Mitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA (mtDNA) could contribute to the neurodegenerative process. We recently screened families with mutations in the major ALS causative genes, namely C9orf72, SOD1, FUS, and TARDBP, observing reduced methylation levels of the mtDNA regulatory region (D-loop) only in peripheral lymphocytes of SOD1 carriers. However, until now no studies investigated the potential role of mtDNA methylation impairment in the sporadic form of ALS, which accounts for the majority of disease cases. The aim of the current study was to investigate the D-loop methylation levels and the mtDNA copy number in sporadic ALS patients and compare them to those observed in healthy controls and in familial ALS patients. Pyrosequencing analysis of D-loop methylation levels and quantitative analysis of mtDNA copy number were performed in peripheral white blood cells from 36 sporadic ALS patients, 51 age- and sex-matched controls, and 27 familial ALS patients with germinal mutations in SOD1 or C9orf72 that represent the major familial ALS forms. In the total sample, D-loop methylation levels were significantly lower in ALS patients compared to controls, and a significant inverse correlation between D-loop methylation levels and the mtDNA copy number was observed. Stratification of ALS patients into different subtypes revealed that both SOD1-mutant and sporadic ALS patients showed lower D-loop methylation levels compared to controls, while C9orf72-ALS patients showed similar D-loop methylation levels than controls. In healthy controls, but not in ALS patients, D-loop methylation levels decreased with increasing age at sampling and were higher in males compared to females. Present data reveal altered D-loop methylation levels in sporadic ALS and confirm previous evidence of an inverse correlation between D-loop methylation levels and the mtDNA copy number, as well as differences among the major familial ALS subtypes. Overall, present results suggest that D-loop methylation and mitochondrial replication are strictly related to each other and could represent compensatory mechanisms to counteract mitochondrial impairment in sporadic and SOD1-related ALS forms.

中文翻译：

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散发性肌萎缩侧索硬化症中线粒体 D 环甲基化水平降低。

在神经退行性疾病中经常报道线粒体失调和异常的表观遗传机制，包括肌萎缩侧索硬化 (ALS)，一些研究人员认为线粒体 DNA (mtDNA) 的表观遗传失调可能导致神经退行性疾病。我们最近筛选了主要 ALS 致病基因突变的家族，即 C9orf72、SOD1、FUS 和 TARDBP，仅在 SOD1 携带者的外周淋巴细胞中观察到 mtDNA 调节区 (D-loop) 的甲基化水平降低。然而，直到现在还没有研究调查 mtDNA 甲基化损伤在散发性 ALS 中的潜在作用，ALS 占大多数疾病病例。本研究的目的是调查散发性 ALS 患者的 D-loop 甲基化水平和 mtDNA 拷贝数，并将它们与健康对照和家族性 ALS 患者中观察到的进行比较。在来自 36 名散发性 ALS 患者、51 名年龄和性别匹配的对照以及 27 名具有 SOD1 或 C9orf72 生发突变的家族性 ALS 患者的外周白细胞中进行 D-loop 甲基化水平的焦磷酸测序分析和 mtDNA 拷贝数的定量分析。代表主要的家族性 ALS 形式。在总样本中，与对照组相比，ALS 患者的 D-loop 甲基化水平显着降低，并且观察到 D-loop 甲基化水平与 mtDNA 拷贝数之间存在显着的负相关。将 ALS 患者分为不同亚型显示，与对照组相比，SOD1 突变型和散发性 ALS 患者的 D-loop 甲基化水平较低，而 C9orf72-ALS 患者的 D-loop 甲基化水平与对照组相似。在健康对照中，而不是在 ALS 患者中，D-loop 甲基化水平随着采样年龄的增加而降低，而男性的 D-loop 甲基化水平高于女性。目前的数据揭示了散发性 ALS 中 D-loop 甲基化水平的改变，并证实了 D-loop 甲基化水平与 mtDNA 拷贝数之间呈负相关的先前证据，以及主要家族性 ALS 亚型之间的差异。总体，