Whole Genome Bisulfite sequencing: Allele-specific DNA methylation is increased in cancers and its dense mapping in normal plus neoplastic cells increases the yield of disease-associated regulatory SNPs

Background: Mapping of allele-specific DNA methylation (ASM) can be a post-GWAS strategy for localizing regulatory sequence polymorphisms (rSNPs). However, the advantages of this approach, and the mechanisms underlying ASM in normal and neoplastic cells, remain to be clarified. Results: We performed whole genome methyl-seq on diverse normal cells and tissues and three types of cancers (multiple myeloma, lymphoma, glioblastoma multiforme). After excluding imprinting, the data pinpointed 15,114 high-confidence ASM differentially methylated regions (DMRs), of which 1,842 contained SNPs in strong linkage disequilibrium or coinciding with GWAS peaks. ASM frequencies were increased 5 to 9-fold in cancers vs. matched normal tissues, due to widespread allele-specific hypomethylation and focal allele-specific hypermethylation in poised chromatin. Cancers showed increased allele switching at ASM loci, but destructive SNPs in specific classes of CTCF and transcription factor (TF) binding motifs were similarly correlated with ASM in cancer and non-cancer. Rare somatic mutations in these same motif classes tracked with de novo ASM in the cancers. Allele-specific TF binding from ChIP-seq was enriched among ASM loci, but most ASM DMRs lacked such annotations, and some were found in otherwise uninformative “chromatin deserts”. Conclusions: ASM is increased in cancers but occurs by a shared mechanism involving rSNPs in CTCF and TF binding sites in normal and neoplastic cells. Dense ASM mapping in normal plus cancer samples reveals candidate rSNPs that are difficult to find by other approaches. Together with GWAS data, these rSNPs can nominate specific transcriptional pathways in susceptibility to autoimmune, neuropsychiatric, and neoplastic diseases.

研究背景：等位基因特异性DNA甲基化（allele-specific DNA methylation, ASM）的图谱绘制，可作为定位调控序列多态性（regulatory sequence polymorphisms, rSNPs）的全基因组关联研究（Genome-Wide Association Study, GWAS）后分析策略。然而，该方法的应用优势，以及正常细胞与肿瘤细胞中ASM的潜在调控机制，仍有待进一步阐明。
研究结果：本研究对多种正常细胞、组织以及三类癌症（多发性骨髓瘤（multiple myeloma）、淋巴瘤（lymphoma）、多形性胶质母细胞瘤（glioblastoma multiforme））开展了全基因组甲基化测序。在排除印记调控区域后，本研究的数据精准鉴定出15114个高置信度的ASM差异甲基化区域（differentially methylated regions, DMRs），其中1842个区域携带强连锁不平衡（linkage disequilibrium）的单核苷酸多态性（single nucleotide polymorphism, SNP），或与GWAS显著位点重合。与匹配的正常组织相比，癌症样本中的ASM发生频率提升了5至9倍，这一现象源于广泛的等位基因特异性低甲基化，以及预启动染色质（poised chromatin）区域内的局灶性等位基因特异性高甲基化。癌症样本在ASM位点上的等位基因转换现象更为显著，但特定类别CCCTC结合因子（CCCTC-binding factor, CTCF）与转录因子（transcription factor, TF）结合基序中的破坏性SNP，在癌症与非癌样本中均与ASM存在相似的相关性。上述同一类别的基序中罕见的体细胞突变，与癌症中新发的ASM存在显著关联。基于染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）得到的等位基因特异性TF结合位点，在ASM位点中显著富集，但多数ASM DMRs并未携带此类注释信息，且部分ASM DMRs位于原本被认为无调控活性的“染色质荒漠（chromatin deserts）”区域内。
研究结论：癌症中ASM的发生频率显著升高，但其调控机制与正常细胞共享同一通路，均涉及CTCF与TF结合位点内的rSNPs。对正常与癌症样本开展高密度ASM图谱绘制，可发现其他方法难以识别的候选rSNPs。结合GWAS数据，此类候选rSNPs可帮助明确自身免疫性疾病、神经精神疾病以及肿瘤易感性相关的特定转录调控通路。