The interaction between a leflunomide-response methylation site (cg17330251) and variant (rs705379) on response to leflunomide in patients with rheumatoid arthritis

Feng Zhao ¹ Yu Lan Chen ¹ Haina Liu ¹ Lei Jin ² Xin Feng ³ Bingbing Dai ⁴ Meng Chen ¹ Qiao Wang ¹ Yuxin Yao ¹ Ruobing Liao ¹ Junyi Zhao ¹ Bingjia Qu ¹ Ying Song ¹ Lingyu Fu ^1*

• ¹ The First Affiliated Hospital of China Medical University, Shenyang, China
• ² Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China
• ³ First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning Province, China
• ⁴ Dalian Municipal Central Hospital, Dalian, Liaoning Province, China

Objectives: This research aims to reveal the mechanisms of the effect of the Paraoxonase 1 (PON1) gene on response to leflunomide (LEF) in rheumatoid arthritis (RA) patients, in terms of single nucleotide polymorphism (SNP), DNA methylation levels.Methods: A total of 240 RA patients enrolled were categorized into the good response group and the non-response group according to the difference in DAS28 scores between baseline and six months after LEF administration. The identified LEF-response cytosine-phosphate-guanines (CpGs) island(cg17330251) and its internal SNPs (rs705379, etc.) located at the PON1 promoter were detected by Sanger sequencing and methyl target sequencing.Results: A total of 12 CpG sites at cg17330251 could be identified in our RA patients. There were significant difference between the responders and non-responders in nine CpG sites: cg17330251_2, cg17330251_3, cg17330251_4, cg17330251_6, cg17330251_7, cg17330251_8, cg17330251_9, cg17330251_10, cg17330251_12, (OR (95CI%) = 0.492 (0.250, 0.969), 0.478 (0.243, 0.940), 0.492 (0.250, 0.969), 0.461 (0.234, 0.907), 0.492 (0.250, 0.969), 0.437 (0.225, 0.849), 0.478 (0.243, 0.941), 0.421 (0.212, 0.836), 0.424 (0.213, 0.843), P < 0.05, respectively). At all these nine CpG sites, the proportions of low methylation levels in the responders were higher than those in the nonresponders (P < 0.05). In a dominant model, there was a significant difference in rs705379 wildtype CC and mutant genotypes (CT+TT) between the responders and non-responders (P < 0.05). The average methylation level of 12 CpG sites was lowest in rs705379-CC (median 0.229, IQR 0.195-0.287), then rs705379-CT (median 0.363, IQR 0.332-0.395), and rs705379-TT (median:0.531, IQR:0.496-0.557). The average methylation levels of 12 CpG sites were significantly negative correlated with ΔDAS28 (r = -0.13, P < 0.05). The Logistic regression indicated that combined effect of rs705379, DNA methylation of the PON1 gene (OR (95CI%) = 1.277 (1.003, 1.626)), systemic inflammation index (SIRI) (OR (95CI%) = 1.079 (1.018, 1.143)) served as protective factors on response to LEF in RA patients.The RA patients with SNP-rs705379-CC, the low methylation level of PON1-cg17330251 and more SIRI would be susceptible of response to LEF and more suitable to choose LEF treatment.