应用全外显子组测序结合代谢组学探索中国汉族人群胸椎黄韧带骨化症的致病基因

Thoracic ossification of ligamentum flavum (TOLF) is one of the most causes of adult disability and brings high burden to the family and the society, and with a prevalence of about 3.8%. Genetic factors are probably important causes of TOLF. In addition, many metabolic disorders resulted from with some genetic mutations are closely associated with TOLF. According to the literature, some progress has been made in genetic etiology, however, the disease-causing gene are still not clear. Locating disease-causing gene and exploring early diagnosis methods and therapeutic targets are the focus of TOLF research. The present project is based on the previous proteomics and metabolomics research, using the strategy of combining family trios with sporadic cases, and following the “integrate omics” concept. For 15 family trios with highly phenotype consistence, differences in metabonomics will be found with UPLC-MC technology and whole exome sequencing will be completed with next-generation sequencing technology. Then the data of the two omics will be integrated and deeply analyzed so as to locate the disease-causing gene mutations in Chinese Han population with TOLF. Then the mutations will be confirmed by polymerase chain reaction (PCR) in large-scale sporadic cases and healthy subjects. As results, the causative gene mutations associated with TOLF will be located and the relationship between genotype and clinical phenotype will be revealed. This project is the extension and expansion of preliminary innovative work of the applicant, aiming to investigate the mechanism of TOLF, expecting to provide the theory basis for the early diagnosis, surveillance and effective intervention in early stage of TOLF.

胸椎黄韧带骨化症（thoracic ossification of ligamentum flavum，TOLF），是一种严重致残性疾病，发病率约3.8%。已有的研究结果证实遗传因素可能是其重要病因，遗传变异导致的多种代谢异常可能与TOLF的发生发展密切相关。目前TOLF的遗传学研究已取得一定进展，但仍未明确TOLF致病基因。明确TOLF的致病基因，以期探索早期诊断方法和病因治疗靶点是当前TOLF的研究热点。本研究拟在前期蛋白质组学及代谢组学研究的基础上，采用核心家系结合散发病例研究策略，遵循“整合组学”理念，对15个表型高度一致的TOLF家系运用UPLC-MC技术进行代谢组学研究，并结合全外显子组测序，筛检与TOLF发病可能相关的基因，并进一步在大样本的散发病例及健康对照人群中进行验证，最终确定TOLF的致病基因，并进行基因型-临床表型关联分析，深入揭示胸椎黄韧带骨化症的发病机制。

主要研究内容：本研究中我们首先收集我院自2009年至今就诊的胸椎黄韧带骨化患者基本信息，同时搜集影像学及临床资料，对这些资料进行初步分析，筛选患者，排除存在后纵韧带骨化、椎体骨折致椎管狭窄等干扰因素，同时排除各种影响检测结果的合并症，包括：糖尿病及各种代谢性疾病。而后收集符合标准的患者的血样标本，采用超高效液相色谱-质谱（UPLC-MC）技术及全外显子技术对患者的代谢组及基因组的情况进行分析。.本研究的重要结果：.1..经过代谢组学的初步筛查（包括正离子和负离子两种模式）其中.a).正离子模式共检测出7057个代谢化合物，经过临床筛选后找出10个代谢差异物。其中包括：维生素D3代谢物，溶血磷脂酰胆碱，促黑激素抑制素，次黄嘌呤，苯丙氨酸，脂肪酸胆碱，谷氨酰脯氨酸，酰基肉毒碱等。.b).负离子模式中检测出6062个代谢化合物，经过筛选后存在6个差异最明显的代谢物，这些代谢物中尿酸、胆固醇酯及dTDP存在临床意义。.2.. 经过对影像学资料的初步分析我们发现了黄韧带骨化患者中硬脊膜骨化与椎管狭窄之间存在一定联系。.3..通过对24名送检患者的基因组DNA进行提取、扩增后，进行分析筛选。.a).选择了52个与OPLL和OLF相关的候选基因进行进一步研究。.b).利用GnomAD v2.1大规模人群数据库对AF>0.01的位点进行剔除，最终得到252个SNP数据库。.c).在进行相关文献报道的筛选，最后锁定8个与TSS相关的SNP.本研究的创新点：.1).本研究首次发现尿酸及溶血性磷脂酸胆碱的浓度对于黄韧带骨化存在一定关系；.2).从对影像学资料搜集过程中我们发现椎管狭窄的严重程度在黄韧带骨化患者中与硬脊膜骨化关系密切。.3).通过常规遗传分析，8个位于候选基因的位点被确定为 TSS 潜在的相关变异其中：PKLR 基因上的 rs201267482，该 SNP 位于与 TSS 表型相关；SNTG1 基因与 TSS 相关； .本研究的关键技术:UPLC-MS检测及Illumin全外显子测序。.本研究的科学价值及社会价值：为实现胸椎管狭窄黄韧带骨化的早期发现、早期干预及治疗提供科学依据。

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