高原适应性相关基因的鉴定及其功能和分子机制研究

Previous studies have suggested that high altitude adaptation (HAA) has an obvious genetic basis. However, the underlying genetic basis of HAA has not yet been fully characterized in the native Tibetans by the platform of whole genome sequencing (WGS). The native Tibetans have lived in the Qinghai-Tibetan Plateau for hundreds of generations, with an average elevation exceeding 4,500 metres and experiencing oxygen concentrations about 40% lower than at sea level, and shown successful adaptation to high altitude. In the previous study, we have sequenced 48 native Tibetans and 50 Han individuals living at sea level using WGS. Then, we performed the comparative genomics analysis based on the neutral theory of molecular evolution. In addition to confirming previously reported EGLN1, EPAS1, and other 19 HAA-related genes, we have identified several new candidates including MCUR1. Our preliminary functional studies showed that knockdown of MCUR1 in the human erythroleukemic cell line K562 can inhibit erythrocyte differentiation of the cells. In this proposal, based on these previous results, we will perform the molecular evolutionary analyses comprehensively, and systematically conduct population comparisons between the genomes of the native Tibetans and those of the Han individuals living at low altitude. The main goals in this proposal are to catalog HAA-related genetic variants, and identify HAA-related genes, pathways and networks systematically. Furthermore, we will investigate the biological functions of the newly identified HAA-related candidate variants and genes by both in vitro and in vivo assays. In addition, we will reveal the underlying molecular mechanisms of the candidates in HAA. This proposal is expected to construct a more comprehensive understanding of the genetic basis and molecular mechanisms for HAA, and may provide aid to develop new approaches for prevention, diagnosis and treatment of high altitude related diseases.

高原适应性具有明显的遗传学基础。目前尚未见在世居高原藏族人群中采用全基因组测序技术鉴定高原适应性相关基因的研究报道。本研究前期对48例高原藏族和50例平原汉族个体开展了全基因组测序和基于分子进化理论的比较基因组学研究。除了重复出以往已报道的EGLN1和EPAS1等21个高原适应性相关基因之外，我们还新发现了MCUR1等一批新的候选基因。前期初步的功能研究显示，敲低MCUR1可显著抑制K562细胞系向红系分化。本项目拟在前期研究基础上，综合运用多种“中性理论检验”的分子进化分析方法，更加系统地比较我国高原藏族和平原汉族人群之间的基因组序列变异，全面绘制与高原适应性相关的遗传变异图谱和基因/通路/网络；针对新发现的候选高原适应相关遗传变异和基因，在细胞和动物模型中探索其生物学功能以及潜在的分子机制。期望通过本项研究，进一步阐明高原适应性的遗传学和分子机理，为高原病新的防诊治措施的研发提供基础。

本项目在前期研究基础上，综合运用多种“中性理论检验”的分子进化分析方法，系统地比较我国高原藏族和平原汉族人群之间的基因组序列变异，全面绘制了与高原适应性相关的遗传变异图谱和基因/通路/网络；针对新发现的候选高原适应相关基因MCUR1，在细胞和动物模型中，系统揭示了其在低氧条件下促进红系生成的生物学功能，并阐明了相应的分子机制；鉴定出rs61644582是影响MCUR1表达的功能性遗传变异。.通过近五年的研究，我们基本完成了预期的研究目标。①绘制了与高原适应性相关的遗传变异图谱；②揭示了候选高原适应性相关基因显著富集的组织类型和生物学通路；③鉴定出一系列新的与高原适应性相关的候选基因，其中，最为显著的新的候选基因是MCUR1；④通过采用基于CRISPER/Cas9技术的Mcur1基因敲除小鼠的单细胞测序和流式分选实验，揭示了Mcur1缺失可显著降低造血干祖细胞（HSPCs）红系分化的倾向性。⑤通过多种细胞模型，证实了MCUR1缺失在低氧或常氧条件下具有减弱红系生成的作用；⑤机制上，MCUR1缺失可显著降低线粒体Ca2+的摄取，增加胞浆Ca2+水平，进而通过CAMKK2-AMPK-mTOR途径减弱红细胞生成；⑥鉴定出rs61644582是影响MCUR1表达的功能性遗传变异。.通过本项研究，我们系统揭示了人体在高原适应过程中受到正向自然选择的新的基因组区域/基因。进一步，通过多种细胞/动物模型和系列实验，阐明了新的高原适应性候选基因MCUR1是新的红系生成过程的调节因子。其他候选基因在高原适应性中的功能和机制同样值得今后进一步的研究。MCUR1等候选高原适应性相关基因的功能的揭示，为未来探索新型有效的高原相关疾病的预防和治疗策略提供了理论依据。