Carlson, Paula

AbstractObjectiveWe identified a pedigree over five generations with 49 members, some of whom had chronic megacolon presenting in adolescence or adulthood. We aimed to assess the genetic cause of chronic megacolon through clinical and DNA studies.DesignAfter ethical approval and informed consent, family members provided answers to standard bowel disease questionnaires, radiological or surgical records, and DNA (buccal mucosal scraping). Exome DNA sequencing of colon tissue or blood DNA from seven family members with colon or duodenal dilatation, or no megacolon (n = 1) was carried out. Sanger sequencing was performed in 22 additional family members to further evaluate candidate variants. The study focused on genes of potential relevance to enteric nerve (ENS) maturation and Hirschsprung's disease or megacolon, based on the literature (GFRA1, NKX2‐1, KIF26A, TPM3, ACTG2, SCN10A, and C17orf107 [CHRNE]) and other genetic variants that co‐segregated with megacolon in the six affected family members.ResultsInformation was available in all except five members alive at time of study; among 30 members who provided DNA, six had definite megacolon, one megaduodenum, seven significant constipation without bowel dilatation, and 16 normal bowel function by questionnaire. Among genes studied, SEMA3F (g.3:50225360A>G; c1873A>G) was found in 6/6 family members with megacolon. The SEMA3F gene variant was assessed as potentially pathogenic, based on M‐CAP in silico prediction. SEMA3F function is associated with genes (KIT and PDGFRB) that impact intestinal pacemaker function.ConclusionFamilial chronic megacolon appears to be associated with SEMA3F, which is associated with genes impacting enteric nerve or pacemaker function.

AbstractBackgroundWeight loss in response to the long‐acting GLP‐1 receptor (GLP1R) analog, liraglutide, is correlated with delay in gastric‐emptying (GE). The aim of this pilot study was to assess whether specific genetic variants in GLP1R or TCF7L2 are associated with delayed GE and weight loss in obese patients treated with liraglutide or the short‐acting GLP‐1 agonist, exenatide.MethodsWe evaluated in obese individuals the associations of genetic variations of GLP1R (rs6923761) and TCF7L2 (rs7903146) on GE T1/2 and weight from two trials that evaluated separately exenatide, 5 μg BID for 30 days, or liraglutide, 3 mg daily for 5 weeks. Data were analyzed using the dominant genetic model and intention‐to‐treat analysis.Key ResultsThere was a significant correlation between changes in weight and GE T1/2 (rs = −.382, P = .004). GLP1R rs6923761 minor allele A (AA_AG) carriers who received either exenatide or liraglutide had greater delay in GE T1/2 relative to baseline (117.9 ± 27.5 [SEM] minutes and 128.9 ± 38.32 minutes) compared to GG genotype (95.8 ± 30.4 minutes and 61.4 ± 21.4 minutes, respectively; P = .11). There was a non‐significant difference in weight loss based on GLP1R rs6923761 genotype after 5 weeks of treatment. There were no significant correlations with TCF7L2 (rs7903146) genotype.Conclusions & InferencesThe minor A allele of GLP1R (rs6923761) is associated with greater delay in GE T1/2 in response to liraglutide and exenatide. These studies provide data to plan pharmacogenetics testing of the hypothesis that GLP1R (rs6923761) influences weight loss in response to GLP1R agonists.

SummaryBackgroundThe serum biomarkers, elevated 7αC4 (C4) and decreased FGF19, have been proposed as screening tests for bile acid diarrhoea.AimTo analyse prevalence, specificity and reproducibility of fasting C4 and FGF19 in identifying bile acid diarrhoea in patients with irritable bowel syndrome with predominant diarrhoea or functional diarrhoea (summarised as IBS‐D).MethodsWe prospectively studied fasting serum C4 and FGF19 in 101 IBS‐D patients; we reviewed data from 37 of the 101 patients with prior fasting serum C4 and FGF19 and from 30 of the 101 patients with prior faecal bile acids per 48 hours. We compared results with normal values (C4 ≥52.5 ng/mL [n=184], FGF‐19 ≤61.7 pg/mL [n=50]). We used Spearman correlation and Bland‐Altman plots to appraise reproducibility.ResultsAmong the 101 patients, there was a negative correlation between serum C4 and FGF19 (Rs=−.342, P=.0005). Bile acid diarrhoea was diagnosed in 10 patients based on elevated serum C4 levels (mean 23.5±23.1 [SD] ng/mL) and 21 patients based on decreased FGF19 levels (121.6±84.2 pg/mL). With replicate tests in patients with stable IBS‐D, 78% of C4 and 70% of FGF19 measurements remained concordant, with 3% and 11% respectively consistently positive for bile acid diarrhoea in the 101 patients. Compared to 48 hours faecal bile acids, specificity for C4 and FGF19 was 83% and 78%, respectively. Bland‐Altman plots demonstrated greater reliability of C4 than FGF19.ConclusionsAmong 101 patents with IBS‐D, fasting FGF19 and C4 levels had good specificity and negative predictive value, suggesting utility as screening tests to exclude bile acid diarrhoea.

AbstractSerum‐derived bovine immunoglobulin/protein isolate (SBI), an oral nutritional therapy, is efficacious in diverse diarrheal diseases. In an open‐label study in 15 patients with irritable bowel syndrome‐diarrhea (IBS‐D), we evaluated effects of SBI (5.0 g, twice a day) for 8 weeks on safety, on bowel function and abdominal pain, tryptophan metabolism (K:T ratio), intestinal permeability (13C‐mannitol and lactulose excretion), bile acid synthesis (fasting serum FGF‐19 and C4), duodenal and stool microbiome, and the expression of 90 genes related to inflammation, immune function, and tight junctions in duodenal mucosa. Statistical analysis (paired tests, baseline vs. treatment) was based on intention to treat (ITT) principles. One of 15 Caucasian patients (13F, 2M, age 40.3 ± 2.3y, BMI 34.3 ± 3.0 kg/m2) withdrew without completing studies. There were improvements in stools/day (decrease, P < 0.001), ease of passage (P = 0.035), and evacuation (P = 0.004) with SBI therapy. Worst pain severity was numerically reduced in last 2 weeks' treatment (P = 0.078). Duodenal mucosal mRNA expression; serum C4, FGF‐19, and KT ratio; small bowel or colon permeability; and stool microbiome were not significantly different after SBI therapy, compared to baseline. In duodenal brushings, there was considerable microbiota structure difference (β diversity analysis P = 0.072, UniFrac) and, on taxonomic analysis, increased abundance of Proteobacteria Burkholderiales, Firmicutes Catonella, and unclassified genus organisms with SBI therapy. Thus, SBI therapy for 8 weeks in IBS‐D patients is associated with improved bowel function; the mechanism of benefit is unclear, though there were microbiota structure differences in duodenal brushings. Further studies in patients with low‐grade inflammation and intestinal barrier dysfunction at baseline are indicated.

Prior studies in with irritable bowel syndrome with diarrhea (IBS-D) patients showed immune activation, secretion, and barrier dysfunction in jejunal or colorectal mucosa. We measured mRNA expression by RT-PCR of 91 genes reflecting tight junction proteins, chemokines, innate immunity, ion channels, transmitters, housekeeping genes, and controls for DNA contamination and PCR efficiency in small intestinal mucosa from 15 IBS-D and 7 controls (biopsies negative for celiac disease). Fold change was calculated using 2(−ΔΔCT) formula. Nominal P values ( P < 0.05) were interpreted with false detection rate (FDR) correction ( q value). Cluster analysis with Lens for Enrichment and Network Studies (LENS) explored connectivity of mechanisms. Upregulated genes (uncorrected P < 0.05) were related to ion transport (INADL, MAGI1, and SONS1), barrier (TJP1, 2, and 3 and CLDN) or immune functions (TLR3, IL15, and MAPKAPK5), or histamine metabolism (HNMT); downregulated genes were related to immune function (IL-1β, TGF-β1, and CCL20) or antigen detection (TLR1 and 8). The following genes were significantly upregulated ( q < 0.05) in IBS-D: INADL, MAGI1, PPP2R5C, MAPKAPK5, TLR3, and IL-15. Among the 14 nominally upregulated genes, there was clustering of barrier and PDZ domains (TJP1, TJP2, TJP3, CLDN4, INADL, and MAGI1) and clustering of downregulated genes (CCL20, TLR1, IL1B, and TLR8). Protein expression of PPP2R5C in nuclear lysates was greater in patients with IBS-D and controls. There was increase in INADL protein (median 9.4 ng/ml) in patients with IBS-D relative to controls (median 5.8 ng/ml, P > 0.05). In conclusion, altered transcriptome (and to lesser extent protein) expression of ion transport, barrier, immune, and mast cell mechanisms in small bowel may reflect different alterations in function and deserves further study in IBS-D.

The mucosal gene expression in rectosigmoid mucosa (RSM) in irritable bowel syndrome with diarrhea (IBS-D) is unknown. Our objectives were, first, to study mRNA expression [by RT2 PCR of 19 genes pertaining to tight junctions, immune activation, intestinal ion transport and bile acid (BA) homeostasis] in RSM in IBS-D patients ( n = 47) and healthy controls ( n = 17) and study expression of a selected protein (PDZD3) in 10 IBS-D patients and 4 healthy controls; second, to assess RSM mRNA expression according to genotype and fecal BA excretion (high ≥2,337 μmol/48 h); and third, to determine whether genotype or mucosal mRNA expression is associated with colonic transit or BA parameters. Fold changes were corrected for false detection rate for 19 genes studied ( P < 0.00263). In RSM in IBS-D patients compared with controls, mRNA expression of GUC2AB, PDZD3, and PR2Y4 was increased, whereas CLDN1 and FN1 were decreased. One immune-related gene was upregulated (C4BP4) and one downregulated (CCL20). There was increased expression of a selected ion transport protein (PDZD3) on immunohistochemistry and Western blot in IBS-D compared with controls ( P = 0.02). There were no significant differences in mucosal mRNA in 20 IBS-D patients with high compared with 27 IBS-D patients with normal BA excretion. GPBAR1 ( P < 0.05) was associated with colonic transit. We concluded that mucosal ion transport mRNA (for several genes and PDZD3 protein) is upregulated and barrier protein mRNA downregulated in IBS-D compared with healthy controls, independent of genotype. There are no differences in gene expression in IBS-D with high compared with normal fecal BA excretion.

ObjectiveTo examine the association of gene variants of uncoupling proteins (UCP)‐2 and −3 with obesity and gastrointestinal (GI) traits.MethodsIn 255 overweight or obese adults, the associations of gene variants in UCP‐2 (−3474, rs659366) and UCP‐3 (rs1626521, rs2075577, rs15763) with body weight (BW) and GI traits were studied. Gene variants were genotyped by TaqMan® assay. The associations of genotypes with BW and GI traits (gastric emptying, gastric volume, satiety by buffet meal, satiation by nutrient drink test and GI hormones) were assessed using ANOVA corrected for false detection rate (FDR).ResultsA novel UCP‐3 gene variant, rs1626521, was identified; it was associated with BW (P = 0.039), waist circumference (P = 0.035), and significantly higher postprandial gastric volume (P = 0.003) and calories ingested at buffet meal (P = 0.006, both significant with FDR). In a subgroup of 11 participants, rs1626521 was also associated with reduced mitochondrial bioenergetics efficiency in skeletal muscle (P = 0.051). In an in vitro study in HEK293 cells, rs1626521 reduced UCP‐3 protein expression (P = 0.049). Associations detected between other genotypes and GI traits were nonsignificant with FDR.ConclusionsA newly identified functional variant (rs1626521) in UCP‐3 affects postprandial gastric functions and satiety and may contribute to weight gain and alter human mitochondrial function.

AbstractBackgroundA valid biomarker is ‘an indicator of normal biologic or pathogenic processes, or pharmacological responses to a therapeutic intervention’. There is no validated biomarker for irritable bowel syndrome (IBS). The aim of the study was to assess ability of three quantitative traits to identify treatable processes to discriminate between IBS‐diarrhea (IBS‐D) patients, IBS‐constipation (IBS‐C) patients and healthy volunteers (HV).MethodsIn 30 HV, 30 IBS‐C patients and 64 IBS‐D patients, we characterized bowel symptoms and quantitated pathophysiological mechanisms: bile acid (BA) synthesis (serum C4 and FGF19), fecal BA and fat, colonic transit (CT), and intestinal permeability (IP). We used multiple logistic regression and receiver‐operating characteristic (ROCAUC) to appraise three factors (fecal BA, CT, and IP) individually and in combination to identify discriminant targets for treatment in IBS.Key ResultsThere were significant associations between the three subgroups and symptoms reflecting bowel function and the quantitative traits. There were significant associations between fecal BA and CT at 48 h (r = 0.43; p < 0.001) and between fecal BA and IP (r = 0.23; p = 0.015). Individually, fecal BA and CT48 (but not IP) were significant independent predictors for distinguishing HV from IBS. In combination, they discriminated HV from IBS‐D patients (ROCAUC 0.70), HV from IBS‐C patients (ROCAUC 0.73), and IBS‐C patients from IBS‐D patients (ROCAUC 0.86). Colonic transit and fecal BA excretion together discriminate between healthy volunteers and IBS‐C patients or IBS‐D patients, or between the IBS subgroups with 75–90% specificity at 60% sensitivity.Conclusions & InferencesColonic transit and fecal BA individually and together constitute useful biomarkers to identify treatable mechanisms in IBS and to differentiate subgroups of IBS.

The pathobiology of irritable bowel syndrome (IBS) is multifaceted. We aimed to identify candidate genes predisposing to quantitative traits in IBS. In 30 healthy volunteers, 30 IBS-constipation, and 64 IBS-diarrhea patients, we measured bowel symptoms, bile acid (BA) synthesis (serum 7α-hydroxy-4-cholesten-3-one and FGF19), fecal BA and fat, colonic transit (CT by scintigraphy), and intestinal permeability (IP by 2-sugar excretion). We assessed associations of candidate genes controlling BA metabolism ( KLB rs17618244 and FGFR4 rs351855), BA receptor ( GPBAR1 rs11554825), serotonin (5-HT) reuptake ( SLC6A4 through rs4795541 which encodes for the 44-bp insert in 5HTTLPR), or immune activation ( TNFSF15 rs4263839) with three primary quantitative traits of interest: colonic transit, BA synthesis, and fecal BA excretion. There were significant associations between fecal BA and CT at 48 h ( r = 0.43; P < 0.001) and IP ( r = 0.23; P = 0.015). GPBAR1 genotype was associated with CT48 ( P = 0.003) and total fecal BA [ P = 0.030, false detection rate (FDR) P = 0.033]. Faster CT48 observed with both CC and TT GPBAR1 genotypes was due to significant interaction with G allele of KLB, which increases BA synthesis and excretion. Other univariate associations ( P < 0.05, without FDR correction) observed between GPBAR1 and symptom phenotype and gas sensation ratings support the role of GPBAR1 receptor. Associations between SLC6A4 and stool consistency, ease of passage, postprandial colonic tone, and total fecal BA excretion provide data in support of future hypothesis-testing studies. Genetic control of GPBAR1 receptor predisposing to pathobiological mechanisms in IBS provides evidence from humans in support of the importance of GPBAR1 to colonic motor and secretory functions demonstrated in animal studies.