Corticotropin-Releasing Hormone Receptor 2 Gene Variants in Irritable Bowel Syndrome

Background Corticotropin-releasing hormone (CRH) plays an important role in the pathophysiology of irritable bowel syndrome (IBS) and regulates the stress response through two CRH receptors (R1 and R2). Previously, we reported that a CRHR1 gene polymorphism (rs110402, rs242924, and rs7209436) and haplotypes were associated with IBS. However, the association between the CRHR2 gene and IBS was not investigated. We tested the hypothesis that genetic polymorphisms and haplotypes of CRHR2 are associated with IBS pathophysiology and negative emotion in IBS patients. Methods A total of 142 IBS patients and 142 healthy controls participated in this study. Seven single nucleotide polymorphisms (SNPs) of the CRHR2 gene (rs4722999, rs3779250, rs2240403, rs2267710, rs2190242, rs2284217, and rs2284220) were genotyped. Subjects' psychological states were evaluated using the Perceived-Stress Scale, the State-Trait Anxiety Inventory, and the Self-Rating Depression Scale. Results We found that rs4722999 and rs3779250, located in intronic region, were associated with IBS in terms of genotype frequency (rs4722999: P = 0.037; rs3779250: P = 0.017) and that the distribution of the major allele was significantly different between patients and controls. There was a significant group effect (controls vs. IBS), and a CRHR2 genotype effect was observed for three psychological scores, but the interaction was not significant. We found a haplotype of four SNPs (rs4722999, rs3779250, rs2240403, and rs2267710) and two SNPs (rs2284217 and rs2284220) in strong linkage disequilibrium (D′ > 0.90). We also found that haplotypes of the CRHR2 gene were significantly different between IBS patients and controls and that they were associated with negative emotion. Conclusion Our findings support the hypothesis that genetic polymorphisms and haplotypes of CRHR2 are related to IBS. In addition, we found associations between CRHR2 genotypes and haplotypes and negative emotion in IBS patients and controls. Further studies on IBS and the CRH system are warranted.

indices in IBS patients and this response was inhibited by the administration of a CRH antagonist [28]. Therefore, dysfunction of CRH signaling is considered to be related to the pathophysiology of IBS.
CRH plays a stimulatory role in the stress response through the activation of CRHR1, while the specific actions of urocortin 2 and urocortin 3 on CRHR2 may be important for dampening the stress response [4]. In the brain, CRHR1 stimulation causes anxiety, but CRHR2 stimulation induces anxiolysis. In regard to gut motility, CRHR1 stimulation evokes colonic motility, but CRHR2 stimulation inhibits gastric emptying [12]. From these studies, one of the important determinants of the brain-gut interaction in the stress response of IBS patients is signaling via CRHR1 and CRHR2.
The gene encoding CRHR1 is located on chromosome 17q21. 31 and has a length of 51.55 Kb and contains 14 exons. The gene encoding CRHR2 is located on chromosome 7p14.3 and has a length 48.19Kb and contains 16 exons. There are many reports on the association between gene variants of CRH receptors and stress-related diseases. In CRHR1, variation of the gene has been found to be a risk factor for depression after childhood maltreatment [29][30][31][32][33]. A polymorphism of CRHR1 was reported to be related to recurrent major depressive disorder (MDD) [34]. In regard to CRHR2, gene variation may affect the risk of PTSD in women by attenuating the stress response and reducing symptoms of the disorder [35]. A polymorphism in the CRHR2 gene was also associated with MDD [36]. In a previous study from our laboratory, we showed that genetic polymorphisms and haplotypes of CRHR1 (rs110402, rs242924, and rs7209436) mediate IBS and related bowel patterns [37]. However, the association between CRHR2 genotypes and IBS has not been investigated. Therefore, we hypothesized that gene polymorphisms and/or haplotypes of CRHR2 may be associated with IBS pathophysiology and negative emotion in IBS patients.

Methods Subjects
In total, 142 patients (62 males and 80 females) with IBS who were diagnosed at the Department of Psychosomatic Medicine, Tohoku University Hospital, were enrolled in this study (mean age 22.0 ± 0.2 years; range [18][19][20][21][22][23][24][25][26][27][28][29][30][31]. Patients with organic diseases were excluded. In addition, 142 healthy volunteers (74 males and 68 females) were recruited at Tohoku University as controls (mean age 22.0 ± 0.2 years; range [19][20][21][22][23][24][25][26][27][28][29][30][31]. Subjects without any symptoms or signs during a medical interview and physical examination were identified as healthy controls. There was no significant difference in age, sex ratio among the groups. IBS patients were diagnosed according to the Rome III criteria [38]. IBS was defined as recurrent abdominal pain or discomfort for at least 3 days per month in the last 3 months associated with two or more of the following symptoms: improvement with defecation, onset associated with a change in the frequency of defecation, and/or onset associated with a change in the form (appearance) of stools. These criteria were fulfilled for the previous 3 months with symptom onset at least 6 months prior to diagnosis. According to the Rome III criteria, IBS was classified as IBS with diarrhea (D), constipation (C), or mixed symptoms of diarrhea and constipation (M). Unclassified IBS patients were classified as IBS-M. All subjects provided written informed consent and this study was approved by the Tohoku University Ethics Committee. Consecutive patients who agreed to participate in this study were enrolled.
Two hundred and twenty-two of 284 participants' DNA samples were previously reported on polymorphisms of the CRHR1 polymorphic region [37]. There are apparently different from the targeted genes in this study.

SNP selection
We used Generic Genome Browser in International HapMap Project (http://hapmap.ncbi.nlm. nih.gov/index.html.en) to search the allele frequency (minor allele frequency > 0.15 in Japanese population). Furthermore, we restricted seven candidate genes, which were selected from single nucleotide polymorphisms (SNPs) for genotyping based on previous literature [35,36,39]. The loci of these SNPs were searched from the dbSNP in the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/), and are shown in

Genotyping
Peripheral blood was sampled from the forearm vein of each subject with a heparinized syringe. DNA was then extracted from the lymphocytes using a standard protocol [40]. The concentration of each DNA was diluted to 0.2 μg/μL. Seven SNPs (rs4722999, rs3779250, rs2240403, rs2267710, rs2190242, rs2284217, and rs2284220) in the regulatory region of the CRHR2 gene were genotyped using real-time polymerase chain reaction (PCR).
We use the CFX-96 Touch TM Real-time PCR Detection System (Bio-Rad Laboratories, Inc., Tokyo, Japan). PCR amplification was carried out using probe/primer sets for SNP genotyping that were purchased from Applied Biosystems, Tokyo, Japan (set of C1617682310 for rs2240403, C1587286110 for rs2190242, C1596058610 for rs2284217, C1596058010 for rs2284220, C34646010 for rs4722999, C2580980110 for rs3779250, and C1587287110 for rs2267710). PCR amplification was performed using a total volume of 10 μL consisting of 5 μL TaqMan Genotyping Master Mix, 4.4 μL ddH 2 O, 0.5 μL TaqMan SNP Genotyping Assays, and 0.1 μL DNA. After initial denaturation at 95°C for 10 min, amplification was performed for 40 cycles at 95°C for 15 s and 60°C for 1 min. To determine the positive control, sequence analysis of CRHR2 genes was performed using an ABI 3130 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). All procedures were performed according to the manufacturer's instructions. (SDS; [40], [45]). The Japanese versions of STAI, SDS, and PSS have been validated and their reliability has been confirmed [42].

Statistical analysis
The frequencies of genotypes, alleles, and haplotypes of the CRHR2 SNPs were compared between the IBS patients and controls, or between patients with different bowel patterns (normal, constipation, mixed, and diarrhea) using the χ 2 -test. The effects of variation in CRHR2 SNPs and haplotypes on psychological states between the IBS patients and controls were examined with two-way analysis of variance (ANOVA). The effects of variation in CRHR2 haplotypes on psychological states were examined with one-way ANOVA. Statistical analyses were performed using SPSS Statistics version 21.0 software (IBM, Inc., New York, NY). We used Haploview [46] to determine the LD of the SNPs within the CRHR2 gene and test for Hardy-Weinberg equilibrium. Results are expressed as the mean ± standard error, and a P value < 0.05 was considered to be significant.

Results
In the 284 samples, genotype distributions were in Hardy-Weinberg equilibrium for all seven SNPs. The genotype frequencies of the SNPs in the IBS group and healthy controls are listed in Table 1. The allele frequencies of the SNPs in the IBS group, healthy controls, and the subtypes of IBS patients are listed in Table 2. We found that rs4722999 (A) and rs3779250 (B) were significantly associated with IBS in terms of genotype distribution (rs4722999: P = 0.037; rs3779250: P = 0.017) (Fig 2). For these two SNPs, there was no significant difference in the frequency of the minor allele, but the frequency of the major allele was significantly different. Table 3 shows the scores of the psychological scales in the controls and IBS patients. There was no significant difference in the test scores for the three psychological scales between the IBS patients and controls. Fig 3 shows that the three psychological scores were significantly different by CRHR2 genotype or group (IBS patients vs. controls). According to PSS in rs2240403, the IBS patients were significantly higher than the controls (P = 0.018), but there was no significant interaction. According to STAI (state) in rs2190242 in males, there was a significant genotype effect (P = 0.046), but there was no significant interaction. According to SDS in rs2190242 in males and rs2240403 in females, there was a significant genotype effect (rs2190242: P = 0.026; rs2240403: P = 0.031), but there was no significant interaction. According to the selection criteria, four SNPs (rs4722999, rs3779250, rs2240403, and rs2267710) of CRHR2 with strong D 0 (>0.90) were in one linkage block, and two SNPs (rs2284217 and rs2284220) with strong pairwise D 0 (>0.90) were in another block (Fig 1). The selection criteria for haplotypes used in the haplotype analyses were adjacent SNPs with pairwise D 0 > 0.90. Haplotype analysis of the IBS group and healthy controls is shown in Table 4. We found that three blocks were positively associated with IBS patients (T-C-C-C: P = 0.017; T-C-C-T: P = 0.017; C-T-C-C: P = 0.043). All haplotypes were found more frequently in the IBS patients than in the controls.
We tested whether the psychological scale scores were different by haplotypes using Oneway ANOVA. The result was shown in

The carriers with four haplotypes (T-T-T-T, C-T-T-T, C-C-T-T, and T-C-T-T) had significantly lower scores than the others in
all psychological scales. The four haplotype carriers were all the same individuals. In addition, in PSS, two haplotypes (C-T-C-T and C-C-C-T) were similarly significant.
We tested whether the psychological scale scores were different by haplotypes between the IBS patients and controls using two-way ANOVA. The result was shown in Table 5. In PSS, group effect (IBS vs. controls) was significant in six haplotypes. However, there was no group

Discussion
The present study is the first to show an association between IBS and SNPs/haplotypes of the CRHR2 gene in the Japanese population. These data suggest that CRHR2 gene polymorphisms and haplotypes are related to the pathophysiology of IBS. Loss of CRHR2 leads to increased inflammation, delayed healing, and exacerbates the inflammatory insult [47][48][49]. Akiba et al. reported that peripheral CRHR2 activation induces colonic hyperemia through nitric oxide synthesis without involving prostaglandin synthesis or sensory nerve activation and may protect the colonic mucosa [50]. In a recent study, low grade inflammation was observed in the colonic mucosa of IBS patients [51]. We found that rs4722999 and rs3779250 were positively associated with IBS in terms of genotype frequency. Especially, for both SNPs, CT carriers were observed significantly more often in IBS patients. Ishitobi et al. investigated the association of six SNPs of CRHR2 with MDD and panic disorder [36]. They reported that rs3779250 was associated with MDD, but rs4722999 and the other SNPs were not [36]. In this study, major allele carriers were observed frequently in MDD patients. Similarly, in our study, the frequency of the major allele of rs3779250 was significantly different between the patients and controls. Like IBS, MDD is worsened by psychosocial stress with dysfunction of the HPA axis. From these results, the major allele of rs3779250 may be a risk allele for stress-related diseases. However, the C allele of rs3779250 is observed in approximately 77% of the normal population. Therefore, the increased frequency of the major allele of rs3779250 alone cannot explain the relationship between CRHR2 SNPs and IBS. Further study of other factors (e.g., other genetic polymorphisms) of IBS is required. Psychological scales and CRHR2 SNPs in IBS patients and controls. PSS: two-way ANOVA showed that the IBS patients with rs2240403 (P = 0.018) had significantly higher perceived stress scale scores than the controls. STAI (state): rs2190242 in males, genotype effect was significant (P = 0.046). SDS: rs2190242 in males and rs2240403 in females, genotype effect was significant (rs2190242: P = 0.026; rs2240403: P = 0.031). In all scale scores, there was no significant group × genotype interaction. In this study, there was no significant difference in perceived stress, anxiety, and depression between the IBS patients and controls. Our previous report [52] and the results of others [53] showed that these factors are higher in IBS patients than in controls. By contrast, IBS patients were found to be neither depressive nor anxious in another study from our laboratory [54]. Therefore, IBS patients in this study were psychologically close to normal, but were physically stressed. This notion is supported by the fact that statistical analysis of stress, anxiety, and depression including CRHR2 genotypes with group (IBS patients vs. controls) showed significant differences. The relatively complex association between genotypes and IBS suggests a modifying role for CRHR2 in the pathophysiology of stress.
In seven SNP which we selected, six SNPs are in the intronic region. Only rs2240403 is in the exon region, but it is the synonymous SNP which does not cause amino acid substitution. In this study, there were significant differences in the genotype distribution of both groups in rs472299 and rs377950. In addition, there were significant interaction in association between genotype and group (IBS patients vs. controls) in psychological condition in rs2190242 and rs2284220. These SNPs are in intronic region, and the domain is spliced during transcription to mRNA. Therefore those SNPs are less likely to have an influence on the receptor expression directly. However, the SNPs influencing modulation of the psychological condition that is in vicinity of these SNP in the linkage disequilibrium in IBS may exist.
We found a haplotype of four SNPs (rs4722999, rs3779250, rs2240403, and rs2267710) and two SNPs (rs2284217 and rs2284220) in strong LD (D 0 > 0.90). In this study, SNPs and haplotypes of the CRHR2 gene were significantly different between IBS patients and controls. Sato  [56]. Therefore, haplotypes of both CRHR1 and CRHR2 may be related to the features of IBS. We found that the psychological state varied from CRHR2 genotypes and haplotypes in IBS patients and controls. Especially, we discovered that the group with one of four haplotypes (T-T-T-T, C-T-T-T, C-C-T-T, and T-C-T-T) in CRHR2 showed a significantly lower score in perceived stress, anxiety, and depression in comparison with the group that did not have one of these haplotypes. In these four haplotypes, the common part is the T-T haplotype that consists of the T allele of rs2240403 and T allele of rs2267710. From this finding, it is suggested that having both these T alleles acts protectively against perceived stress and anxiety. To the best of our knowledge, this analysis of these four haplotypes of the CRHR2 gene has not been performed before [35,36]. This is a novel finding and further studies investigating the association between the T-T haplotype and other stress-related disorders are warranted.
This study has several limitations. First, the sample size was relatively small. Particularly, there were small numbers of subjects with the minor allele of rs2240403 and the data had a wide standard error. However, our study has the strength of a higher precision of genome analyses than a genome-wide association study. Moreover, recently published genomic studies [57,58] contained smaller number of participants than our study. Replication with a larger sample is necessary. Second, all subjects in this study were Japanese. Villafuerte et al. reported that the CRHR2 gene was unlikely to be involved in the genetic liability underlying HPA axis dysfunction and mood disorders [59]. Therefore, there may be ethnic differences in the function of CRHR2. Third, the molecular biologic differences in the CRHR2 gene polymorphisms examined in this study are not known. It is necessary to investigate any changes of function in vivo and behavior associated with the different CRHR2 gene polymorphisms in the future. Despite some limitations, the present study is the first to clarify the possible importance of CRHR2 in the pathophysiology of IBS. In conclusion, our findings support the hypothesis that genetic polymorphisms and haplotypes of CRHR2 are associated with IBS. Further studies of IBS and the CRH system are warranted.

Acknowledgments
We thank Dr. Masaaki Kato, Naoko Shimakura, and Risa Ando, Department of Neurology, Tohoku University Graduate School of Medicine; members of the Department of Behavioral Medicine, Tohoku University Graduate School of Medicine, for their help with this study.