Association Study Of KLB, FGF21 And FGFR Gene Polymorphisms With Non-alcoholic Fatty Liver Disease And Obesity In Han Population In East China

Background Non-alcoholic fatty liver disease(NAFLD)is characterized by excessive fat deposited in the liver in the form of triglycerides.Obesity is usually defined as abnormal and/or excessive accumulation of fat.With changes of eating patterns and physical activity patterns in social,the incidences of NAFLD and obesity are gradually increasing.The elements of the genes and the environments effect NAFLD.The incidence of NAFLD is significantly different among different ethnic groups in different regions,as well as among different ethnic groups in the same region.These suggest that the incidence of NAFLD is related to genetic factors.Current studies have identified that patstatin-like phospholipase domain-containing 3(PNPLA3)and transmembrane 6 superfamily member 2(TM6SF2)are associated with nonalcoholic steatohepatitis(NASH)occurrence and liver injury.However,the incidences of NAFLD and NASH must be jointly affected by multiple genetic factors.The gene polymorphisms that have been found now are far from able to explain the pathogenesis of NAFLD.Therefore,more single-nucleotide polymorphisms(SNPs)need to be studied to clarify the pathogenesis of NAFLD and search the valuable genetic targets.Klotho beta(KLB)gene is located in Chr4p14,encoding human Klotho beta(KLB)protein.KLB is a single-chain transmembrane protein.By binding Fibroblast growth factor FGF 21 and FGF19 to Fibroblast growth factor(FGFR),KLB plays a key role in regulating the metabolism of energe and the metabolism of substances in the liver.Therefore,FGF21-KLB-FGFR signaling system may be involved in the development and progression of NAFLD.Several studies have also found that FGF21 gene polymorphisms are associated with nutrient intake,and KLB gene polymorphisms are associated with coronary atherosclerotic heart disease and alcohol intake.So we speculated that the SNPs of FGF21,KLB and FGFR were related to NAFLD and obesity.Part 1 The association study of non-alcoholic fatty liver disease,obesity and KLB gene in Han Chinese residing in East ChinaObjective 1.To study the association between KLB gene polymorphisms and nonalcoholic fatty liver disease and obesity.2.To search the SNPs related to NAFLD and obesity,and the genetic targets related to drug therapy.Methods From January 2018 to October 2018,a total of 1688 subjects were recruited from the Department of Physical Examination Center,the Affiliated Hospital of Xuzhou Medical University.The subjects with history of significant alcohol consumption,recent long-term drug use and the patients with hepatitis B virus or hepatitis C virus were excluded.All subjects were divided into NAFLD group and Non-NAFLD group according to the ultrasound results of digestive system.According to BMI,the subjects were divided into the Obese group with BMI≥25kg/m2 and the Non-obese group with BMI<25kg/m2.Furthermore,the subjects were divided into Obese with NAFLD group,the Obese without NAFLD group,Non-obese with NAFLD group and Non-obese without NAFLD group.All subjects were measured height and weight on empty stomach,BMI was calculated and biochemical examination was performed.At the same time,whole blood samples were taken to separate genomic DNA,primers were designed,and the amplified product was detected by Matrix-assisted laser desorption ionization-Time of Flight(MALDI-TOF)mass spectrometer and the SNPs was analyzed.Plink 1.9 software was applied for the analysis of the associations,The genotype-phenotype association analyses were performed using three covariate-adjusted models(dominant,recessive and additive)with age and sex as random effects.Quantitative-traits association analyses were used to assess the interaction between the factors for NAFLD and the genotypes of each SNP through multiple linear regression analyses in all subjects.Results 1.The A-allele frequency of KLB SNP rs7670903 was higher in the obese than non-obese group(P = 0.0003)and correlated with higher BMI(P = 0.0005).2.The G-allele frequency of KLB rs7674434 and T-allele frequency of rs12152703 were higher in the obese with NAFLD than obese without NAFLD group in the dominant model P = 0.004 and P = 0.006 respectively,and in additive model P = 0.005 and P = 0.007 respectively.3.In the NAFLD group,the G-allele of rs7674434 and T-allele of rs12152703 still had strong positive correlations with ALT levels(P = 0.005 and P = 0.008,respectively)Conclusions 1.In the Chinese population residing East China,SNP rs767090 of KLB is strongly correlated with obesity,and the G allele of rs7670903 is the risk gene for obesity.2.In the obese population,SNPs rs7674434 and rs12152703 of KLB were strongly correlated with NAFLD,then the G allele of rs7674434 and T allele of rs12152703 were risk genes for NAFLD.3.Meanwhile,SNPs rs7674434 and rs12152703 of KLB were associated with liver inflammation in NAFLD patients.4.SNP rs12152703 of KLB is in complete linkage disequilibrium with rs7674434,they are ht SNPs of KLB.5.KLB gene polymorphisms are involved in the pathogenesis of NAFLD and may be therapeutic targets for NAFLD.Part 2 The association study of non-alcoholic fatty liver disease,obesity and FGF21,FGFR gene polymorphisms in Han Chinese residing in East ChinaObjective 1.To study the association between FGF21 and FGFR gene polymorphisms and non-alcoholic fatty liver disease and obesity.2.To search the SNPs related to NAFLD and obesity,and the genetic targets related to drug therapy.Methods The subjects were selected according to the inclusion and exclusion criteria and divided into groups according to the ultrasound results of digestive system and BMI.BMI of all subjects was calculated and underwent biochemical examination.Serum was taken from all subjects and serum FGF21 was detected by ELISA.At the same time,whole blood samples were taken to separate genomic DNA,primers were designed,and the amplified product was detected by a MALDI-TOF mass spectrometer and the correlations were analyzed.Results 1.In the NAFLD group,the C-allele frequency of FGF21 SNP rs838136 was significantly lower in the Obese with NAFLD group than in the Non-obese with NAFLD group(P=0.007).2.In the obese population,the C-allele frequency of FGF21 SNP rs838136 in the Obese with NAFLD group was significantly lower than that in the Obese without NAFLD group(P=0.045).3.The G allele of FGF21 SNP rs499765 was significantly positively correlated with the level of serum FGF21(P=0.049).Conclusions 1.FGF21 gene polymorphism is associated with obesity in NAFLD patients,and the C allele of SNP rs838136 may be a protective gene for obese risk in NAFLD patients.2.SNP rs499765 of FGF21 was correlated with serum FGF21 level.3.FGF21 and KLB gene polymorphisms were correlated with NAFLD in the population with the presence of environmental risk factors for obesity,suggesting that subsequent studies on FGF21 and KLB gene polymorphism need to be carried based on population stratification.