and it has been suggested that CB1 receptors form heteromers with dopamine D2 and adenosine A2A receptors (Ehlers et al., 2007). Although we do not know the functional significance of the minor variant of rs2023239 in GTS, we put forward the hypothesis that it could be related to faulty cannabinoid transmission due to lower expression of the CB1 receptor or reduced receptor’s affinity for the endocannabinoid ligand. This, in turn, could lead to reduction of adenosinergic signaling and over-activity of dopaminergic transmission, and finally aggravate tics. Another possible mechanism is a direct enhancement of dopaminergic transmission as a consequence of limited endocannabinoid activity, without an involvement of adenosine signaling.

In conclusion, the obtained results indicate that the C allele of the rs2023239 polymorphism of CNR1 gene is a risk factor of GTS in the Polish population, associated with the occurrence of tics, but not with the co-existing psychiatric symptoms. The results also support the hypothesis of an ECS involvement in the pathogenesis of GTS. Nevertheless, our finding is preliminary and needs to be replicated in an independent cohort. Further research is needed to determine functional significance of the variant.

The following limitations of the study have to be addressed: 1) the analyzed variants can be in linkage disequilibrium with true risk conferring variants located outside analyzed regions; 2) the study group was relatively small; 3) the patients were evaluated for co-morbid disorders only once (one-time registration) and it cannot be excluded that psychiatric disorders could develop over time and additionally it was impossible to measure the YGTSS in all patients; there is a possibility of false negative results in rs2180619, rs806379, and rs1049353 CNR1 polymorphisms; 4) some clinical data, especially on adult patients, were subject to a recall bias, ; 5) we have not included the comparison with the variants previously associated with GTS and this should be taken into consideration in the future studies; and 6) our finding needs to be replicated using study groups from different populations.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. These are also included in Supplementary Material .

The study has been approved by the Ethics Committee of the Medical University of Warsaw (KB/2/2007, KB/53/A/2010, KB/63/A/2018) and has therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All participants aged ≥18 years signed an informed consent form prior to their inclusion in the study, and legal guardians gave a written consent on behalf of individuals under 18.

Research project: Conception (PJ), Organization (PJ, CŻ), Execution (JF), Creation of electronic database (TD, NS, PJ), Clinical examination: (PJ), Manuscript Preparation: Writing of the first draft (NS, PJ, JF, KS), Review and Critique (CŻ, PJ, KS, JF, NS, TD).

This study was kindly supported by a grant provided by Medical University of Warsaw (1WC/NM1/17) and by a grant funded by National Science Center (UMO-2016/23/B/NZ2/03030).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.