Model Systems for PXE

摘要 这项与之竞争的续期拨款申请《PXE模型系统》(AR R0155225)围绕以下内容展开 弹性假黄瘤(PXE)是遗传性异常矿化疾病的典范，表现在 皮肤、眼睛和心血管系统有相当高的发病率和死亡率。PXE由以下原因引起 ABCC6基因突变，编码一种主要在肝脏表达的外排转运蛋白。我们最新的数据， 主要基于对作为PXE模型的Abcc6-/-小鼠的实验，已经表明 PXE是一种位于基因组/环境界面的代谢紊乱。 PXE的临床特征之一是相当大的家庭内和家庭间的异质性。我们以前的 在约300名患者中建立直接基因/表型相关性的尝试已经完成 没有成功，表明遗传和环境因素对表型进行了调节。此应用程序具有 两个具体的调查领域。第一个集中在表型修饰基因的鉴定上 通过数量性状座位分析进行小鼠基因组学研究。这些分析利用了我们最近的 发现四个近交系小鼠的基因组中含有相同的ABCC6突变，但 表型表达具有很高的变异性。这些小鼠品系，在Abcc6-/-小鼠和它们的野生- 对应的类型，将用于建立信息性十字，这些十字将用于矿化分析 并通过基因分型分析扫描数量性状基因座。已确定的候选基因 PXE表型的调节将通过功能分析和组织阵列表达谱来验证。我们的 研究还将集中在一种特定的突变小鼠，Enpp1-/-，我们最近开发了它作为一种模型 一种严重的异位矿化障碍，婴儿期泛发性动脉钙化，与临床重叠 PXE的特点。Enpp1-/-小鼠将与Abcc6-/-小鼠杂交，发育的克隆具有双 杂合子和纯合子/杂合子复合突变将被分析表型。 调制。 第二个调查领域将集中在确定错义突变的后果 ABCC6基因，特别是关于功能性转运蛋白活性的丧失(由昆虫细胞，Sf9， 由内向外的囊泡系统)和突变蛋白在肝脏中的细胞内运输(如在In 活体小鼠灌流系统)。错义突变的致病本质也将在一项新的研究中得到验证 斑马鱼吗啡拆卸系统。最后，我们将尝试分子的鉴定(S) 由ABCC6通过多种途径进行生理运输。 总而言之，这些研究旨在确定导致发病机制的遗传因素。 从ABCC6基因突变到外周组织异位矿化的细节和鉴定 可通过药物干预的具体步骤来治疗这一目前难以治愈的疾病。

ABSTRACT This competing renewal application for the grant, "Model Systems for PXE" (AR R0155225) revolves around pseudoxanthoma elasticum (PXE), the paradigm of heritable aberrant mineralization disorders, manifesting in the skin, eyes, and the cardiovascular system with considerable morbidity and mortality. PXE is caused by mutations in the ABCC6 gene, encoding an efflux transporter expressed primarily in the liver. Our recent data, based primarily on experimentation on Abcc6-/- mice, which serve as a model for PXE, have suggested that PXE is a metabolic disorder at the genome/environment interface. One of the clinical features of PXE is considerable intra- and interfamilial heterogeneity. Our previous attempts to establish direct genotype/phenotype correlations in a cohort of ~300 patients have been unsuccessful, suggesting phenotypic modulation by genetic and environmental factors. This application has two specific areas of investigation. The first one focuses on identification of phenotypic modifier genes by mouse genomics through quantitative trait loci analysis. These analyses take advantage of our recent discovery of four inbred mouse strains that harbor the same ABCC6 mutation in their genome, yet the phenotypic expression is highly variable. These mouse strains, in the context of Abcc6-/- mice and their wild- type counterparts, will be used to set up informative crosses which will be analyzed for mineralization phenotypes and scanned for quantitative trait loci by genotyping analysis. The identified candidate genes for modulation of PXE phenotype will be verified by functional assays and by tissue array expression profiles. Our studies will also focus on a specific mutant mouse, Enpp1-/-, which we have recently developed as a model for a severe ectopic mineralization disorder, generalized arterial calcification of infancy, with overlapping clinical features of PXE. The Enpp1-/- mice will be crossed with Abcc6-/- mice, and the developed colonies with double heterozygous as well as homozygous/heterozygous compound mutations will be analyzed for phenotypic modulation. The second area of investigation will focus on identification of consequences of missense mutations in the ABCC6 gene, particularly with respect to loss of functional transporter activity (assayed by an insect cell, Sf9, inside-out vesicle system) and intracellular trafficking of the mutant protein in the liver (as assessed in an in vivo mouse perfusion system). The pathogenic nature of missense mutations will also be verified in a novel zebrafish morpholino knock-down system. Finally, we will attempt the identification of the molecule(s) physiologically transported by ABCC6, by a number of approaches. Collectively, these studies are designed to identify genetic factors that contribute to the pathomechanistic details leading from mutations in the ABCC6 gene to ectopic mineralization of peripheral tissues and to identify specific steps amenable to pharmacologic intervention towards treatment of this, currently intractable disease.