Genetic Influences in Non-alcoholic Fatty Liver Disease in Obese Children
Date Issued
2011
Date
2011
Author(s)
Lin, Yu-Cheng
Abstract
Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of conditions ranging from simple hepatic steatosis to potentially fatal nonalcoholic steatohepatitis and cirrhosis. Concurrent with the epidemic of childhood obesity, pediatric NAFLD is a growing global problem. The pathogenesis of NAFLD is multifactorial; it is strongly associated with obesity and insulin resistance. Not all children with obesity will develop NAFLD, suggesting that genetic factors may contribute to the NAFLD susceptibility.
NAFLD is a complex metabolic disease that is strongly associated with obesity and insulin resistance. Day and James proposed a two-hit hypothesis. Fat accumulation in the liver is the first hit. Additional hits, including oxidative stress, lipotoxicity, adipocytokines, etc., are potential mediators in inducing persistent liver injury. Candidate genes studied in our studies were selected based on the two-hit hypothesis.
With regard to the genetic susceptibility to the first hit, PNPLA3 rs738409 single nucleotide polymorphism (SNP), causing a nonsynonymous sequence variation I148M, had a strong association with increased liver fat content in adults. In our study, we hypothesized that the presence of the PNPLA3 rs738409 G allele would increase the susceptibility of NAFLD in obese Taiwanese children. We recruited a total of 520 obese children aged 6-18 years. Their PNPLA3 rs738409 genotypes-CC, CG, or GG-were detected by the 5''-nuclease assay. The effects of the PNPLA3 rs738409 G allele on pediatric NAFLD were evaluated based on liver ultrasonography findings and mean serum alanine aminotransferase levels in these children. We found that NAFLD was present in 19.6% of the obese children. In comparison to the subjects with CC alleles, the risk of NAFLD was increased by 2.96-fold (95% confidence interval, 1.57-5.59, P = 0.0008) in the subjects with CG alleles and by 5.84-fold (95% confidence interval, 2.59-13.16; P < 0.0001) in those with GG alleles. Variant PNPLA3 rs738409 genotypes were associated with increases in mean serum alanine aminotransferase level of 4.77 IU/L (P = 0.0435) in subjects with CG alleles and of 10.86 IU/L (P < 0.0001) in those with GG alleles compared with subjects with CC alleles. In conclusion, the variant PNPLA3 rs738409 genotypes increased the risk of NAFLD in our population of obese Taiwanese children. The effect of the G allele on pediatric NAFLD followed a dominant genetic model.
With regard to the genetic susceptibility to the second hit, oxidative stress plays an important role in the pathogenesis of NAFLD. Variants in the UGT1A1 gene contribute to increased bilirubin levels, and bilirubin can act as an antioxidant. In our study, we hypothesized that variant UGT1A1 genotypes would reduce the risk for NAFLD development. We recruited 234 obese children 6 to 13 years of age. 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. We found that 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 = 0.021). 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 (OR) of 0.31 [95% confidence interval (CI): 0.11-0.91; P = 0.033], but variant UGT1A1*28 genotypes were not significantly associated with the occurrence of NAFLD. In conclusion, 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.
The heritability of pediatric NAFLD, as estimated by family aggregation study, is high. We genotyped 24 selected SNPs in 11 NAFLD-related candidate genes which may be involved in the first hit or second hit on the pathogenesis of NAFLD . We examined the associations between SNPs and the risk of pediatric NAFLD in obese children with conditioning on the effect of PNPLA3 rs738409 polymorphism. We selected these 24 SNPs by a pathway-driven approach, including autophagy (ATG16L1, PIK3C3, IRGM), toll-like receptor (CD14, TOLLIP), inflammatory (TNF-α), fatty acid metabolism (PPARG, PPARGC1A), and adiponectin signaling (ADIPOQ, ADIPOR1, ADIPOR2) pathways. SNPs were chosen based on minor allele frequency higher than 5% among Han Chinese. NAFLD was determined by liver ultrasonography. Associations between SNPs and pediatric NAFLD were examined using multiple logistic regression models. We found that a total of 95 cases and 91 controls were studied. The two groups matched each other in terms of age, gender and body mass index. With conditioning on the effects of waist circumference, triglyceride, adiponectin and PNPLA3 rs738409 polymorphism, one PPARGC1A SNP (rs8192678) was significantly associated with an increased risk for pediatric NAFLD OR, 2.21; 95% CI, 1.04–4.69; P=0.0389) and one TNF-α SNP (rs1799964) was significantly associated with a decreased risk for pediatric NAFLD (OR, 0.49; 95% CI, 0.24–0.99; P=0.0487). We further genotyped all 520 participants and analyzed the effect of PPARGC1A rs8192678 SNP variants on pediatric NAFLD. With conditioning on the effects of gender, adujsted BMI, waist circumference, adiponectin and PNPLA3 rs738409 polymorphism, variant PPARGC1A rs819267 genotypes increased the risk for pediatric NAFLD by 90% (OR, 1.9; 95% CI, 1.02–3.57; P=0.045). In conclusion, the variant PPARGC1A rs8192678 and TNF-α rs1799964 genotypes significantly modified the risk of NAFLD independent of the effect of PNPLA3 rs738409 polymorphism in our population of obese Taiwanese children.
Excessive cholesterol accumulation in the liver is involved in the deterioration of NAFLD. The underlying mechanism was unknown. We used an oleic acid-induced steatotic Huh7 cell model to investigate the changes in the expression levels of key cholesterol metabolism genes. We found that the mRNA expression of HMG-CoA reductase was increased by 19% in 0.5 mM oleic acid treated steatotic Huh7 cells (P = 0.024). HMG-CoA reductase is the rate-limiting key enzyme in the regulation of cholesterol biosynthesis in liver. We proved that the excessive cholesterol accumulation in the NAFLD liver is induced by increased expression of HMG-CoA reductase gene. Our results suggest that cholesterol is a possible lipid mediator linking the first hit and the second hit on the pahtogenesis of NAFLD.
In summary, we focused on finding associations between pediatric NAFLD and variants in candidate genes encoding proteins involved in NAFLD pathogenesis. Our findings may contribute to the understanding of the genetic susceptibility to pediatric NAFLD. Considering the lack of data in Asian children, the results of our genetic association studies in obese Taiwanese children could be valuable for global comparisons with data from other ethnic groups, including Hispanic, African, and North American or European origin.
Subjects
children
non-alcoholic fatty liver disease
cholesterol
steatotic cell model
SDGs
Type
thesis
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