A murine model for human factor H R1210C mutation-related diseases

描述(由申请人提供)：因子H(FH)是替代途径(AP)补体的血浆抑制物。它由20个短共识重复(SCR)结构域组成。Fh的补体调节功能位于N端的SCR1-4，而Fh的两个C端SCR(SCR 19和20)对于在宿主细胞特异的多阴离子成分的背景下与表面沉积的C3b/C3d相互作用至关重要。人FH的SCR19-20基因突变破坏了其与宿主细胞的相互作用，并易发生非典型溶血性尿毒症综合征(AHUS)。在aHUS患者中发现的各种FH的SCR19-20突变中，R1210C突变尤其令人感兴趣，因为最近的遗传学研究发现，该突变也是老年性黄斑变性(AMD)发展过程中的一种高穿透性突变。人FH的R1210C变异体与C3b/C3d、肝素和内皮细胞的结合减少，但R1210不能跨物种保守(D1210在小鼠，G1210在大鼠，P1210在牛和猪)。我们推测，人类fh基因1210位氨基酸变为半胱氨酸，而不是简单的精氨酸丢失，是导致aHUS和AMD风险的关键因素。在这项探索性拨款中，我们提出了两个具体的目标来检验这一假设。具体目的1.我们将在体外对人和小鼠的Fh进行1210位的突变研究，并测试小鼠D1210C突变与人R1210C突变一样会损害与C3b、肝素和内皮细胞的结合的预测。另一方面，我们预测，人Fh中的R1210突变到R1210D、R1210G和R1210P(分别对小鼠、大鼠和牛/猪残留物)，以及小鼠Fh中的D1210突变到D1210R、D1210G和D1210P(分别对人、大鼠和牛/猪残留物)是可以容忍的，并且不会对C3b、肝素和内皮细胞结合产生功能影响。具体目的2.我们将通过基因靶向产生D1210C突变小鼠，并确定该小鼠是否患上了类似于人类AMD的AHUS和/或视网膜变性。这些研究将为Fh C-末端结构域的结构/功能知识提供新的线索，并确定该区域的特定突变如何在不同的组织和物种中对Fh功能产生不同的影响。此外，它们将帮助我们了解两种与FH相关的人类疾病的发病机制，并为测试针对这些疾病的抗补体疗法提供急需的小鼠模型。

DESCRIPTION (provided by applicant): Factor H (fH) is a plasma inhibitor of the alternative pathway (AP) complement. It is composed of 20 short consensus repeat (SCR) domains. The complement regulatory function of fH is located in the N-terminal SCR1-4, whereas the two C-terminal SCRs of fH (SCR 19 and 20) are critical for interacting with surface deposited C3b/C3d in the context of host cell-specific polyanionic constituents. Mutations in SCR19-20 of human fH impair its interaction with host cells and predispose to the development of atypical hemolytic uremic syndrome (aHUS). Among the various SCR19-20 mutations of fH that are found in aHUS patients, the R1210C mutation is of particular interest as a recent genetic study has identified this mutation to be also a high-penetrant mutation for the development of age-related macular degeneration (AMD). The R1210C variant of human fH showed reduced binding to C3b/C3d, heparin and endothelial cells, yet R1210 is not conserved across species (D1210 in mouse, G1210 in rat, P1210 in cows and pigs). We hypothesize that the change of amino acid at position 1210 of human fH to cysteine, rather than the simple loss of arginine, is critical for conferring the risk of aHUS and AMD. In this exploratory grant, we propose two specific aims to test this hypothesis. Specific Aim 1. We will perform in vitro mutagenesis studies of human and mouse fH at residue 1210 and test the prediction that a mouse D1210C mutant will, like human R1210C mutant, have impaired binding to C3b, heparin and endothelial cells. On the other hand, we predict that mutations of R1210 in human fH to R1210D, R1210G and R1210P (to mouse, rat and cow/pig residue, respectively), and mutations of D1210 in mouse fH to D1210R, D1210G and D1210P (to human, rat, and cow/pig residue, respectively), are tolerated and produce no functional consequences for C3b-, heparin- and endothelial cell-binding. Specific Aim 2. We will generate by gene targeting a D1210C mutant mouse and determine if this mouse develops aHUS and/or retinal degeneration resembling human AMD. These studies will shed new light on structure/function knowledge of the fH C-terminal domain and establish how specific mutations in this domain may differentially affect fH function in different tissues and species. Furthermore, they will help us understand the pathogenesis of two fH-related human diseases and provide a much- needed mouse model for testing anti-complement therapies for these pathologies.