The Role Of Vitamin D Level And VDR Genetic Variation In The Risk Assessment Of Non-alcoholic Fatty Liver Disease

Section 1 The relationship between the level of 25-hydroxyvitamin D₃ and non-alcoholic fatty liver disease[Background]In recent years,the prevalence of non-alcoholic fatty liver disease（NAFLD）has increased rapidly,which has become the first chronic liver disease in China.In the early stage of NAFLD,patients often have no symptoms or feel slight discomfort.With the development of NAFLD,it can increase the risk of fibrosis,cirrhosis and HCC,and accelerate the development of hypertension,type 2 diabetes and metabolic syndrome.The pathogenesis of NAFLD has not been fully elucidated.The relationship between vitamin D（VD）and liver diseases such as NAFLD,viral hepatitis,cirrhosis and liver cancer has drawn increasing attention in recent years.It has been proved that VD level is related to the risk of NAFLD,but the results are still inconsistent.[Objectives]To explore the relationship between serum 25（OH）D₃level and the risk of NAFLD by comparing the serum 25-hydroxyvitamin D₃[25（OH）D₃]level between NAFLD and non-NAFLD groups,and the prevalence of NAFLD among different25（OH）D₃level groups,respectively.[Methods]A cross-sectional design was performed in this study.519 people who were followed up in a community hospital of Nanjing in July 2018 were included in the study,including 183 NAFLD patients and 336 non-NAFLD patients.A self-designed questionnaire was used to conduct a baseline survey of the subjects,and the serum 25（OH）D₃level was detected by enzyme-linked immunosorbent assay（ELISA）kit.[Results]There was significant difference in 25（OH）D₃level between NAFLD and non NAFLD groups（P<0.05）.Among all the subjects,297（57.2%）were deficient in VD,124（23.9%）were insufficient in VD and 98（18.9%）were sufficient in VD.There was significant difference in NAFLD prevalence among the three groups with different VD levels（P<0.05）,and the multiple comparison showed that the prevalence of NAFLD in VD deficiency was significantly higher than that of VD insufficiency and VD sufficiency(all P_Bonferroni<0.017),respectively.Pearson linear correlation analysis showed that age,body mass index（BMI）,direct bilirubin（DBIL）,triglyceride（TG）,high density lipoprotein cholesterol（HDL-C）and fasting blood glucose were correlated with 25（OH）D₃（all P<0.05）.Multiple liner regression analysis indicated that age was negatively correlated with 25（OH）D₃（P=0.001）,and HDL-C was positively correlated with 25（OH）D₃（P=0.003）.Logistic regression analysis showed that VD sufficiency（crude OR=0.464,95%CI=0.280-0.768,P=0.003）and VD insufficiency（crude OR=0.358,95%CI=0.220-0.580,P<0.001）had a significantly reduced risk of NAFLD compared to VD deficiency.With the increase of VD level,the risk of NAFLD decreased,showing a significant dose-response relationship(P_trend<0.001).After adjusting for gender,age,BMI and systolic blood pressure（model 2）,the risk of NAFLD in VD sufficiency（adjusted OR=0.568,95%CI=0.305-0.812,P=0.008）and VD insufficiency（adjusted OR=0.389,95%CI=0.214-0.708,P=0.002）were all significantly lower than VD deficiency,and the risk was decreasing significantly with the increase of VD level(P_trend=0.014).When adjusting for the above factors and DBIL,TG,HDL-C,fasting blood glucose,the risk of NAFLD still decreased significantly with the increase of VD level(VD sufficiency vs.VD deficiency:adjusted OR=0.610,95%CI=0.304-0.993,P=0.047;VD sufficiency vs.VD deficiency:adjusted OR=0.371,95%CI=0.186-0.741,P=0.005;P_trend=0.045).[Conclusions]VD deficiency was more common in this population and may associated with an increased risk of NAFLD.This may provide a theoretical basis for exploring the pathogenesis and prevention of NAFLD.Section 2The role of vitamin D receptor genetic variation in the risk assessment of non-alcoholic fatty liver disease[Background] The prevalence of NAFLD is increasing year by year and there is no specific treatment drug,which brings a high burden.Therefore,it is of great significance for early prevention and individualized health management of NAFLD to explore the risk factors and then to evaluate the risk of NAFLD and screen the high-risk population.It is believed that a combination of environmental exposure factors and genetic susceptibility factors contribute to the pathogenesis and development of NAFLD.A growing body of research suggests that VD is associated with the risk of NAFLD,cirrhosis and hepatocellular carcrinoma.Vitamin D receptor（VDR）,as a key gene in the metabolism of VD,can participate in the processes of glucose metabolism,bile acid metabolism,hepatitis,liver fibrosis and hepatocyte proliferation,which are all related to NAFLD.Therefore,we speculated that the genetic variation of VDR may be related to the development and progression of NAFLD by affecting VD level and its biological effect.It is necessary to study the combined effects of VDR genetic variation and environmental behavioral factors on NAFLD for further comprehensive risk assessment and individualized health management.[Objectives] To explore the association between VDR genetic variation and the risk of NAFLD and to provide reference for elucidating the genetic susceptibility mechanism and screening biomarkers for risk assessment of NAFLD.[Methods] A total of 2987 people from a community in Nanjing were enrolled in this case-control study,including 1097 NAFLD patients and 1890 controls matched by gender and age.Eight potential functional single nucleotide polymorphisms（SNPs）（rs3782905,rs3847987,rs11574129,rs2228570,rs11568820,rs739837,rs7975232,rs11168287）in VDR gene were genotyped using Taq Man allelic discrimination assay on the ABI 7900 HT Real-Time PCR System.The relationships between VDR SNPs and the risk of NAFLD were analyzed by co-dominant model,dominant model,recessive model,and additive model,respectively.We performed the receiver-operating characteristic（ROC）curve and computed area under the curve（AUC）to assess the predictive power of the combined effects of positive SNPs,environmental behavior factors and related indicators for NAFLD risk.[Results] After adjusting for gender and age,logistic regression analyses showed that VDR rs2228570-A variant（AA: adjusted OR=0.750,95%CI=0.650-0.903,P=0.009;dominant model: adjusted OR=0.844,95%CI=0.716-0.994,P=0.043;recessive model:adjusted OR=0.809,95%CI=0.672-0.973,P=0.025;additive model: adjusted OR=0.868,95%CI=0.780-0.965,P=0.009）and rs11168287-A variant（GA: adjusted OR=0.830,95%CI=0.706-0.974,P=0.023;dominant model: adjusted OR=0.849,95%CI=0.729-0.988,P=0.035）all significantly reduced the risk of NAFLD in different models.Further analyses of the combined effects of the significantly associated SNPs indicated that the more favorable alleles the subjects carried,the lower susceptibility to NAFLD,suggesting a significant locus-dosage effect of the combined genotyped on NAFLD risk(P_trend=0.028).Stratified analysis showed that the combined protective effect of VDR rs2228570-A and rs11168287-A was still prominent in subgroups of male（adjusted OR=0.857,95CI%=0.741-0.991,P=0.037）,subjects≤40 years（adjusted OR=0.802,95CI%=0.667-0.964,P=0.019）,non-hypertension（adjusted OR=0.846,95CI%=0.724-0.988,P=0.035）,non-hyperglycemia（adjusted OR=0.862,95CI%=0.750-0.992,P=0.039）and non-low HDL-C（adjusted OR=0.0.824,95CI%=0.693-0.980,P=0.028）.The heterogeneity test discovered no significant differences among all the subgroups（P>0.05）.Multiple stepwise regression analysis showed that gender,age,abdominal obesity,hypertension,high TG,low HDL-C,ALT,exercise time and rs2228570-rs11168287 protective allele were independent influencing factors for NAFLD.The three combined factors for assessing NAFLD risk were constructed based on the above factors,respectively.The combined factor 1 had higher predictive efficiency than the combined factor 3 in NAFLD risk assessment（combined factor 1 vs.3=0.788 vs.0.785,95%CI=0.001-0.006,P=0.014）.[Conclusions] Our results suggested that VDR variants（rs2228570-A and rs11168287-A）might contribute to a decreased susceptibility to NAFLD in Chinese Han population,and the combined factor that contained SNPs information and exercise time was more effective than the combined factor without both of them in predicting the risk of NAFLD,which may contribute to a more comprehensive risk assessment of NAFLD and health management.