Contribution of ADD3 and the HLA Genes to Biliary Atresia Risk in Chinese.

Nonsyndromic biliary atresia (BA) is a rare polygenic disease, with autoimmunity, virus infection and inflammation thought to play roles in its pathogenesis. We conducted a genome-wide association study in 336 nonsyndromic BA infants and 8900 controls. Our results validated the association of rs17095355 in ADD3 with BA risk (odds ratio (OR) = 1.70, 95% confidence interval (95% CI) = 1.49-1.99; p = 4.07 × 10-11). An eQTL analysis revealed that the risk allele of rs17095355 was associated with increased expression of ADD3. Single-cell RNA-sequencing data and immunofluorescence analysis revealed that ADD3 was moderately expressed in cholangiocytes and weakly expressed in hepatocytes. Immuno-fluorescent staining showed abnormal deposition of ADD3 in the cytoplasm of BA hepatocytes. No ADD3 auto-antibody was observed in the plasma of BA infants. In the HLA gene region, no variants achieved genome-wide significance. HLA-DQB1 residue Ala57 is the most significant residue in the MHC region (OR = 1.44, 95% CI = 1.20-1.74; p = 1.23 × 10-4), and HLA-DQB1 was aberrantly expressed in the bile duct cells. GWAS stratified by cytomegalovirus (CMV) IgM status in 87 CMV IgM (+) BA cases versus 141 CMV IgM (-) BA cases did not yield genome-wide significant associations. These findings support the notion that common variants of ADD3 account for BA risk. The HLA genes might have a minimal role in the genetic predisposition of BA due to the weak association signal. CMV IgM (+) BA patients might not have different genetic risk factor profiles compared to CMV IgM (-) subtype.

Biallelic ANKS6 null variants cause notable extrarenal phenotypes in a nephronophthisis patient and lead to hepatobiliary abnormalities by YAP1 deficiency.

The ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) gene, encoding an inversin compartment protein of the primary cilium, was recently reported as a pathogenic gene of nephronophthisis (MIM PS256100). Extrarenal manifestations are frequently observed in this disease, however, potential genotype-phenotype correlations and the underlying mechanisms remain poorly understood. Here we described an infant with kidney failure, hepatobiliary abnormalities, and heart disease, in whom whole exome sequencing identified compound heterozygous variants in ANKS6, including a novel nonsense variant p.Trp458* and a recurrent splicing variant c.2394+1G > A. mRNA expression studies showed that the splicing variant caused aberrant mRNA splicing with exon 13 skipping and the biallelic variants were predicted to cause loss of ANKS6 function. We systematically characterized the clinical and genetic spectra of the disease and revealed that biallelic null variants in ANKS6 cause more severe kidney disease and more extrarenal manifestations, thus establishing a clear genotype-phenotype correlation for the disease. Further evaluations showed that ANKS6 deficiency reduced YAP1 expression in the patient's bile duct epithelium and ANKS6 promotes YAP1 transcriptional activity in a dose-dependent manner, indicating that loss of ANKS6 function causes hepatobiliary abnormalities through YAP1 deficiency during biliary morphogenesis and development, which may offer new therapeutic targets.

Association analysis and functional follow-up identified common variants of JAG1 accounting for risk to biliary atresia.

Background: Biliary atresia (BA) is a destructive, obliterative cholangiopathy characterized by progressive fibro-inflammatory disorder and obliteration of intra- and extrahepatic bile ducts. The Jagged1 (JAG1) gene mutations have been found in some isolated BA cases. We aim to explore the association of common variants in JAG1 with isolated BA risk in the Chinese Han population. Methods: We genotyped 31 tag single nucleotide polymorphisms covering the JAG1 gene region in 333 BA patients and 1,665 healthy controls from the Chinese population, and performed case-control association analysis. The expression patterns of JAG1 homologs were investigated in zebrafish embryos, and the roles of jag1a and jag1b in biliary development were examined by morpholino knockdown in zebrafish. Results: Single nucleotide polymorphisms rs6077861 [P Allelic = 1.74 × 10-4, odds ratio = 1.78, 95% confidence interval: 1.31-2.40] and rs3748478 (P Allelic = 5.77 × 10-4, odds ratio = 1.39, 95% confidence interval: 1.15-1.67) located in the intron region of JAG1 showed significant associations with BA susceptibility. The JAG1 homologs, jag1a and jag1b genes were expressed in the developing hepatobiliary duct of zebrafish, especially at 72 and 96 h postfertilization. Knockdown of both jag1a and jag1b led to poor biliary secretion, sparse intrahepatic bile duct network and smaller or no gallbladders compared with control embryos in the zebrafish model. Conclusion: Common genetic variants of JAG1 were associated with BA susceptibility. Knockdown of JAG1 homologs led to defective intrahepatic and extrahepatic bile ducts in zebrafish. These results suggest that JAG1 might be implicated in the etiology of BA.

Intestinal Reg4 deficiency confers susceptibility to high-fat diet-induced liver steatosis by increasing intestinal fat absorption in mice.

Background & Aims: Regenerating gene family member 4 (REG4) is a novel marker for enteroendocrine cells and is selectively expressed in specialised enteroendocrine cells of the small intestine. However, the exact roles of REG4 are largely unknown. In this study we investigate the effects of REG4 on the development of dietary fat-dependent liver steatosis and the mechanisms involved. Methods: Mice with intestinal-specific Reg4 deficiency (Reg4 ΔIEC ) and Reg4-floxed alleles (Reg4 fl/fl ) were generated to investigate the effects of Reg4 on diet-induced obesity and liver steatosis. Serum levels of REG4 were also measured in children with obesity using ELISA. Results: Reg4 ΔIEC mice fed a high-fat diet demonstrated significantly increased intestinal fat absorption and were prone to obesity and hepatic steatosis. Importantly, Reg4 ΔIEC mice exhibit enhanced activation of adenosine monophosphate-activated protein kinase (AMPK) signalling and increased protein abundance of the intestinal fat transporters, as well as enzymes involved in triglyceride synthesis and packaging at the proximal small intestine. Moreover, REG4 administration reduced fat absorption, and decreased the expression of intestinal fat absorption-related proteins in cultured intestinal cells possibly via the CaMKK2-AMPK pathway. Serum REG4 levels were markedly lower in children with obesity with advanced liver steatosis (p <0.05). Serum REG4 levels were inversely correlated with levels of liver enzymes, homeostasis model assessment of insulin resistance, low-density lipoprotein cholesterol, and triglycerides. Conclusions: Our findings directly link Reg4 deficiency with increased fat absorption and obesity-related liver steatosis, and suggest that REG4 may provide a potential target for prevention and treatment of liver steatosis in children. Impact and Implications: Hepatic steatosis is a key histological feature of non-alcoholic fatty liver disease, which is the leading chronic liver disease in children leading to the development of metabolic diseases; however, little is known about mechanisms induced by dietary fat. Intestinal REG4 acts as a novel enteroendocrine hormone reducing high-fat-diet-induced liver steatosis with decreasing intestinal fat absorption. REG4 may be a novel target for treatment of paediatric liver steatosis from the perspective of crosstalk between intestine and liver.

Up-Regulation of microRNA-424 Causes an Imbalance in AKT Phosphorylation and Impairs Enteric Neural Crest Cell Migration in Hirschsprung Disease.

Insights into the role of microRNAs (miRNAs) in disease pathogenesis have made them attractive therapeutic targets, and numerous miRNAs have been functionally linked to Hirschsprung disease (HSCR), a life-threatening genetic disorder due to defective migration, proliferation, and colonization of enteric neural crest cells (ENCCs) in the gut. Recent studies have demonstrated that miR-424 strongly inhibits migration in a variety of cell types and its potential target RICTOR is essential for neural crest cell development. We therefore sought to interrogate how miR-424 and RICTOR contribute to the pathogenesis of HSCR. We utilized HSCR cases and human neural cells to evaluate the miR-424-mediated regulation of RICTOR and the downstream AKT phosphorylation. We further developed an ex vivo model to assess the effects of miR-424 on ENCC migration and proliferation. Then, single-cell atlases of gene expression in both human and mouse fetal intestines were used to determine the characteristics of RICTOR and AKT expression in the developing gut. Our findings demonstrate that miR-424 levels are markedly increased in the colonic tissues of patients with HSCR and that it regulates human neural cell migration by directly targeting RICTOR. Up-regulation of miR-424 leads to decreased AKT phosphorylation levels in a RICTOR-dependent manner, and this, in turn, impairs ENCC proliferation and migration in the developing gut. Interestingly, we further identified prominent RICTOR and AKT expressions in the enteric neurons and other types of enteric neural cells in human and mouse fetal intestines. Our present study reveals the role of the miR-424/RICTOR axis in HSCR pathogenesis and indicates that miR-424 is a promising candidate for the development of targeted therapies against HSCR.

Loss-of-function variants within LMOD1 actin-binding site 2 cause pediatric intestinal pseudo-obstruction by impairing protein stability and actin nucleation.

The leiomodin1 (LMOD1) gene, encoding a potent actin nucleator, was recently reported as a potential pathogenic gene of megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS, OMIM 619362). However, only a single patient has been reported to have LMOD1 mutations, and the underlying pathogenic mechanism remains unknown. Here, we described a male infant with LMOD1 mutations presenting typical symptoms of pediatric intestinal pseudo-obstruction (PIPO) but without megacystis and microcolon. Two compound heterozygous missense variants (c.1106C>T, p.T369M; c.1262G>A, p.R421H) were identified, both affecting highly conserved amino acid residues within the second actin-binding site (ABS2) domain of LMOD1. Expression analysis showed that both variants resulted in significantly reduced protein amounts, especially for p.T369M, which was almost undetectable. The reduction was only partially rescued by the proteasome inhibitor MG-132, indicating that there might be proteasome-independent pathways involved in the degradation of the mutant proteins. Molecular modeling showed that variant p.T369M impaired the local protein conformation of the ABS2 domain, while variant p.R421H directly impaired the intermolecular interaction between ABS2 and actin. Accordingly, both variants significantly damaged LMOD1-mediated actin nucleation. These findings provide further human genetic evidence supporting LMOD1 as a pathogenic gene underlying visceral myopathy including PIPO and MMIHS, strengthen the critical role of ABS2 domain in LMOD1-mediated actin nucleation, and moreover, reveal an unrecognized role of ABS2 in protein stability.

Association Analysis of Variants of DSCAM and BACE2 With Hirschsprung Disease Susceptibility in Han Chinese and Functional Evaluation in Zebrafish.

Hirschsprung disease (HSCR) has a higher incidence in children with Down syndrome (DS), which makes trisomy 21 a predisposing factor to HSCR. DSCAM and BACE2 are close together on the HSCR-associated critical region of chromosome 21. Common variants of DSCAM and rare variants of BACE2 were implicated to be associated with sporadic HSCR. However, the submucosal neuron defect of DS mouse model could not be rescued by normalization of Dscam. We aimed to explore the contribution of DSCAM and BACE2 to the development of the enteric nervous system (ENS) and HSCR susceptibility. We genotyped 133 tag single-nucleotide polymorphisms (SNPs) in DSCAM and BACE2 gene region in 420 HSCR patients and 1,665 controls of Han Chinese. Expression of DSCAM and BACE2 homologs was investigated in the developing gut of zebrafish. Overexpression and knockdown of the homologs were performed in zebrafish to investigate their roles in the development of ENS. Two DSCAM SNPs, rs430255 (P Addtive = 0.0052, OR = 1.36, 95% CI: 1.10-1.68) and rs2837756 (P Addtive = 0.0091, OR = 1.23, 95% CI: 1.05-1.43), showed suggestive association with HSCR risk. Common variants in BACE2 were not associated with HSCR risk. We observed dscama, dscamb, and bace2 expression in the developing gut of zebrafish. Knockdown of dscama, dscamb, and bace2 caused a reduction of enteric neurons in the hindgut of zebrafish. Overexpression of DSCAM and bace2 had no effects on neuron number in the hindgut of zebrafish. Our results suggested that common variation of DSCAM contributed to HSCR risk in Han Chinese. The dysfunction of both dscams and bace2 caused defects in enteric neuron, indicating that DSCAM and BACE2 might play functional roles in the occurrence of HSCR. These novel findings might shed new light on the pathogenesis of HSCR.

Common variation of the NSD1 gene is associated with susceptibility to Hirschsprung's disease in Chinese Han population.

BACKGROUND: Hirschsprung's disease (HSCR) is the most common congenital cause of intestinal obstruction in children. Sotos syndrome (SoS) is an overgrowth disorder with constipation and sometimes accompanied by HSCR. NSD1 gene mutation is the main cause of SoS. We aimed to investigate association of NSD1 common single nucleotide polymorphisms (SNPs) with HSCR susceptibility in Chinese Han population. METHOD: We genotyped 15 SNPs encompassing NSD1 gene region in 420 HSCR patients and 1665 controls on Fludigm EP1 platform. Association analysis was performed between cases and controls. RESULT: Rs244709 was the most associated SNP with HSCR susceptibility of the sample set (PAllelic = 9.69 × 10-5, OR = 1.37, 95% CI: 1.17-1.61). Gender stratification analysis revealed that NSD1 SNPs were associated with HSCR in males, but not in females. The nonsynonymous coding SNP rs28932178 in NSD1 exon 5 represented the most significant signal in males (PAllelic = 6.43 × 10-5, OR = 1.42, 95% CI: 1.20-1.69). The associated SNPs were expression quantitative trait loci (eQTLs) of nearby genes in multiple tissues. NSD1 expression levels were higher in aganglionic colon tissues than ganglionic tissues (P = 3.00 × 10-6). CONCLUSION: NSD1 variation conferred risk to HSCR in males, indicating SoS and HSCR may share common genetic factors. IMPACT: This is the first study to reveal that NSD1 variation conferred risk to Hirschsprung's disease susceptibility in males of Chinese Han population, indicating Sotos syndrome and Hirschsprung's disease may share some common genetic background. This study indicates more attention should be paid to the symptom of constipation in patients with Sotos syndrome. Our results raise questions about the role of NSD1 in the development of enteric nervous system and the pathogenesis of Hirschsprung's disease.

Association of Variants in PLD1, 3p24.1, and 10q11.21 Regions With Hirschsprung's Disease in Han Chinese Population.

Background and Aims: Hirschsprung's disease (HSCR) is a rare genetically heterogeneous congenital disorder. A recent study based on whole genome sequencing demonstrated that common variants at four novel loci, which contained two intronic variants on CASQ2 and PLD1, and intergenic variants located between SLC4A7 and EOMES at 3p24.1, and between LINC01518 and LOC283028 at 10q11.21, were associated with HSCR susceptibility. To validate these associations with HSCR susceptibility, we performed a case-control study in a Han Chinese sample set. Methods: We selected four previously identified single nucleotide polymorphisms (SNPs) for replication, along with tag SNPs to cover the four associated regions. In total, 61 SNPs were genotyped in 420 HSCR patients and 1,665 healthy controls from the Han Chinese population. Results: None of the 14 tag SNPs in the CASQ2 gene region, including the previously associated rs9428225, showed an association with HSCR. Among the 24 tag SNPs from the SLC4A7-EOMES region at 3p24.1, rs2642925 [odds ratio (OR) = 1.41, 95% confidence interval (95% CI) = 1.10-1.79; P Additive = 0.007] and the previously associated SNP rs9851320 showed a suggestive association (OR = 1.22, 95% CI = 1.01-1.47; P Additive = 0.042). A non-synonymous SNP, rs2287579, in PLD1 showed a suggestive association with HSCR susceptibility (OR = 1.71, 95% CI = 1.18-2.46; P Additive = 0.004). Additionally, the previously associated PLD1 SNP rs12632766 showed a suggestive significance (OR = 1.20, 95% CI = 1.01-1.42, P Additive = 0.038). In the LINC01518-LOC283028 region at 10q11.21, three SNPs meet the study-wide significance threshold. Rs17153309 was the most associated SNP (OR = 1.60, 95% CI = 1.34-1.90; P Additive = 1.13 × 10-7). The previously associated SNP rs1414027 also showed significant association (OR = 1.43, 95% CI = 1.20-1.70, P Additive = 3.92 × 10-5). Two associated SNPs at 10q11.21 (rs1414027 and rs624804) were expression quantitative trait loci in digestive tract tissues from GTEx databases. Conclusions: Our results confirmed that variants of the LINC01518-LOC283028 region were associated with HSCR in the Han Chinese population. Additionally, the susceptibility of SNPs in the LINC01518-LOC283028 region were associated with the expression levels of nearby genes. These results provide new insight into the pathogenesis of HSCR.

Common variants of NRG1 and ITGB4 confer risk of Hirschsprung disease in Han Chinese population.

BACKGROUND: Hirschsprung disease (HSCR) is a neurodevelopmental disorder with a strong genetic component. Common variants of NRG1 contributed to HSCR risk in Asians, and rare variants of ERBB2 and ITGB4 were found to be associated with HSCR. ERBB2 and ITGB4 are partners of Nrg1/ErbB pathway, which is important in HSCR pathogenesis. We aimed to investigate whether common variants in NRG1, ERBB2 and ITGB4 were associated with HSCR in Chinese Han population. METHODS: We genotype 17 single nucleotide polymorphisms (SNPs) of NRG1, ERBB2 and ITGB4 in 420 HSCR patients and 1665 controls, and performed association analysis. RESULTS: We validated associations of two NRG1 SNPs rs7835688 (PAllelic = 2.2 × 10-20, OR = 2.21, 95%CI = 1.86-2.62) and rs16879552 (PAllelic = 5.6 × 10-9, OR = 1.57, 95%CI = 1.35-1.83) with risk to HSCR. SNP rs3744000 located 5' upstream of ITGB4 showed association with HSCR (PAllelic = 2.4 × 10-3, OR = 1.27, 95%CI = 1.09-1.49). Four SNPs of ERBB2 exhibited no association. CONCLUSIONS: Our results suggested that common variation of ITGB4 and NRG1 conferred risk to HSCR in Chinese Han population, which further highlighted Nrg-1/ErbB pathway involving in the pathogenesis of HSCR.

Association of common variation in ADD3 and GPC1 with biliary atresia susceptibility.

Biliary atresia (BA) is an idiopathic neonatal cholestatic disease. Recent genome-wide association study (GWAS) revealed that common variation of ADD3, GPC1, ARF6, and EFEMP1 gene was associated with BA susceptibility. We aimed to evaluate the association of these genes with BA in Chinese population. Twenty single nucleotide polymorphisms (SNPs) in these four genes were genotyped in 340 BA patients and 1,665 controls. Three SNPs in ADD3 were significantly associated with BA, and rs17095355 was the top SNP (PAllele = 3.23×10-6). Meta-analysis of published data and current data indicated that rs17095355 was associated with BA susceptibility in Asians and Caucasians. Three associated SNPs were expression quantitative trait loci (eQTL) for ADD3. Two GPC1 SNPs in high linkage disequilibrium (LD) showed nominal association with BA susceptibility (PAllele = 0.03 for rs6707262 and PAllele = 0.04 for rs6750380), and were eQTL of GPC1. Haplotype harboring these two SNPs almost reached the study-wide significance (P = 0.0035). No association for ARF6 and EFEMP1 was found with BA risk in the current population. Our study validated associations of ADD3 and GPC1 SNPs with BA risk in Chinese population and provided evidence of epistatic contributions of genetic factors to BA susceptibility.

Genetic variants in RET, ARHGEF3 and CTNNAL1, and relevant interaction networks, contribute to the risk of Hirschsprung disease.

Hirschsprung disease (HSCR), the most common enteric neuropathy, stands as a model for complex genetic disorders. It has recently been demonstrated that both ARHGEF3 and CTNNAL1 map to the RET-dependent HSCR susceptibility loci. We therefore sought to explore whether genetic variants within RET, ARHGEF3 and CTNNAL1, and their genetic interaction networks are associated with HSCR. Taking advantage of a strategy that combined the MassArray system and gene-gene interaction analysis with case-control study, we interrogated 38 polymorphisms within RET, ARHGEF3 and CTNNAL1 in 1015 subjects (502 HSCR cases and 513 controls) of Han Chinese origin. There were statistically significant associations between 20 genetic variants in these three genes and HSCR. Haplotype analysis also revealed some significant global P values, i.e. RET_ rs2435357-rs752978-rs74400468-rs2435353-rs2075913-rs17028-rs2435355 (P = 3.79×10-58). Using the MDR and GeneMANIA platforms, we found strong genetic interactions among RET, ARHGEF3, and CTNNAL1 and our previously studied GAL, GAP43, NRSN1, PTCH1, GABRG2 and RELN genes. These results offer the first indication that genetic markers of RET, ARHGEF3 and CTNNAL1 and relevant genetic interaction networks confer the altered risk to HSCR in the Han Chinese population.

MicroRNA-4516-mediated regulation of MAPK10 relies on 3' UTR cis-acting variants and contributes to the altered risk of Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a life-threatening congenital disorder in which the enteric nervous system is completely missing from the distal gut. Recent studies have shown that miR-4516 markedly inhibits cell migration, and as one of its potential targets, MAPK10 functions as a modifier for developing HSCR. We thus aimed to evaluate the role of miR-4516 and MAPK10 in HSCR and how they contribute to the pathogenesis of HSCR. METHODS: We examined 13 genetic variants using the MassArray system in a case-control study (n=1015). We further investigated miR-4516-mediated regulation of MAPK10 in HSCR cases and human neural cells, the effects of cis-acting elements in MAPK10 on miR-4516-mediated modulation and cell migration process. RESULTS: Three positive 3' UTR variants in MAPK10 were associated with altered HSCR susceptibility. We also showed that miR-4516 directly regulates MAPK10 expression, and this regulatory mechanism is significantly affected by the 3' UTR cis-acting elements of MAPK10. In addition, knock-down of MAPK10 rescued the effect of miR-4516 on the migration of human neural cells. CONCLUSION: Our findings indicate a key role of miR-4516 and its direct target MAPK10 in HSCR risk, and highlight the general importance of cis- and posttranscriptional modulation for HSCR pathogenesis.

CELF1/p53 axis: a sustained antiproliferative signal leading to villus atrophy under total parenteral nutrition.

Intestinal villus atrophy is a major complication of total parenteral nutrition (TPN). Our previous study revealed that TPN-induced villus atrophy is accompanied by elevated expression of CUGBP, Elav-like family member 1 (CELF1); however, its mechanism of action has not been fully understood. Herein, we report a pivotal role of CELF1/p53 axis, which induces a sustained antiproliferative signal, leading to suppressed proliferation of intestinal epithelial cells (IECs). By using a rat model of TPN, we found synchronous upregulation of CELF1 and p53 in jejunum mucosa, accompanied by a 51% decrease in crypt cell proliferation rate. By using HCT-116 cells as an IEC model in vitro, we found that the expression of CELF1 altered dynamically in parallel to proliferation rate, suggesting a self-adaptive expression pattern in IECs in vitro. Furthermore, ectopic overexpression of CELF1 elicited a significant antiproliferative effect in HCT-116, Caco-2, and IEC-6 cells, whereas knockdown of CELF1 elicited a significant proproliferative effect. Moreover, cell-cycle assay revealed that ectopic overexpression of CELF1 induced sustained G2 arrest and G1 arrest in HCT-116 and IEC-6 cells, respectively, which could be abolished by p53 silencing. Mechanistically, polysomal profiling and nascent protein analysis revealed that regulation of p53 by CELF1 was mediated through accelerating its protein translation in polysomes. Taken together, our findings revealed a sustained suppression of IEC proliferation evoked by CELF1/p53 axis, which may be a potential therapeutic target for the treatment of TPN-induced villus atrophy.-Yan, J.-K., Zhang, T., Dai, L.-N., Gu, B.-L., Zhu, J., Yan, W.-H., Cai, W., Wang, Y. CELF1/p53 axis: a sustained antiproliferative signal leading to villus atrophy under total parenteral nutrition.

Common genetic variants in GAL, GAP43 and NRSN1 and interaction networks confer susceptibility to Hirschsprung disease.

Hirschsprung disease (HSCR) is a severe multifactorial genetic disorder. Microarray studies indicated GAL, GAP43 and NRSN1 might contribute to the altered risk in HSCR. Thus, we focused on genetic variations in GAL, GAP43 and NRSN1, and the gene-gene interactions involved in HSCR susceptibility. We recruited a strategy combining case-control study and MassArray system with interaction network analysis. For GAL, GAP43 and NRSN1, a total of 18 polymorphisms were assessed in 104 subjects with sporadic HSCR and 151 controls of Han Chinese origin. We found statistically significant differences between HSCR and control groups at 5 genetic variants. For each gene, the haplotypes combining all polymorphisms were the most significant. Based on SNPsyn, MDR and GeneMANIA analyses, we observed significant gene-gene interactions among GAL, GAP43, NRSN1 and our previous identified RELN, GABRG2 and PTCH1. Our study for the first time indicates that genetic variants within GAL, GAP43 and NRSN1 and related gene-gene interaction networks might be involved in the altered susceptibility to HSCR in the Han Chinese population, which might shed more light on HSCR pathogenesis.

Soybean Oil-Based Lipid Emulsion Increases Intestinal Permeability of Lipopolysaccharide in Caco-2 Cells by Downregulation of P-Glycoprotein via ERK-FOXO 3a Pathway.

BACKGROUND AND AIMS: Elevated intestinal permeability of lipopolysaccharide (LPS) is a major complication for patients with parenteral nutrition (PN), but the pathogenesis is poorly understood. Intestinal P-glycoprotein (P-gp) is one of the efflux transporters that contribute to restricting the permeability of lipopolysaccharide via transcellular route. P-gp expression may be regulated by PN ingredients, and thus this study sought to investigate the effect of PN on the expression of P-gp and to elucidate the underlying mechanism in vitro. METHODS: Caco-2 cells were treated with PN ingredients. Changes in P-gp expression and function were determined and the role of ERK-FOXO 3a pathway was studied. Transport studies of FITC-lipopolysaccharide (FITC-LPS) across Caco-2 cell monolayers were also performed. RESULTS: Among PN ingredients, soybean oil-based lipid emulsion (SOLE) exhibited significant inhibitory effect on P-gp expression and function. This regulation was mediated via activation of ERK pathway with subsequent nuclear exclusion of FOXO 3a. Importantly, P-gp participated in antagonizing the permeation of FITC-LPS (apical to basolateral) across Caco-2 cell monolayers. SOLE significantly increased the permeability of FITC-LPS (apical to basolateral), which was associated with impaired P-gp function. CONCLUSIONS: The expression and function of intestinal P-gp is suppressed by SOLE in vitro.

CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function.

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.