Participants and study design
In the present study 2,393 participants aged above 45 years were a subset of the 16,320 participants from Haian County among the 70,458 participants of the Nantong Chronic Diseases Study (NCDS); a cohort study of people (aged 18-90 years) living in Nantong China. Baseline recruitment for the NCDS was conducted between 2017–2018. The eligible residents of six communities in Haian were invited to participate and 12,533 people who had no prior history of cancer were enrolled in the study (response rate: 76.8%), and 2,393 middle-aged and elderly people were selected, with a response rate of 96.7%. Information on socio-demographic characteristics, lifestyle factors, personal medical history and the family history of chronic diseases were collected by trained interviewers at the in-person interview. Participants were asked to provide a fasting blood sample. The study protocol was approved by the Institutional Review Boards of Nantong University and the Nantong Centers for Disease Control. All participants provided written informed consent.
Anthropometric and biochemical measurements
Anthropometric measurements of weight, height, waist and hip circumferences (WC and HC) were taken twice according to a standard protocol. If the difference between the first two measurements was greater than 1 cm for circumference or 1 kg for weight, a third measurement was taken. The average of the two closest measurements were applied in the present study. From these measurements, the waist-hip ratio (WHR) and the body mass index (BMI) were calculated, BMI was calculated as weight in kg divided by the square of height in meters.
A 10 ml blood sample was drawn into an EDTA vacutainer tube, stored in a portable Styrofoam box with ice packs (0–4 oC) and were processed within 6 hours. Serum 25(OH)D concentration was assayed by enzyme linked immunosorbent assay (ELISA).
We defined 25(OH)D < 25 nmol/L as severe deficiency, 25 to < 50 nmol/L as deficiency, 50 to < 75 nmol/L as insufficiency and ≥ 75 nmol/L as sufficient [29]. Furthermore, fasting blood glucose (FBG) and blood lipids (triglyceride (TG) and high-density lipoprotein cholesterol (HDL-c)) were measured. Insulin level was measured by chemiluminescent immunoassay (CLIA). Homeostasis model assessment of insulin resistance (HOMR-IR) was calculated based on the formula: HOMR-IR= (FBG(mmol/L)×Insulin(μIU/mL))/22.5. Blood pressure comprising systolic blood pressure (SBP) and DBP were taken twice with the interval time of more than 3 minutes. If the difference between the first two measurements was larger than 10 mmHg, a third measurement was taken; the average of the two closest measurements was applied in this study. Other demographic information, such as education, income, lifestyles factors (such as physical activity, smoking and drinking status), personal medical history, family history of chronic diseases, and vitamin D and calcium supplements were collected using a standard questionnaire.
Diagnostic criteria for MS and T2D
MS was defined based on joint interim statement of the International Diabetes Federation criteria [30] by adopting the Asian criteria for WC as having ≥ 3 of the following metabolic abnormalities: Central obesity: WC ≥ 85 cm for Chinese men and ≥ 80 cm for Chinese women; abnormal fasting serum TG ≥ 1.7 mmol/L or taking TG lowering medication; abnormal fasting serum HDL-c < 1.3 mmol/L for Chinese women and < 1.0 mmol/L for Chinese men or under treatment to raise HDL-c levels; abnormal blood pressure (hypertension): SBP ≥ 130 mmHg, DBP ≥ 85 mmHg or on antihypertensive medication; abnormal fasting serum glucose (diabetes) ≥ 5.6 mmol/L or on anti-diabetic medication.
T2D was defined as FBG ≥ 7.0 mmol/L and/or 2 hours oral glucose tolerance test (2h-OGTT) ≥ 11.1 mmol/L and/or treatment with anti-diabetic medication and/or previously diagnosed diabetes by physicians [31].
SNPs selection and genotyping
Four vitamin D-related SNPs: two synthesis SNPs (DHCR7/NADSYN1-rs12785878 and CYP2R1-rs10741657), one transport SNP (GC-rs2282679) and one metabolism SNP (CYP24A1-rs6013897) were selected on the basis of a recent MR study of Asian population [32]. These SNPs were significantly associated with plasma 25(OH)D concentration in previous genome-wide studies [18] and also used in Mendelian analyses in studies from China [23, 24]. All four SNPs were on the Hardy-Weinberg equilibrium HWE (P > 0.05), and frequency of the alleles was > 0.05.
Genotyping was performed on the iPLEX™ Sequenom MassARRAY® platform. Polymerase chain reaction (PCR) and extension primers were designed by using the MassARRAY Assay Design 3.0 software (Sequenom, Inc). PCR and extension reactions were performed according to the manufacturer’s instructions, and extension product sizes were determined by mass spectrometry using the Sequenom iPLEX system. On each 96-well plate, two negative controls (water), two blinded duplicates, and two samples were included.
Genetic Scores
We assumed an additive genetic model for the SNPs with scores of 0, 1 or 2 for genotypes containing 0, 1 or 2 alleles, respectively based on the relationship between SNPs and circulating vitamin D levels. We calculated genetic scores for two synthesis SNPs (DHCR7-rs12785878+CYP2R1-rs10741657), three SNPs (DHCR7-rs12785878+CYP2R1-rs10741657+GC-rs2282679) and all four SNPs.
Statistical analysis
Normally distributed continuous variables were expressed as mean ± standard deviation (SD) and compared using ANOVA test, non-normally distributed continuous variables were expressed as median (interquartile range (IQR)) and analyzed using Wilcoxon rank sum test, and categorical variables were expressed as percentage and analyzed by Pearson chi-square test between diabetes cases and diabetes non-cases. Odds Ratios (ORs) and 95% confidence intervals (CI) were estimated using logistic regression models to analyze the association between serum 25(OH)D concentration and its determined genetic scores and MS and T2D adjusted for confounders. All analyses were performed using SAS (version 9.3; SAS Institute, Cary, NC) and P < 0.05 was considered statistically significant and were based on two-sided probability.