TPH gene polymorphisms are associated with disease perception and quality of life in women with irritable bowel syndrome.

The total sample included 199 women with IBS. Demographic characteristics are shown in Table 1 . Only unrelated Caucasian women were included in the analysis. Participants in this study were relatively well educated, with 66% reporting a bachelor’s degree or higher. None of the TPH gene polymorphisms were statistically significantly associated with current psychological distress as measured by GSI scores in women with IBS. Similarly no associations were found with the individual subscales of the BSI. Likewise, we found no association between TPH gene polymorphisms and lifetime history of mental health disorders measured by CIDI in the IBS group. These results are shown in Tables 2 and 3 . In contrast, SNP1 and SNP3 in the TPH1 gene were associated with IBS-related negative cognitions ( Table 4 ). These associations did not change substantially when controlling for GSI and mean score of GI symptoms (abdominal pain, diarrhea, constipation, bloating, and intestinal gas). The SNP5 of TPH2 gene did not show any association with IBS-related cognitions. TPH1, but not TPH2, gene polymorphisms were associated with QOL scores, in particular the mental health and energy subscales ( Table 5 ). These associations were strengthened if GSI and mean GI symptom score were controlled for in the analysis. Since use of antidepressant or antianxiety medications could affect reports of psychological symptoms, analyses were repeated while excluding all subjects taking such medications. The results did not change substantially from those reported here, with one exception: In the subset of patient not taking antidepressant or antianxiety medications, there was a significant (p=0.018) association between SNP4 on the THP2 gene and GSI, with GSI being higher in GG than in GT subjects.

In this exploratory study, we examined possible associations between TPH gene polymorphisms and psychosocial factors in women with IBS. We found significant associations of TPH1 , but not TPH2 polymorphisms, with IBS-related cognitions and IBS-specific QOL. Interestingly, the associations between TPH1 SNPs and IBS-QOL were strengthened once GSI and the severity of GI symptoms were controlled for, while those between TPH1 SNPs and IBS-related cognitions remained with modest changes. This might indicate that the link between TPH1 polymorphisms and cognitions or illness impact on QOL was not mediated by current psychological or GI distress. CS-FBD is a cognitive scale which was developed to evaluate the belief systems of patients as they relate to their functional bowel disorders ( Toner et al., 1998 ). IBS-QOL was designed for use in patients with IBS to evaluate their life quality associated with the symptoms of IBS ( Hahn et al., 1997 ). Dysfunctional cognition or negative cognitions have been considered important factors impacting symptom severity and emotional consequences of living with IBS. In this context, the biopsychosocial model of IBS provides a theoretical framework for explaining how cognition might affect IBS symptoms ( Drossman et al., 2006 ; Levy et al., 2006 ). Within this model, genetic predispositions influence physiological dysfunction (e.g., disturbance in motility and/or visceral sensitivity) and the way in which the individual reacts cognitively to recurrent GI symptoms (e.g. catastrophizing). It is well known that 5-HT modulates developmental states ( Nakamura & Hasegawa, 2007 ). Since TPH is the rate-limiting enzyme in the 5-HT synthesis, variations in TPH genes may lead to dysfunction of the serotonin system. A recent study demonstrated that SNP rs453771 of the TPH1 gene is positively associated with cerebrospinal fluid (CSF) concentrations of the major serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in healthy European Americans ( Andreou et al., 2010 ). The levels of 5-HIAA were higher in those with the minor allele (G/G and A/G) as compared to those with the A/A genotype. However, another study has failed to find this association in US patients with major depression ( Galfalvy et al., 2009 ). TPH2 gene polymorphisms have also been found to be associated with 5-HIAA concentrations in the CSF ( Zhou et al., 2005 ). In addition, acute tryptophan depletion on brain-gut responses and emotional arousal give support to the idea that serotonin availability plays an important role in IBS-related cognitions ( Kilkens, Honig, van Nieuwenhoven, Riedel, & Brummer, 2004 ; Labus et al., 2011 ). Although TPH2 is expressed in the brain and has been shown to be linked to a number of disorders associated with cognition, our results found no association between negative cognitions about IBS and the TPH2 gene SNP. It may be that cognitive measures related to depression, cognitive control, and hyperactivity are influenced to a greater degree than the negative appraisal of IBS measured in this sample. However, our data do provide evidence for the association of TPH1 gene polymorphisms with negative cognition about IBS symptoms, especially as they relate to perception of pain and anxiety over bowel movements. We also saw that TPH gene polymorphisms influence IBS-related QOL. For SNP1, the allele (A) we found to be associated with better cognition and higher QOL has previously been associated with lower serotonin levels ( Andreou et al., 2010 ). This is in keeping with prior findings that inhibition of TPH, which would also cause lower serotonin levels, leads to symptom improvement in patients with IBS ( Camilleri, 2011 ). Several studies have shown associations between TPH1 SNPs and personality traits or psychiatric disorders. The SNP1 of TPH1 gene has been linked to suicidal behavior in patients with mood disorders ( Abbar et al., 2001 ; Bellivier, Chaste, & Malafosse, 2004 ), anger-aggression-related traits ( Rujescu et al., 2002 ) and schizophrenia ( Watanabe, Nunokawa, Kaneko, & Someya, 2007 ). SNP4 has been shown to moderate the influence of social support on depressive symptoms ( Jokela, Raikkonen, Lehtimaki, Rontu, & Keltikangas-Jarvinen, 2007 ) and has been associated with bipolar disorder and alcohol dependence (Chen et al.). TPH2 gene polymorphisms have been investigated for associations with various cognitive or/and emotional and behavioral traits ( Waider, Araragi, Gutknecht, & Lesch, 2011 ). The minor (T) allele carriers of TPH2 gene SNP5 were reported to have a greater functional MRI (fMRI) response of the amygdala, a structure critically involved in the modulation of emotional behaviors, to emotional stimuli( Brown et al., 2005 ; Canli, Congdon, Gutknecht, Constable, & Lesch, 2005 ). SNP5 has been also associated with affective disorders ( Harvey et al., 2004 ) and the modulation of negative emotionality such as neuroticism and harm avoidance ( Gutknecht et al., 2007 ; Reuter, Kuepper, & Hennig, 2007 ). However, we saw no associations between SNP5, psychological distress, lifetime prevalence of psychiatric disorders, IBS-related cognitions, or quality of life. One reason for our negative finding might be the combination of our small sample size and the relatively low minor allele frequency for this SNP (18%) which might have made subtle effects difficult to detect. Another possible explanation is that different cognitive mechanisms with different etiologies come into play in IBS as opposed to primarily psychiatric problems. Also, only one SNP was tested for THP2. Even though SNP5 has been tested in its association with many psychiatric and behavioral studies and showed high LD with other functional TPH2 SNPs which are associated with CSF5-HIAA levels, it does not cover all of the haploblocks of TPH2 gene. Therefore further studies are suggested to test associations between other TPH2 SNPs and psychosocial factors in IBS. Our study has several limitations. Due to our small sample size our report of genetic associations must be interpreted with caution until confirmed in a larger sample. Because of the exploratory nature of our study, results were presented without correction for multiple testing. Because of our small sample size we focused on the main effects of genetic polymorphisms on psychosocial factors in IBS without taking possible gene-environment interaction into account. Nonetheless, studies on depression give evidence that often genetic vulnerabilities only reveal themselves in the context of social stressors ( Caspi et al., 2003 ; Grabe et al., 2005 ). This is particularly relevant in IBS, since a history of psychological trauma and higher levels of daily stress have been repeatedly shown to be positively associated with GI symptoms ( Jarrett et al., 1998 ). Therefore, further studies are needed to examine possible interactions between stress, life events, and TPH gene polymorphisms on psychological distress and IBS symptoms. In conclusion, in this exploratory hypothesis-generating study we report a possible association of variants in the TPH1 gene with negative cognitions and reduced QOL in women with IBS. These associations were independent of experienced levels of GI distress, thus indicating a possible mechanism whereby genetic factors might influence the onset and course of IBS by limiting patients’ ability to cope with their symptoms. This study reinforces the concept of IBS as a biopsychosocial illness and indicates the necessity of individualized approaches in providing nursing care such as cognitive behavioral treatment tailored to the patients’ appraisal of their illness.

DNA samples and survey data were assembled from three studies of IBS carried out in Washington State ( Jarrett et al., 2009 ; Jarrett et al., 2007 ; Motzer, Jarrett, Heitkemper, & Tsuji, 2002 ). All participants had a prior diagnosis of IBS made by a health care provider and currently met Rome III criteria ( Drossman et al., 2006 ). Recruitment was conducted through community advertisement. Subjects were excluded if they 1) had a history of co-existing GI pathology (e.g., inflammatory bowel disease) or surgery (e.g., bowel resection), renal or reproductive pathology (e.g., endometriosis), severe fibromyalgia, infectious disease (e.g., hepatitis B or C), or severe cardiovascular disease; or 2) were currently taking the following medications more than 3 days a week: antibiotics, anticholinergics, cholestyramine, narcotics, colchicines, docusate, an enema preparation, iron supplements, or laxatives. The protocol for this study was approved by the University of Washington’s institutional review board and all the patients gave written informed consent. The HapMap project ( http://www.hapmap.org ) results showed the minor allele frequencies of all the selected SNPs are different across ethnicity and our data also showed similar patterns with the HapMap results (data not shown). The only ethnic group with a large enough sample size for a reasonable analysis was Caucasians, thus in this report analysis is restricted to only those women who identified themselves as Caucasian. This resulted in an analysis set of 199 women with IBS, of which 20% (n = 41) met criteria for constipation-predominant, 44% (n = 88) met criteria for diarrhea-predominant and 26% (n = 52) met criteria for mixed IBS. Genomic DNA was extracted from buffy coat preparations ( Miller, Dykes, & Polesky, 1988 ) using Puregene DNA Purification kits (Gentra Systems Inc., Minneapolis, MN). Genotyping was done using Applied Biosystems (ABI, Foster City, CA) TaqMan custom genotyping assays and an ABI 7300 Real-time PCR System as shown in our previous report ( Jun et al., 2011 ). The minor allele frequencies in our sample for SNP1 to SNP5 were 0.39, 0.44, 0.21, 0.42, and 0.18, respectively. The BSI (Brief Symptom Inventory) has 53 items that ask how much a certain problem has bothered the subject over the last 7 days ( Derogatis, 1993 ). Each item of the BSI is rated on a 5-point scale that ranged from 0 ( not at all ) to 4 ( extremely ) and profiled into 9 subscales: somatization, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism, and a global index (Global Severity Index, GSI) as a mean score of all items. The internal consistency in this sample, as measured by Cronbach’s alpha, was α =0.94 for the Global Severity Index, α =0.79 for Anxiety, and α =0.79 for Depression. History of mental disorders was assessed using the World Health Organization Composite International Diagnostic Interview (CIDI). This instrument is a fully structured diagnostic interview administered via a computer to derive diagnoses of DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4 th edition) and ICD-10 (International Classification of Disease, 10 th revision) mental disorders. The CIDI mood-disorders module (depression as single and recurrent episode, dysthymia and mania), anxiety-disorders module (panic disorder with our without agoraphobia, agoraphobia, generalized anxiety disorder, obsessive-compulsive disorder, phobias including social phobia, and post-traumatic stress disorder) and suicidal ideation module (recurrent thoughts of death, suicidal ideation and suicide attempt) were used in this study ( Andrews & Peters, 1998 ). The Cognitive Scale for Functional Bowel Disorders (CS-FBD) contains items related to cognitions about bowel function and personal characteristics relevant to IBS ( Toner et al., 1998 ). Each item is rated on a 7-point scale, ranging from 1 ( strongly disagree ) over 4 ( neither agree/disagree ) to 7 ( strongly agree ). Typical items are “I often worry there might not be a toilet available when I need it” and “I often feel this abdominal pain will never go away”. We included 6 items in addition to the original 25 in order to calculate 2 subscales: pain and bowel performance anxiety. The 6 additional items had been part of the original version and the correlation between the extended version and the standard version was 0.99 in our study. A total cognitive score was the mean of all 31 items with higher scores indicating more negative cognition regarding IBS symptoms and consequences. For this study, the internal consistency was α = 0.942 for the total score, α = 0.685 for pain, α = 0.941 for bowel performance anxiety. The IBS specific quality of life (IBS-QOL) is a 31- item questionnaire with 9 subscales: emotional, mental health, sleep, energy, physical functioning, diet, social role, physical role, and sexual relations ( Hahn, Kirchdoerfer, Fullerton, & Mayer, 1997 ). Example items are “How often during the past 4 weeks did your IBS make you feel worried?” scored from 0 “None of the time” to 6 “all of the time”; or “During the past 4 weeks, how much of the time did you feel emotionally worn out and tired because of IBS?” scored from 1 “Every day” to 5 “None”. The scales were transformed to standard scales of 0 ( poor quality of life ) to 100 ( highest quality of life ). A total score was computed by averaging all except sexual relations because this was missing for a large portion of the sample. Internal consistency for subscales ranged from α = 0.745 to α = 0.916. The mean score of GI symptoms was measured by a daily dairy which subjects filled in for 28 days ( Jun et al., 2011 ). Symptoms include abdominal pain, diarrhea, constipation, bloating, and intestinal gas. They were rated from 0 (not present), 1 (mild), 2 (moderate), 3 (severe) and 4 (very severe). Since some participants did not complete all of the questionnaires, there are differences in the number of subjects reported for each questionnaire. Hardy-Weinberg equilibrium was tested using Pearson’s Chi-square tests. Analysis of covariance (ANCOVA) was used to assess the association of BSI scores and CS-FBD scores with individual SNPs, adjusting for age as a covariate because scores were correlated with age. Associations with IBS-QOL scores of individual SNPs were tested by analysis of variance (ANOVA). We tested 30 hypotheses and 5 SNPs; to adjust for multiple comparisons, a p-value of less than 0.05/150 = 0.0003 would be considered to be significant. Because of the exploratory nature of our study, however, the results were presented without correction for multiple testing and should be interpreted with this in mind. All p-values were two-sided and p < 0.05 was considered statistically significant.