Correlation Between Physical Activity, Genome-wide Copy Number Variation And Non-alcoholic Fatty Liver Disease

Objective(1)To explore the relationship between physical activity and non-alcoholic fatty liver disease(NAFLD)in a Chinese population.(2)To detect genome-wide copy number variation in patients with NAFLD.(3)To validate the CNV region associated with NAFLD and then explore the association of CNV and disease severity and interaction between CNV and physical activity.This study will provide substantial evidence on the prevention and treatment of NAFLD and provide new clues to elucidate its genetic susuceptibility.Methods(1)A hospital-based case-control study was conduct and a Unified structured physical activity questionnaires were administered among the collected cases and controls.The intensity of physical activity was defined in terms of Metabolic Equivalent of Energy(MET),the dose of physical activity was quantified by the frequency and duration and presented in the form of MET-minutes per week.Unconditional logistic model was applied to explore the role of physical activity in the risk of NAFLD.(2)A highresolution Agilent ACGH platform was used to perform genome-wide copy number analysis in patients with NAFLD,then gene annotation analysis software(KOBAS)was utilized to conduct a enrichment analysis of pathways,diseases and functions involved in genes in the CNV region,finally,combined with the database such as PubMed,the CNV regions related to NAFLD were screened.(3)Peripheral blood samples were collected from patients and controls.TaqMan? probe-based real-time fluorescence quantitative PCR methods was used to validate the screened CNV region associated with NAFLD in a large sample.Results(1)After adjusting for hypertension,diabetes,body mass index(BMI),fasting blood glucose,energy intake and sedentary time,the total amount of physical activity was found to be inversely associated with NAFLD in a dose-dependent manner in males.(>3180 MET-min/week vs.<1441 MET-min/week: OR = 0.60,95% CI = 0.40-0.91,P for trend=0.01).In addition,both moderate-and vigorous-intensity physical activity were effective in reducing the risk of NAFLD,independent of confounding variables in males(Moderate-intensity physical activity: ≥685 MET-min/week vs.none: OR = 0.58,95%CI= 0.40-0.86,P for trend=0.01;Vigorous-intensity physical activity: ≥961 MET-min/week vs.none: OR = 0.63,95% CI = 0.41-0.95,P for trend=0.02).(2)9 CNV regions associated with NAFLD susceptibility were finally found,Chr3: 57199594-57590187,Chr7: 54813380-55274871,chr17: 38688567-38738474,chr19: 56370486-56416408,chr22: 24347959-24390254,chr14: 74001651-74022324,chr12: 57897795-57918452,chr1:65935075-65959904,chr20:33470663-33495348,these regions contain the following NAFLD-related genes: APPL1,EGFR,CCR7,NLRP4,GSTT1,ACOT1,DDIT3,LEPR,ACCS2.(3)Since NAFLD is a chronic inflammatory disease,there is an inflammation-related gene-NLRP4 in the CNV region of chr19:56370486-56416408.Therefore,the region with NLRP4 was targeted,and applied with a large sample size verification.The results showed that the frequency of NLRP4 copy number deletion was higher in the case group than in the control group(P < 0.05).Copy number deletion deletion in NLRP4 gene increased the risk of NAFLD(OR = 4.49,95% CI =1.3-15.52).Further analysis revealed no association between the copy number deletion and the severity of the disease.Considering the low mutation frequency of NLRP4,the interaction analysis between physical activity and NLRP4 copy number variation did not performed.Conclusions(1)Physical activity was inversely associated with risk of NAFLD in a dose-dependent manner in males.Vigorous-and moderate-intensity physical activity were both beneficial to NAFLD,independent of confounding variables.(2)9 CNV regions associated with NAFLD susceptibility: Chr3:57199594-57590187,chr7: 54813380-55274871,chr17: 38688567-38738474,chr19: 56370486-56416408,chr22: 24347959-24390254,chr14:74001651-74022324,chr12:57897795-57918452,chr1:65935075-65959904,chr20:33470663-33495348.(3)The copy number deletion of NLRP4 in chr19:56370486-56416408 may increase the risk of NAFLD,but no association with the disease severity.