HTR3A and HTR3E gene polymorphisms and diarrhea predominant irritable bowel syndrome risk: evidence from a meta-analysis

Several studies have reported an association between serotonin receptor type 3 (5-HT3) subunit genes HTR3A (rs1062613) and HTR3E (rs62625044) and diarrhea predominant irritable bowel syndrome (IBS-D). However, the results remain inconclusive and controversial, particularly for the data derived from different ethnicities and genders. Therefore, we performed a meta-analysis to evaluate this association. All eligible case-control studies that met the search criteria were retrieved from multiple databases, and five case-control studies were included for detailed evaluation. The pooled odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated to assess the strengths of the associations of HTR3A (rs1062613) and HTR3E (rs62625044) polymorphisms with IBS-D risk. Our results revealed statistically significant associations of the HTR3A (rs1062613, C/T) polymorphism with a decreased risk of IBS-D in all genetic models. Additionally, the HTR3E (rs62625044, G/A) polymorphism was also found to be significantly associated with a decreased risk of IBS-D in the allele and recessive models. Subgroup analysis revealed that these associations held true especially for Asians and female. In conclusion, this meta-analysis suggested that the C allele of HTR3A (rs1062613) and the G allele of HTR3E (rs62625044) are associated with a decreased risk of IBS-D.


INTRODUCTION
Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorder characterized by abdominal discomfort, pain, and altered bowel habits; it may considerably reduce patients' quality of life and work productivity, which affects more than 7 percent of people all around the world [1,2]. According to the recurrent symptoms, IBS patients can experience constipation (IBS-C), diarrhea (IBS-D), or both (IBS-M) [3,4]. IBS is a multi-factorial disorder disease associated with biological and psychosocial factors [5][6][7][8][9]. However, the etiology of IBS remains largely unknown. Genetic predisposition has been demonstrated in classical family/twin studies and epidemiological surveys, but unequivocal susceptibility genes have yet to be identified [2,10]. Recently, several genetic association studies identified the serotonin receptor type 3 (5-HT 3 ) subunit genes HTR3A and HTR3E polymorphisms as being significantly associated with IBS ( particularly IBS-D) [11][12][13][14].

Meta-Analysis
The 5-HT 3 receptor is a Cys-loop ligand-gated ion channel composed of five subunits [15], which are encoded by the HTR3A, HTR3B, HTR3C, HTR3D and HTR3E genes [16][17][18]. Different HTR3 variants have been reported to be associated with schizophrenia [19], depression [20], anxiety [21], autism [22], obsessive compulsive disorder (OCD) [23], drug abuse and addiction [24,25]. Recently, the associations between these two HTR3 polymorphisms (HTR3A and HTR3E) and the risk of IBS-D have been intensively investigated. However, the community is still unable to reach a consensus, particularly regarding the data from different ethnicities and genders [11][12][13][14]26]. Such inconsistencies are generally due to ethnic differences or variations in sample size. Hence, we designed this metaanalysis to quantify the overall genetic effects of the HTR3A and HTR3E polymorphisms on the risk of IBS-D.

Characteristics of studies
As showed in Figure 1, 5 studies involving 1,287 cases and 1,418 controls were ultimately included in the present meta-analysis. Regarding the HTR3A (rs1062613, C/T) polymorphism, 5 studies were available and included a total of 1,178 cases and 1,124 controls. Regarding the HTR3E (rs62625044, G/A) polymorphism, 5 studies involving a total of 1,276 cases and 1,389 controls were available. All of these included studies were hospital-based, of which 3 from China, 1 from Germany, 1 from USA. Additionally, all of the included studies were of high quality, as indicated by the Newcastle-Ottawa scale (NOS) scores of each study being above 6 points (see Supplementary  Table 1), and the genotype distributions in all of the controls were consistent with Hardy-Weinberg equilibrium (HWE), except one [6]. Studies with controls not in HWE were also considered for the meta-analysis, but they were excluded in the sensitivity analysis. The main characteristics of the included studies were summarized in Table 1.

Power analysis
Before implementation of this meta-analysis, statistical power was assessed with the assumptions:α err prob = 0.05, OR = 1.30 (corresponding to a "weak to moderate" gene effect) for the two single nucleotide polymorphisms (SNPs), and minor allele frequencies (MAF) of HTR3A (rs1062613, C/T) and HTR3E (rs62625044, G/A) were estimated from the 1000 Genomes. The present samples indicated that 97.8% power for HTR3A (rs1062613, C/T) (MAF = 0.25), and 17.5% power for HTR3E (rs62625044, G/A) (MAF = 0.01). The power analysis indicated that these recruited samples could provide sufficient power in identifying the association between HTR3A (rs1062613, C/T) polymorphism and IBS-D.

Quantitative synthesis
As illustrated in Figures 2-3, different genetic models of HTR3A (rs1062613, C/T) and HTR3E (rs62625044, G/A) were used in our analysis.
The C allele of rs1062613 was found to be significantly associated with a decreased risk of IBS-D.  Table 2.

Sensitivity analysis
Sensitivity analysis of the summary odds ratio coefficients on the relationships of the two SNPs and the risk of IBS-D is computed by omitting each study in turn. The corresponding pooled ORs were not significantly altered after excluding each eligible study at a time (Figures 4-5).

Publication bias
No evidence of publication bias was detected regarding the ORs of the two SNPs in this study by either Begg's or Egger's test (Table 3).

DISCUSSION
Recently, several genetic association studies identified a novel association between HTR3 (HTR3A and HTR3E) and IBS-D. In Europe, Kapeller J [11] found that the novel HTR3E variant c.*76G>A (rs62625044) is significantly associated with female IBS-D in two independent cohorts from the UK and Germany, while the HTR3A variant c.-42C>T (rs1062613) is only associated with the risk for IBS-D in UK, but not in Germany population. In Asia, Gu et al. [13] demonstrated that genetic polymorphisms in HTR3A and HTR3E are associated with the risk for D-IBS in Chinese population, especially in women. However, another studies [26] revealed that the HTR3A polymorphism loci rs1062613 may be not associated with IBS risk in American population. Due to relatively small samples from different populations, these studies demonstrated inconsistent results. Therefore, we performed a metaanalysis to estimate the association between HTR3A and HTR3E polymorphisms and the risk for IBS-D. To the best of our knowledge, this is the first meta-analysis to explore the relationships between HTR3A and HTR3E gene polymorphisms and IBS-D susceptibility. Five casecontrol studies [11][12][13][14]26] with a total of 1,287 IBS-D patients and 1,418 healthy controls were included in our meta-analysis, which was giving a greater power to detect IBS-D risk associated with HTR3A and HTR3E gene polymorphisms.
In our meta-analysis, the main finding was that the C allele of HTR3A (rs1062613) decreases the risk of IBS-D in all of the comparison models, and the G allele of HTR3E (rs62625044) was also observed to have lower IBS-D risk in the allele and recessive models. Besides that, subgroup analyses by ethnicity indicated that SNP of rs62625044 (G/A) was significantly associated with a decreased risk of IBS-D in both Asian and non-Asian population, while the SNP of rs1062613(C/T) was only associated with the risk for IBS-D in Asian population. Moreover, analyses stratified by different gender showed a significant association between rs1062613(C/T) polymorphism and risk of IBS-D in both female and male population, while the SNP of rs62625044 (G/A) was only associated with risk of IBS-D in female population. Our results have some differences from the previous studies [11,26]. A possible explanation for this phenomenon is that the  Additionally, the genotype distributions in all of the controls were consistent with HWE, except one for one study reported by Kapeller et al. [11]. However, the association was not significant change when excluded the study. The NOS results indicated that the included studies were credible. Moreover, sensitivity analysis was conducted, and it did not significantly alter the combined ORs. Additionally, no evidence of publication bias was identified by either Begg's or Egger's tests. Taken together, the results of this meta-analysis are reliable and stable.
There were some limitations in the current study. Firstly, the sample sizes of the studies included in our meta-analysis were relatively small, especially in the subgroup of non-Asians, which may lead to a false negative result. Therefore, the negative results of the non-Asians should be interpreted with caution. Moreover, although meta-analysis has the benefit to overcome this limitation and may generate more precise results, the combined sample sizes in our meta-analysis were still inadequate to detect the association between rs62625044 polymorphism and IBS-D risk because power calculation for the pooled sample sizes was less than 80%, which might attenuate the statistical power to detect a slight effect and increase the chance of opportunity bias. Second, due to the limited data, we did not carry out subgroup analysis to other factors, which may participate in the progression of IBS-D, such as age, lifestyle and social    psychology. Thirdly, only articles in English and Chinese language were included; thus, studies written in other languages were neglected. Finally, although we performed a systematic searching strategy to identify eligible studies, there was still probability that few studies so called "grey literatures" were not included.
In conclusion, this meta-analysis suggested that the C allele of HTR3A (rs1062613) and the G allele of HTR3E (rs62625044) are associated with a decreased risk of IBS-D. One evidence indicates that these HTR3 polymorphisms can remarkably up-regulate the expression of 5-HT 3 receptors, which have been proved to cause the gastric functional disorders including emesis, eating disorders and IBS-D, et al. [27], as illustrated in Supplementary Figure 1. Due to the above-mentioned limitations, a well-designed large-scale study that includes ethnicities, genders and psychosocial factors is required to confirm the findings of the current meta-analysis.

Search strategy
In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [28], we searched the related literature of the electronic records of the PubMed, Science Direct, Embase, WANFANG databases and Chinese National Knowledge Infrastructure (CNKI) published through 30 March 2017. The search terms included the following key words: ("Irritable bowel syndrome" or "IBS") AND ("polymorphism" or "allele" or "genetic" or "gene" or "mutation" or "variant") AND ("serotonin type 3" or "serotonin receptor 3" or "5-HT 3 " or "HTR3A" or "HTR3E"). Furthermore, the references of all retrieved articles were also checked by hand to identify additional potential studies. The languages were limited to English and Chinese.

Inclusion criteria
The inclusion criteria were as follows: (1) studies of the association between HTR3A or HTR3E polymorphism and IBS; (2) cohort or case-control study (3) provided sufficient data of allele and genotype frequencies of SNPs or required information could be calculated; and (4) if serial studies on the same population were published, only the most recent study was included. Additionally, we excluded reviews, abstracts, and redundant and animal studies.

Data extraction
Two independent investigators extracted relevant data from all included studies on the basis of the inclusion criteria, and a third investigator verified them. The following information from eligible studies was extracted: the first author ' s name, publication year, country of origin, ethnicity, sample size, genotyping method, and the HTR3A and HTR3E genotype distributions and alleles in the case and control groups.

Quality assessment
The quality of included studies were assessed by two investigators independently on the basis of Newcastle-Ottawa Scale (NOS) [29], which based on three aspects: selection, comparability and exposure. Studies with a score of 5 points or higher were considered to be of high quality.

Statistical analysis
The HWE of the genotype distributions in the controls of the include studies were tested by the Chisquare test, P < 0.05 was considered statistically significant. Studies with the controls not in HWE were subjected to a sensitivity analysis [30]. The power analysis was calculated by using the Power and Sample Size Program software [31]. The associations of the HTR3A and HTR3E polymorphisms with the risk of IBS were assessed by the pooled ORs with the corresponding 95% CIs under the following genetic models: allele model, codominant model, dominant model, and recessive model. The heterogeneity between studies was determined by the Cochrane's Q-statistic test [32], and the inconsistency was quantified with the I 2 statistic. When I 2 > 50% or P Q ≤ 0.1, which suggest substantial heterogeneity, a random-effects model (DerSimonian-Laird method) [33] was used; otherwise, the fixed-effects model (Mantel-Haenszel method) [28] was applied. Sensitivity analysis was conducted by sequentially omitting each study to validate the reliability of the results. Publication bias was examined with Begg's funnel plot and Egger's test [34], and P < 0.05 was considered statistically significant. All analyses were conducted using the RevMan 5.1 and STATA 12.0 software packages.