DNA methylation on type-specific human skeletal muscle fibers

Purpose: Reduced Representation Bisulfite Sequencing (RRBS) DNA input requirements become a challenge when working with small pools of tissue-specific cell types. We describe an application of the RRBS method to assess DNA methylation on low-DNA input from human slow-twitch (MHC I) and fast-twitch (MHC IIa) skeletal muscle fibers. Methods : Fiber-type specific (MHC I and MHC IIa muscle fibers) total DNA was extracted from vastus lateralis muscle biopsies of 8 young physically active men (~25 yrs). A total of 16 DNA samples were generated : 8 DNA samples from pure MHC I and 8 DNA samples from pure MHC IIa muscle fibers. An equal quantity of DNA (4 ng) from each sample was combined to generate a "pooled" DNA sample representing all 8 subjects for each fiber type. Two fiber-type specific "pooled" samples of 32 ng of DNA were generated for library construction and sequencing, creating a Type 1 (MHC I muscle fibers) and Type 2a (MHC IIa muscle fibers) sample. Sequencing was performed using the HiSeq 2500 (Illumina) with 50 bp paired-end read parameters. Minimum sequencing read coverage of 5 (5x) was used as the cutoff for CpG-sites inclusion in the DNA methylation analysis. Fisher’s exact test was performed on CpG-sites that overlapped (i.e. identified in both samples) Type 1 and Type 2a samples to obtain p-values that indicate the likelihood of the site being a differentially methylated CpG-site (DMS). DMS with p<0.05 were classified as hypermethylated or hypomethylated if they were more or less methylated than the Type 1 sample, which was used as the reference sample. Results: The 32 ng of DNA from fiber-type specific muscle samples (Type 1 and 2a) used in this study ensured similar sequencing quality as compared to other studies using greater DNA input (>50 ng). Mapping ratios of ~47% and bisulfite conversion rates of ~97-98% were obtained.The unique and best alignment was successfully assessed for each of 17,376,728 CpG-sites in the Type 1 sample and 17,006,993 in the Type 2a sample, which represents ~30% of the total CpG number in the human genome. We identified 143,160 differentially methylated CpG-sites (DMS) across 14,046 genes among MHC I and MHC IIa muscle fibers. The analysis revealed that some genes predominantly expressed in MHC I were hypermethylated in MHC IIa muscle fibers. Conclusion: This study validates a low-DNA input RRBS method for human skeletal muscle samples to investigate the methylation patterns at a fiber-type specific level. These are the first fiber-type specific methylation data reported from human skeletal muscle. Considering the metabolic and structural differences between MHC I and MHC IIa muscle fibers, this technique could provide novel insights into the skeletal muscle methylation profile in relation to health, performance, disease or disuse. Comparison of DNA methylation profiles in slow-twitch (MHC I) and fast-twitch (MHC IIa) human skeletal muscle fibers.

研究目的：在处理少量组织特异性细胞群样本时，还原代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing, RRBS）对DNA投入量的要求成为一大挑战。本研究阐述了将RRBS方法应用于人类慢收缩（MHC I）与快收缩（MHC IIa）骨骼肌纤维的低投入DNA样本，以评估其DNA甲基化水平的研究方案。
研究方法：从8名年轻体力活动男性（年龄约25岁）的股外侧肌活检标本中提取纤维亚型特异性（MHC I与MHC IIa型肌纤维）总DNA。最终共获得16份DNA样本：8份来自纯MHC I型肌纤维，8份来自纯MHC IIa型肌纤维。将每份样本的等量DNA（4 ng）进行混合，以制备代表每种纤维亚型全部8名受试者的「混合」DNA样本。最终为建库与测序制备2份纤维亚型特异性混合DNA样本（每份含32 ng DNA），分别命名为1型（MHC I型肌纤维）与2a型（MHC IIa型肌纤维）样本。测序采用Illumina HiSeq 2500平台，设置50 bp双端读段参数。以最低测序读段覆盖度5（5x）作为CpG位点（CpG-site）纳入DNA甲基化分析的临界阈值。对在1型与2a型样本中均被检测到（即两个样本中共有的）的CpG位点进行费希尔精确检验（Fisher’s exact test），以获取p值，用以判断该位点是否为差异甲基化CpG位点（differentially methylated CpG-site, DMS）。将p<0.05的DMS按照甲基化水平相对于作为参照的1型样本的高低，分为高甲基化与低甲基化两类。
研究结果：本研究中使用的纤维亚型特异性肌样本（1型与2a型）的32 ng DNA投入量，确保了与其他使用更高DNA投入量（>50 ng）的研究相当的测序质量。本研究获得了约47%的比对率与约97%~98%的亚硫酸氢盐转化率。成功对1型样本中的17,376,728个CpG位点与2a型样本中的17,006,993个CpG位点进行了唯一最优比对，该数量约占人类基因组总CpG位点数的30%。本研究在MHC I型与MHC IIa型肌纤维的14,046个基因中共鉴定出143,160个差异甲基化CpG位点（DMS）。分析结果显示，部分主要在MHC I型肌纤维中表达的基因在MHC IIa型肌纤维中呈现高甲基化状态。
研究结论：本研究验证了一种适用于人类骨骼肌样本的低投入DNA量RRBS方法，可用于在纤维亚型特异性层面探究甲基化模式。这是首次公开报道的人类骨骼肌纤维亚型特异性甲基化数据。鉴于MHC I型与MHC IIa型肌纤维在代谢与结构上的差异，该技术可为骨骼肌甲基化谱与健康、运动表现、疾病或失用的关联研究提供全新视角。
人类慢收缩（MHC I）与快收缩（MHC IIa）骨骼肌纤维的DNA甲基化谱比较