Ethics statement:
All participant has signed an informed consent form prior to undergoing the examination. If the subjects had no ability to sign, the family members signed on behalf of the patients.The study was approved by Ethics Committee of China Rehabilitation Research Center(No.2017-075-1),all methods were carried out in accordance with relevant guidelines and regulations. This study adheres to the ethical principles outlined in the Helsinki Declaration.
Participants
This was a prospective, two-center, cross-sectional observational study conducted in the Orthopedic ward and outpatient department. We calculated the sample size based on the prevalence rate of KOA in the elderly in China, and the final result was 204 people according to the sampling error of 20% of the morbidity. The subjects consisted of 103 KOA patients and 128 healthy volunteers who were all recruited at the Beijing Bo' ai and Chao-Yang Hospitals between January 2017 and September 2018. The KOA group included 17 males and 86 females who were diagnosed with KOA based on American College of Rheumatology criteria[14]. The diagnostic criteria for KOA include knee pain plus at least 3 out of the 7 following items: ①age 50 and above, ② knee stiffness less than 30 minutes, ③knee crepitus, ④knee swelling, ⑤bony tenderness, ⑥The local temperature of the knee joint was not normal,⑦Imaging examination revealed osteophyte formation. The 128 healthy subjects had no history of tumors, immunopathy, or osteoarthritis. The control samples which included 23 males and 105 females, were admitted to the medical center of the same hospitals. The KOA group was selected from patients who had Grade 3 or Grade 4 OA on knee plain radiographs.
The inclusion criteria of the patient group: 1) Han nationality; 2) having lived in the north of China longer than twenty years; 3) aged 50–80; 4)BMI < 30kg/m2; 5) definite KOA disease; 6) volunteer to join the study.
The inclusion items of the control group: 1) Han nationality; 2) living and working in Northern China over two decades; 3) between 50 and 75 years old; 4) BMI༜30kg/m2; 5) no history of knee disease.
Exclusion criteria of patient group included: 1) Age less than 50 or older than 75; 2) Unwilling to sign an informed consent form; 3) long-term migrant workers who have been away over 5 years, or the cumulative resident period is shorter than two decades; 4) Various secondary OA, such as previous knee trauma or surgery, congenital developmental diseases, etc; 5) Inflammatory joint disease (gouty arthritis, rheumatoid arthritis, systemic lupus erythematosus, hemophilia arthritis, etc.); 6) history of diabetes, heart disease, infectious disease, tumors, mental illness and other serious mental diseases or do not cooperate; 7)history of corticosteroid use; 8) limb paralysis after cerebral infarction;9) BMI ≥ 30kg/m2; 10) incompatible diagnostic criteria of knee joint osteoarthritis.
The exclusion criteria for control group included: 1)Age less than 50 and older than 80; 2) Unwilling to sign an informed consent form; 4) BMI ≥ 30kg/m2.
Clinical data and sample collection
We collected background data from all subjects, including age, gender, weight, BMI and history of relevant diseases. 2ml blood samples were drawn from a peripheral vein of all participants in 5ml vacutainer tubes containing EDTA (ethylene diamine tetraacetic acid) as an anticoagulant. The samples were packaged separately and stored in a -80℃refrigerator for future use.
The samples were sorted on ice bags in batches, and We followed the instructions in the QIAamp DNA Mini Kit (Kaj) operation protocol for genomic DNA extraction. DNA was removed from the centrifuge column with 1.5µl deionized water, and preserved at -20℃in a refrigerator for future analysis. The concentration of the blood samples were measured using a NanoDrop™ 1000 ultraviolet spectrophotometer (Thermo Scientific, Waltham, MA, USA) to obtain DNA purity and DNA concentration based on A260/A280 ratios. The elution buffer was applied to calibrate the instruments. Specimens were thoroughly mixed and tested at room temperature. DNA was stored in a 4℃ fridge after detecting genomic integrity with agarose gel electropho-resis.
DNA amplification and genotyping
The COL6A4P1 gene fragment amplified by PCR was sequenced using the first generation method (Shanghai Majorbio bio Pharm Technology Co., Ltd.). The four mutation sites(SNP rs11718863, rs7639618, rs7651842 and rs7639807) are all contained in the COL6A4P1 gene and close together. One primer is enough. Four SNPs in COL6A4P1 gene were genotyped using primers: forward primer5’-AGGCTGCCTGC CATTATTACTT- 3’, and reverse primer sequence was5’-CCCATGCTGTT TCCTTTGAACA − 3’. This set of primers can cover the four SNP we want to study. The 25µl reaction system included 1µl of both forward and reverse primers, 5µl of DNA templates, 12.5µL of 2×Taq PCR Mastermix (50 mM Tris-HCl, pH 8.7, 100 mM KCl, 4 mM MgCl2, 0.1 U Taq Platinum polymerase/µL, 500µM dNTP each), and 5.5µl of sterilized water (DNase /RNase-free). We set the annealing temperature between 54℃-59 ℃ and performed quantitative PCR detection using agarose gel electrophoresis. Our results supported a 57℃ annealing temperature as the ideal temperature. The size of the PCR were about 363bp, The results were detected with agarose gel electrophoresis. SNP alleles and genotypes were analyzed from the sequencing results.
Statistical Analysis
SPSS 27.0 (IBM Corp., Armonk, USA) was used for statistical analysis. Genotypes and allele frequencies of each group were directly counted. The independent segregation of the alleles was confirmed by the Hardy- Weinberg equilibrium (HWE) test. The independent-Sample T-test or Pearson's chi-squared test were used to assess the difference in baseline data between the two groups. Data conforming to normal distribution were denoted as mean ± standard deviation, and chi square test was adopted to evaluate the difference in genotype distribution and sex ratio between the patient group and the control group. A multivariate logistic regression was used to assess the association between SNP and KOA. Due to the age difference between the two groups of patients, we regarded age as the covariate to conduct logistic regression again. The difference was statistically significant at P < 0.05.