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Open Access

Peer-reviewed

Research Article

Identification of putative transcriptomic biomarkers in irritable bowel syndrome (IBS): Differential gene expression and regulation of TPH1 and SERT by vitamin D

  • Aleksandra Grozić,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America

    ⨯
  • Keaton Coker,

    Roles Formal analysis, Visualization, Writing – review & editing

    Affiliation School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America

    ⨯
  • Christopher M. Dussik ,

    Contributed equally to this work with: Christopher M. Dussik, Marya S. Sabir

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – review & editing

    Affiliation School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America

    ⨯
  • Marya S. Sabir ,

    Contributed equally to this work with: Christopher M. Dussik, Marya S. Sabir

    Roles Investigation, Writing – review & editing

    Affiliation School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America

    ⨯
  • Zhela Sabir,

    Roles Conceptualization

    Affiliation School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America

    ⨯
  • Arianna Bradley,

    Roles Investigation

    Affiliation School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America

    ⨯
  • Lin Zhang,

    Roles Investigation, Writing – review & editing

    Affiliation School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America

    ⨯
  • Jin Park,

    Roles Formal analysis, Investigation, Methodology, Supervision, Visualization, Writing – review & editing

    Affiliation Biodesign Institute, Arizona State University, Tempe, AZ, United States of America

    ⨯
  • Steven Yale,

    Roles Conceptualization, Funding acquisition, Project administration, Writing – review & editing

    Affiliation Department of Medicine, University of Central Florida College of Medicine, Orlando, FL, United States of America

    ⨯
  • Ichiro Kaneko,

    Roles Formal analysis

    Affiliations School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America, Department of Basic Medical Sciences, University of Arizona College of Medicine, Phoenix, AZ, United States of America

    ⨯
  • Maryam Hockley,

    Roles Data curation, Investigation

    Affiliation School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America

    ⨯
  • Lucinda A. Harris,

    Roles Project administration, Writing – review & editing

    Affiliation Mayo Clinic Division of Gastroenterology & Hepatology, Alix School of Medicine, Mayo Clinic, Scottsdale, AZ, United States of America

    ⨯
  • Tisha N. Lunsford,

    Roles Writing – review & editing

    Affiliation Mayo Clinic Division of Gastroenterology & Hepatology, Alix School of Medicine, Mayo Clinic, Scottsdale, AZ, United States of America

    ⨯
  • Todd R. Sandrin,

    Roles Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing

    Affiliations School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America, Julie Ann Wrigley Global Futures Laboratory, Arizona State University, Tempe, AZ, United States of America

    ⨯
  • Peter W. Jurutka

    Roles Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing

    Peter.Jurutka@asu.edu

    Affiliations School of Mathematical and Natural Sciences, Arizona State University, Phoenix, AZ, United States of America, Department of Basic Medical Sciences, University of Arizona College of Medicine, Phoenix, AZ, United States of America

    ⨯

Abstract

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders and affects approximately 4% of the global population. The diagnosis of IBS can be made based on symptoms using the validated Rome criteria and ruling out commonly occurring organic diseases. Although biomarkers exist for “IBS mimickers” such as celiac disease and inflammatory bowel disease (IBD), no such test exists for IBS. DNA microarrays of colonic tissue have been used to identify disease-associated variants in other gastrointestinal (GI) disorders. In this study, our objective was to identify biomarkers and unique gene expression patterns that may define the pathological state of IBS. Mucosal tissue samples were collected from the sigmoid colon of 29 participants (11 IBS and 18 healthy controls). DNA microarray analysis was used to assess gene expression profiling. Extraction and purification of RNA were then performed and used to synthesize cDNA. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was employed to identify differentially expressed genes in patients diagnosed with IBS compared to healthy, non-IBS patient-derived cDNA. Additional testing probed vitamin D-mediated regulation of select genes associated with serotonergic metabolism. DNA microarray analyses led to the identification of 858 differentially expressed genes that may characterize the IBS pathological state. After screening a series of genes using a combination of gene ontological analysis and RT-qPCR, this spectrum of potential IBS biomarkers was narrowed to 23 genes, some of which are regulated by vitamin D. Seven putative IBS biomarkers, including genes involved in serotonin metabolism, were identified. This work further supports the hypothesis that IBS pathophysiology is evident within the human transcriptome and that vitamin D modulates differential expression of genes in IBS patients. This suggests that IBS pathophysiology may also involve vitamin D deficiency and/or an irregularity in serotonin metabolism.

Citation: Grozić A, Coker K, Dussik CM, Sabir MS, Sabir Z, Bradley A, et al. (2022) Identification of putative transcriptomic biomarkers in irritable bowel syndrome (IBS): Differential gene expression and regulation of TPH1 and SERT by vitamin D. PLoS ONE 17(10): e0275683. https://doi.org/10.1371/journal.pone.0275683

Editor: Michal Zmijewski, Medical University of Gdańsk, POLAND

Received: May 23, 2022; Accepted: September 21, 2022; Published: October 20, 2022

Copyright: © 2022 Grozić et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: Todd Sandrin (T.S.), Peter Jurutka (P.J.), and Steven Yale (S.Y.) received funding from the Wisconsin Network for Health Research (WiNHR) via the Marshfield Clinic Research Foundation in grant award number AWD00025284 AGR 11/09/11. The URL for WiNHR is https://ictr.wisc.edu/groups/wisconsin-network-for-health-research-winhr/ and the Marshfield Clinic Foundation URL is https://www.marshfieldclinic.org/health-system/research The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Irritable bowel syndrome (IBS) is a disorder of gut-brain interaction (DGBI) that affects approximately 4% of the global population but estimates vary based on nonuniform study methodology [1]. Patients with IBS often display symptoms such as altered stool forms, constipation, bloating, and abdominal pain with overlapping co-morbidities including depression, anxiety, chronic fatigue, insomnia, sexual dysfunction, and fibromyalgia, collectively impairing quality of life [2, 3]. A 2013 systematic review estimated the direct and indirect health care costs of IBS to be between $22–30 billion [4].

Symptom-based criteria along with the judicious use of testing have improved the diagnosis of IBS but the average patient has often seen several physicians and undergone extensive costly testing before clearly being told they had IBS. Additionally, the lack of reliable biomarkers to help make an accurate diagnosis has added to the challenge of arriving at a diagnosis in a timely fashion [5–7]. The diagnostic tool, Rome III criteria, was recently revised into the Rome IV criteria. The Rome IV criteria define IBS as abdominal pain with altered bowel habits as the predominant symptom at least once a week in the last 3 months [8]. The term abdominal pain replaces the prior term of “discomfort”, which was deemed to be nonspecific and lacking in cultural sensitivity as it has variable meanings in different languages. Additionally, the Bristol Stool Scale is used for subtyping IBS according to stool form and frequency into its predominant bowel habit [8]. Despite extensive research, the pathophysiology of IBS remains elusive. Various mechanisms have been proposed including increased visceral sensitivity, alterations in intestinal motility and permeability, immune dysfunction, autonomic nervous system dysregulation, alterations in the gut microbiome, brain-gut axis alterations, and genetic and psychosocial factors [2].

The role of genetics in IBS has been investigated in pedigree, genome-wide association, and international twin studies [9–19]. In addition to identifying genetic risk loci for IBS, genetic polymorphisms have been associated with specific phenotypic characteristics of the syndrome [9, 10, 20–22]. Studies suggest that environmental triggers, including bacterial infection, air and water pollution, and radiation contribute to the development of IBS presumably in a susceptible person [23, 24]. Research suggests that environmental pollutants alter microbiome composition, an increasingly recognized plausible factor in IBS etiology [23, 25, 26]. Because the GI tract is sensitive to particulate matter (PM) and smoking, exposure to air pollution may lead to oxidative damage of colonic mucosa or exacerbate inflammation, presenting a risk factor for IBS [23, 24, 27, 28].

Beyond genetic and environmental factors, patients with more severe forms of IBS report higher psychosocial problems [13, 29–34]. Anxiety and depression occur in a significantly greater number of patients with IBS compared to patients without IBS [35, 36]. A positive correlation has been shown between anxiety, depression, and the severity of IBS symptoms [13, 37]. Recent work has shown that the application of cognitive behavioral therapy (CBT) can significantly reduce reported symptom severity, reduce pain severity, and improve the quality of life in patients with IBS [38].

Interestingly, the association between IBS and psychosocial problems may be accounted for due to alteration in the intestinal microbiota in IBS patients. Certain taxa of microbiota have been associated with specific IBS symptoms, while IBS symptom severity correlates with gut microbiota instability [13, 39–42]. For example, the bacterial family Actinomycetaceae was less prevalent in the GI tracts of IBS patients that are depressed compared to IBS patients who do not have depression [13]. Another example is how the distribution of gut bacterial phyla Clostridiales has been observed to distinguish IBS subtypes [43].

Moreover, evidence suggests impaired serotonergic signaling in IBS patients [44]. Serotonin (5-hydroxytryptamine, 5-HT) is one of the most-studied neurotransmitters in IBS due to its abundance within cells throughout the GI tract (~95%) and its role in the brain-gut axis [44–46]. Enterochromaffin cells (EC cells) synthesize approximately 90% of serotonin, with the remainder produced within the myenteric plexus [47]. Patients with IBS have more EC cells than controls [44, 48, 49]. Serotonin is secreted by EC cells via a variety of stimuli and plays a pivotal role in modulating the immune system and pathophysiological responses such as intestinal muscle contractility and inflammatory responses [13, 50]. Serotonin transporter (SERT) and tryptophan hydroxylase-1 (TPH1), the rate-limiting enzyme in serotonin production, is found in low levels in patients with IBS [51]. Other studies have shown disrupted enteric serotonergic signaling in IBS [52].

A specific connection between serotonergic signaling and IBS is not clear. However, the use of selective serotonin reuptake inhibitors (SSRIs) as a SERT antagonist employed in the treatment of depression, has also been shown to alleviate IBS symptoms [53]. The biosynthetic pathway of serotonin is a multistep process with tryptophan hydroxylase (TPH) acting as the rate-limiting enzyme. Evidence suggests that the expression of TPH1, the main isozyme of tryptophan hydroxylase, might be modulated by dihydroxycholecalciferol (vitamin D) [54]. In another study, tryptophan hydroxylase was stimulated by calcitriol (1,25D), the active hormonal vitamin D metabolite, in serotonergic neurons [55]. Vitamin D plays a crucial role in inflammatory processes and immunomodulatory activity [56]. Moreover, vitamin D deficiency has been associated with depression, a significantly reported co-morbidity among IBS patients, particularly those with IBS-D [13, 57, 58]. The role of vitamin D in the pathogenesis of IBS is emerging, and vitamin D supplementation has been proposed as a potential treatment for IBS [13, 59–62].

Several studies have attempted to identify specific candidate genes involved in the pathogenesis of IBS [44]. Some studies have shown dysregulated serotonergic signaling and blood serotonin levels [44]. Patients with the IBS-D subtype have higher blood serotonin levels [44, 63], while lower blood serotonin levels occur in the IBS-C subtypes [44, 52, 64]. More specifically, an analysis in blood and mucosal biopsies of 5-HT/5 HIAA ratios suggested defects in serotonin release in IBS-C subtypes and irregularities in serotonin uptake in IBS-D [44, 52, 64]. The SERT gene, also known as SLC6A4, has been studied extensively in relation to IBS; however, there is a lack of consensus regarding the association between IBS and SERT [44, 51, 65, 66].

In related GI disorders such as IBD, DNA microarrays of colonic tissue have successfully identified variants associated with the disease states [67]. Similarly, DNA microarrays have been used with IBS samples to identify distinguishing genetic characteristics between IBS and non-IBS individuals, but with less successful results. In this present study, several genes from our previous work using DNA microarrays and gene ontology (GO) analysis were screened using RT-qPCR to identify distinguishing biomarkers and a unique gene expression profile that may elucidate the pathological state of IBS [68]. Such a gene expression profile might serve as a foundation for the development of a diagnostic panel for IBS. Additionally, we sought to ascertain the effect of active vitamin D (1,25D) in HCT-116 human colorectal cells in regulating key genes associated with IBS. We propose that the current study may help improve the efficiency of IBS diagnosis, as well as identify novel genes involved in IBS pathogenesis, thus leading to a more timely and efficient method of disease management along with more effective targeted treatments.

Methods

Patient cohort characteristics

Patients were recruited for participation from a rural Midwestern multispecialty group practice. Mucosal tissue samples were collected from the sigmoid colon of 29 participants in the manner described by Costello et al. [67]. The experimental group consisted of 11 patients (eight females and three males) ranging from 41 to 58-years-old diagnosed with IBS-C, IBS-D, or IBS-M using the Rome III criteria. The control group included 18 patients (12 females and six males) ranging from 47 to 60-years-old, who did not meet diagnostic Rome III criteria for IBS. Patients enrolled in this study provided written informed consent. All experimental procedures were approved by the Marshfield Clinic Research Foundation and Arizona State University Institutional Research Board (#YAL10308 and #1106006538 respectively) in accordance with institutional and national research committee ethical standards.

Gene expression profiling and ontology enrichment analysis

Mucosal tissue samples from the sigmoid colon of IBS and control patients were processed for DNA microarray analysis and GO pathway enrichment as previously described [68]. Differential expression of 858 genetic features between the two experimental groups and GO terms with a p-value <0.05 were identified and scrutinized for further investigation in this study.

Tissue: RNA extraction, purification, and quality control

Colonic tissue samples were weighed and then suspended in 700 μL of lysis buffer. An ultrasonic converter was placed on ice for three minutes, rinsed with DI water, and dried with a Kimwipe followed by 5 x 10 seconds of sonication on ice (setting 3 on Fisher Scientific Sonic Dismembrator, Model 100). The ultrasonic converter was placed on ice for 30 seconds between sonication rounds to avoid specimen degradation. RNA was extracted and purified under RNase-free conditions in accordance with the manufacturer’s protocol for the Aurum Total RNA Mini kit (Bio-Rad, Hercules, CA). A spectrophotometer (Eppendorf BioPhotometer) was employed to obtain A260/280, A260/230 ratios and concentrations for analysis of RNA quality and yield.

Cell culture: RNA extraction, purification, and quality analysis

Human colorectal cancer cells (HCT-116) were obtained from the American Type Culture Collection in Manassas, VA, and used to investigate fold-induction by 1,25-dihydroxyvitamin D3 (1,25D) on IBS biomarkers of interest in colonic epithelial cells. The complete growth medium used contained DMEM/high glucose (including 4.00 mM L-glutamine, 4500 mg/L glucose, and sodium pyruvate) supplemented with 10% fetal bovine serum and 1X penicillin/streptomycin. Cells were plated at 800,000 per well, induced with either 10nM (1,25D) or ethanol (vehicle control), and incubated at 37°C with 5% CO2 air atmosphere for 24 hours. Extraction and purification of RNA were conducted under RNase-free conditions and in accordance with the manufacturer’s protocol of the Aurum Total RNA Mini kit (Bio-Rad, Hercules, CA). A spectrophotometer (Eppendorf BioPhotometer) was employed to assess RNA quality and yield.

RT-qPCR of sample tissue and HCT-116 cells

RT-qPCR was employed to examine the gene expression of potential IBS biomarkers. The results were compared to the control cohort to test the external validity of the DNA microarray data. RNA was extracted and purified from patient mucosal tissue samples and HCT-116 cells. Spectroscopy was employed to obtain A260 and A280 values to assess RNA yield and quality. Next, 1 μg RNA was reverse transcribed to produce 20 μL of first-strand complementary DNA (cDNA) using the iScript cDNA Synthesis Kit (Bio-Rad). Reaction mixtures for RT-qPCR comprised 1.5 μL of cDNA, 3.5 μL of the appropriate primer, and 5 μL of SYBR Green Master Mix (Roche Applied Science, Indianapolis, IN, USA). Various aggregate mixtures of pooled control samples were tested for experimental optimization and reproducibility. The control RNA used in this study was an RNA aggregate of eight patients who did not meet the diagnostic criteria for IBS. The RT-qPCR data for each gene of interest is reported as a global standard error mean (SEM) relative to pooled non-IBS patients. Gene percent accuracy is reported for suggested IBS biomarkers for a diagnostic assay using Eq 1. For HCT-116 cells, the RT-qPCR data are reported as a fold change of gene expression in the presence of the active form of vitamin D (1,25D) compared to the expression in cells dosed with an ethanol vehicle control.

(1)

Statistical analyses

All genes reported in Table 2 had a minimum of two independent RT-qPCR experiments (and usually 3 or more) performed with a minimum of three to six replicate PCR wells per patient and gene when analyzing IBS gene expression, with "fold change" computed as experimental (IBS) group relative to control (non-IBS) patients. All analyses were performed in a blinded fashion. Agilent’s Feature Extraction software (v. 11.0.1.1) was utilized to process DNA microarray data and normalized using limma (Ritchie et al. 2015) [69]. The data were used to identify differentially expressed genes through a combination of limma linear model t-test, Mann-Whitney U-test, and two-tailed student’s t-test with a p-value ≤ 0.05 considered statistically significant.

Results

Gene expression profiling and serotonergic pathways in IBS patients

Mucosal tissue biopsy samples in the sigmoid colon of patients diagnosed with IBS and non-IBS patients (control) were profiled using DNA microarray analysis. Patient demographics are outlined in Table 1. As previously shown, 858 genetic features were identified with differential expression levels between IBS patients and patients not diagnosed with IBS [68]. Statistical analysis further narrowed the results of the DNA microarray results to 200 differentially expressed genes between the groups to be externally validated by RT-qPCR (Fig 1). GO analysis of these identified genetic features revealed that among the top 25 (ranked by increasing p-value) GO terms, seven were involved with serotonin metabolism [68].

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Fig 1. Workflow and DNA microarray analysis.

Workflow in the acquisition of an IBS genetic “fingerprint” from patient sample to transcriptomic analysis. Differentially expressed genes from mucosal tissue biopsy samples in the sigmoid colon of patients diagnosed with irritable bowel syndrome (IBS) and non-IBS patients (control). The heat map (adapted from Dussik et al.) displayed differential expression of 858 genetic features between the two experimental groups (p-value < 0.05) [68]. Pronounced clusters of similar color and intensity are also visible with blue indicative of individual biomarkers with lower expression levels and red illustrating higher levels of expression.

https://doi.org/10.1371/journal.pone.0275683.g001

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Table 1. Demographical and IBS subtype designations of study population.

https://doi.org/10.1371/journal.pone.0275683.t001

Potential IBS biomarkers

The genes selected for further analysis by RT-qPCR were identified based on the following criteria: significance in the GO analysis, microarray statistical significance, function in relation to IBS pathophysiology, and high differential expression between the two groups in gene expression profiling. As a result of these criteria, 29 genes were selected and tested using RT-qPCR in 11 IBS-derived RNA, as well as control RNA from an aggregate of eight asymptomatic patients (S1 Table). From the initial 29 selected genes, the 23 reported in Fig 2 have a minimum of two independent RT-qPCR plate replicates. Next, RT-qPCR-measured fold change of the filtered 23 genes was compared to DNA microarray fold change followed by calculation of percent accuracy for each replicate per gene calculated using Eq 1 (Table 2). Of the 23 filtered genes tested using RT-qPCR, only genes with an overall minimum 70 percent accuracy and function related to IBS pathophysiology were considered for a proposed IBS biomarker assay. As a result of these criteria, seven genes of interest were identified as potential IBS biomarkers with an average accuracy of 84.2% (Table 2).

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Fig 2. Gene expression fold change in IBS patients relative to control patients using RT-qPCR testing.

In total, 29 genes of interest were evaluated with only 23 being reported here (only those genes with at least two independent RT-qPCR plate replicates are included). Red coloration is indicative of lower levels of expression while green represents higher expression levels with SEM being reported.

https://doi.org/10.1371/journal.pone.0275683.g002

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Table 2. Putative IBS biomarker genes with calculated accuracy using DNA microarray and RT-qPCR as a validation tool.

https://doi.org/10.1371/journal.pone.0275683.t002

Vitamin D-meditated regulation of IBS biomarkers of interest in human colorectal cells

Given the prevalence of significantly altered serotonergic-related pathway expression from GO analysis, serotonin’s close association with the GI tract, and the positive correlation between vitamin D and serotonin synthesis via TPH1 induction, a selection of differentially expressed genes in IBS patients (Fig 2) was assessed for regulation by vitamin D in human colonic cells [45, 70, 71]. HCT-116 colorectal cells were treated with 10−8 M 1,25D for 24 hours, and RT-qPCR was employed to analyze mRNA expression levels of four potential IBS genetic biomarkers (Fig 3). The four specific IBS-candidate genes were selected using microarray data based on significance (p < 0.05) and differential regulation in IBS patients relative to controls as indicated by the RT-qPCR data (Fig 2). TPH1 and SERT were selected based upon their association with serotonin (and thus vitamin D), while TDRD6 and FLT4 were selected to provide a complete spectrum of differential gene expression and to further validate and extend our previous data of these potential IBS biomarkers [68]. As shown, TPH1 and FLT4 represent more extreme repression and induction, respectively, while SERT and TDRD6 displayed more moderate levels of repression and induction relative to the controls (Fig 2). All IBS biomarkers tested for regulation by vitamin D appeared influenced by the presence of 10−8 M 1,25D. More specifically, gene regulation by vitamin D resulted in TDRD6 repression by 0.49-fold (p < 0.0005), FLT4 repression by 0.62-fold (p < 0.0005), SERT induction by 3.18 (p < 0.005), and TPH1 induction by 1.48-fold (p < 0.0005) (Fig 3). RT-qPCR patient data indicated an induced expression pattern in TDRD6 and FLT4, while TPH1 and SERT exhibited a repressed expression pattern in the IBS cohort (Fig 2).

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Fig 3. IBS-Associated gene regulation by 1,25D.

The mRNA levels of each gene were determined using quantitative real-time polymerase chain reaction (RT-qPCR) for a total of three to six replicates. Values are reported as fold change relative to pooled control. SEM reported on bar plots. Ranges of p-values are represented as: *P < 0.005; **P < 0.0005, t-Test: Two-Sample Assuming Equal Variance.

https://doi.org/10.1371/journal.pone.0275683.g003

Discussion

DNA microarray analysis revealed 858 genetic features with differential expression between IBS and control patient populations, providing evidence that IBS pathophysiology may be linked to and/or reflected in the transcriptome [72, 73]. Differential expression of genes in IBS patients better clarifies the IBS pathophysiological state. Therefore, GO interaction analysis was performed to assess patterns of differentially expressed genes in IBS on biochemical pathways to identify which pathways might be disrupted or modified in the IBS disease state. Differentially expressed genes between the IBS and the control cohort were grouped into pathways that consisted of five or more genes with similar functions and/or shared interactions. The pathways most influenced by IBS were serotonin uptake, positive regulation of glycogen (starch) synthase activity, negative regulation of serotonin secretion, SA node cell action potential, and Type II Na+/Pi co-transporters. The serotonergic pathway was most prevalent among differentially expressed genes, consistent with the role that serotonin metabolism may play a role in the pathogenesis of IBS [68]. This association between IBS and serotonin is supported as some serotonergic agents have been successfully implemented in evidenced-based therapeutic regimes in IBS [74, 75].

In total, 29 genes were identified for more extensive testing based upon the following criteria: differential expression between IBS patients and control in the DNA microarray, GO analysis, and primary gene function (with an emphasis on serotonergic signaling/metabolism). To validate data from DNA microarray, RT-qPCR was employed. Differentially expressed genes between IBS and control patients identified by DNA microarray analysis were compared to the RT-qPCR measured fold change in colonic tissue from 11 IBS patients compared to non-IBS patient controls to calculate a percent accuracy for each gene. RT-qPCR analyses revealed seven genes with expression patterns sufficiently consistent to indicate the viability of an IBS diagnostic tool. The seven potential genes, six repressed and one up-regulated (underlined), were SLC19A1, PPARD, TPH1, CD36, VIP, SERT, and VSIG2. Collectively, these putative genetic biomarkers could be used to more efficiently screen for IBS with an average accuracy of 84.2% in our blinded analysis (Table 2).

It is interesting to note that the IBS microarray data suggests repression of MUC20 and induction of FOLR2, MAOA, HTR4, FHL1, PSEN1, MMP26, and GC while further investigation with RT-qPCR contradicted the preliminary microarray data (S1 Table). The use of RT-qPCR as a validation tool for microarray analysis suggests that MUC20 is induced and FOLR2, MAOA, HTR4, FHL1, PSEN1, MMP26, and GC are in fact repressed in IBS patients. However, an important note is that the fold changes of MMP26 and GC as described by RT-qPCR were calculated from a single plate replicate (S1 Table) and warrant further study before a clear expression pattern in IBS patients is identified.

Associations between IBS subtypes and SERT polymorphisms have also been studied [44, 76, 77]. A meta-analysis that included 12 studies and 2,068 IBS patients did not find an association between SERT polymorphisms and IBS [78]. However, another meta-analysis of 25 studies and 3,443 IBS patients did find a positive association between IBS and SERT polymorphisms, but only among East Asian and not Caucasian populations [79]. TPH, the rate-limiting enzyme that catalyzes serotonin biosynthesis, has two isoforms: TPH1 and TPH2. There appears to be an association between TPH1 and IBS severity [44]. Additionally, colonic mucosa levels in IBS patients have been found to have significantly reduced TPH1 mRNA levels [44]. Given the apparent importance of TPH1 and SERT in relation to the IBS disease state, both genes were a primary focus for testing.

The expression level of TPH1, one of the main isoforms of tryptophan hydroxylase, was among the genes explored using RT-qPCR to determine whether IBS patients exhibited repressed GI serotonin production by indirect means. In the IBS cohort, 10 out of 11 patients showed decreased levels of TPH1 when compared to controls [68]. There is evidence to suggest vitamin D plays a role in controlling the rate of serotonin synthesis in vivo by modulating the expression of TPH1 [54]. Indeed our group found that approximately 82 percent of sampled patients with IBS exhibited serum 25-hydroxyvitamin D levels below the optimal clinical level [68]. Expanding upon this, we hypothesized that the active hormonal metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), may influence the expression of other putative IBS candidate genetic biomarkers, similar to the role of vitamin D in modulating TPH1 expression.

To test the effect of vitamin D, HCT-116 colorectal cells were treated with 10−8 M 1,25D for 24 hours, and RT-qPCR was employed to test the expression patterns of four candidate IBS genes (TDRD6, FLT4, SERT, and TPH1). As previously described, TPH1 and SERT were selected for their role in serotonergic metabolism, and TDRD6 and FLT4 were chosen to provide a spectrum of varying expression levels in IBS patients. Specifically, TPH1 and FLT4 represented greater differential expression levels in repression and induction, respectively in IBS patients relative to control. Meanwhile, SERT and TDRD6 represented more moderate differential expression in repression and induction respectively (Fig 2). Vitamin D significantly regulated the expression of all four genes in the opposite direction than what was observed in the IBS cohort. Significantly, the reversal of gene expression in human colonic cells by 1,25D relative to IBS patients in both the microarray and RT-qPCR data suggests that vitamin D may be effective at reversing biomarker gene expression in IBS patients and thus alleviate symptoms (Figs 2 and 3).

Serotonin secretion by enterochromaffin cells is correlated with GI motility via smooth muscle contractility [46, 80–82]. In the context of IBS, IBS-D patients would be associated with elevated serotonin production, IBS-C with depressed serotonin production, and IBS-M with alternating or intermediate serotonin production. The IBS cohort of this study consisted of one IBS-C patient, one unknown, four IBS-D patients, and the remaining IBS-M. Due to the cohort consisting of only one IBS-C patient, and four IBS-D patients, we expected to see intermediate to elevated serotonin production as an average. However, approximately 90 percent of the IBS patient tissue exhibited repressed TPH1 expression levels relative to controls.

The significance of this finding can be explained based on the analysis conducted by Sabir et al. They measured serotonin production in response to vitamin D in RN46A-B14, a serotonergic cell line [70]. The authors reported induction of TPH1 mRNA and the increased synthesis of serotonin by vitamin D, suggesting that 5-HT production occurs in response to vitamin D in the large intestine. Reduced levels of TPH1 in IBS-D patients result from negative feedback by enteric 5-HT signaling, while decreased TPH1 levels in other IBS subtypes may be due to vitamin D insufficiency. Further support is based on a finding in our study of increased levels of expression of SERT and TPH1 in HCT-116 colorectal cells when treated with 1,25D. We previously reported a trend of decreased serum vitamin D levels in IBS patients relative to the control cohort [68]. Therefore, the irregular serotonin production that generally describes the IBS pathological state may be a result of vitamin D deficiency.

Tazzyman et al. reported decreased serum 25(OH)D levels among IBS patients and noted a positive correlation between vitamin D supplementation and quality of life [83]. The authors found that IBS-C patients were the most responsive to the vitamin D supplementation resulting in improvement across nearly all IBS symptoms. Since IBS-C patients are thought to have diminished serotonin production, the responsiveness of IBS-C patients to vitamin D is consistent with vitamin D increasing TPH1 serotonin levels in the gut and our hypothesis that induction of the TPH1 gene in the gut by vitamin D may play a role in IBS pathophysiology [55, 84].

Though Tazzyman et al. reported a positive correlation between vitamin D supplementation and quality of life, the contradictory findings reported by Williams et al. found no improvement in IBS symptoms with vitamin D supplementation [83, 85]. Differences in measuring vitamin D in serum blood samples via immunoassay and dry blood spotting via LC-MS-MS assay could explain these contradictory findings. Our study suggests that, in a controlled, in vitro model, the active metabolite of vitamin D is capable of reversing potentially aberrant/pathological IBS gene expression with emphasis on serotonergic metabolism given the GO analysis highlighting serotonergic signaling and the markedly significant increase in SERT and TPH1 expression in HCT-116 colorectal cells when treated with 1,25D. It is important to note that all four genes tested for regulation by vitamin D were influenced by the presence of 10−8 M vitamin D. Thus, these findings may inform future treatment decisions and research into patients diagnosed with IBS.

Of particular note in this study was the analysis of gene expression by RT-qPCR in IBS patients relative to controls, evaluation of the accuracy of the proposed biomarkers, and assessment of the regulatory role of vitamin D in genes associated with serotonin metabolism. Outside of reversal of aberrant gene expression by vitamin D, we have also identified seven putative biomarker genes that may be of use in the diagnosis of IBS. Building the bridge between clinical and nonclinical uses of vitamin D in the treatment of IBS and serotonergic signaling will likely require further research not only at the genomic and transcriptomic levels, but also in the proteomic, metabolomic, and microbiome realms of IBS pathophysiology via a multiomic approach [68, 86]. Limitations of this study include a small IBS patient sample of varying subtypes rather than of a single subtype. Such nonspecific sampling may lead to trends in data that are not reflected among a single IBS subtype, thus obscuring the development of more targeted therapies. Future work should focus on such multiomics-based approaches to understanding IBS and an expansion of sample pools to include greater patient numbers and diversity to enable subsampling and analysis of multiple IBS subtypes.

Supporting information

S1 Table. Complete 29 genes tested by RT-qPCR.

Complete 29 genes tested in RT-qPCR analysis compared to IBS Microarray values. Genes with a minimum of 2 replicates are graphically reported in Fig 2. Fold change reported in IBS Microarray and RT-qPCR data relative to pooled control patients.

https://doi.org/10.1371/journal.pone.0275683.s001

(DOCX)

Acknowledgments

The authors extend their gratitude to the Marshfield Clinic Research Institute for providing colonic tissue specimens. We also thank the Marshfield Clinic Research Foundation and the ASU New College Undergraduate Inquiry and Research Experiences (NCUIRE) program for support.

References

  1. 1. Sperber AD, Bangdiwala SI, Drossman DA, Ghoshal UC, Simren M, Tack J, et al. Worldwide Prevalence and Burden of Functional Gastrointestinal Disorders, Results of Rome Foundation Global Study. Gastroenterology. 2021;160(1):99–114.e3. Epub 20200412. pmid:32294476.
  2. 2. Chey WD, Kurlander J, Eswaran S. Irritable bowel syndrome: a clinical review. JAMA. 2015;313(9):949–58. pmid:25734736.
  3. 3. Shiha MG, Aziz I. Review article: Physical and psychological comorbidities associated with irritable bowel syndrome. Aliment Pharmacol Ther. 2021;54 Suppl 1:S12–S23. pmid:34927759.
  4. 4. Nellesen D, Yee K, Chawla A, Lewis BE, Carson RT. A systematic review of the economic and humanistic burden of illness in irritable bowel syndrome and chronic constipation. J Manag Care Pharm. 2013;19(9):755–64. pmid:24156644.
  5. 5. Cash BD, Chey WD. Irritable bowel syndrome—an evidence-based approach to diagnosis. Aliment Pharmacol Ther. 2004;19(12):1235–45. pmid:15191504.
  6. 6. Ford AC, Moayyedi P, Lacy BE, Lembo AJ, Saito YA, Schiller LR, et al. American College of Gastroenterology monograph on the management of irritable bowel syndrome and chronic idiopathic constipation. Am J Gastroenterol. 2014;109 Suppl 1:S2–26; quiz S7. pmid:25091148.
  7. 7. Lacy BE, Moreau JC. Diarrhea-predominant irritable bowel syndrome: Diagnosis, etiology, and new treatment considerations. J Am Assoc Nurse Pract. 2016;28(7):393–404. pmid:27436200.
  8. 8. Lacy BE, Patel NK. Rome Criteria and a Diagnostic Approach to Irritable Bowel Syndrome. J Clin Med. 2017;6(11). Epub 20171026. pmid:29072609; PubMed Central PMCID: PMC5704116.
  9. 9. Ek WE, Reznichenko A, Ripke S, Niesler B, Zucchelli M, Rivera NV, et al. Exploring the genetics of irritable bowel syndrome: a GWA study in the general population and replication in multinational case-control cohorts. Gut. 2015;64(11):1774–82. Epub 2014/09/23. pmid:25248455.
  10. 10. Holliday EG, Attia J, Hancock S, Koloski N, McEvoy M, Peel R, et al. Genome-wide association study identifies two novel genomic regions in irritable bowel syndrome. Am J Gastroenterol. 2014;109(5):770–2. pmid:24797007.
  11. 11. Levy RL, Jones KR, Whitehead WE, Feld SI, Talley NJ, Corey LA. Irritable bowel syndrome in twins: heredity and social learning both contribute to etiology. Gastroenterology. 2001;121(4):799–804. pmid:11606493.
  12. 12. Locke GR, Zinsmeister AR, Talley NJ, Fett SL, Melton LJ. Familial association in adults with functional gastrointestinal disorders. Mayo Clin Proc. 2000;75(9):907–12. pmid:10994826.
  13. 13. Foxx-Orenstein AE. New and emerging therapies for the treatment of irritable bowel syndrome: an update for gastroenterologists. Therap Adv Gastroenterol. 2016;9(3):354–75. Epub 2016/02/21. pmid:27134665; PubMed Central PMCID: PMC4830102.
  14. 14. Morris-Yates A, Talley NJ, Boyce PM, Nandurkar S, Andrews G. Evidence of a genetic contribution to functional bowel disorder. Am J Gastroenterol. 1998;93(8):1311–7. pmid:9707057.
  15. 15. Svedberg P, Johansson S, Wallander MA, Hamelin B, Pedersen NL. Extra-intestinal manifestations associated with irritable bowel syndrome: a twin study. Aliment Pharmacol Ther. 2002;16(5):975–83. pmid:11966507.
  16. 16. Bengtson MB, Rønning T, Vatn MH, Harris JR. Irritable bowel syndrome in twins: genes and environment. Gut. 2006;55(12):1754–9. Epub 2006/09/28. pmid:17008364; PubMed Central PMCID: PMC1856463.
  17. 17. Lembo A, Zaman M, Jones M, Talley NJ. Influence of genetics on irritable bowel syndrome, gastro-oesophageal reflux and dyspepsia: a twin study. Aliment Pharmacol Ther. 2007;25(11):1343–50. pmid:17509102.
  18. 18. Saito YA. The role of genetics in IBS. Gastroenterol Clin North Am. 2011;40(1):45–67. pmid:21333900; PubMed Central PMCID: PMC3056499.
  19. 19. Saito YA, Larson JJ, Atkinson EJ, Ryu E, Almazar AE, Petersen GM, et al. The role of 5-HTT LPR and GNbeta3 825C>T polymorphisms and gene-environment interactions in irritable bowel syndrome (IBS). Dig Dis Sci. 2012;57(10):2650–7. Epub 20120802. pmid:22855291; PubMed Central PMCID: PMC3912694.
  20. 20. Camilleri M, Klee EW, Shin A, Carlson P, Li Y, Grover M, et al. Irritable bowel syndrome-diarrhea: characterization of genotype by exome sequencing, and phenotypes of bile acid synthesis and colonic transit. Am J Physiol Gastrointest Liver Physiol. 2014;306(1):G13–26. Epub 2013/11/07. pmid:24200957; PubMed Central PMCID: PMC3920085.
  21. 21. Grasberger H, Chang L, Shih W, Presson AP, Sayuk GS, Newberry RD, et al. Identification of a functional TPH1 polymorphism associated with irritable bowel syndrome bowel habit subtypes. Am J Gastroenterol. 2013;108(11):1766–74. Epub 2013/09/24. pmid:24060757; PubMed Central PMCID: PMC4067697.
  22. 22. Wouters MM, Lambrechts D, Knapp M, Cleynen I, Whorwell P, Agréus L, et al. Genetic variants in CDC42 and NXPH1 as susceptibility factors for constipation and diarrhoea predominant irritable bowel syndrome. Gut. 2014;63(7):1103–11. Epub 2013/09/16. pmid:24041540.
  23. 23. Marynowski M, Likońska A, Zatorski H, Fichna J. Role of environmental pollution in irritable bowel syndrome. World J Gastroenterol. 2015;21(40):11371–8. pmid:26523104; PubMed Central PMCID: PMC4616213.
  24. 24. Tan TK, Saps M, Lin CL, Wei CC. Is Long-term Ambient Air Pollutant Exposure a Risk Factor for Irritable Bowel Syndrome in Children? A 12-year Longitudinal Cohort Study. J Neurogastroenterol Motil. 2019;25(2):241–9. pmid:30982240; PubMed Central PMCID: PMC6474697.
  25. 25. Kish L, Hotte N, Kaplan GG, Vincent R, Tso R, Gänzle M, et al. Environmental particulate matter induces murine intestinal inflammatory responses and alters the gut microbiome. PLoS One. 2013;8(4):e62220. Epub 2013/04/24. pmid:23638009; PubMed Central PMCID: PMC3634745.
  26. 26. Choi JJ, Eum SY, Rampersaud E, Daunert S, Abreu MT, Toborek M. Exercise attenuates PCB-induced changes in the mouse gut microbiome. Environ Health Perspect. 2013;121(6):725–30. Epub 2013/04/26. pmid:23632211; PubMed Central PMCID: PMC3672930.
  27. 27. Sobczak M, Fabisiak A, Murawska N, Wesołowska E, Wierzbicka P, Wlazłowski M, et al. Current overview of extrinsic and intrinsic factors in etiology and progression of inflammatory bowel diseases. Pharmacol Rep. 2014;66(5):766–75. Epub 2014/04/29. pmid:25149979.
  28. 28. Rurgo S, Vaino V, Andreozzi M, Pagliaro M, Senneca P, Di Giorgio G, et al. Predictors of abdominal pain severity in patients with constipation-prevalent irritable bowel syndrome. J Basic Clin Physiol Pharmacol. 2022. Epub 20220606. pmid:35659327.
  29. 29. Afari N, Ahumada SM, Wright LJ, Mostoufi S, Golnari G, Reis V, et al. Psychological trauma and functional somatic syndromes: a systematic review and meta-analysis. Psychosom Med. 2014;76(1):2–11. Epub 2013/12/12. pmid:24336429; PubMed Central PMCID: PMC3894419.
  30. 30. Bradford K, Shih W, Videlock EJ, Presson AP, Naliboff BD, Mayer EA, et al. Association between early adverse life events and irritable bowel syndrome. Clin Gastroenterol Hepatol. 2012;10(4):385–90.e1-3. Epub 2011/12/16. pmid:22178460; PubMed Central PMCID: PMC3311761.
  31. 31. Chitkara DK, van Tilburg MA, Blois-Martin N, Whitehead WE. Early life risk factors that contribute to irritable bowel syndrome in adults: a systematic review. Am J Gastroenterol. 2008;103(3):765–74; quiz 75. Epub 2008/01/02. pmid:18177446; PubMed Central PMCID: PMC3856200.
  32. 32. Drossman DA, Chang L, Bellamy N, Gallo-Torres HE, Lembo A, Mearin F, et al. Severity in irritable bowel syndrome: a Rome Foundation Working Team report. Am J Gastroenterol. 2011;106(10):1749–59; quiz 60. Epub 2011/07/12. pmid:21747417.
  33. 33. Halland M, Almazar A, Lee R, Atkinson E, Larson J, Talley NJ, et al. A case-control study of childhood trauma in the development of irritable bowel syndrome. Neurogastroenterol Motil. 2014;26(7):990–8. Epub 2014/05/11. pmid:24813232.
  34. 34. Knight JR, Locke GR, Zinsmeister AR, Schleck CD, Talley NJ. Family history of mental illness or alcohol abuse and the irritable bowel syndrome. J Psychosom Res. 2015;78(3):237–41. Epub 2014/12/03. pmid:25582802; PubMed Central PMCID: PMC4505618.
  35. 35. Koloski NA, Jones M, Kalantar J, Weltman M, Zaguirre J, Talley NJ. The brain—gut pathway in functional gastrointestinal disorders is bidirectional: a 12-year prospective population-based study. Gut. 2012;61(9):1284–90. Epub 2012/01/10. pmid:22234979.
  36. 36. Ladabaum U, Boyd E, Zhao WK, Mannalithara A, Sharabidze A, Singh G, et al. Diagnosis, comorbidities, and management of irritable bowel syndrome in patients in a large health maintenance organization. Clin Gastroenterol Hepatol. 2012;10(1):37–45. Epub 2011/08/24. pmid:21871250; PubMed Central PMCID: PMC3242893.
  37. 37. Rey de Castro NG, Miller V, Carruthers HR, Whorwell PJ. Irritable bowel syndrome: a comparison of subtypes. J Gastroenterol Hepatol. 2015;30(2):279–85. pmid:25091059.
  38. 38. Lackner JM, Gudleski GD, Radziwon CD, Krasner SS, Firth RS, Naliboff BD, et al. Cognitive flexibility improves in cognitive behavioral therapy for irritable bowel syndrome but not nonspecific education/support. Behav Res Ther. 2022;154:104033. Epub 20220110. pmid:35653934.
  39. 39. Carroll IM, Ringel-Kulka T, Keku TO, Chang YH, Packey CD, Sartor RB, et al. Molecular analysis of the luminal- and mucosal-associated intestinal microbiota in diarrhea-predominant irritable bowel syndrome. Am J Physiol Gastrointest Liver Physiol. 2011;301(5):G799–807. Epub 2011/07/07. pmid:21737778; PubMed Central PMCID: PMC3220325.
  40. 40. Codling C, O’Mahony L, Shanahan F, Quigley EM, Marchesi JR. A molecular analysis of fecal and mucosal bacterial communities in irritable bowel syndrome. Dig Dis Sci. 2010;55(2):392–7. Epub 2009/08/20. pmid:19693670.
  41. 41. Durbán A, Abellán JJ, Jiménez-Hernández N, Artacho A, Garrigues V, Ortiz V, et al. Instability of the faecal microbiota in diarrhoea-predominant irritable bowel syndrome. FEMS Microbiol Ecol. 2013;86(3):581–9. Epub 2013/08/23. pmid:23889283.
  42. 42. Durbán A, Abellán JJ, Jiménez-Hernández N, Salgado P, Ponce M, Ponce J, et al. Structural alterations of faecal and mucosa-associated bacterial communities in irritable bowel syndrome. Environ Microbiol Rep. 2012;4(2):242–7. Epub 2012/03/01. pmid:23757279.
  43. 43. Gargari G, Taverniti V, Gardana C, Cremon C, Canducci F, Pagano I, et al. Fecal Clostridiales distribution and short-chain fatty acids reflect bowel habits in irritable bowel syndrome. Environ Microbiol. 2018;20(9):3201–13. Epub 20180924. pmid:29749705.
  44. 44. Makker J, Chilimuri S, Bella JN. Genetic epidemiology of irritable bowel syndrome. World J Gastroenterol. 2015;21(40):11353–61. pmid:26525775; PubMed Central PMCID: PMC4616211.
  45. 45. Camilleri M. Serotonin in the gastrointestinal tract. Curr Opin Endocrinol Diabetes Obes. 2009;16(1):53–9. pmid:19115522; PubMed Central PMCID: PMC2694720.
  46. 46. Margolis KG, Cryan JF, Mayer EA. The Microbiota-Gut-Brain Axis: From Motility to Mood. Gastroenterology. 2021;160(5):1486–501. Epub 20210122. pmid:33493503; PubMed Central PMCID: PMC8634751.
  47. 47. Gershon MD. 5-Hydroxytryptamine (serotonin) in the gastrointestinal tract. Curr Opin Endocrinol Diabetes Obes. 2013;20(1):14–21. pmid:23222853; PubMed Central PMCID: PMC3708472.
  48. 48. Ahonen A, Kyösola K, Penttilä O. Enterochromaffin cells in macrophages in ulcerative colitis and irritable colon. Ann Clin Res. 1976;8(1):1–7. pmid:937988.
  49. 49. Kyösola K, Penttilä O, Salaspuro M. Rectal mucosal adrenergic innervation and enterochromaffin cells in ulcerative colitis and irritable colon. Scand J Gastroenterol. 1977;12(3):363–7. pmid:867000
  50. 50. Coates MD, Mahoney CR, Linden DR, Sampson JE, Chen J, Blaszyk H, et al. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome. Gastroenterology. 2004;126(7):1657–64. pmid:15188158.
  51. 51. Kerckhoffs AP, ter Linde JJ, Akkermans LM, Samsom M. SERT and TPH-1 mRNA expression are reduced in irritable bowel syndrome patients regardless of visceral sensitivity state in large intestine. Am J Physiol Gastrointest Liver Physiol. 2012;302(9):G1053–60. Epub 2012/02/09. pmid:22323131.
  52. 52. Dunlop SP, Coleman NS, Blackshaw E, Perkins AC, Singh G, Marsden CA, et al. Abnormalities of 5-hydroxytryptamine metabolism in irritable bowel syndrome. Clin Gastroenterol Hepatol. 2005;3(4):349–57. pmid:15822040.
  53. 53. Ford AC, Quigley EM, Lacy BE, Lembo AJ, Saito YA, Schiller LR, et al. Effect of antidepressants and psychological therapies, including hypnotherapy, in irritable bowel syndrome: systematic review and meta-analysis. Am J Gastroenterol. 2014;109(9):1350–65; quiz 66. Epub 2014/06/17. pmid:24935275.
  54. 54. Patrick RP, Ames BN. Vitamin D hormone regulates serotonin synthesis. Part 1: relevance for autism. FASEB J. 2014;28(6):2398–413. Epub 2014/02/20. pmid:24558199.
  55. 55. Kaneko I, Sabir MS, Dussik CM, Whitfield GK, Karrys A, Hsieh JC, et al. 1,25-Dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D. FASEB J. 2015;29(9):4023–35. Epub 2015/06/12. pmid:26071405.
  56. 56. Haussler MR, Whitfield GK, Haussler CA, Hsieh J-C, Jurutka PW. Chapter 8—Nuclear Vitamin D Receptor: Natural Ligands, Molecular Structure–Function, and Transcriptional Control of Vital Genes. 2011 2011 [cited 20 May 2011]. In: Vitamin D [Internet]. Vitamin D: Elsevier. Third. [cited 20 May 2011]; [137–70]. Available from: https://www.sciencedirect.com/science/article/pii/B9780123819789100083?via%3Dihub.
  57. 57. Fond G, Loundou A, Hamdani N, Boukouaci W, Dargel A, Oliveira J, et al. Anxiety and depression comorbidities in irritable bowel syndrome (IBS): a systematic review and meta-analysis. Eur Arch Psychiatry Clin Neurosci. 2014;264(8):651–60. Epub 2014/04/06. pmid:24705634.
  58. 58. Hoang MT, Defina LF, Willis BL, Leonard DS, Weiner MF, Brown ES. Association between low serum 25-hydroxyvitamin D and depression in a large sample of healthy adults: the Cooper Center longitudinal study. Mayo Clin Proc. 2011;86(11):1050–5. pmid:22033249; PubMed Central PMCID: PMC3202994.
  59. 59. Nwosu BU, Maranda L, Candela N. Vitamin D status in pediatric irritable bowel syndrome. PLoS One. 2017;12(2):e0172183. Epub 2017/02/13. pmid:28192499; PubMed Central PMCID: PMC5305242.
  60. 60. Cantorna MT, McDaniel K, Bora S, Chen J, James J. Vitamin D, immune regulation, the microbiota, and inflammatory bowel disease. Exp Biol Med (Maywood). 2014;239(11):1524–30. Epub 2014/03/25. pmid:24668555; PubMed Central PMCID: PMC4176535.
  61. 61. Karaahmet F, Basar Ö, Yüksel I, Coban S, Yuksel O. Letter: vitamin D supplementation and the irritable bowel syndrome. Aliment Pharmacol Ther. 2013;37(4):499. pmid:23336687.
  62. 62. Sprake EF, Grant VA, Corfe BM. Vitamin D3 as a novel treatment for irritable bowel syndrome: single case leads to critical analysis of patient-centred data. BMJ Case Rep. 2012;2012. Epub 2012/12/13. pmid:23239770; PubMed Central PMCID: PMC4544096.
  63. 63. Bearcroft CP, Perrett D, Farthing MJ. Postprandial plasma 5-hydroxytryptamine in diarrhoea predominant irritable bowel syndrome: a pilot study. Gut. 1998;42(1):42–6. pmid:9505884; PubMed Central PMCID: PMC1726971.
  64. 64. Atkinson W, Lockhart S, Whorwell PJ, Keevil B, Houghton LA. Altered 5-hydroxytryptamine signaling in patients with constipation- and diarrhea-predominant irritable bowel syndrome. Gastroenterology. 2006;130(1):34–43. pmid:16401466.
  65. 65. Camilleri M, Andrews CN, Bharucha AE, Carlson PJ, Ferber I, Stephens D, et al. Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome. Gastroenterology. 2007;132(1):17–25. Epub 20061117. pmid:17241856; PubMed Central PMCID: PMC2474784.
  66. 66. Yeo A, Boyd P, Lumsden S, Saunders T, Handley A, Stubbins M, et al. Association between a functional polymorphism in the serotonin transporter gene and diarrhoea predominant irritable bowel syndrome in women. Gut. 2004;53(10):1452–8. pmid:15361494; PubMed Central PMCID: PMC1774243.
  67. 67. Costello CM, Mah N, Häsler R, Rosenstiel P, Waetzig GH, Hahn A, et al. Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays. PLoS Med. 2005;2(8):e199. Epub 2005/08/23. pmid:16107186; PubMed Central PMCID: PMC1188246.
  68. 68. Dussik CM, Hockley M, Grozić A, Kaneko I, Zhang L, Sabir MS, et al. Gene Expression Profiling and Assessment of Vitamin D and Serotonin Pathway Variations in Patients With Irritable Bowel Syndrome. J Neurogastroenterol Motil. 2018;24(1):96–106. pmid:29291611; PubMed Central PMCID: PMC5753908.
  69. 69. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47. Epub 20150120. pmid:25605792; PubMed Central PMCID: PMC4402510.
  70. 70. Sabir MS, Dussik CM, Kaneko I, Whitfield GK, Haussler MR, Jurutka PW. Modulation of Serotonin Synthesis, Reuptake, and Degradation by 1,25-Dihydroxyvitamin D: Potential Relevance for Neuropsychiatric Disorders. The FASEB Journal. 2016;30. Epub 1 Apr 2016.
  71. 71. Manocha M, Khan WI. Serotonin and GI Disorders: An Update on Clinical and Experimental Studies. Clin Transl Gastroenterol. 2012;3:e13. Epub 2012/04/26. pmid:23238212; PubMed Central PMCID: PMC3365677.
  72. 72. Aerssens J, Camilleri M, Talloen W, Thielemans L, Göhlmann HW, Van Den Wyngaert I, et al. Alterations in mucosal immunity identified in the colon of patients with irritable bowel syndrome. Clin Gastroenterol Hepatol. 2008;6(2):194–205. pmid:18237869; PubMed Central PMCID: PMC2453689.
  73. 73. Videlock EJ, Mahurkar-Joshi S, Hoffman JM, Iliopoulos D, Pothoulakis C, Mayer EA, et al. Sigmoid colon mucosal gene expression supports alterations of neuronal signaling in irritable bowel syndrome with constipation. Am J Physiol Gastrointest Liver Physiol. 2018;315(1):G140–G57. Epub 20180322. pmid:29565640; PubMed Central PMCID: PMC6109711.
  74. 74. Gershon MD, Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology. 2007;132(1):397–414. pmid:17241888.
  75. 75. Zheng Y, Yu T, Tang Y, Xiong W, Shen X, Jiang L, et al. Efficacy and safety of 5-hydroxytryptamine 3 receptor antagonists in irritable bowel syndrome: A systematic review and meta-analysis of randomized controlled trials. PLoS One. 2017;12(3):e0172846. Epub 20170314. pmid:28291778; PubMed Central PMCID: PMC5349445.
  76. 76. Van Kerkhoven LA, Laheij RJ, Jansen JB. Meta-analysis: a functional polymorphism in the gene encoding for activity of the serotonin transporter protein is not associated with the irritable bowel syndrome. Aliment Pharmacol Ther. 2007;26(7):979–86. pmid:17877505.
  77. 77. Dai C, Zheng CQ, Jiang M. Letter: serotonin transporter gene polymorphisms and the irritable bowel syndrome. Aliment Pharmacol Ther. 2013;37(6):657–8. pmid:23406411.
  78. 78. Areeshi MY, Haque S, Panda AK, Mandal RK. A serotonin transporter gene (SLC6A4) polymorphism is associated with reduced risk of irritable bowel syndrome in American and Asian population: a meta-analysis. PLoS One. 2013;8(9):e75567. Epub 2013/09/19. pmid:24069428; PubMed Central PMCID: PMC3777956.
  79. 79. Zhang ZF, Duan ZJ, Wang LX, Yang D, Zhao G, Zhang L. The serotonin transporter gene polymorphism (5-HTTLPR) and irritable bowel syndrome: a meta-analysis of 25 studies. BMC Gastroenterol. 2014;14:23. Epub 2014/02/10. pmid:24512255; PubMed Central PMCID: PMC3926682.
  80. 80. Chen JJ, Li Z, Pan H, Murphy DL, Tamir H, Koepsell H, et al. Maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high-affinity serotonin transporter: Abnormal intestinal motility and the expression of cation transporters. J Neurosci. 2001;21(16):6348–61. pmid:11487658; PubMed Central PMCID: PMC6763151.
  81. 81. Gershon MD. Review article: serotonin receptors and transporters—roles in normal and abnormal gastrointestinal motility. Aliment Pharmacol Ther. 2004;20 Suppl 7:3–14. pmid:15521849.
  82. 82. Nozawa K, Kawabata-Shoda E, Doihara H, Kojima R, Okada H, Mochizuki S, et al. TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells. Proc Natl Acad Sci U S A. 2009;106(9):3408–13. Epub 2009/02/11. pmid:19211797; PubMed Central PMCID: PMC2651261.
  83. 83. Tazzyman S, Richards N, Trueman AR, Evans AL, Grant VA, Garaiova I, et al. Vitamin D associates with improved quality of life in participants with irritable bowel syndrome: outcomes from a pilot trial. BMJ Open Gastroenterol. 2015;2(1):e000052. Epub 2015/12/21. pmid:26719813; PubMed Central PMCID: PMC4691664.
  84. 84. De Ponti F. Pharmacology of serotonin: what a clinician should know. Gut. 2004;53(10):1520–35. pmid:15361507; PubMed Central PMCID: PMC1774227.
  85. 85. Williams CE, Williams EA, Corfe BM. Vitamin D supplementation in people with IBS has no effect on symptom severity and quality of life: results of a randomised controlled trial. Eur J Nutr. 2021. Epub 20210730. pmid:34328539.
  86. 86. Zhang J, Zhu S, Ma N, Johnston LJ, Wu C, Ma X. Metabolites of microbiota response to tryptophan and intestinal mucosal immunity: A therapeutic target to control intestinal inflammation. Med Res Rev. 2021;41(2):1061–88. Epub 20201110. pmid:33174230.