Mosavi Ezatollah, Bandehpour Mojgan, Mostafazadeh Amrollah, YousefGhahari Behnaz, Majidi Fateme, Zali Hakimeh, Kazemi Bahram
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Int J Mol Cell Med. 2024;13(2):171-185. doi: 10.22088/IJMCM.BUMS.13.2.171.
In a system biology-based study, we previously reported that IL-6 and IL6R -specific m-RNA levels were elevated in leukocytes of patients with Rheumatoid arthritis (RA). Here, the association of toll-like receptor4 (TLR4) rs 141534085 and cytochrome P450 family 51 subfamily A member 1(CYP51A1) rs6 with tumor necrosis factor-α (TNF- α), rheumatoid factor (RF)- and Anti- cyclic citrullinated peptide (anti-CCP) antibody -positivity was investigated in almost the same subjects. Forty-six patients and 48 normal subjects were recruited in this study. The blood leucocytes TLR4 rs 141534085 and CYP51A1 rs6 -comprising DNA sequences were amplified by using tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) technique and the PCR products were checked by Sanger DNA sequencing method. ELISA method was used to determine plasma levels of TNF- α, anti-CCP antibody and RF positivity of plasma was evaluated through a latex agglutination test. The TNF- α level was significantly higher in the patient group than control subjects (p= 0.001). Moreover, we were not able to find any correlation between TNF-α levels and RF as well as anti-CCP antibodies when we used the K/ Fisher's exact test and Pearson test respectively. Our DNA sequencing data revealed the following new mutations in TLR4 rs141534085 comprising regions: A>T in position 1050, T>A in position 1052, and C>A in position 1054; and for CYP51A1 rs6 encompassing region, the new mutations were; G>A in position 21680, the T nucleotide was inserted in position 21762 and the G nucleotide was inserted in position 21763, G>T in position 21764. The data of this study showed that both TLR4 rs141534085 and CYP51A1 rs6 related DNA regions should be considered as hotspot areas in RA pathogenicity. Moreover, these data indicated that, TNF- α did not alter the production of anti-CCP and RF pathogenic antibodies in patients with long-term RA.
在一项基于系统生物学的研究中，我们之前报道过类风湿关节炎（RA）患者白细胞中白细胞介素6（IL-6）和白细胞介素6受体（IL6R）特异性mRNA水平升高。在此，我们在几乎相同的受试者中研究了Toll样受体4（TLR4）rs141534085和细胞色素P450家族51亚家族A成员1（CYP51A1）rs6与肿瘤坏死因子-α（TNF-α）、类风湿因子（RF）及抗环瓜氨酸肽（抗CCP）抗体阳性之间的关联。本研究招募了46例患者和48名正常受试者。采用四引物扩增不应性突变系统聚合酶链反应（T-ARMS-PCR）技术扩增血液白细胞中包含TLR4 rs141534085和CYP51A1 rs6的DNA序列，并用桑格DNA测序法检查PCR产物。采用酶联免疫吸附测定（ELISA）法测定血浆TNF-α水平，通过乳胶凝集试验评估血浆抗CCP抗体和RF阳性情况。患者组TNF-α水平显著高于对照组（p = 0.001）。此外，当我们分别使用卡方/费舍尔精确检验和皮尔逊检验时，未发现TNF-α水平与RF以及抗CCP抗体之间存在任何相关性。我们的DNA测序数据显示，在包含TLR4 rs141534085的区域有以下新突变：第1050位A>T、第1052位T>A、第1054位C>A；对于包含CYP51A1 rs6的区域，新突变有：第21680位G>A、第21762位插入T核苷酸、第21763位插入G核苷酸、第21764位G>T。本研究数据表明，TLR4 rs141534085和CYP51A1 rs6相关的DNA区域均应被视为RA发病机制中的热点区域。此外，这些数据表明，TNF-α不会改变长期RA患者中抗CCP和RF致病抗体的产生。
