Genetic and transcriptional analysis of inflammatory bowel disease-associated pathways in patients with GUCY2C-linked familial diarrhea.

OBJECTIVE: Activating mutations in the GUCY2C gene, which encodes the epithelial receptor guanylate cyclase C, cause diarrhea due to increased loss of sodium chloride to the intestinal lumen. Patients with familial GUCY2C diarrhea syndrome (FGDS) are predisposed to inflammatory bowel disease (IBD). We investigated whether genes in the guanylate cyclase C pathway are enriched for association with IBD and reversely whether genetic or transcriptional changes associated with IBD are found in FGDS patients. METHODS: (1) A set of 27 genes from the guanylate cyclase C pathway was tested for enrichment of association with IBD by Gene Set Enrichment Analysis, using genome-wide association summary statistics from 12,882 IBD patients and 21,770 controls. (2) We genotyped 163 known IBD risk loci and sequenced NOD2 in 22 patients with FGDS. Eight of them had concomitant Crohn's disease. (3) Global gene expression analysis was performed in ileal tissue from patients with FGDS, Crohn's disease and healthy individuals. RESULTS: The guanylate cyclase C gene set showed a significant enrichment of association in IBD genome-wide association data. Risk variants in NOD2 were found in 7/8 FGDS patients with concomitant Crohn's disease and in 2/14 FDGS patients without Crohn's disease. In ileal tissue, downregulation of metallothioneins characterized FGDS patients compared to healthy controls. CONCLUSIONS: Our results support a role of guanylate cyclase C signaling and disturbed electrolyte homeostasis in development of IBD. Furthermore, downregulation of metallothioneins in the ileal mucosa of FGDS patients may contribute to IBD development, possibly alongside effects from NOD2 risk variants.

Does the low FODMAP diet improve symptoms of radiation-induced enteropathy? A pilot study.

RATIONALE: Patients with radiation-induced enteropathy (RE) after cancer treatment show similar symptoms as patients with irritable bowel syndrome (IBS). The low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) diet (LFD) is a widespread management strategy for IBS. We aimed to investigate if there may be a positive effect of LFD on symptoms and health-related quality of life (HRQOL) in patients with RE. METHODS: In an open non-controlled pilot study, 11 patients (all female) with RE-related IBS symptoms were recruited largely based on own initiative. All followed LFD for four weeks. IBS Severity Scoring System (IBS-SSS) and IBS Symptom Questionnaire (IBS-SQ) were used to assess symptoms. Short Form Nepean Dyspepsia Index (SF-NDI) and 12-item Short Form Health Survey (SF-12) evaluated HRQOL. A three day food record was used to estimate baseline intake of FODMAPs and to reveal dietary changes. RESULTS: FODMAP intake was successfully reduced, although LFD was found a burdensome intervention. IBS symptoms improved significantly based on mean total score of IBS-SSS and IBS-SQ, which changed from 310.2 ± 60.7 to 171.4 ± 107.2 (p = .001) and 27.4 ± 4.1 to 15.7 ± 10.1 (p = .002). HRQOL improved based on SF-NDI total score (30.5 ± 9.4 to 18.3 ± 8.2, p = .001) and based on mental (p = .047) and physical (p = .134) score of SF-12. Main additional dietary changes were reduced intake of energy, carbohydrates, and fiber. CONCLUSION: Our findings from this small-scaled pilot study indicate that the LFD may alleviate symptoms and improve HRQOL in patients with RE. Further controlled studies with larger sample size should be conducted to verify our results and hopefully enable implementation of LFD as a future part of the management strategy for RE.

An activating gucy2c mutation causes impaired contractility and fluid stagnation in the small bowel.

OBJECTIVE: Familial GUCY2C diarrhoea syndrome (FGDS) is caused by an activating mutation in the GUCY2C gene encoding the receptor guanylate cyclase C in enterocytes. Activation leads to increased secretion of fluid into the intestinal lumen. Twenty percent of the patients have increased risk of Crohn's disease and intestinal obstruction (CD, 20%) and the condition resembles irritable bowel syndrome with diarrhoea. We aimed to describe fluid content, contractility, peristaltic activity and bowel wall thickness in the intestine in fasting FGDS patients, using ultrasound, with healthy volunteers serving as controls. METHODS: Twenty-three patients with FGDS and 22 healthy controls (HC) were examined with a Logiq E9 scanner in a fasting state. Bowel wall thickness was measured and fluid-filled small bowel loops were counted using three-dimensional (3D) magnetic positioning navigation. The HC ingested 500 ml PEG solution, an electrolyte balanced, non-absorbable solution, in order to investigate the contractions of the small bowel. RESULTS: The fasting 23 FGDS patients had significantly higher number of fluid-filled small bowel segments compared to 22 fasting HC, p < 0.001. A high number of non-occlusive contractions in the ileum was observed, which was significant when compared to HC after ingesting PEG solution, p < 0.016. An increase in intestinal wall thickness or other signs of CD were not observed. CONCLUSIONS: FGDS is characterised by multiple, fluid-filled small bowel loops with incomplete contractions and fluid stagnation in fasting state. These findings may play a role in the increased risk of bowel obstruction as well as IBS-like symptoms observed in these patients.

Colonic Mucosal Epigenome and Microbiome Development in Children and Adolescents.

Epigenetic and microbiome changes during pediatric development have been implicated as important elements in the developmental origins of inflammatory bowel diseases (IBDs) including Crohn's disease (CD) and ulcerative colitis (UC), which are linked to early onset colorectal cancer (CRC). Colonic mucosal samples from 22 control children between 3.5 and 17.5 years of age were studied by Infinium HumanMethylation450 BeadChips and, in 10 cases, by 454 pyrosequencing of the bacterial 16S rRNA gene. Intercalating age-specific DNA methylation and microbiome changes were identified, which may have significant translational relevance in the developmental origins of IBD and CRC.

Influence of mineral and vitamin supplements on pregnancy outcome.

The literature was searched for publications on minerals and vitamins during pregnancy and the possible influence of supplements on pregnancy outcome. Maternal iron (Fe) deficiency has a direct impact on neonatal Fe stores and birth weight, and may cause cognitive and behavioural problems in childhood. Fe supplementation is recommended to low-income pregnant women, to pregnant women in developing countries, and in documented deficiency, but overtreatment should be avoided. Calcium (Ca) deficiency is associated with pre-eclampsia and intra-uterine growth restriction. Supplementation may reduce both the risk of low birth weight and the severity of pre-eclampsia. Gestational magnesium (Mg) deficiency may cause hematological and teratogenic damage. A Cochrane review showed a significant low birth weight risk reduction in Mg supplemented individuals. Intake of cereal-based diets rich in phytate, high intakes of supplemental Fe, or any gastrointestinal disease, may interfere with zinc (Zn) absorption. Zn deficiency in pregnant animals may limit fetal growth. Supplemental Zn may be prudent for women with poor gastrointestinal function, and in Zn deficient women, increasing birth weight and head circumference, but no evidence was found for beneficial effects of general Zn supplementation during pregnancy. Selenium (Se) is an antioxidant supporting humoral and cell-mediated immunity. Low Se status is associated with recurrent abortion, pre-eclampsia and IUGR, and although beneficial effects are suggested there is no evidence-based recommendation for supplementation. An average of 20-30% of pregnant women suffer from any vitamin deficiency, and without prophylaxis, about 75% of these would show a deficit of at least one vitamin. Vitamin B6 deficiency is associated with pre-eclampsia, gestational carbohydrate intolerance, hyperemesis gravidarum, and neurologic disease of infants. About 25% of pregnant women in India are folate deficient. Folate deficiency may lead to congenital malformations (neural tube damage, orofacial clefts, cardiac anomalies), anaemia and spontaneous abortions, and pre-eclampsia, IUGR and abruption placentae. Pregestational supplementation of folate prevents neural tube defects. A daily supplemental dose of 400 µg/day of folate is recommended when planning pregnancy. In developing countries diets are generally low in animal products and consequently in vitamin B12 content. An insufficient supply may cause reduced fetal growth. In vegetarian women, supplementation of vitamin B12 may be needed. Vitamin A deficiency is prevalent in the developing world, impairing Fe status and resistance to infections. The recommended upper limit for retinol supplements is 3000 IU/day. Vitamin A supplementation enhances birth weight and growth in infants born to HIV-infected women. Overdosing should be avoided. Low concentrations of vitamin C seem to increase the development of pre-eclampsia, and supplementation may be beneficial. Supplementation with vitamin D in the third trimester in vitamin D deficient women seems to be beneficial. The use of vitamins E, although generally considered "healthy", may be harmful to the pregnancy outcome by disrupting a physiologic oxidative gestational state and is consequently not recommended to prevent pre-eclampsia. Further studies on specific substances are needed as the basis for stratified, placebo-controlled analyses.

Familial diarrhea syndrome caused by an activating GUCY2C mutation.

BACKGROUND: Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS: We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS: We identified a heterozygous missense mutation (c.2519G→T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS: Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest [Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.).

Time patterns of changes in biomarkers, symptoms and histopathology during pelvic radiotherapy.

Acute radiation proctitis was evaluated before, during and after radiotherapy (RT) for prostate cancer. The main aims of the study were to examine changes related to the increasing radiation dose, and identify surrogate markers of gastrointestinal (GI) reaction to radiation. Twenty consecutive prostate cancer patients scheduled for 7 weeks of conformal RT were prospectively included in a longitudinal study assessing symptoms, inflammation in rectal mucosa biopsies, and blood and stool samples at four time points (before RT and 2, 6 and 11 weeks after start of RT). Blood samples were examined for acute phase response-related markers, fatty acids (FAs), vitamin E and leukotriene B(4) (LTB(4)). Lactoferrin, calprotectin and S100A12 were measured in stool samples and FAs in biopsies from rectal mucosa. The increase in histopathological inflammation reached a maximum 2 weeks after start of RT. Symptoms of GI toxicity increased with higher radiation dose and had not returned to pre-treatment level 4 weeks after RT. Lactoferrin concentrations in stool increased significantly at week 6. Significant decreases of vitamin E, leukocyte count, hemoglobin and some groups of FAs were discovered, while a few FAs increased significantly during the study period. Time courses vary between the selected indicators of acute radiation proctitis. The biopsy grading of inflammatory changes were most intense 2 weeks into the treatment period while symptoms continued to increase until week 6. Lactoferrin in stool samples could be a non-invasive marker of GI inflammation during RT. A transient decrease in vitamin E and some FAs during RT warrants further studies.

Profiles and time course of acute radiation toxicity symptoms during conformal radiotherapy for cancer of the prostate.

Symptoms of gastrointestinal toxicity are dose-limiting for pelvic radiotherapy (RT). Existing toxicity registrations (RTOG/EORTC) are helpful in defining maximal tolerated doses, but tend to underestimate the total toxicity burden by excluding several minor complaints. We have applied a more detailed and quantitative recording of symptoms and related these scores to RT-induced endoscopic and histopathologic changes. Prevalence and severity of specific toxicity symptoms were recorded before, during (weeks 2 and 6) and 2 and 8 weeks after RT in 96 patients undergoing external beam RT for localized prostate cancer. RTOG/EORTC acute toxicity and ad hoc total toxicity scores (TTS) were recorded. TTS scores were calculated by adding scores based on visual analog scale (VAS) grading of individual symptoms Fifty of the patients also underwent sequential proctoscopy with mucosal biopsy. Individual symptoms increased, but differed in prevalence and intensity during and after RT TTS increased during the entire treatment course in spite of normalizing histopathologic and endoscopic changes from week 2 onwards. Twenty-seven patients had no RTOG/EORTC toxicity, four had grade 3 and none had grade 4 toxicity. All patients with grade 0 had increased TTS. Thus, TTS appeared more sensitive than RTOG/EORTC scoring. The study demonstrates that multiple toxicity symptoms contribute to total toxicity in response to pelvic RT. TTS is a feasible and sensitive method for detecting and quantifying acute toxicity and unveils morbidity which remains hidden with the RTOG/EORTC score system. The development and timing of symptoms may give clues to pathogenesis, treatment, and prophylaxis.

Clinical significance of increased gelatinolytic activity in the rectal mucosa during external beam radiation therapy of prostate cancer.

PURPOSE: Rectal toxicity (proctitis) is a dose-limiting factor in pelvic radiation therapy. Mucosal atrophy, i.e., net extracellular matrix degradation, is a prominent feature of radiation proctitis, but the underlying mechanisms are not known. We prospectively examined changes in matrix metalloproteinase (MMP)-2 and MMP-9 (gelatinase A and B) in the rectal mucosa during radiation therapy of prostate cancer, as well as the relationships of these changes with symptomatic, structural, and cellular evidence of radiation proctitis. METHODS AND MATERIALS: Seventeen patients scheduled for external beam radiation therapy for prostate cancer were prospectively enrolled. Symptoms of gastrointestinal toxicity were recorded, and endoscopy with biopsy of the rectal mucosa was performed before radiation therapy, as well as 2 and 6 weeks into the treatment course. Radiation proctitis was assessed by endoscopic scoring, quantitative histology, and quantitative immunohistochemistry. MMP-2 and MMP-9 were localized immunohistochemically, and activities were determined by gelatin zymography. RESULTS: Symptoms, endoscopic scores, histologic injury, and mucosal macrophages and neutrophils increased from baseline to 2 weeks. Symptoms increased further from 2 weeks to 6 weeks, whereas endoscopic and cellular evidence of proctitis did not. Compared to pretreatment values, there was increased total gelatinolytic activity of MMP-2 and MMP-9 at 2 weeks (p = 0.02 and p = 0.004, respectively) and 6 weeks (p = 0.006 and p = 0.001, respectively). Active MMP-2 was increased at both time points (p = 0.0001 and p = 0.002). Increased MMP-9 and MMP-2 at 6 weeks was associated with radiation-induced diarrhea (p = 0.007 and p = 0.02, respectively) and with mucosal neutrophil infiltration (rho = 0.62). CONCLUSIONS: Pelvic radiation therapy causes increased MMP-2 and MMP-9 activity in the rectal mucosa. These changes correlate with radiation-induced diarrhea and granulocyte infiltration and may contribute to abnormal connective tissue remodeling in radiation proctitis.