Xi Zhengrui, Rainero Innocenzo, Rubino Elisa, Pinessi Lorenzo, Bruni Amalia C, Maletta Raffaele G, Nacmias Benedetta, Sorbi Sandro, Galimberti Daniela, Surace Ezequiel I, Zheng Yonglan, Moreno Danielle, Sato Christine, Liang Yan, Zhou Ye, Robertson Janice, Zinman Lorne, Tartaglia Maria Carmela, St George-Hyslop Peter, Rogaeva Ekaterina
Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, 60 Leonard Street, Toronto, Ontario, Canada M5T 2S8.
Neurology I, Rita Levi Montalcini Department of Neuroscience, University of Torino, Torino, Italy.
Hum Mol Genet. 2014 Nov 1;23(21):5630-7. doi: 10.1093/hmg/ddu279. Epub 2014 Jun 6.
The G₄C₂-repeat expansion in C9orf72 is a common cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). C9orf72 transcription is reduced in expansion carriers implicating haploinsufficiency as one of the disease mechanisms. Indeed, our recent ALS study revealed that the expansion was associated with hypermethylation of the CpG-island (5'of the repeat) in DNA samples obtained from different tissues (blood, brain and spinal cord). However, the link between FTLD and methylation of the CpG-island is unknown. Hence, we investigated the methylation profile of the same CpG-island by bisulfite sequencing of DNA obtained from blood of 34 FTLD expansion carriers, 166 FTLD non-carriers and 103 controls. Methylation level was significantly higher in FTLD expansion carriers than non-carriers (P = 7.8E-13). Our results were confirmed by two methods (HhaI-assay and sequencing of cloned bisulfite PCR products). Hypermethylation occurred only in carriers of an allele with >50 repeats, and was not detected in non-carriers or individuals with an intermediate allele (22-43 repeats). As expected, the position/number of methylated CpGs was concordant between the sense and anti-sense DNA strand, suggesting that it is a stable epigenetic modification. Analysis of the combined ALS and FTLD datasets (82 expansion carriers) revealed that the degree of methylation of the entire CpG-island or contribution of specific CpGs (n = 26) is similar in both syndromes, with a trend towards a higher proportion of ALS patients with a high methylation level (P = 0.09). In conclusion, we demonstrated that hypermethylation of the CpG-island 5'of the G₄C₂-repeat is expansion-specific, but not syndrome-specific (ALS versus FTLD).
C9orf72基因中G₄C₂重复序列的扩增是额颞叶痴呆（FTLD）和肌萎缩侧索硬化症（ALS）的常见病因。C9orf72转录在扩增携带者中减少，提示单倍剂量不足是疾病机制之一。事实上，我们最近的ALS研究表明，在从不同组织（血液、大脑和脊髓）获取的DNA样本中，扩增与CpG岛（重复序列的5'端）的高甲基化有关。然而，FTLD与CpG岛甲基化之间的联系尚不清楚。因此，我们通过对34例FTLD扩增携带者、166例FTLD非携带者和103例对照者血液中提取的DNA进行亚硫酸氢盐测序，研究了同一CpG岛的甲基化谱。FTLD扩增携带者的甲基化水平显著高于非携带者（P = 7.8E-13）。我们的结果通过两种方法（HhaI检测和克隆的亚硫酸氢盐PCR产物测序）得到了证实。高甲基化仅发生在等位基因重复数>50的携带者中，在非携带者或中等等位基因（22-43个重复）个体中未检测到。正如预期的那样，有义链和反义链DNA上甲基化CpG的位置/数量是一致的，这表明它是一种稳定的表观遗传修饰。对ALS和FTLD联合数据集（82例扩增携带者）的分析表明，两种综合征中整个CpG岛的甲基化程度或特定CpG（n = 26）的贡献相似，ALS患者甲基化水平高的比例有升高趋势（P = 0.09）。总之，我们证明了G₄C₂重复序列5'端CpG岛的高甲基化是扩增特异性的，但不是综合征特异性的（ALS与FTLD）。
