Ethics approval and consent to participate
The study was approved by the local ethics committees of Fujian Medical University (ethics number 2014096). All methods were performed in accordance with the relevant guidelines and regulations.In addition, each subject gave written informed consent before participation in the study.
Study design
We conducted a case-control study in a health examination centre at the Affiliated Nanping First Hospital of Fujian Medical University from April 2015 to August 2017. Patients newly diagnosed with NAFLD using ultrasonography in accordance with the “Guidelines for the diagnosis and treatment of nonalcoholic fatty liver disease in China18” were included in the study. Hepatic ultrasonography examination was performed by trained ultrasonographists who were blinded to the clinical and laboratory data. Hepatic steatosis was diagnosed by characteristic echo patterns according to conventional criteria, such as the evidence of diffuse hyper-echogenicity of the liver relative to the kidneys, ultrasound beam attenuation, and poor visualization of intrahepatic structures.
Outcome--eligibility of NAFLD cases and controls
All participants were of Chinese Han ethnicity. The exclusion criteria were as follows: (a) daily alcohol intake of > 40 g (men) and > 20 g (women); (b) a history of other liver diseases, including drug-induced liver disease, viral hepatitis, autoimmune hepatitis, total parenteral nutrition, and hepatolenticular degeneration; (c) taking hypolipidaemic or weight reduction drugs, (d) age < 18 or > 70 years old, (e) non-resident of Nanping, or (f) not of Han ethnicity.
The controls were randomly selected from the same centre during the study period. Their eligibility criteria were identical to those of the cases, except for the requirement of a diagnosis of liver steatosis ; they were frequency-matched with cases by age (within 5-year intervals), gender, ethnicity, and region of origin The study was approved by the local ethics committee of Fujian Medical University (ethics number 2014096). In addition, All subjects who participated in this study provided written informed consent and all methods were performed in accordance with the relevant guidelines and regulations.
Data measurements and data collection
Potential confounders
Trained interviewers performed a comprehensive medical history on each participant that included eliciting information about their demographic and socio-economic characteristics (e.g., age, gender, education, income, marriage status and history of diabetes, hypertension, hyperlipidemiaand ), lifestyle habits (e.g., smoking, drinking (alcohol), tea drinking, and physical activity), anthropometric assessment (e.g., height, body weight and blood pressure). The data were obtained from participants using structured questionnaires during face-to-face interviews.
Clinical data
The biochemical tests for serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) of each participant were performed according to standard clinical laboratory methods carried out in an accredited laboratory at Nanping First Hospital.
Exposure-CES1copy number and serum CES1 levels
DNA extraction
Peripheral blood was collected from all subjects in vacuum blood tubes containing EDTA-K2. Genomic DNA was isolated from 2 mL of whole blood using a whole blood genomic DNA extraction kit, according to the manufacturer’s instructions (Catalogue No DP1101/DP1102, Bioteke Corporation, Beijing, China). The concentration and purity of DNA were detected using a Beckman DU800 UV-vis spectrophotometer (Beckman, Franklin Lakes, NJ, USA) and genomic DNA was stored at −80 °C before CNVs genotype detection.
Taqman gene copy number assay (qPCR)
The good quality extracted DNA (OD260/OD280=1.7–2.0) was diluted to a final concentration of 5 ng/μL. The Applied Biosystems protocols for the TaqMan real-time quantitative polymerase chain reaction (qPCR) method were used to assess CNVs (16q12.2: Assay Hs00114970_cn) in every sample. Each reaction (20 μL) contained 10 μL of master mix, 1 μL of TaqMan Copy Number Assay, 1 μL of TaqMan Copy Number Reference Assay, 4 μL of nuclease free water, and 4 μL of genomic DNA (5ng/μL). Quantitative real-time PCR (qPCR) was performed used the following programme: 1 PCR cycle at 95 °C for 10 min; followed by 40 cycles at 95 °C for 15 sec and 60 °C for 1 min. Negative controls were introduced for every run to ensure the genotyping quality. Each sample was run in quadruplicate, and all the above reagents were obtained from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). The CNVs assay was performed using an ABI 7500 Real-time PCR system. The Taqman copy number assay results were analysed using CopyCaller` Software version 2.0 (Thermo Fisher Scientific). The copy numbers called with 50% confidence were included in the final analysis.
Measurement of serum CES1 levels
Fasting blood samples were collected from each subject and then stored at −80 °C. When performing the assay, samples were brought to room temperature. Serum CES1 levels were measured using an enzyme-linked immunosorbent assay (ELISA) (Catalogue No. ml057683; Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s recommendations. The intra-assay coefficient of variation (CV) of the kit was less than 10%, and the inter-assay CV was less than 12%.
Statistical analyses
Categorical and continuous variables were compared between patients with NAFLD and controls using the Chi-squared test and the independent Mann–Whitney U test as appropriate. The distribution of CES1 copy numbers between patients with NAFLD and control subjects was compared using the Chi-squared test.
According to our experimental results, two genomic copies of CES1 were considered to be most common in the healthy population, and therefore this copy number was set as the reference. Unconditional logistic regression models were used to compute the odds ratios (ORs) and their 95% confidence intervals (CIs) for the association between NAFLD risk and the various copy numbers of CES1, using the two genomic copy numbers as the reference category. The following known independent risk factors for NAFLD were included in all models: age, sex, education level, occupational status, income, marital status, smoking status, tea drinking status, exercise, history of diabetes, hyperlipidaemia, and hypertension. We evaluated the influence of CNVs status across strata of other potential predictors and confounders, comparing subjects with the CNVs loss or gain with CNVs neutral cases. We also evaluated interactions between CNVs loss or gain with age, sex, marital status. Data analyses were conducted using SPSS version 20.0.0.1 (IBM Corp., Armonk, NY, USA), and a P value < 0.05 was considered statistically significant.