Abstract

Background

Mitochondria play an important role in cell survival, function and lineage differentiation. Changes in mitochondrial DNA (mtDNA) may control mitochondrial functions and thus may impart an alternative cellular state thereby leading to a disease condition in the body. Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease wherein immune cells become self-reactive causing joint inflammation, swelling and pain in patients. The changes in mtDNA may alter cellular functions thereby directing the immune cells towards an inflammatory phenotype in RA. Therefore, it becomes pertinent to identify changes in mtDNA sequence in immune cells of RA patients to understand the pathogenesis and progression of RA.

Methods

mtDNA from peripheral blood mono-nuclear cells (PBMCs) of 23 RA patients and 17 healthy controls (HCs) were sequenced using next-generation sequencing (NGS). Further, single nucleotide polymorphisms (SNPs) and other variable changes in mtDNA hypervariable and coding regions, amino acid changes with a putative impact on disease, levels of heteroplasmy, copy number variations and haplogroup analysis in RA patients and HCs were analysed and compared to identify any association of mtDNA changes and RA disease.

Results

A total of 382 single nucleotide mtDNA variants were observed, 91 (23.82%) were present in hypervariable region and 291 (76.18%) in coding region of patients and HC. The variant 513 GCA > ACA, with G present in HVR-III, known to control the mitochondrial translation function, was significantly present in RA patients. The CYTB gene had larger number of SNPs in HC samples while RNR2 was more variable in RA patients. A non-synonymous heteroplasmy in ND1 gene was found at a single nucleotide position 3533 in an increased number of RA patients as compared to the controls. A significant increase in mtDNA duplication and a higher frequency of the haplogroup U was also characteristic of RA. Also, the presence of SNPs in mitochondrial tRNA genes at two positions 12308 A > G and 15924 A > G were found to be pathogenic.

Conclusion

We herein observed an altered mtDNA sequence in immune cells of RA patients and thus a possible role of mitochondrial genome in the development of RA. The observed nucleotide changes in mtDNA control region, RNR2 gene, increased heteroplasmy and mtDNA duplication in RA patients may alter sites for transcription factor binding thereby influencing mtDNA gene expression, as well as copy numbers thereby affecting the mitochondrial proteins and their functions. These changes in mtDNA could be one of the probable reasons among many leading to the progression of RA.

中文翻译：

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类风湿关节炎患者线粒体基因组差异

摘要

背景

线粒体在细胞存活、功能和谱系分化中起重要作用。线粒体 DNA (mtDNA) 的变化可能会控制线粒体功能，因此可能会赋予另一种细胞状态，从而导致体内疾病。类风湿性关节炎 (RA) 是一种慢性炎症性自身免疫性疾病，其中免疫细胞变得自我反应，导致患者出现关节炎症、肿胀和疼痛。mtDNA 的变化可能会改变细胞功能，从而将免疫细胞导向 RA 的炎症表型。因此，识别 RA 患者免疫细胞中 mtDNA 序列的变化以了解 RA 的发病机制和进展就变得很重要。

方法

使用二代测序 (NGS) 对 23 名 RA 患者和 17 名健康对照 (HC) 的外周血单核细胞 (PBMC) 的 mtDNA 进行测序。此外，分析并比较了单核苷酸多态性 (SNP) 和 mtDNA 高变区和编码区的其他可变变化、对疾病有推定影响的氨基酸变化、异质性水平、拷贝数变异和单倍群分析，以识别mtDNA 变化与 RA 疾病的任何关联。

结果

共观察到 382 个单核苷酸 mtDNA 变异，91 个（23.82%）存在于高变区，291 个（76.18%）存在于患者和 HC 的编码区。变异 513 GCA > ACA，其中 G 存在于 HVR-III 中，已知控制线粒体翻译功能，在 RA 患者中显着存在。CYTB 基因在 HC 样本中具有更多的 SNP，而 RNR2 在 RA 患者中的变化更大。与对照组相比，在数量增加的 RA 患者中，在单核苷酸位置 3533 处发现 ND1 基因中的非同义异质性。mtDNA 复制的显着增加和单倍群 U 的更高频率也是 RA 的特征。此外，发现线粒体 tRNA 基因中 12308 A > G 和 15924 A > G 两个位置的 SNP 具有致病性。

结论

我们在此观察到 RA 患者免疫细胞中 mtDNA 序列的改变，因此线粒体基因组在 RA 的发展中可能发挥作用。在 RA 患者中观察到的 mtDNA 控制区、RNR2 基因的核苷酸变化、异质性增加和 mtDNA 重复可能会改变转录因子结合位点，从而影响 mtDNA 基因表达，以及拷贝数，从而影响线粒体蛋白及其功能。mtDNA 的这些变化可能是导致 RA 进展的许多可能原因之一。