Model Systems for PXE

描述（由申请人提供）：这一竞争性的赠款续期申请，“PXE模型系统”（AR R 0155225）围绕弹性假黄瘤（PXE），遗传性异常矿化障碍的范例，表现在皮肤，眼睛和心血管系统，具有相当大的发病率和死亡率。PXE是由ABCC 6基因突变引起的，ABCC 6基因编码主要在肝脏中表达的外排转运蛋白。我们最近的数据，主要是基于实验Abcc 6-/-小鼠，这是一个模型的PXE，已经表明，PXE是一种代谢紊乱的基因组/环境界面。PXE的临床特征之一是相当大的家族内和家族间异质性。我们以前试图在一个约300例患者的队列中建立直接的基因型/表型相关性，但没有成功，这表明遗传和环境因素对表型的调节。本申请有两个具体的调查领域。第一个是通过数量性状位点分析，利用小鼠基因组学技术鉴定表型修饰基因。这些分析利用了我们最近发现的四种近交系小鼠品系，它们的基因组中含有相同的ABCC 6突变，但表型表达是高度可变的。在Abcc 6-/-小鼠及其野生型对应物的背景下，这些小鼠品系将用于建立信息杂交，其将分析矿化表型并通过基因分型分析扫描数量性状基因座。将通过功能测定和组织阵列表达谱来验证所鉴定的用于调节PXE表型的候选基因。我们的研究还将集中在一个特定的突变小鼠，Enpp 1-/-，我们最近开发的一个严重的异位矿化障碍，广义的婴儿动脉钙化，与重叠的PXE临床特征的模型。Enpp 1-/-小鼠将与Abcc 6-/-小鼠杂交，并分析具有双杂合以及纯合/杂合复合突变的发育集落的表型调节。调查的第二个领域将集中在识别ABCC 6基因中的错义突变的后果，特别是关于功能性转运蛋白活性的丧失（通过昆虫细胞Sf 9，由内而外的囊泡系统测定）和突变蛋白在肝脏中的细胞内运输（如在小鼠体内灌注系统中评估的）。错义突变的致病性也将在一种新的斑马鱼吗啉敲除系统中得到验证。最后，我们将尝试通过多种方法鉴定ABCC 6生理转运的分子。总的来说，这些研究旨在确定遗传因素，导致从ABCC 6基因突变到外周组织异位矿化的病理机制细节，并确定适合于治疗这种目前难治性疾病的药物干预的具体步骤。

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.