Genetic Associations of Serotoninergic and GABAergic Genes in an Extended Collection of Early-Onset Obsessive-Compulsive Disorder Trios

Abstract

Objective:

Obsessive-compulsive disorder (OCD) is a debilitating neuropsychiatric disorder whose etiology includes important genetic contributions. In a previous transmission disequilibrium study in which 75 complete trios were included, single-nucleotide polymorphisms (SNPs) in serotoninergic and GABAergic genes were associated with early-onset OCD. Our aim was to assess those findings in an extended collection of early-onset OCD trios.

Methods:

A transmission disequilibrium test for SNPs in HTR1B (rs2000292), SLC18A1 (rs6586896), GAD1 (rs3791860), and GAD2 (rs8190748) was performed in a total of 101 early-onset OCD trios, from which 26 trios were newly recruited for the purpose of the present analysis.

Results:

All the SNPs were overtransmitted from parents to OCD probands (p < 0.012, significant after Bonferroni correction).

Conclusions:

These results are consistent with the previous findings and constitute more evidence of the role of genetic factors related to serotoninergic and GABAergic pathways in the pathophysiology of early-onset OCD.

Introduction

Obsessive-compulsive disorder (OCD) is a debilitating neuropsychiatric disorder characterized by recurrent, persistent, and intrusive thoughts or images that cause distress or anxiety (i.e., obsessions), and repetitive behavior aimed at reducing this feeling of anxiety (i.e., compulsions) (American Psychiatric Association 1994). OCD is estimated to affect 1%–3% of the population, with 30%–50% of patients developing it in childhood (Stewart et al. 2004; Ruscio et al. 2010).

The precise etiology of OCD is unknown, but there is a clear evidence of genetic susceptibility, particularly in children and adolescents. Twin and family studies have shown a high degree of heritability, around 50%, of this psychiatric disorder (Tambs et al. 2009; Mataix-Cols et al. 2013). Particularly, early-onset OCD has been associated with a higher prevalence of OCD in the first-degree relatives, suggesting higher familiarity in this OCD subtype (Taylor 2011), which might represent a more severe subtype of the disorder (Rosario-Campos et al. 2001). Therefore, it is important to characterize better early-onset OCD to improve treatment and prognostic of these pediatric patients.

Genetic association studies have contributed to our current knowledge of the pathophysiology and pharmacology of OCD (Brandl et al. 2012; Pauls et al. 2014). However, more research is needed to confirm results and reduce discrepancies. A previous transmission disequilibrium test (TDT) study conducted by our group, in which 266 single-nucleotide polymorphisms (SNPs) in 35 candidate genes were analyzed in early-onset OCD probands, showed differences in allele transmission for some of the polymorphisms (Mas et al. 2014). Those results mainly involved genes coding for the serotonin receptor 1B (HTR1B), the vesicular monoamine transporter (SLC18A1), and the glutamate decarboxylase isoenzymes (GAD1 and GAD2).

The serotoninergic pathway became a leading target for research into the neurobiology of OCD largely because of the remarkable therapeutic effects of serotonin reuptake inhibitors on obsessions and compulsions (Greist et al. 1995). Different genes related to this neurotransmitter system have been consistently associated with OCD (Taylor 2016). HTR1B and SLC18A1 are important candidate serotoninergic genes as they are directly related to the serotonin release. Despite being little explored in this psychiatric disorder, these genes have been associated with OCD and different related endophenotypes in previous genetic studies (Mundo et al. 2002; Camarena et al. 2004; Kim et al. 2009; Atmaca et al. 2010; Mas et al. 2013, 2014). Besides the serotonin pathway, other neurotransmitter systems may also be involved in OCD pathology. Interestingly, gamma aminobutyric acid (GABA) abnormalities have recently been reported (Richter et al. 2012; Simpson et al. 2012; Russo and Pietsch 2013). Moreover, there is evidence for the GABA type B receptor 1 gene (GABBR1) as a susceptibility factor for OCD (Zai et al. 2005). GAD1 and GAD2 are pivotal GABAergic candidate genes as they are responsible for the GABA synthesis. However, no other authors have explored polymorphisms in these genes for OCD.

The aim of the present study was to assess our previous findings in an extended collection of early-onset OCD trios.

Methods

Subjects

Child and adolescent patients meeting the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) diagnostic criteria for OCD (American Psychiatric Association 1994) and their parents were recruited for the purposes of this study. Only complete trios (father, mother, and OCD proband) were included in the analyses. Recruitment was performed in two periods. First, from 2010 to 2013, 75 complete trios were recruited at the Department of Child and Adolescent Psychiatry and Psychology of the Hospital Clínic in Barcelona. Those trios were included in a previous TDT study (Mas et al. 2014). Then, from 2013 to 2015, 26 new child and adolescent OCD patients and their parents were also recruited at the same department. Thus, a total of 101 complete trios were included in the present TDT study.

All the participants were Caucasian, as determined by self-reported ancestries. Subjects who mentioned non-European grandparents were excluded. As described elsewhere (Mas et al. 2014), the patients and their parents were interviewed with the Spanish version (Ulloa et al. 2006) of the semi-structured diagnostic interview K-SADS-PL (Schedule for Affective Disorders and Schizophrenia for School-Age Children–Present and Lifetime Version) (Kaufman et al. 1997) to assess the current and past psychopathology.

The sociodemographic and clinical characteristics of the OCD probands are shown in Table 1. The age of onset was defined as the age at which the patients first displayed significant distress or impairment associated with obsessive-compulsive symptoms. OCD severity was measured using the CY-BOCS scale (Children's Yale-Brown Obsessive Compulsive Scale) (Scahill et al. 1997). Four obsessive-compulsive symptom dimensions were computed: aggressive, sexual, and religious obsessions and checking compulsions; symmetry, ordering, counting, and arranging obsessions and compulsions; contamination obsessions and cleaning compulsions; and hoarding (Leckman et al. 1997).

Table 1.

Clinical Data of the Early-Onset Obsessive-Compulsive Disorder Probands Included in the Present Study

	All
N	101
Gender, male, n (%)	50 (49.5)
Age (mean ± SD)	14.6 ± 2.4
Age at onset (mean ± SD)	11.9 ± 2.9
Current CY-BOCS score (mean ± SD)	18.8 ± 8.1
Maxim CY-BOCS score (mean ± SD)	26.4 ± 6.5
Dimensions, n (%)
Harm/checking	59 (58.4)
Washing/cleaning	23 (22.8)
Symmetry/ordering	19 (18.9)
Comorbidities, n (%)
Anxiety disorder	24 (23.8)
ADHD	12 (11.9)
Tic disorders	11 (10.9)
Eating disorders	9 (8.9)
ODD	1 (1.0)
MDD	1 (1.0)
Family history of OCD, n (%)
First grade	45 (44.5)
Second grade	9 (8.9)

ADHD, attention-deficit hyperactivity disorder; CY-BOCS, Children's Yale-Brown Obsessive Compulsive Scale; MDD, major depression disorder; OCD, obsessive-compulsive disorder; ODD, oppositional defiant disorder; SD, standard deviation.

All the procedures were approved by the hospital's ethics committee (Ethics No. 2009/4884). Written informed consent was obtained from all the parents, and verbal informed consent was given by all the subjects following an explanation of the procedures involved.

Sample preparation

Blood samples were collected from the individuals in EDTA (K2EDTA BD Vacutainer EDTA tubes; Becton Dickinson, Franklin Lakes, NJ), and genomic DNA was extracted using the MagNA Pure LC DNA isolation Kit III and an LC MagNA Pure system (Roche Diagnostics GmbH, Mannheim, Germany). The DNA concentration was determined by absorbance (ND1000; NanoDrop, Wilmington, DE).

SNP selection, genotyping, and quality control

The results of our previous TDT study (Mas et al. 2014) showed several SNPs in HTR1B (rs9359271, rs2000292, rs6296, rs6298, and rs4140535), SLC18A1 (rs6586896 and rs7013199), GAD1 (rs1420385 and 3791860), and GAD2 (rs2236418, rs7908975, rs992990, rs8190748, and rs3781108) with a differential allele transmission to OCD probands. To assess these four genes in an extended collection of early-onset OCD trios, we particularly selected the most significantly differently transmitted SNP from each of these genes: HTR1B_rs2000292, SLC18A1_rs6586896, GAD1_rs3791860, and GAD2_rs8190748. These SNPs were genotyped using TaqMan genotyping assays (Applied Biosystems, Foster City, CA) on a 7500 Real-Time PCR System (Applied Biosystems, Warrington, United Kingdom). All the analysis showed consistent clustering and high genotype rates (100% of samples), and no Mendelian errors were detected.

Statistics

Sample size and statistical power calculations were performed with the function trio.power implemented in the trio R-package. Given the sample size, and assuming a 5% level of significance, statistical power ranged from 84% to 91% when polymorphisms with minor allele frequencies from 0.1 to 0.4 were analyzed, respectively. The analysis was completed with the TDT implemented in Haploview 4.2, using the parentTDT option to include parents' diagnoses in the analysis, as previously described (Mas et al. 2014). Briefly, the TDT assesses the distortion in the transmission of alleles from a heterozygous parent to an affected offspring. An allele that is preferentially transmitted to the affected offspring could increase the risk of disease. Given a complete trio, the parents will be informative if at least one of them (father and/or mother) is heterozygote for a particular SNP. Therefore, the T test statistic considers all heterozygous parents and compares the number of transmissions of each allele.

To avoid false-positive results, we applied the Bonferroni correction for multiple testing (significant p < 0.012, as four SNPs were assessed).

Results

The sociodemographic and clinical characteristics of the 101 early-onset OCD probands are shown in Table 1. A total of 50 males and 51 females with a mean age of 14 years were included. Checking was the most frequently observed dimension. Comorbidities were present in about 57% of OCD probands. Significant differences were observed between males and females in incidence of tics (18.0% vs. 3.9%, respectively; p = 0.02) and eating disorders (2.0% vs. 15.7%, respectively; p = 0.01).

The recruitment of 26 new early-onset OCD trios increased the sample size by 35% and therefore the statistical power of the present analysis. All four SNPs analyzed were overtransmitted from parents to OCD probands (p < 0.012, significant after Bonferroni correction; Table 2). In our previous analysis, the SNPs that reached the lowest p values were HTR1B rs2000292 and GAD2 rs8190748 (p = 0.0001), for which an overtransmission of the minor (allele A) and major (allele A) frequency alleles, respectively, was observed. In both cases, alleles were transmitted to about 75% of OCD probands. In the present analysis, these same alleles were also overtransmitted. However, the percentages of OCD probands who received the alleles from the heterozygous parents were slightly lower (about 67%), and the p values were higher than those in the first study (p = 0.0034 and p = 0.0008, respectively). This is in part due to the risk alleles of both SNPs were transmitted to about 50% of the new recruited OCD probands (HTR1B rs2000292: 10 transmitted and 12 untransmitted; GAD2 rs8190748: 13 transmitted and 12 untransmitted).

Table 2.

Genetic Association Study Using Parent Transmission Disequilibrium Test Analysis of 101 Early-Onset Obsessive-Compulsive Disorder Trios

					All (101 trios ^a )				Male (50 trios ^a )				Female (51 trios ^a )
Gene	SNP	Chromosome position	Alleles	Frequency	T	U	Statistic	p	T	U	Statistic	p	T	U	Statistic	p
HTR1B	rs2000292	6:77457228	G/A	0.235	41	22	8.56	0.0034	19	10	5.44	0.0196	22	12	3.27	0.070
SLC18A1	rs6586896	8:20147130	T/C	0.110	28	8	9.52	0.0020	14	2	10.89	0.0009	14	6	1.50	0.221
GAD1	rs3791860	2:170832911	C/G	0.431	60	40	8.81	0.0030	33	16	5.25	0.0218	27	24	0.01	0.891
GAD2	rs8190748	10:26280826	A/G	0.688	56	27	11.13	0.0008	26	13	3.93	0.0474	30	14	7.36	0.006

Frequency of the allele analyzed is shown in bold.

Informative parents (heterozygous father and/or heterozygous mother) correspond to the sum of individuals with the transmitted allele plus individuals with the untransmitted allele.

SNP, single-nucleotide polymorphism; T, transmitted; U, untransmitted.

Regarding GAD1 rs3791860 and SLC18A1 rs6586896, although percentages of OCD probands who received the risk alleles from the informative parents were similar in both studies (about 60% and 80%, respectively), lower p values were detected when the whole sample has been analyzed. While GAD1 rs3791860 was only nominally associated in the previous analysis (p = 0.0186), the overtransmission of the minor frequency allele (allele G) reached significance after the Bonferroni correction in the present study (p = 0.003). The minor frequency allele (C) of SLC18A1 rs6586896 was overtransmitted 3.5-fold and showed a slightly reduction in the p-value (p = 0.002) than in the previous analysis (p = 0.0031). Although no significance was reached when only the new 26 recruited families were considered, again similar percentages of OCD probands received the risk alleles of GAD1 rs3791860 (65%: 17 transmitted and 9 untransmitted) and SLC18A1 rs6586896 (72%: 8 transmitted and 3 untransmitted).

As in the previous analysis, differences between genders were also observed here and certain polymorphisms only reached the significance (p < 0.012) in only one gender. While HTR1B rs2000292 showed a lower p-value (p = 0.019) in males than females (p = 0.07); the lower p-value for GAD2 rs8190748 was observed in females (p = 0.006 compared with p = 0.047 for males, Table 2). Moreover, for GAD1 rs3791860 and SLC18A1 rs6586896, significant p values were only observed in males (p = 0.021 and p = 0.0009, respectively).

Discussion

According to our previous findings, the results of this TDT study show significant associations between polymorphisms in genes related to both serotoninergic (HTR1B and SLC18A1) and GABAergic (GAD1 and GAD2) pathways and early-onset OCD. All four SNPs were selected in our previous study (Mas et al. 2014) by a tagging strategy. They have been very little explored in the literature, and there has been no functionality associated with any of them. These SNPs could be in linkage disequilibrium (LD) with the causal variants but serve as markers of genetic risk regions for early-onset OCD.

HTR1B rs2000292 is located in the downstream region and was associated for the first time in our previous TDT study (Mas et al. 2014). Recently, this SNP has also been related to alcohol dependence (Wu et al. 2016). It is in high LD with two synonymous variants (rs6296 and rs6298) that we reported to be associated with inositol concentration in the anterior cingulate cortex in pediatric OCD patients (Ortiz et al. 2015). Studies have also reported associations between other HTR1B polymorphisms and OCD (Mundo et al. 2002; Kim et al. 2009) as well as other endophenotypes, such as orbitofrontal cortex volume (Atmaca et al. 2010), and the severity of obsessions as measured using the Y-BOCS (Camarena et al. 2004). In fact, in a recent meta-analysis, Taylor (2013) identified a trend for the involvement of HTR1B in OCD. Although the apparent evidence for the role of HTR1B in OCD, the highly complex genetic and epigenetic regulation of HTR1B expression (Gassó et al. 2017) makes it necessary to perform more studies to identify the real causal HTR1B polymorphisms involved in this pathology. Meanwhile, few studies have considered the genetic variability of SLC18A1 in relationship with psychiatric disorders. However, it is interesting that the association found between this gene and both anorexia nervosa and OCD was also found in pediatric patients (Mas et al. 2013). It has to be noted that the same SNP associated here, SLC18A1 rs6586896, was also related to glutamate plus glutamine (Glx) concentration in the anterior cingulate cortex in pediatric OCD patients (Ortiz et al. 2015). Although it is an intronic SNP, it is located in the binding site of Spi1 (www.genome.ucsc.edu). This transcription factor could play a crucial role in microgliogenesis (Satoh et al. 2014), which seems to be involved in the OCD pathophysiology (Frick and Pittenger 2016). Importantly, other key genes involved in the serotoninergic system, such as HTR2A (encoding the serotonin receptor 2A) or SLC6A4 (encoding the serotonin transporter), have consistently been associated with OCD in recent studies and also meta-analysis (Walitza et al. 2014; Cengiz et al. 2015; Taylor 2016; Tükel et al. 2016).

Recent findings indicate GABA abnormalities in OCD. Neuroimaging studies have shown cortical inhibitory dysfunctions (Richter et al. 2012) and reduced GABA levels in the medial prefrontal cortex (Simpson et al. 2012) in patients with the disease. Moreover, GABA plasma levels have been related to the severity of OCD symptoms (Russo and Pietsch 2013). However, few genetic studies of OCD have focused on genes related to the GABAergic system (Zai et al. 2005; Gratacòs et al. 2009; Richter et al. 2009), and our previous TDT study was the first to explore the genetic variability of GAD1 and GAD2 in relation to the disorder. Besides providing further more evidence for the association with GAD2, our present analysis also provides significant results for GAD1, indicating that, in fact, genetic factors affecting both isoenzymes responsible for GABA synthesis could be involved in the pathophysiology of OCD.

Gender differences that have been consistently reported in OCD (Mathis et al. 2011) and that were found and widely discussed in our previous study (Mas et al. 2014) were also observed in the present analysis for all four genes assessed. High comorbidity was found in the OCD probands, according to its prevalence in early-onset OCD (Ortiz et al. 2016). As reported in our previous analysis (Mas et al. 2014), gender differences were also observed in the comorbidity profile, with males having higher incidence of tics and females higher incidence of eating disorders.

Despite the evidence discussed above, it should be noted that there are none or very few candidate gene studies that have evaluated the genes associated here and that these studies have important limitations, such as reduced sample size and number of assessed genetic variants. Moreover, genome wide association studies (GWAS) in OCD have not identified significant signals in the genetic regions studied here (Stewart et al. 2013; Mattheisen et al. 2015).

It is believed that a high number of loci with small effect size are involved in OCD. Although candidate genes studies have reported associations with several genes (Taylor 2013, 2016), there are discrepancies and lack of replicability for these genetic associations, which is, in part, due to high heterogeneity across different studies. Consortia therefore play an increasing role in achieving very large sample sizes with enough statistical power to deal with this heterogeneity (Kraft et al. 2009). However, given the strictness of multiple comparison correction, GWAS have identified no or very few genetic variants associated with OCD at genome-wide significance level (Stewart et al. 2013; Mattheisen et al. 2015). It is know that clinical heterogeneity tends to increase with the number of data collection sites. Therefore, it is also interesting to prolong recruitment by independent research teams to keep homogeneity in extended cohorts that enable previous associations to be replicated, thereby demonstrating the reliability of those findings.

Our sample compromises only early-onset OCD patients, so it represents a homogeneous clinical population. The recruitment of new 26 trios did not enable us to perform a replication study; instead of that an analysis in an extended collection of early-onset OCD trios was conducted, which is also valuable given the difficulties to increase the number of complete early-onset OCD trios. Despite increasing the number of complete trios, sample size is still the main limitation of this study. Therefore, further research with larger cohorts is necessary to confirm these associations.

Conclusion

These results are consistent with our previous findings and provide more evidence of the role of genetic factors related to the serotoninergic and GABAergic pathways in the pathophysiology of early-onset OCD.

Clinical Significance

Our results emphasize the importance of considering genetic pleiotropy to study psychiatric disorders and highlights its appropriateness, especially when predicting common symptoms and clinically relevant endophenotypes, such as age of onset.

Footnotes

Acknowledgments

Our special thanks go to the participating subjects and their families. The authors thank the Language Advisory Service of the University of Barcelona, Spain, for article revision.

Disclosures

No competing financial interests exist.

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