Molecular genetic methods
DNA was extracted from whole blood using Qia-amplification DNA extraction kit (Qiagen, USA). 200 ul EDTA whole blood was used for extraction of DNA by DNA extraction kit according to the protocol provided with the kit. The extracted DNA samples were subjected to DNA quantitation and purity assessment using the NanoDrop® (ND)-1000 spectrophotometer (NanoDrop Technologies, Inc. Wilmington, USA). Genotyping was performed using real-time PCR with TaqMan allelic discrimination assay (Applied Biosystems, USA). The DNA used is (100 μg); this study is consistent with the declaration of Helsinki. Each participant gave a written consent before experiment. A predesigned primer/probe set for the two genotypes was used (Applied Biosystems, USA). Probes were synthesized with reporter dye FAM or VIC covalently linked at the 5/end and a quencher dye MGB linked to the 3/end of the probe (Applied Biosystems, USA) (rs2910164 (C/G), rs3746444 (T/C), rs12537(C/T), rs767649 (A/T), rs3027898 (A/C) and rs1748033 (C/T)). Real-time PCR was performed using a Rotor gene Q Real-Time PCR System (Qiagen, Valencia, CA, USA) with the following conditions: after a denaturation time of 10 min at 95 °C, 45 cycles at 92 °C for 15 s then 60 °C for 90 s for annealing and extension were carried out and fluorescence was measured at the end of every cycle and at the endpoint.
Statistical methods
The Chi-square test (χ2-test) was used to analyze genotype and allele distributions of cases and controls. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated as well. Hardy–Weinberg equilibrium (HWE) test was applied on the controls. If the HWE p value was less than 0.001 [17, 18], this indicates a deviation in the population from HWE and the corresponding SNP was excluded from the study. The ratio of the males versus females between the cases and controls was analyzed by proportion test using STATA software, and it showed that the ratio is not statistically significant as the p value was equals to 0.068 which passes the defined 0.05 threshold.
The Chi-square test is a formal statistical test used to analyze categorical data to verify the statistical significance of the results. Generally, the lower the χ2 value, the greater the likelihood that there is no significant difference between cases and controls. To be sure that the Chi-square result gives a real statistically significant difference, the p value should be looked up. A low p value indicates a low expectation of finding these results by coincidence, while a high p value means a high probability of finding these results by chance.
In case of p value of 1, it means that the two groups were not different at all. Odds ratio is one of the most popular measures of the strength of association between a disease severity and a biomarker SNP. OR is used to determine the probability of disease severity presence versus the disease severity absence in exposed and unexposed individuals.
Confidence interval (CI) is a formula that shows how to use a sample data to calculate an interval that estimates a point estimate (OR). A large CI marks a low level of precision of the OR, while a narrower CI indicates a reliable OR. When the two values of CI are less than 1, this indicates a protective association. When the two values of CI are greater than 1, this indicates a susceptible association.
The used equations for calculating OR, CI, and Chi-square test are as follows:
For the allelic odds ratio:
$${text{OR}}_{A} = frac{{m_{12} m_{21} }}{{m_{11} m_{22} }}$$
(1)
where m11 and m21 indicate allele a in cases and controls, respectively, and m12 and m22 refer to allele A in cases and controls, and the equation is to calculate the OR for allele A.
The following formulas are used for a 95% confidence interval (CI):
$${text{Upper}},{text{95% }},{text{CI}} = e^{{left[ {ln left( {{text{OR}}} right) + 1.96 times sqrt {left( {frac{1}{{m_{11} }} + frac{1}{{m_{12} }} + frac{1}{{m_{21} }} + frac{1}{{m_{22} }}} right)} } right]}}$$
(2)
$${text{Lower}},{text{95% }},{text{CI}} = e^{{left[ {ln left( {{text{OR}}} right) – 1.96 times sqrt {left( {frac{1}{{m_{11} }} + frac{1}{{m_{12} }} + frac{1}{{m_{21} }} + frac{1}{{m_{22} }}} right)} } right]}}$$
(3)
Chi-square test formula:
$$chi^{2} = mathop sum limits_{i} frac{{left( {O_{i} – E_{i} } right)^{2} }}{{E_{i} }}$$
(4)
where Oi represents the observed frequency and Ei represents the expected frequency.
Study population
RA patients were diagnosed by physician investigators and followed the 1987 American College of Rheumatology (ACR) criteria. DAS28 (Disease Activity Score in 28 Joints), which is a validated score for established RA, was used as a measure for disease activity.
The observed controls had no signs of RA, including morning joint stiffness, citrulline antibody, positive rheumatoid factor (RF), or the findings of rheumatoid nodules. Furthermore, the patients with other inflammatory disorders or autoimmune diseases unrelated to RA were not included.
Subjects
The study consists of 52 RA patients, 85% of them are females, and 49 controls that 67% of whom are females. In cases, the mean age of females’ ± standard deviation (SD) was 40 ± 9.6 years. The mean age of males was 37 ± 16.64 years. In the controls, the mean age of females was 40 ± 9.92 years, while the mean age of males was 37.2 ± 10.7 years. The average disease duration of RA in females was 6.63 ± 4.29 years, while the duration of RA in males was 9.06 ± 8.25.
The study was approved by the Ethical Committee of Faculty of Medicine, Cairo University, and an oral and written informed consent was obtained from all participants.
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