Genetic association analysis of 5‐HTR2A gene variants in eating disorders in a Mexican population

Abstract Introduction The 5‐HTR2A gene has been implicated as candidate gene for eating disorders. The aim of the present study was to analyze the association of rs6311 and rs6313 polymorphisms of 5‐HTR2A gene with eating disorders in Mexican population, and to evaluate if the polymorphisms of 5‐HTR2A gene were associated with comorbidities in eating behavior. Methods We conducted a case–control analysis with 460 subjects. We included 168 patients with eating disorders and 292 controls; two polymorphisms of 5‐HTR2A gene were genotyped. We assessed the association by allele, genotype, and inheritance models. Psychiatric comorbidities were analyzed by genotype in patients with eating disorders. Results We found an association between rs6311 and eating disorders in a Mexican population by allele (OR = 8.09; 95% CI = 5.99–11.03; p = 2.2e‐16) and genotype (OR = 76.14; 95% CI = 35.61–177.18; p = 2.2e‐16). Individuals who carried GG genotype showed increased risk for suicide attempted (OR = 2.14; CI = 1.10–4.26; p = 0.035) as comorbidity associated with eating disorders. No positive associations were observed for rs6313 polymorphism. Conclusion Our results showed an association of rs6311 (A1438G) polymorphism of 5‐HTR2A gene with eating disorders, and these polymorphic variants could increase the risk of psychiatric comorbidities. However, more studies are required to replicate the results and to reach to a conclusive association between eating disorders and rs6311.


| INTRODUC TI ON
Eating disorders are multifactorial and chronic psychiatric conditions associated with aberrant eating behavior and disturbances in body image perception. The most common disorders are anorexia nervosa (AN), bulimia nervosa (BN), and binge eating disorder (BED) (American Psychiatric Association, 2013). The recent literature reports a lifetime prevalence ranging from 0.8% to 3.6% (Mustelin et al., 2016;Stice, Marti, & Rohde, 2013).
The etiology of eating disorders is unknown, however historical studies from family, twin, and adoption studies have suggested a genetic risk on eating disorders (Trace, Baker, Penas-Lledo, & Bulik, 2013). In this idea,  demonstrated that in cerebrospinal fluid (CSF) of patients with anorexia nervosa, there are lower concentrations of the degradation's products of serotonin (5-HT) metabolites. Moreover, by Positron-emission tomography (PET) imaging was found a diminished activity of 5-HTR2A on orbital frontal cortex in patients recovered from bulimia nervosa (Kaye et al., 2001). This study suggested that HTR2A gene could be associated with eating disorders.
Two SNPs of 5-HTR2A gene have been examined with psychiatric disorders: rs6311 and rs6313. The A1438G (rs6311) polymorphism of 5-HTR2A gene is also known as -1438G/A. The rs6311 is a nucleotide substitution of guanine for adenine at the position -1438 of the promoter region. This variant has been suggested that modulate the 5-HTR2A promotor activity in neuropsychiatric disorders. (Parsons, D'Souza, Arranz, Kerwin, & Makoff, 2004) Furthermore, it was associated with eating disorder, although results vary in different populations (Trace et al., 2013). However, the T102C (rs6313) polymorphism of 5-HTR2A gene, also known as -102C/T, is a substitution of cytosine for thiamine at position -102 in the exon 1; this silent variant has been associated in psychiatric disorders as schizophrenia, obsessive-compulsive disorder (OCD), major depression, and impulsivity in Caucasian and Asian populations (Gonzalez-Castro et al., 2013;Gray et al., 2018;Kaur et al., 2018;Ni et al., 2013;Sinopoli, Burton, Kronenberg, & Arnold, 2017). Nevertheless, up to the moment there are no association studies of 5-HTR2A gene polymorphisms and eating disorders in Mexican population; therefore, we decided to analyze the association of rs6311 and rs6313 polymorphisms of 5-HTR2A gene with eating disorders in this population, by (1) a case-control study, and (2) evaluating if the polymorphisms of 5-HTR2A gene were associated with comorbidities in eating behavior.

| Subjects and clinical evaluation
The control group included 292 subjects recruited randomly at the Blood Donor Center in the municipality of Comalcalco, Tabasco; all subjects approved a medical examination prior to blood donation.
Trained psychiatrics evaluated via a face-to-face interview to ensure a mentally healthy group. As criteria inclusion all the individuals were descendent from Mexican parents and grandparents. The subjects with concomitant illness or neurological disease, and psychiatric antecedents on relatives were excluded. Eighty-four subjects were included initially. However, to increase the statistical power TA B L E 1 Distribution of allelic and genotypes frequencies of polymorphisms rs6311 and rs6313 in patient with eating disorders we selected subjects with age older than 30 years and with a body mass index (BMI) >24 kg/m 2 . These subjects originally belonged to another case-control study previously reported (Lopez-Narvaez et al., 2015); and the genotypification was previously done along with the original 84 subjects.
For the case group we recruited two samples. The first sample of patients included 94 patients recruited from May 2014 to August 2015.
The second sample (n = 69) has been recruited from 2016 to date. Interview for Children and Adolescent). In the same interview, the Questionnaire on Eating and Weight Pattern-Revised (QEWP-R) was applied, this in order to identify the characteristics of binge episodes.
As criteria inclusion, only Mexican subjects descending from Mexican parents and grandparents were included. Subjects whose missing data were found on the questionnaires; and whose parents or tutors withdrew their consent for participation in the study were excluded. Finally, a total of 168 patients were incorporated in our study.

| Ethical statement
All subjects participating in the present study were given verbal and written information related to the research objectives and procedures. The subjects read and signed an informed consent in order to participate in the study; whereas for underage subjects, we ensure that informed consent was given at least for one of his/her parents or tutors. The subjects were informed of anonymity, and not economical remuneration was given from the researchers to require their participation. This study was in accordance with the principles

| Blood sample collection and genotyping
The blood sample was collected in EDTA tubes for further conser- We selected two SNPs of 5-HTR2A gene; the rs6311 variant was selected because this polymorphism has been reported associated

| Statistical analysis
The clinical characteristics were reported by frequencies and per-

| Clinical characteristics
In the present study, a total of 460 subjects were included in the analysis.

| Comparison of genotypes and allele frequencies
The  For the polymorphisms rs6313 we found a lack of association between this polymorphism and eating disorders in a Mexican population.

| Analysis of comorbidities by genotypes
Then, we evaluated the association of rs6311 and comorbidities in eating disorders using the recessive model of inheritance. We found an association of G/G genotype of rs6311 in patients with suicide risk (OR = 2.14; CI = 1.10-4.26; p = 0.035). The analysis of psychiatric comorbidities among patients with eating disorders and polymorphisms rs6311 is shown in Table 3.

| D ISCUSS I ON
We analyzed the association of polymorphism rs6311 and rs6313 of

5-HTR2A gene with eating disorders in a sample of Mexican patients
and comorbidities according to genotypes in a case-control study in eating behavior.
First, we observed an association between rs6311 and eating disorder in our population in study. Similar to our results, Nishiguchi et al. (2001) found association of allele G in patients with eating disorders in Japanese population. This result was supported in a Canadian population (Bruce et al., 2005;Steiger et al., 2008).
In contrast to our result, in Italian population, Ricca et al. (2002) evaluated obese patients with/without BED and reported a lack of association of rs6311 polymorphism with BED. Furthermore, authors reported a lack of association of eating disorder in American, Caucasian, Polish, French, and Japanese population (Ando et al., 2001;Enoch et al., 1998;Fuentes et al., 2004;Kipman et al., 2002;Nacmias et al., 1999;Rybakowski et al., 2003Rybakowski et al., , 2006Ziegler et al., 1999). Also, other studies reported that the allele associated to eating disorders is the A-allele; and the population studied with association of A-allele include Japanese, German, and British population (Campbell, Sundaramurthy, Markham, & Pieri, 1998;Gorwood et al., 2002;Hinney, Ziegler, Nothen, Remschmidt, & Hebebrand, 1997;Kang et al., 2017;Ricca et al., 2004). The discrepancy in our results could be adjudicated to the ethnical race.
Other possible cause of discrepancy in our study could be the small size sample included in the studies, possibly overlapping another positive association. In the reports, the range of sample in the reports goes from 75 to 316 patients. Then more studies are necessary but increasing the size of sample. Also, it is necessary that the studies evaluate according to the diagnostic. As in our study, we observed in our Mexican sample that G-allele carriers of rs6311 have at least ninefold times risk of anorexia nervosa and binge eating, and sevenfold times risk of bulimia nervosa.
Second, we found a significate association of lifetime comorbidities (suicide risk) with rs6311 polymorphism. Similar to our result, Nishiguchi et al. (2001) reported borderline personality disorder (BPD) as comorbidity in patients with anorexia nervosa and bulimia nervosa. The G-allele (rs6311) was found associated with BPD in Japanese population. Despite the little information on comorbidities TA B L E 2 Analysis of 5-HTR2A polymorphisms rs6311 and rs6313 as a function of the inheritance model in patients with eating disorders and controls  (Racine, Culbert, Larson, & Klump, 2009). In this sense, altered behavior like perfectionism or obsessive-compulsive behavior are traits that persist even after recovery (Gorwood, Kipman, & Foulon, 2003). These findings supported the hypothesis that behavioral traits could be expressed before the onset of the disease. However, genetic variants on specific brain regions as cortical and limbic structures , represent a genetic risk to the phenotypic traits in patients with eating disorders. Other hypothesis that could explain the possible association of rs6311 and psychiatric comorbidities in patients with eating disorders, could be a share genetic factor. Although psychiatric disorders are polygenetic, patients with comorbidities, like suicide risk, major depression episode (Wade, Fairweather-Schmidt, Zhu, & Martin, 2015), and obsessive-compulsive disorder (Enoch et al., 1998), were analyzed in twin studies, and the presence of a common share genetic factor was suggested as risk factor to other psychiatric disorders; nevertheless, more studies are required in order to enrich the knowledge of possible genetic interaction among associated comorbidities in patients with eating disorders (Anttila et al., 2018).
However, it has been proposed that comorbidity lies on the dysregulation of mood; it could lead to a chronic progression of a full/sub-threshold bipolar disorder or psychotic disorders on early adulthood (Skjelstad, Malt, & Holte, 2010 genes involved on eating disorders. We acknowledge that only patients with AN, BN, and BED were included in the analysis; and other diagnoses within the broad of eating behavior (pica and rumination) and endophenotypes (compulsivity and impulsivity) were not assessed. We recognized that the number of cases of each eating disorder is not homogeneous and the positive association between rs6311 polymorphism and eating disorders could be overestimated.
In conclusion, our study found that rs6311 polymorphism of 5-HTR2A gene is associated with eating disorders in Mexican population. Suicide risk as comorbidity was associated with rs6311 in patients with eating disorders. However, more studies like GWAS and epigenetic studies, with larger samples are needed to gain a deep insight comprehension into the association of 5-HTR2A polymorphisms and eating disorders.

ACK N OWLED G M ENT
The authors thank Obed Rubio (Psychiatrist), for his contribution in the diagnoses.