Associations between Single-Nucleotide Polymorphisms in Corticotropin-Releasing Hormone-Related Genes and Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common functional disorder with distinct features of stress-related pathophysiology. A key mediator of the stress response is corticotropin-releasing hormone (CRH). Although some candidate genes have been identified in stress-related disorders, few studies have examined CRH-related gene polymorphisms. Therefore, we tested our hypothesis that single-nucleotide polymorphisms (SNPs) in CRH-related genes influence the features of IBS. Methods: In total, 253 individuals (123 men and 130 women) participated in this study. They comprised 111 IBS individuals and 142 healthy controls. The SNP genotypes in CRH (rs28364015 and rs6472258) and CRH-binding protein (CRH-BP) (rs10474485) were determined by direct sequencing and real-time polymerase chain reaction. The emotional states of the subjects were evaluated using the State-Trait Anxiety Inventory, Perceived Stress Scale, and the Self-rating Depression Scale. Results: Direct sequencing of the rs28364015 SNP of CRH revealed no genetic variation among the study subjects. There was no difference in the genotype distributions and allele frequencies of rs6472258 and rs10474485 between IBS individuals and controls. However, IBS subjects with diarrhea symptoms without the rs10474485 A allele showed a significantly higher emotional state score than carriers. Conclusions: These results suggest that the CRH and CRH-BP genes have no direct effect on IBS status. However, the CRH-BP SNP rs10474485 has some effect on IBS-related emotional abnormalities and resistance to psychosocial stress.


Introduction
Irritable bowel syndrome (IBS) is a widespread functional disorder of the lower gastrointestinal tract that is primarily characterized by abdominal pain and altered bowel habits (diarrhea and/ or constipation) [1]. IBS is associated with impaired quality of life and excessive use of health care resources and has long been considered to be a stress-related disorder [2,3]. Co-morbidity their predominant bowel pattern: constipation, diarrhea, or mixed. There was no significant difference in age, sex ratio, and IBS status between the groups. This study was approved by the Ethics Committee of Tohoku University Graduate School of Medicine. All participants gave their written informed consent.
Among the 253 participants, 190 were included in a study of the polymorphisms of the 5-hydroxytryptamine transporter [37] and CRH-R1 polymorphic region [32]. However, those genes were completely different from the genes targeted in this study. The hypotheses in this study and the previous studies are different. The previous analysis does not impact on this study.

Psychometric Tests
The emotional states of the subjects were rated using the Self-rating Depression Scale (SDS) [38,39], Perceived Stress Scale (PSS) [40,41], and State-Trait Anxiety Inventory (STAI) [42,43]. The SDS is a rating of affective, psychological, and somatic symptoms associated with depression, the PSS measures the perception of stress, and the STAI is a rating of two types of anxiety: state anxiety and trait anxiety.

Genotyping
Whole blood (10mL) was sampled from the median cubital vein of all subjects. DNA was extracted from the white blood cells using a standard protocol [32]. SNPs in CRH (rs28364015 and rs6472258) [22,33] and CRH-BP (rs10474485) [33] were selected by considering previous reports, and minor allele frequencies were determined from a database [NCBI: http://www. ncbi.nlm.nih.gov/snp/]. CRH is encoded by the CRH gene, which is located on chromosome 8 and spans 2.34 kb. rs28364015 and rs6472258 are also located on chromosome 8 (position 67253453 and 67259033, respectively). CRH rs28364015 is a 5 0 untranslated region variant, and rs6472258 is an intergenic variant. Further, CRH-BP is encoded by the CRH-BP gene, which is located on chromosome 5 and spans 16.76 kb. CRH-BP rs10474485 is located on chromosome 5: (position 76306609). rs10474485 is also intronic and is a non-coding transcript variant.
Similarly, rs6472258 and rs10474485 TaqMan probe sets were designed, and the participants were genotyped by real-time PCR. However, the TaqMan assay failed for rs28364015 because of the frequency of the C allele (0%). The PCR reactions consisted of 200 nM of each primer, 200 nM fluorogenic probe, 1× SsoAdvanced Probes Supermix, and distilled water (up to 10 μL). Automated sequencing was performed on a CFX 96 TM Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Tokyo, Japan).

Statistical Analysis
We used Haploview [44] to confirm the Hardy-Weinberg equilibrium. The effects of the genotypes or alleles of each SNP were compared between the IBS patients and controls using the χ 2 test and Odds Ratio (OR) (95% confidence interval [CI]). Two-way analysis of variance with a post hoc test was performed to determine the association between the SNPs and the psychometric tests. Results are expressed as the mean ± SD. We considered p < 0.05 to be statistically significant, whereas a p-value < 0.1 was considered to be tendentially but not statistically, significant. Statistical analyses were performed using SPSS Statistic 22.0 (IBM, Inc., New York, NY).

Results
The genotype frequencies of the selected CRH and CRH-BP gene variations in the IBS patients and controls are shown in Table 2. There was no significant difference in the genotype frequencies of rs6472258 (p = 0.990, χ 2 test) and rs10474485 (p = 0.969, χ 2 test) between the patients and controls. Similarly, there was no significant difference in the allele frequencies of the rs6472258 T allele (p = 0.675, χ 2 test) or G allele (p = 0.890, χ 2 test), and rs10474485 C allele (p = 0.816, χ 2 test) or A allele (p = 0.986, χ 2 test) between the IBS patients and controls. Moreover, the results of the additional analysis (OR [95% CI]) in rs6472258, alleles are not associated with IBS, with an OR of 1.036 (95% CI, 0.629-1.71) for G allele and 1.015 (95% CI, 0.524-1.97) for T allele. Similarly, the allelic OR (95% CI) of rs10474485 were 1.000 (95% CI, 0.976-1.025) for A allele and 0.914 (95% CI, 0.427-1.956). There was no difference in statistical results between genotype or allele frequency and OR.
The psychometric scores of the IBS patients and controls are shown in Table 3. For the SDS, the score of the IBS patients was higher than that of the controls (p = 0.016). In addition, for the PSS, the score of the IBS patients was higher than that of the controls (p = 0.028). The STAI score was not significantly different between the two groups (State, p = 0.111; Trait, p = 0.115).
In the analysis of all subjects (IBS patients + controls), rs10474485 A allele non-carriers showed a higher PSS score than carriers (F[1, 507] = 6.459, p = 0.012). There was a significant group (IBS vs. control) effect with the SDS (p = 0.014) and PSS (p = 0.030) scores. The mean (± SD) PSS score of A allele carriers was 25.56 (± 8.359), whereas that of non-carriers was 28.48 (± 9.303). The other psychometric scores were not significantly different (SDS, p = 0.112; State, p = 0.140; Trait, p = 0.143). In contrast, we analyzed the associations between rs6472258 (genotype and allele) and the psychometric scores, but there was no significant difference.
In addition, in analysis of the male subjects alone, significant group (IBS/control) × rs10474485 genotype (F[2, 167] = 3.180, p = 0.045) (Fig 2A) and group (IBS/control) × rs10474485 A allele (F[1, 309] = 5.821, p = 0.017) (Fig 2B) interaction were observed in the SDS score. However, there was no difference in the emotional scores of the male subjects with respect to the A allele ( Fig 3A). In the female subjects, significant differences were observed in the PSS (F[1,360] = 4.931, p = 0.028) and State (F[1, 520] = 4.557, p = 0.035) scores with respect to the rs10474485 A allele (Fig 3B). The mean (± SD) PSS score of female A allele carriers was 26.87 (± 7.983), whereas that of non-carriers was 30.42 (± 8.843). The mean (± SD) State score of A allele carriers was 42.74 (± 10.091), whereas that of non-carriers was 46.72 (± 10.962). Analyses of rs28364015 did not indicate a significant difference in males or females. As for the CRH SNP rs6472258, there was a nonsignificant trend for a difference in the SDS score between G allele carriers and non-carriers (all subjects, F[1, 244] = 3.837, p = 0.051; male  In the present study, all subjects (n = 40) whose DNA was analyzed with direct sequencing had the rs28364015 TT genotype and no subjects had the C allele. Therefore, we excluded rs28364015 from the analysis of the real-time PCR data.

Discussion
In the current study, we analyzed CRH-related peptide genetic variations in controls and patients with IBS, which is one of the representative disorders with stress-related pathophysiology. On the basis of previous studies and reports of the sexually dimorphic expression of CRHrelated genes [45][46][47][48], sex differences would be expected in our study. Indeed, in the present study, the CRH-BP SNP rs10474485 had a significant effect on the PSS score, especially in female subjects. In addition, there was a significant gene × group interaction in the SDS score of males. It has been suggested that sex differences are important when considering genetic influences on IBS. Some previous studies suggested that the functions of hormone and genetic variations were connected to sex [45][46][47][48][49]. For example, a role for 5-hydroxytryptamine receptor 2B genotypes in impulsivity was shown in male subjects [45], and transcriptional regulation of the human CRH-BP gene promoter by estrogen receptors was reported [49]. Similarly, it is thought that the association between differences in sex hormones and gene polymorphisms are important. As CRH plays a dominant role in the HPA axis, the effect of sex differences on hormone and hypothalamic function must be investigated. For the CRH SNP rs6472258, a tendency for a difference in the SDS score between G allele carriers and non-carriers was found, which may have influenced the differences in the SDS score in male subjects. Significant results might have been observed with more subjects.
Some studies have suggested that CRH-related gene variations are not associated with IBS per se, but that the IBS subgroup or patients' comorbid anxiety is associated with polymorphisms [50][51][52]. IBS subjects with diarrhea showed a higher response (namely, more inhibition) than healthy controls to a CRH receptor antagonist [53]. Therefore, IBS subjects with diarrhea might be more responsive to CRH or CRH-related peptides than healthy controls.
Here, the IBS subjects with diarrhea symptoms (diarrhea or a mix) showed significant differences in some emotion states according to the presence of the rs10474485 SNP. It is suggested that these CRH-BP polymorphisms might more strongly influence IBS patients with diarrhea symptoms. CRH is a major mediator to activate HPA axis in stress response. In contrast, CRH-BP reduces the effect of CRH [28]. If rs10474485 A allele make the CRH-BP effects increase, CRH function may be more interfered with. Thereby, it is suggested that HPA axis activity attenuated, and stress response decreased. As a result, it is thought that psychometric scores might decrease when individuals have A allele.
In the present study, the CRH G and CRH-BP A alleles were minor alleles. According to previous studies of stress-related disorders, CRH-related gene-specific polymorphisms appeared to protect against adult depressive symptoms [54,55]. Subjects without the minor alleles of the selected SNPs showed higher psychometric scores than minor allele carriers. CRH exaggerates various stress responses, such as anxiety. Conversely, human CRH-BP binds to CRH and prevent activation of CRH receptors, thereby, reduce high activity with CRH [56]. In rodents, CRH-BP cells are concentrated within a hypothalamic circuit involved in mediating neuroendocrine and autonomic responses to stress [57]. CRH-BP expression downregulates the HPA axis by interfering with CRH, and the stress responses converge [28].
Here, CRH-BP rs10474485 was not directly related to IBS. However, the selected CRH-BP polymorphism was found to be in linkage disequilibrium with the CRH-BP SNPs rs10055255 (r 2 = 0.410, D 0 = 0.928) and rs1875999 (r 2 = 0.410, D 0 = 0.928) in the 1000 Genomes Project of Japanese individuals in Tokyo [the 1000 Genomes Browsers: http://browser.1000genomes.org/ ]. Homozygosity of the rs10055255 minor allele (TT) was associated with fewer incidences of post-traumatic stress disorder and depressive symptoms in post-intensive care units [58]. Under a condition of stress manipulation, individuals homozygous for the rs10055255 minor allele (TT) were noted to show high stress-induced alcohol craving, tension, and negative mood compared with their pre-stress condition. During the neutral imagery condition, there was no effect, but the post-stress rating was lower than that of pre-stress [59]. Thus, this SNP (rs10055255) may only show the differential effects of stress responses under a stressful situation. However, because our study was not conducted under stress conditions, the rs10055255 SNP was not selected. Nonetheless, it is suggested that CRH-BP polymorphisms influence stress responses during stress manipulation. In addition, the rs1875999 major allele (T) was more common in unipolar patients [60], and its minor allele (C) was associated with both heroin and cocaine addiction [61]. At present, the functionality of rs1875999 is unknown and the directionality of the role of the alleles in the stress response is not consistent. Accordingly, we did not select this SNP in this study. However, it is thought that this SNP has an inhibitory role in the development of stress-related neuropsychiatric disorders. Therefore, it is conceivable that a functional increase may occur in the CRH-BP produced by the CRH-BP rs10474485 minor allele and that the activity of the HPA axis may decrease. In other words, there may be protective effects of minor CRH/CRH-BP gene variants on the stress response.
Although identified in the rhesus monkey [22], the genetic variant of the rs28364015 SNP has not been identified in the Japanese population. The present study is the first to try to identify this polymorphism in Japanese individuals. However, the results were negative. Regardless of the limited number of subjects (40 individuals), the possibility of finding this variation is considered to be very low.
There are some limitations to this study. First, we did not have a sufficient number of samples to investigate SNPs. A future direction would be to collect more samples and to explore more SNPs in CRH, CRH-BP, CRH-R1, and CRH-R2. Moreover, because the average age of all subjects was low (22.0 ± 2.2 years), it is thought that the present data could not be extrapolated to the entire IBS population. However, a meta-analysis study showed that there was not significant difference in the prevalence of IBS between different age bands [62]. Therefore, it is thought that the subjects used in the present study were representative as an IBS group. Second, IBS severity was not explored. In the future, the association between disease severity and polymorphisms should be explored. Third, the rs6472258 and rs10474485 SNPs selected in this study are intronic. The rs6472258 SNP is located upstream of CRH gene exon 1. Similarly, rs10474485 is located downstream of CRH-BP gene exon 7 (Ensembl Genome Browser: http:// www.asia.ensembl.org/). The effects of these areas on the transcriptional activity and protein expression of the CRH/CRH-BP genes are not clear. However, in a previous study, CRH receptor-1 SNPs located in the intron around exon 2 were associated with IBS [32]. Therefore, there may be a possibility that intronic SNPs can regulate the function of the CRH/CRH-BP genes. In any future study, the role of CRH genes in the endophenotype of IBS should be explored.
In conclusion, our study suggests that CRH-BP gene polymorphisms had some effect on IBS-related psychological abnormalities. In contrast, we could not detect the associations between the CRH/CRH-BP genes and IBS status per se. Further evidence for a general role of CRH and CRH-BP gene polymorphisms in stress-related disorders is required.