Center for Public Health Genomics, University of Virginia, Charlottesville, Virginia, USA.
Department of Family Medicine, University of Virginia, Charlottesville, Virginia, USA.
Cancer Med. 2023 Jun;12(12):13551-13572. doi: 10.1002/cam4.6048. Epub 2023 May 10.
Lynch syndrome (LS) is a hereditary condition that increases the risk of colorectal (CRC) and extracolonic cancers that exhibit microsatellite instability (MSI-H). MSI-H is driven by defective mismatch repair (dMMR), and approximately 15% of nonhereditary CRCs also exhibit MSI-H. Here, we aimed to better define mechanisms underlying tumor initiation in LS and MSI-H cancers through multi-omic analyses of LS normal colon organoids and MSI-H tumors.
Right (n = 35) and left (n = 23) colon organoids generated from normal colon biopsies at routine colonoscopy of LS and healthy individuals were subjected to Illumina EPIC array. Differentially methylated region (DMR) analysis was performed by DMRcate. RNA-sequencing (n = 16) and bisulfite-sequencing (n = 15) were performed on a subset of right colon organoids. CRISPR-cas9-mediated editing of MMR genes in colon organoids of healthy individuals was followed by quantitative PCR of MSH4. The relationship between MSH4 expression and tumor mutational burden was further explored in three independent tumor data sets.
We identified a hypermethylated region of MSH4 in both the right and left colon organoids of LS versus healthy controls, which we validated using bisulfite-sequencing. DMR analysis in three gastrointestinal and one endometrial data set revealed that this region was also hypermethylated in MSI-H versus microsatellite stable (MSS) tumors. MSH4 expression was increased in colon organoids of LS versus healthy subjects and in publicly available MSI-H versus MSS tumors across four RNA-seq and four microarray data sets. CRISPR-cas9 editing of MLH1 and MSH2, but not MSH6, in normal colon organoids significantly increased MSH4 expression. MSH4 expression was significantly associated with tumor mutational burden in three publicly available data sets.
Our findings implicate DNA methylation and gene expression differences of MSH4 as a marker of dMMR and as a potential novel biomarker of LS. Our study of LS colon organoids supports the hypothesis that dMMR exists in the colons of LS subjects prior to CRC.
林奇综合征（LS）是一种遗传性疾病，会增加结直肠癌（CRC）和表现出微卫星不稳定（MSI-H）的结外癌症的风险。MSI-H 是由缺陷错配修复（dMMR）驱动的，大约 15%的非遗传性 CRC 也表现出 MSI-H。在这里，我们旨在通过对 LS 正常结肠类器官和 MSI-H 肿瘤的多组学分析，更好地定义 LS 和 MSI-H 癌症中肿瘤起始的机制。
从 LS 和健康个体常规结肠镜检查的正常结肠活检中生成右（n=35）和左（n=23）结肠类器官，并进行 Illumina EPIC 芯片分析。通过 DMRcate 进行差异甲基化区域（DMR）分析。对右结肠类器官的一部分进行 RNA 测序（n=16）和亚硫酸氢盐测序（n=15）。对健康个体的结肠类器官进行 CRISPR-cas9 介导的 MMR 基因编辑，然后对 MSH4 进行定量 PCR。在三个独立的肿瘤数据集中进一步探索 MSH4 表达与肿瘤突变负担之间的关系。
我们在 LS 与健康对照的右和左结肠类器官中均发现 MSH4 的高甲基化区域，并用亚硫酸氢盐测序进行了验证。在三个胃肠道和一个子宫内膜数据集的 DMR 分析中，我们发现该区域在 MSI-H 与微卫星稳定（MSS）肿瘤中也呈高甲基化。MSH4 表达在 LS 与健康个体的结肠类器官中增加，在四个 RNA-seq 和四个微阵列数据集中的 MSI-H 与 MSS 肿瘤中增加。在正常结肠类器官中 CRISPR-cas9 编辑 MLH1 和 MSH2，但不编辑 MSH6，显著增加了 MSH4 的表达。在三个公开可用的数据集中，MSH4 表达与肿瘤突变负担显著相关。
我们的研究结果表明 MSH4 的 DNA 甲基化和基因表达差异是 dMMR 的标志物，也是 LS 的潜在新型生物标志物。我们对 LS 结肠类器官的研究支持 dMMR 存在于 LS 个体的结肠中，早于 CRC 的假说。
