Genetic variants of the mechanosensitive ion channel Piezo1 in red blood cells - their role in human physiology and evolution

该项目旨在建立红细胞Piezo1基因多态与通道活性、变形性、红细胞携氧能力以及最终组织氧合之间的可能联系。实现这一目标将支持或反对Piezo1变体在适应不断变化的环境条件和现代人(MH)自然选择中可能扮演的角色的假设。为了验证我们的假设，我们的目标是对携带mh_Piezo1序列(编码307G)的人诱导多能干细胞(IPSC)进行基因组编辑，并用古老的arcPiezo1变体(编码307S)取代它。体外扩增修饰的IPSC的RBC(Arc_RBC)将使我们能够研究Arc_Piezo1通道的电生理特性及其对红细胞生成的影响，并最终与未修饰的RBC(MH_RBC)进行比较。此外，将在具有基因或药物修饰的Piezo1活性的小鼠模型中系统地研究Piezo1改变对RBC特性、氧气输送和组织氧合的影响。我们将使用功能增益(GOF)小鼠模型。为了初步了解Piezo1变异在人类组织氧合中的作用，将对携带已知Piezo1 GOF突变的遗传性干细胞症(HX)患者进行运动后Piezo1通道功能和红细胞特性以及组织氧合测量。我们不打算研究HX的病理生理学，但我们将利用病理性的Piezo1突变及其在体内应激条件下对组织氧合的影响。类似的实验将在一组匹配的健康对照组中进行。最后，对潜在的环境决定因素和相关的选择性过程进行数学建模是区分人口统计学效应和选择性效应的可行方法，在我们的案例中，用来阐明影响红细胞生理(通道功能、钙信号、细胞流动特性和氧气输送能力)的Piezo1基因多态性的人口统计学图景和历史。已经提出了从有限数量的个体的二进制或有序的基因/表型数据进行估计的各种方法，包括近似贝叶斯计算(ABC)方法。我们将根据我们的实验结果，通过模拟潜在选择机制的效果，使用后者来测试我们的两个假设。

This project aims to establish the possible links between erythroid Piezo1 polymorphisms and channel activity, deformability, oxygen carrying capacity of red blood cells (RBC), and finally tissue oxygenation. Reaching this aim will support or disapprove the hypothesis of the possible role of Piezo1 variants in adaptation to the changing environmental conditions and natural selection in modern humans (mh). To test our hypothesis, we aim to perform genome editing of human-induced pluripotent stem cells (iPSC) carrying the mh_Piezo1 sequence (coding 307G) and replace it with the archaic arc_Piezo1 variant (coding 307S). Ex vivo amplification of RBC from modified iPSC (arc_RBC) will allow us to investigate the electrophysiological arc_Piezo1 channel properties and its impact on erythropoiesis and, finally, RBC function in comparison to the unmodified RBC (mh_RBC). Furthermore, in vivo effects on RBC properties, oxygen delivery, and tissue oxygenation caused by Piezo1 alterations will be investigated systemically in a mouse model with genetically or pharmacologically modified Piezo1 activity. We will use a gain of function (GOF) mouse model. To get initial insight into the role of Piezo1 variants in human tissue oxygenation, Piezo1 channel function and RBC properties as well as tissue oxygenation measurements after physical exercise will be performed in a small cohorts of Hereditary Xerocytosis (HX) patients carrying known Piezo1 GOF mutations. We do not aim to investigate the pathophysiology of HX, but we will take advantage of the pathological Piezo1 mutations and their effect on tissue oxygenation under stress conditions in vivo. Comparable experiments will be performed in a matched group of healthy controls. Finally, mathematical modelling of potential environmental determinants and associated selective processes is a viable approach to distinguish demographic from selective effects, in our case, to elucidate the demographic landscape and history of Piezo1 polymorphisms affecting RBC physiology (channel function, Calcium signalling, cell flow properties, and oxygen delivery capacity). Various methods have been proposed for estimations from binary or ordinal genotypic/phenotypic data from a limited number of individuals, including Approximate Bayesian Computation (ABC) methods. We will use the latter to test our two hypotheses by simulating the effects of potential selection mechanisms based on our experimental results.