Functional characterization of genetic variants in the apical sodium-dependent bile acid transporter (ASBT; SLC10A2).

BACKGROUND AND AIM: The major transporter responsible for bile acid uptake from the intestinal lumen is the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). Analysis of the SLC10A2 gene has identified a variety of sequence variants including coding region single nucleotide polymorphisms (SNPs) that may influence bile acid homeostasis/intestinal function. In this study, we systematically characterized the effect of coding SNPs on SLC10A2 protein expression and bile acid transport activity. METHODS: Single nucleotide polymorphisms in SLC10A2 from genomic DNA of ethnically-defined healthy individuals were identified using a polymerase chain reaction (PCR)-based temperature gradient capillary electrophoresis (TGCE) system. A heterologous gene expression system was used to assess transport activity of SLC10A2 nonsynonymous variants and missense mutations. Total and cell surface protein expression of wild-type and variant ASBT was assessed by Western blot analysis and immunofluorescence confocal microscopy. Expression of ASBT mRNA and protein was also measured in human intestinal samples. RESULTS: The studies revealed two nonsynonymous SNPs, 292G>A and 431G>A, with partially impaired in vitro taurocholate transport. A novel variant, 790A>G, was also shown to exhibit near complete loss of taurocholate transport, similar to the previously identified ASBT missense mutations. Examination of ASBT protein expression revealed no significant differences in expression or trafficking to the cell surface among variants versus wild-type ASBT. Analysis of ASBT mRNA and protein expression in human intestinal samples revealed modest intersubject variability. CONCLUSIONS: Genome sequencing and in vitro studies reveal the presence of multiple functionally relevant variants in SLC10A2 that may influence bile acid homeostasis and physiology.

The human proton-coupled folate transporter (hPCFT): modulation of intestinal expression and function by drugs.

Folic acid is a vitamin essential for thymidylate and purine synthesis. The human proton-coupled folate transporter (hPCFT) has recently been identified as a pH-dependent folic acid transporter, and mutations in this transporter have been linked to hereditary folic acid malabsorption. In this study, we assessed hPCFT-mediated transport activity in vitro, intersubject variability of intestinal expression in relation to blood folates, and the relationship of proton-pump inhibitor (PPI) therapy on hPCFT expression in vivo. We created a Madin-Darby canine kidney strain II (MDCKII) cell line stably expressing hPCFT to evaluate its drug substrates and inhibitors. Intestinal pinch biopsies (duodenum, ileum, colon) were collected from patients undergoing routine endoscopy procedures, and expressed levels of hPCFT were determined by RT-PCR. When assessed using MDCKII-hPCFT cells, folic acid and methotrexate were found to be high-affinity hPCFT substrates. Sulfasalazine and pyrimethamine were noted to inhibit hPCFT activity with Ki values of 42.3 and 161.7 micromol/l, respectively. hPCFT was localized to the brush-border membrane of enterocytes with highest expression in the duodenum and reduced levels in the ileum and colon. When we assessed hPCFT expression in a subset of patients who were receiving PPI therapy, a near 50% reduction in duodenal hPCFT mRNA expression was noted. These results suggest that hPCFT transporter activity can be modulated by many drugs in clinical use, and expression of this transporter in the gastrointestinal tract is higher in the duodenum than more distal sites (duodenum > ileum > colon). Importantly, we note that PPI drug use appears to be associated with reduced hPCFT expression in vivo.

Validation of the Rockall scoring system for outcomes from non-variceal upper gastrointestinal bleeding in a Canadian setting.

AIM: To validate the Rockall scoring system for predicting outcomes of rebleeding, and the need for a surgical procedure and death. METHODS: We used data extracted from the Registry of Upper Gastrointestinal Bleeding and Endoscopy including information of 1869 patients with non-variceal upper gastrointestinal bleeding treated in Canadian hospitals. Risk scores were calculated and used to classify patients based on outcomes. For each outcome, we used chi2 goodness-of-fit tests to assess the degree of calibration, and built receiver operating characteristic curves and calculated the area under the curve (AUC) to evaluate the discriminative ability of the scoring system. RESULTS: For rebleeding, the chi2 goodness-of-fit test indicated an acceptable fit for the model [chi2 (8) = 12.83, P = 0.12]. For surgical procedures [chi2 (8) = 5.3, P = 0.73] and death [chi2 (8) = 3.78, P = 0.88], the tests showed solid correspondence between observed proportions and predicted probabilities. The AUC was 0.59 (95% CI: 0.55-0.62) for the outcome of rebleeding and 0.60 (95% CI: 0.54-0.67) for surgical procedures, representing a poor discriminative ability of the scoring system. For the outcome of death, the AUC was 0.73 (95% CI: 0.69-0.78), indicating an acceptable discriminative ability. CONCLUSION: The Rockall scoring system provides an acceptable tool to predict death, but performs poorly for endpoints of rebleeding and surgical procedures.

Intestinal CYP3A4 and midazolam disposition in vivo associate with VDR polymorphisms and show seasonal variation.

Vitamin D, whose levels vary seasonally with sunlight, is activated to 1α,25-dihydroxyvitamin D(3) that binds the vitamin D receptor (VDR) and transcriptionally regulates intestinal CYP3A4 expression. We genotyped VDR polymorphisms and determined their associations with intestinal CYP3A4 and with midazolam pharmacokinetics, and whether intestinal CYP3A4 levels/activity varied seasonally. The VDR BsmIG > A (rs1544410) polymorphism was significantly associated with CYP3A4 jejunal expression/activity, with CYP3A4 duodenal mRNA, and with midazolam area under the curve (AUC). Intestinal CYP3A4 expression/activity was significantly higher in biopsies with the VDR promoter polymorphisms Cdx2-3731G > A and GATA-1012A > G that increase VDR activation of target genes. Duodenal CYP3A4 mRNA was significantly higher between April and September than between October and March. Midazolam p.o. AUC and oral bioavailability trended higher October through March compared to April through September. These data suggest VDR polymorphisms are predictors of intestinal CYP3A4, and that CYP3A4 intestinal expression varies seasonally--likely related to annual changes in UV sunlight and vitamin D levels.

Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe.

Breast cancer resistance protein (BCRP) is an efflux transporter expressed in tissues that act as barriers to drug entry. Given that single nucleotide polymorphisms (SNPs) in the ABCG2 gene encoding BCRP are common, the possibility exists that these genetic variants may be a determinant of interindividual variability in drug response. The objective of this study is to confirm the human BCRP-mediated transport of sulfasalazine in vitro, evaluate interindividual variation in BCRP expression in human intestine and to determine the role of ABCG2 SNPs to drug disposition in healthy patients using sulfasalazine as a novel in vivo probe. To evaluate these objectives, pinch biopsies were obtained from 18 patients undergoing esophagogastro-duodenoscopy or colonoscopy for determination of BCRP expression in relation to genotype. Wild-type and variant BCRP were expressed in a heterologous expression system to evaluate the effect of SNPs on cell-surface trafficking. A total of 17 healthy individuals participated in a clinical investigation to determine the effect of BCRP SNPs on sulfasalazine pharmacokinetics. In vitro, the cell surface protein expression of the common BCRP 421 C>A variant was reduced in comparison with the wild-type control. Intestinal biopsy samples revealed that BCRP protein and mRNA expression did not significantly differ between patients with 34GG/421CC versus patients with 34GG/421CA genotypes. Remarkably, in subjects with 34GG/421CA genotype, sulfasalazine area under the concentration-time curve was 2.4-fold greater compared with 34GG/421CC subjects (P<0.05). This study links commonly occurring SNPs in BCRP with significantly increased oral sulfasalazine plasma exposure in humans. Accordingly, sulfasalazine may prove to have utility as in vivo probe for assessing the clinical impact of BCRP for the disposition and efficacy of drugs.