Model Systems for PXE

描述（由申请人提供）：“PXE的模型系统”（AR R0155225）是一项竞争性续期申请，涉及弹性假性黄瘤（PXE），这是一种遗传性异常矿化疾病的范例，表现在皮肤、眼睛和心血管系统，具有相当高的发病率和死亡率。PXE是由ABCC6基因突变引起的，该基因编码一种主要在肝脏表达的外排转运蛋白。我们最近的数据，主要基于Abcc6-/-小鼠的实验，作为PXE的模型，表明PXE是一种基因组/环境界面的代谢紊乱。PXE的临床特征之一是相当大的家族内和家族间异质性。我们之前试图在约300名患者的队列中建立直接的基因型/表型相关性，但没有成功，这表明表型受遗传和环境因素的调节。这个应用程序有两个特定的调查领域。第一部分是通过数量性状位点分析，利用小鼠基因组学方法鉴定表型修饰基因。这些分析利用了我们最近发现的四种近交小鼠品系，它们的基因组中含有相同的ABCC6突变，但表型表达却高度可变。这些小鼠菌株，在Abcc6-/-小鼠及其野生型小鼠的背景下，将被用来建立信息杂交，用于分析矿化表型，并通过基因分型分析扫描数量性状位点。已确定的PXE表型调节候选基因将通过功能分析和组织阵列表达谱进行验证。我们的研究还将集中在一个特定的突变小鼠，Enpp1-/-，我们最近开发了一个模型，严重异位矿化障碍，婴儿期广泛性动脉钙化，与PXE重叠的临床特征。将Enpp1-/-小鼠与Abcc6-/-小鼠杂交，对双杂合、纯合/杂合复合突变的发育菌落进行表型调控分析。第二个研究领域将集中于鉴定ABCC6基因错义突变的后果，特别是关于功能性转运蛋白活性的丧失（通过昆虫细胞，Sf9，内外囊泡系统进行检测）和肝脏中突变蛋白的细胞内运输（通过小鼠体内灌注系统进行评估）。错义突变的致病性质也将在一个新的斑马鱼morpholino敲除系统中得到验证。最后，我们将尝试通过多种方法鉴定由ABCC6生理运输的分子。总的来说，这些研究旨在确定导致从ABCC6基因突变到外周组织异位矿化的病理机制细节的遗传因素，并确定对这种目前难治性疾病的药物干预治疗的具体步骤。

DESCRIPTION (provided by applicant): 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.