青少年特发性脊柱侧凸正常-患病表型分离同卵双胞胎队列的表观遗传学研究

Adolescent idiopathic scoliosis is a complicated spinal discord for spine surgeons, and is a heavy burden on society and families. It has become the current research hotspot of AIS to explore its early diagnosis and treatment targets. The genesis of scoliosis is related to not only genetics but also epigenetics. However, the molecular mechanism underlining its pathogenesis is not well known. In our previous study, we have set up an AIS cohort and found 5 monozygotic twins, of which one was diagnosed with AIS, and the other was normal. We use WGBS to perform methylation analysis, the difference of whole-genome DNA methylation between the twins was significant, and some of the DMR located nearby SNP. Therefore, we presumed that epigenetics mutation and SNP might have a pivotal role in AIS. Based on our previous study, we hypothesize that epigenetics mutation combing with SNP could lead to the development of AIS. The techniques of WGBS and the strategy of MZ twins will be used to explore the epigenetic and SNP factors of AIS. SNP-DNA methylation-AIS clinical phenotype Regulation network will be built. Up to now, there was no study on the whole genome epigenetic research about AIS. This study will give a new insight into the etiology of AIS and provide a new way for the early diagnosis, treatment, and prevention of AIS.

青少年特发性脊柱侧凸（AIS）是脊柱外科的难题，对社会和家庭造成沉重负担，探索其早期诊断和治疗靶点是当前研究热点。其发生除与遗传因素相关外，表观遗传学也发挥重要作用，但具体机制尚不明确。同卵双胞胎由于其遗传背景完全相同，是表观遗传学理想的研究对象。前期工作中，我们已建立AIS队列，并发现5对AIS正常-患病同卵双胞胎，应用WGBS对其中1对进行测序分析，结果显示两者存在可能与AIS相关的全基因组DNA甲基化水平差异，并发现差异区域部分位于SNP附近。因此，本项目以“全基因组表观遗传学变异结合SNP可能导致AIS的发病”为假说，采用全基因组甲基化测序方法，通过对比表型分离的同卵双胞胎差异，结合SNP并进行大样本及细胞功能验证，寻找并阐明与AIS发生相关的表观遗传学及SNP因素，建立SNP-DNA methylation-AIS临床表型调控网络，为AIS的预防和早期诊治提供新的思路和靶点。

青少年特发性脊柱侧凸（AIS）是脊柱外科的难题，对社会和家庭造成沉重负担，探索其早期诊断和治疗靶点是当前研究热点。其发生除与遗传因素相关外，表观遗传学也发挥重要作用，但具体机制尚不明确。同卵双胞胎由于其遗传背景完全相同，是表观遗传学理想的研究对象。我们通过已建立的AIS队列，应用全基因组亚硫酸氢盐测序法（WGBS），对AIS 正常-患病同卵双胞胎进行测序分析，发现与AIS发病相关的差异甲基化区（DMR），通过生信分析，发现相应基因显著聚类于MAPK信号通路、内分泌耐药等通路，并通过与人类基因组序列对比后发现相关基因与脊柱侧凸显著相关。同时在AIS患者及健康对照人群中，通过多重PCR技术进行测序，检测高致病DMR相关基因变异在散发AIS患者及健康对照中的携带比例，我们发现NDN: TSS-DMR (chr15:23932133-23932304, Hg19)在两组中甲基化水平具有显著性差异，提示该DMR与AIS致病相关。后续我们通过分离骨髓的间充质干细胞，通过构建相关DMR及差异SNP的双荧光素酶报告系统，在细胞水平观察基因甲基化及单核苷酸序列多态性对于基因表达具有显著影响，通过关联性分析，发现SNP及基因甲基化与AIS分型相关，为后续进一步探讨表观遗传学对于不同类型的AIS奠定了良好的前期基础。本项目目前已在国际知名SCI期刊发表论文多篇，部分课题实验结果尚未完成论文撰写及发表，将陆续完成。本课题成功的通过实验发现与青少年特发性脊柱侧凸发病相关的基因甲基化差异，并初步从细胞水平探讨了相关分子机制，为青少年特发性脊柱侧凸的预防和早期诊治提供新的思路和靶点，为下一步进行相关基因及靶向治疗奠定了基础。