Study population
We consecutively enrolled adults who underwent weight management from August 10, 2018, to November 30, 2019, and staff health examinations from January 1 to March 10, 2020, in the Health Management Center of our hospital. Those who were pregnant or lactating, were taking medication known to affect body weight or energy metabolism, or had mental illness, bulimia, anorexia, gastrointestinal disorders or morbid obesity were excluded. We designed an unmatched case‒control study of 466 adults including 226 normal-weight, 168 overweight and 72 obese adults, who provided data on anthropometry and venous blood samples. All participants were independent and unrelated individuals. We obtained the informed consent from each participant. This study was approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province in China. All participants’ identity information was anonymous. All detailed protocols followed the principles of our institutional research ethics committee and were in accordance with the Declaration of Helsinki.
Measurements
Anthropometric data were collected by trained health technicians using a standard protocol. All instruments were validated following the standard methods of the manufacturers. Weight was measured to the nearest 0.1 kg on a balance beam scale, and height was measured to the nearest 0.1 cm with a wall-mounted stadiometer. BMI was calculated as the weight in kilograms divided by the square of the height in metres. Obesity was defined by a BMI ≥ 30 kg/m2, and overweight was defined as 25 kg/m2 ≤ BMI < 30 kg/m2 for adults according to the WHO.
Blood pressure was measured on the right upper arm using an electronic sphygmomanometer (Omron, HBP-9021) with the cuff maintained at heart level after 5 min of rest in a sitting position. Elevated blood pressure was considered with systolic blood pressure (SBP) ≥ 130 mmHg or diastolic blood pressure (DBP) ≥ 85 mmHg or reported use of antihypertensive medication.
Blood specimen collection and biochemical index detection
Approximately 3 mL of peripheral venous blood was collected from subjects that fasted for 12 h in the morning. After nonanticoagulant centrifugation, serum samples were collected for biochemical tests, and blood clots containing leukocytes were left at the bottom precipitation and stored at − 80 °C for DNA extraction. Fasting plasma glucose (FPG) was measured by the hexokinase method. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) were detected by enzyme-coupled colorimetry. All reagents were purchased and blood biochemical indices were determined by an AU5800 automatic biochemical analyser (BECKMAN COULTER). All laboratory equipment was calibrated.
According to 2016 Chinese guidelines for the management of dyslipidaemia in adults13, dyslipidaemia was defined by the presence of one or more of the following component conditions: TC ≥ 5.20 mmol/L (200 mg/dL), LDL-C ≥ 3.40 mmol/L (130 mg/dL), HDL-C < 1.00 mmol/L (40 mg/dL), TG ≥ 1.70 mmol/L (150 mg/dL), or if they were taking anti-dyslipidaemia medication. Impaired fasting glucose (IFG) was diagnosed with FPG ≥ 5.60 mmol/L (100 mg/dL). Abnormalities in either blood pressure, lipids or FPG were considered cardiometabolic abnormalities.
DNA extraction, polymerase chain reaction (PCR) and sequencing for SULT1A2 rs1059491
In this study, we collected only one blood sample tube, which needed to be used simultaneously for biochemical testing, serum protein testing and DNA extraction. The genomic DNA of peripheral blood mainly comes from leukocytes according to the TIANamp N96 Blood DNA Kit (Nano MagBio, Cat. no. 4992450) according to the manufacturer’s instructions and then stored at − 20 °C for PCR analysis. The reaction mixture included with 15 µl 2 × Taq PCR Master Mix, 1 µl genomic DNA, 12 µl ddH2O, 1 µl forwards primer and reverse primer (10 pmol/µl), respectively. The final total volume reached 30 µl. The sequences for the PCR primers are presented in Supplementary Table 1, and the expected size was 920 bp. DNA was amplified under the conditions of preheating at 95 °C for 5 min for initial denaturation, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s, and a final extension at 72 °C for 5 min. Sanger sequencing is the international gold standard for genetic tests and consists of four separate reaction systems, including the target fragment, four deoxyribonucleotides (dNTP), DNA polymerase, and sequencing primers (Supplementary Table 1). The amplified products were sequenced by Sanger sequencing on an ABI 3730xl DNA Analyser, and the sequencing results were analysed by using DNAman (Lynnon Biosoft, America) and Chromas (Technelysium, Australia) software. Genotyping of all samples was performed simultaneously in the same laboratory.
Statistical analysis
We calculated the sample size and power for an unmatched case‒control study using Quanto 1.2.4 software http://hydra.usc.edu/gxe/.Assuming a dominant genetic model, an allele frequency of 0.06, and overweight and obesity prevalence of 50% in the adults of China, we estimated that an enrolment target of 466 participants (233 per group) would provide the study with greater than 80% statistical power to detect a significant odds ratio of 2.3 for risk of overweight and obesity between the different genotype groups at a significance level of 0.05 using a two-tailed test. The anticipated effect size for the risk of obesity was determined based on previous data obtained from northern Chinese children and adolescents11.
Continuous variables are expressed as the mean ± standard deviation (SD). Comparison of variables between groups was performed using one way ANOVA for continuous variables and chi-square tests for categorical variables. Serum TG levels without a normal distribution were described as the geometric mean (interquartile range) and transformed by natural logarithm prior to one-way ANOVA. Hardy–Weinberg equilibrium was performed using the chi-squared test. Assuming a dominant inheritance mode, multivariable logistic regression models were applied to estimate the association between genotype of rs1059491 and overweight combined obesity and related cardiometabolic abnormalities. Sex, age and weight status were adjusted as covariables. The false discovery rate (FDR) approach was used to correct for multiple comparisons. FDR analysis was applied for 18 outcome measures (10 quantitative traits in Table 2, 8 qualitative traits in Table 3) and one variant simultaneously (number of tests: 18 × 1 = 18). In brief, the original P value was considered statistically significant only if it was less than the P value for FDR. All statistical analyses were performed using SPSS Statistics software version 26.0 (IBM Corp., Armonk, New York). Statistical significance was inferred at a two-tailed P value less than 0.05.
Ethical statement
This study involving human participants was reviewed and approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province in China. The studies involving human participants were reviewed and approved by the Ethics Committee of Taizhou Hospital of Zhejiang Province in China. All procedures were performed in accordance with the guidelines of the institutional ethics committee of the authors and adhered to the tenets of the Declaration of Helsinki. All data, including demographic, biochemical, and genetic information, were anonymized. Personal information is not involved in this article.