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

2.1 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 we selected subjects with age older than 30 years and with a body mass index (BMI) >24 kg/m². 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. However, adult patients (n = 5) were included from a different case–control study (Saucedo-Uribe et al., 2019). The patients were evaluated and diagnosed by a medical specialist in psychiatry in the Children's Psychiatric Hospital “Dr. Juan N. Navarro” in Mexico City, Mexico. All the information was gathered in only one psychiatric interview; the diagnoses of eating disorder were based on the DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, 5th edition). Additionally, the comorbidities associated were established in the case of co-occurrence with the MINI Kid Spanish version (Mini International Neuropsychiatric 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.

2.2 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 of the Helsinki Declaration in 1975; and approved by Institutional Review Board at Carracci Medical Group at INMEGEN; this study was in compliance with the Code of Ethic of the World Medical Association.

2.3 Blood sample collection and genotyping

The blood sample was collected in EDTA tubes for further conservation and storage. Genomic DNA was extracted and purified from peripheral white blood cells following the instructions of Promega's kit (Wizard Genomic DNA Purification). Genotyping of 5-HTR2A polymorphism rs6311 and rs6313 for the first sample of patients and control group was determined by polymerase chain reaction end-point method by means of allelic discrimination following the manufacturer's instructions. For quality control in our genotyping analyses, duplicate samples were genotyped randomly. The polymorphisms (rs6311 and rs6313) studied are available from the TaqMan SNP Genotyping Assay made to order by Applied Biosystems. However, for the second sample of patients (included the adult patients) genotypes of polymorphism rs6311 and rs6313 were extracted from a genetic database constructed with information from the Human Psych Array (Saucedo-Uribe et al., 2019).

We selected two SNPs of 5-HTR2A gene; the rs6311 variant was selected because this polymorphism has been reported associated with patients with anorexia nervosa and bulimia nervosa, although with controversial results (Trace et al., 2013). Polymorphism rs6313 was considered because this variant has been studied and associated with psychiatric pathologies like schizophrenia, major depressive disorder, and obsessive-compulsive disorder, although there is limited data about the role of rs6313 polymorphism in patients with eating disorders (Ni et al., 2013; Sun, Xu, Zhou, Zuo, & Liu, 2017; Wade, Bulik, Neale, & Kendler, 2000).

2.4 Statistical analysis

The clinical characteristics were reported by frequencies and percentages for the categorical variables, and with means and standard deviations for the continuous variables. A χ² test was used to calculate the Hardy–Weinberg equilibrium (HWE); HWE and associations were performed online, freely, and available at https://www.snpstats.net. Odds ratio (OR) with a 95% confidence interval (95% CI) was reported for allelic comparisons and genotype frequencies between cases and controls, as well as comparisons of control group versus AN, BN and BED and comorbidities. The five inheritance models (codominant, dominant, recessive, over-dominant, and log-additive) were applied in a logistic regression analysis to estimate the risk of eating disorders between cases and controls. Logistic regression analysis was adjusted by age, gender, and BMI. Statistical analyses were performed with the Statistical Package for the Science Software v.25 (SPSS, Chicago, IL). The level of significance was set at <0.05; and for multiple comparisons the p-value was set at <0.025 using the Bonferroni test. Permutation test was performed in results with statistical differences.

3.1 Clinical characteristics

In the present study, a total of 460 subjects were included in the analysis. The control group include 147 male subjects (50.34%) and 145 female subjects (49.66%). The mean age was 33.97 ± 10.08 years old and the mean BMI was 28.1 ± 4.8 kg/m². For the case group, 168 patients were included, of whom 23.95% were male patients (n = 40) and 76.05% were female patients (n = 127). Of the 168 cases, 17.86% of the patients had the diagnosis of anorexia nervosa (n = 30); 59.52% were patients with bulimia nervosa (n = 100); and 22.62% (n = 38) were patients with binge eating disorder.

When we analyzed the case group in subgroups (AN, BN, BED), the mean age for the case group was 14.5 ± 3.6 years old (AN [14.2 ± 2.8 years old]; BN [14.8 ± 4.3 years old]; BED [13.8 ± 1.7 years old]) and the mean BMI was 23.9 ± 5.5 kg/m² (AN [18.8 ± 2.9 kg/m²]; BN [24 ± 4.7 kg/m²]; BED [27.5 ± 6.0 kg/m²]). The mean z score for BMI in the subgroups was −0.18 ± 1.17 for AN; 0.87 ± 0.81 for BN and 1.43 ± 0.92 for BED. When we grouped the BMI by diagnosis, we observed 49.69% of patients with normal weight (n = 80); the rest of the cases were either in underweight, overweight or obesity (4.35% [n = 7]; 18.63% [n = 30]; 27.33% [n = 44]; respectively).

When the MINI Kid Spanish version was applied, we observed that 90.1% (n = 147) of the cases had life time psychiatric comorbidities associated. The most common comorbidities were major depressive episode (n = 82), suicidality (n = 57), dysthymia disorder (n = 36), attention-deficit/hyperactivity disorder (n = 36), generalized anxiety disorder (n = 29), oppositional defiant disorder (n = 20), and psychotic disorder (n = 12).

3.2 Comparison of genotypes and allele frequencies

The 5-HTR2A polymorphisms rs6311 and rs6313 for the cases and controls satisfied the Hardy–Weinberg equilibrium (rs6311: control group [p = 0.21], case group [p = 0.7]; rs6313: control group [p = 0.16], case group [p = 0.7]).

The comparison of distribution of allelic and genotypes between controls and eating disorder is shown in Table 1. We observed an association between polymorphism of 5-HTR2A and eating disorders by allele (OR = 8.09; 95% CI = 5.99–11.03; p = 2.2e-16) and by genotype (OR = 76.14; 95% CI = 35.61–177.18; p = 2.2e-16) in the study population. In the analysis by diagnostic groups of eating disorders (AN, BN, BED), we found an association between rs6311 by alleles and genotypes with anorexia nervosa, bulimia nervosa, and binge eating disorder. Table 1. For rs6313 there was a lack of association between this polymorphism and eating disorders in the Mexican population.

3.3 Univariate logistic regression analysis of relationship between each SNP and eating disorder for five inherited models

Table 2 shows the inheritance model analysis of 5-HTR2A polymorphisms rs6311 and rs6313 and eating disorders. The genotype G/G was significantly associated with eating disorders in the codominant (OR = 27.19; 95% CI = 7.51–98.46; p = 2.2e-16), recessive (OR = 9.36; CI = 3.12–27.99; p = 2.2e-16), and dominant (OR = 10.66; CI = 3.96–28.68; p = 2.2e-16) models. Likewise, in the additive model (OR = 5.26; CI = 2.77–10.01; p = 2.2e-16) the G allele was demonstrated to be associated with eating disorders. For the polymorphisms rs6313 we found a lack of association between this polymorphism and eating disorders in a Mexican population.

3.4 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.