Pi-Feng Chang

Genetic variants in GCKR and PNPLA3 confer susceptibility to nonalcoholic fatty liver disease in obese individuals

BACKGROUND A genome-wide association study identified variants in or near patatin-like phospholipase domain-containing-3 (PNPLA3), neurocan (NCAN), lysophospholipase-like 1 (LYPLAL1), glucokinase regulatory protein (GCKR), and protein phosphatase 1 regulatory subunit 3b (PPP1R3B) that were strongly associated with nonalcoholic fatty liver disease (NAFLD) in adults of European ancestry. OBJECTIVE We examined these genetic variants in obese children and tested whether their effects on NAFLD are significant in the Taiwanese Han Chinese population. DESIGN We genotyped PNPLA3 rs738409, NCAN rs2228603, LYPLAL1 rs12137855, GCKR rs780094, and PPP1R3B rs4240624 in 797 obese children aged 7-18 y. NAFLD was identified by liver ultrasonography. We analyzed the effect of these genetic variants on NAFLD. RESULTS NAFLD was identified in 24% of the recruited obese children. We found significant associations with NAFLD at variants in PNPLA3 and GCKR but not in NCAN, LYPLAL1, and PPP1R3B. Multiple logistic regression analysis showed that, after control for the effects of age- and sex-adjusted body mass index, waist-to-hip ratio, sex, and PNPLA3 rs738409 polymorphism, the variant GCKR rs780094 TT genotype independently increased the OR of NAFLD by 1.997 (95% CI: 1.196, 3.335; P = 0.008) compared with the CC genotype. Subjects with the variant GCKR rs780094 TT genotype had a higher mean serum alanine aminotransferase concentration than did those with the CC genotype (30.8 ± 34.7 compared with 22.2 ± 18.6 IU/L; P = 0.01). CONCLUSIONS By studying the genetic variants of obese Taiwanese children, we confirmed that the genetic variants in GCKR rs780094 and PNPLA3 rs738409, but not in NCAN rs2228603, LYPLAL1 rs12137855, and PPP1R3B rs4240624, are associated with an increased risk of NAFLD. GCKR and PNPLA3 variants are the common genetic factors that may confer susceptibility to NAFLD in obese individuals across multiple ethnic groups.

Variants in the UGT1A1 Gene and the Risk of Pediatric Nonalcoholic Fatty Liver Disease

OBJECTIVE: Oxidative stress is increased in nonalcoholic fatty liver disease (NAFLD). Variants in the UGT1A1 gene contribute to increased bilirubin levels, and bilirubin can act as an antioxidant. We hypothesize that variant UGT1A1 genotypes reduce the risk for NAFLD development. METHODS: Two hundred thirty-four obese children 6 to 13 years of age were recruited. NAFLD was determined through liver ultrasonography. The UGT1A1 genotypes UGT1A1*6 and UGT1A1*28 were detected. We assessed the effects of UGT1A1 genotypes on pediatric NAFLD. RESULTS: In total, 12% of the obese children had NAFLD. The subjects with NAFLD had lower serum total bilirubin levels (0.25 ± 0.30 mg/dL) than did those without NAFLD (0.36 ± 0.38 mg/dL; P = .021). With conditioning on the effects of age- and gender-adjusted BMI, waist/hip ratio, and adiponectin levels, variant UGT1A1*6 genotypes were a protecting factor for NAFLD, with an estimated adjusted odds ratio of 0.31 (95% confidence interval: 0.11–0.91; P = .033), but variant UGT1A1*28 genotypes were not significantly associated with the occurrence of NAFLD. CONCLUSIONS: Variant UGT1A1*6 genotypes are associated with a lower risk of NAFLD in obese Taiwanese children. The UGT1A1 genotype is a new risk factor for pediatric NAFLD.

Risk Factors for Liver Steatosis in Obese Children and Adolescents

BACKGROUND Concurrent with the recent rise of the incidence in obesity, nonalcoholic fatty liver disease is increasingly prevalent in childhood. The aim of this study was to identify non-invasive biomarkers for liver steatosis in obese children and adolescents. METHODS We used a cross-sectional study to examine risk factors for liver steatosis in obese children and adolescents. Sixty-nine obese subjects aged 6-17 years were recruited. The diagnosis of liver steatosis was made by liver ultrasonography. Anthropometric, serum biochemical variables, and oral glucose tolerance tests were measured. RESULTS Thirty-eight (55.1%) subjects had liver steatosis. Elevated alanine aminotransferase levels (> 30 IU/L in boys and >19 IU/L in girls) were found in 27 (71.1%) of the 38 subjects with liver steatosis. In multivariate logistic regression analysis, liver steatosis was associated with waist circumference and the change of plasma glucose level before and after oral glucose tolerance testing (C-OGTT). For every 5 cm increase in waist circumference, there was an odds ratio of 1.391 for predicting liver steatosis (95% confidence interval: 1.009-1.916, p = 0.044). C-OGTT was the only laboratory variable that independently predicted liver steatosis, with an odds ratio of 1.198 (95% confidence interval: 1.022-1.404, p = 0.026) for each 5 mg/dL of increase. CONCLUSION In this hospital-based sample of obese children and adolescents, liver steatosis was common. Liver steatosis was positively associated waist circumference and C-OGTT. These findings have implications for screening liver steatosis in obese children and adolescents.

A common variant in the peroxisome proliferator–activated receptor-γ coactivator-1α gene is associated with nonalcoholic fatty liver disease in obese children

BACKGROUND The single nucleotide polymorphism in the peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α gene (PPARGC1A) was identified to be associated with nonalcoholic fatty liver disease (NAFLD) in adults. The PPARGC1A gene encodes PGC-1α, which regulates cellular energy metabolism. OBJECTIVE We aimed to test the hypothesis that the PPARGC1A rs8192678 risk A allele would influence the risk of NAFLD in obese children. DESIGN We genotyped PPARGC1A rs8192678 and PNPLA3 rs738409 in 781 obese children aged 7-18 y. NAFLD was determined by ultrasonography. We evaluated the independent influence of the PPARGC1A rs8192678 risk A allele on pediatric NAFLD after control for the effect of the PNPLA3 rs738409 polymorphism. RESULTS A total of 23.3% of the recruited obese children had NAFLD. PNPLA3 rs738409 increased the OR of NAFLD by 1.622 (95% CI: 1.071, 2.457; P = 0.023) in subjects with GC alleles and 2.659 (95% CI: 1.509, 4.686; P < 0.001) for GG alleles, as compared with CC alleles. After control for the effects of age- and sex-adjusted BMI, sex, and PNPLA3 rs738409 polymorphism, the PPARGC1A rs8192678 risk A allele was an independent risk factor for developing NAFLD, with an OR of 1.740 (95% CI: 1.149, 2.637; P = 0.009). Subjects with the PPARGC1A rs8192678 risk A allele had an increase in the mean serum alanine aminotransferase concentration of 5.2 IU/L, as compared with subjects without this allele (P = 0.011). CONCLUSION The PPARGC1A rs8192678 risk A allele is associated with an increased risk of NAFLD, independent of the effect of the PNPLA3 rs738409 polymorphism in our population of obese Taiwanese children.

Prolonged Unconjugated Hyperbiliriubinemia in Breast-fed Male Infants with a Mutation of Uridine Diphosphate-Glucuronosyl Transferase

OBJECTIVE To test the hypothesis that a mutation in uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1) gene of breast-fed infants is a contributory factor to prolonged unconjugated hyperbilirubinemia. STUDY DESIGN Of 125 breast-fed term infants, 35 infants had prolonged unconjugated hyperbilirubinemia; another 90 breast-fed neonates without prolonged jaundice were control infants. The polymerase chain reaction-restriction fragment length polymorphism method was used to detect the known variant sites (promoter area, nucleotides 211, 686, 1091, and 1456) of the UGT1A1 gene. RESULTS Of 35 breast-fed infants with prolonged unconjugated hyperbilirubinemia, 29 had at least 1 mutation of the UGT1A1 gene. Variation at nucleotide 211 was most common. The percentages of the neonates carrying the variant nucleotide 211 were significantly different between the prolonged hyperbilirubinemia group and control neonates. Male breast-fed infants had a higher risk than female infants for prolonged hyperbilirubinemia. CONCLUSIONS Male breast-fed neonates with a variant nucleotide 211 in UGT1A1 have a high risk for developing prolonged hyperbilirubinemia.

Differences of microbiota in small bowel and faeces between irritable bowel syndrome patients and healthy subjects

Abstract Objective Several studies suggested that colonic microbiota have impacts on irritable bowel syndrome (IBS) patients. However, the knowledge about the association of small intestine (SI) microbiota with IBS is limited. We aimed to investigate the gut microbiota composition of SI and stool in IBS patients. Materials and methods Biopsies of jejunum mucosa by balloon-assisted enteroscopy and faecal samples from 28 IBS patients and 19 healthy controls were analysed by next-generation sequencing method. Results The three major phyla in SI microbiota of case/control groups were Proteobacteria (32.8/47.7%), Bacteroidetes (25.2/15.3%), and Firmicutes (19.8/11.2%), and those of stool were Bacteroidetes (41.3/45.8%), Firmicutes (40.7/38.2%), and Proteobacteria (15.4/7.1%). Analysis based on the family level, IBS patients had a higher proportion of Veillonellaceae (mean proportion 6.49% versus 2.68%, p = 0.046) in stool than controls. Prevotellaceae was more abundant in IBS patients than in control group (14.27% versus 6.13%, p = 0.023), while Mycobacteriaceae (0.06% versus 0.17%, p = 0.024) and Neisseriaceae (6.40% versus 8.94%, p = 0.038) was less abundant in IBS patients’ jejunal mucosa than those in controls. This less abundant jejunal Neisseriaceae was associated with more severe IBS (p = 0.03). The ratio of Firmicutes to Bacteroidetes in the stool of IBS-diarrhoea type patients was approximately three-fold higher, and the ratio of Firmicutes to Actinobacter in SI of IBS-mixed type patients was about nine-fold higher than healthy subjects. Conclusion Higher abundance of colonic Veillonellaceae and SI Prevotellaceae, and lower amount of oral cavity normal flora in proximal SI were found in IBS patients. We may manipulate these bacteria in IBS patients in future studies (ClinicalTrial.gov Number NCT01679730).

Risk of Hyperbilirubinemia in Breast-Fed Infants

OBJECTIVE To investigate the risk factors for hyperbilirubinemia in infants who are exclusively breast-fed. STUDY DESIGN A prospective study was conducted to investigate the effects of birth body weight, sex, mode of delivery, glucose-6-phosphate dehydrogenase (G6PD) deficiency, variant UDP-glucuronosyltransferase 1A1 (UGT1A1) gene, and hepatic solute carrier organic anion transporter 1B1 (SLCO1B1) gene on hyperbilirubinemia in neonates who were breast-fed. Hyperbilirubinemia was diagnosed when a full term neonate had a bilirubin level ≧15.0 mg/dL (256.5 μM) in serum at 3 days old. The polymerase chain reaction-restriction fragment length polymorphism method was used as a means of detecting the known variant sites in the UGT1A1 and SLCO1B1 gene. RESULTS Of 252 infants born at term who were exclusively breast-fed, 59 (23.4%) had hyperbilirubinemia. The significant risk factors were a variant nucleotide 211 in UGT1A1 (2.48; 95% CI, 1.29 to 4.76; P = .006), G6PD deficiency (12.24; 95% CI, 1.08 to 138.62; P < .05), and vaginal delivery (3.55; 95% CI, 1.64 to 7.66; P < .001). CONCLUSION Breast-fed neonates who are 211 variants in the UGT1A1, G6PD deficiency, and vaginal delivery are at high-risk for hyperbilirubinemia.

Variants in the autophagy-related gene IRGM confer susceptibility to non-alcoholic fatty liver disease by modulating lipophagy

BACKGROUND & AIMS Autophagy has been shown to be crucial in the regulation of the intracellular lipid stores in hepatocytes. We hypothesize that immunity-related GTPase family M (IRGM) gene (an autophagy-related gene) variants confer the susceptibility to non-alcoholic fatty liver disease (NAFLD) development. METHODS 832 obese children and adolescents aged 6-18years were recruited. NAFLD was determined by liver ultrasonography. We genotyped PNPLA3 rs738409, GCKR rs780094, TM6SF2 rs58542926, six IRGM single nucleotide polymorphisms (rs13361189, rs9637876, rs72553867, rs10065172, rs1000113, and rs11747270). To understand the molecular mechanism, we examined the effects of IRGM knockdown and overexpression on autophagic flux and lipid droplet metabolism in human hepatoma cells. RESULTS 22.8% of recruited obese children and adolescents had NAFLD. Multiple logistic regression analysis revealed that after controlling for the effects of age- and gender-adjusted body mass index, gender, PNPLA3, GCKR, and TM6SF2 polymorphisms, variant IRGM rs10065172 TT genotype independently increased the odds ratio of NAFLD by 2.04 (95% confidence interval 1.22-3.42; p=0.007), as compared to the CC genotype. The predictive model was validated by means of 10-fold cross validation. Functional assay revealed that IRGM knockdown inhibited autophagic flux and increased lipid droplet content in HepG2 and PLC/PRF/5 cells, which were reversed by the autophagy inducer rapamycin administration. Similarly, wortmannin (an autophagy inhibitor) increased intracellular lipid droplet content. In contrast, overexpression of IRGM caused decreased lipid droplet content in HepG2 cells. CONCLUSIONS Our findings suggest that IRGM may contribute to the development of human NAFLD by altering hepatic lipid metabolism through the autophagy pathway. LAY SUMMARY Autophagy is involved in the process of lipid metabolism in hepatocytes. The mechanism of autophagy regulation by IRGM has just been unveiled. This study demonstrates that genetic variants in IRGM confer risk of human non-alcoholic fatty liver disease. The functional studies reveal how IRGM regulates hepatic lipid droplet content.

Identifying term breast-fed infants at risk of significant hyperbilirubinemia.

Background:The aim of this study was to establish a model to identify term breast-fed infants who are at risk of developing significant neonatal hyperbilirubinemia.Methods:A prospective study was designed to investigate the effects of birth weight, mode of delivery, cephalohematoma, glucose-6-phosphate dehydrogenase (G6PD) deficiency, predischarge total serum bilirubin, variant uridine 5’diphospho-glucuronosyltransferase 1A1 (UGT1A1) gene, and hepatic solute carrier organic anion transporter 1B1 (SLCO1B1) gene on significant hyperbilirubinemia in term breast-fed neonates. Significant hyperbilirubinemia was defined as a bilirubin level exceeding the hour-specific phototherapy treatment threshold recommended by the American Academy of Pediatrics in 2004.Results:Of 240 exclusively breast-fed term neonates, 26 (10.8%) had significant hyperbilirubinemia. The predischarge total serum bilirubin on the third day (odds ratio (OR) = 2.63; 95% confidence interval (CI): 1.87–3.70; P < 0.001) and the variant UGT1A1 gene at nucleotide 211 (OR = 5.00; 95% CI: 1.08–23.03; P < 0.05) were significant risk factors. The area under the receiver operating characteristic (ROC) curve of the predictive probability was 0.964 (95% CI: 0.932–0.984; P < 0.0001).Conclusion:Combining the total serum bilirubin on the third day and the variant UGT1A1 gene at nucleotide 211 can predict hyperbilirubinemia well in term breast-fed infants.

Heme oxygenase-1 gene promoter polymorphism and the risk of pediatric nonalcoholic fatty liver disease

Background and objectives:Oxidative stress and the insulin-resistant state are thought to be key components in the pathogenesis of pediatric nonalcoholic fatty liver disease (NAFLD). Heme oxygenase (HO) is important in the defense against oxidative stress. This study aimed to assess the association of HO-1 gene promoter polymorphism and insulin resistance with NAFLD among obese children.Methods:A total of 101 obese children aged 6–17 years were recruited. Anthropometric, serum biochemical variables and biomarkers for glucose and insulin metabolism were measured. We screened the allelic frequencies of (GT)n repeats in the HO-1 gene promoter among these obese children. NAFLD was determined through liver ultrasonography. Because the distribution of numbers of (GT)n repeats was bimodal, we divided the alleles into two classes: class S included shorter (27) repeats, and class L included longer (⩾27) repeats. We assessed the effects of the length of (GT)n repeats in HO-1 gene promoter on pediatric NAFLD.Results:Of the 101 obese subjects, 27 (26.7%) had NAFLD. The alanine aminotransferase level was higher in patients carrying L alleles (L/L and L/S) than patients with S alleles (S/S) (46.2±49.3 IU|−1 versus 30.2±20.1 IU|−1; P=0.027). The significant risk factors for pediatric NAFLD were patients carrying L alleles (L/L and L/S) (odds ratio (OR)=18.84; 95% confidence interval (CI): 1.45–245.22; P=0.025), homeostasis model assessment of insulin resistance (OR=1.40; 95% CI: 1.07–1.83; P=0.014) and age (OR=1.24; 95% CI: 1.03–1.50; P=0.025).Conclusion:In this hospital-based study, the obese children with longer GT repeats in the HO-1 gene promoter and insulin resistance were susceptible to NAFLD.