MOLECULAR GENETICS OF HUMAN ARPKD

常染色体隐性多囊肾病（ARPKD）是先天性肾脏疾病之一；是儿童发病和死亡的重要原因。患儿表现为高血压、进行性肾功能不全和门静脉纤维化。ARPKD的临床谱变化很大，大多数病例出现在婴儿期。这种疾病的遗传缺陷是未知的，其表型的分子机制仍有待阐明。该疾病似乎具有遗传同质性，严重的围产期和较轻的形式都在6p21-12上具有相同的遗传间隔。该提案是一项竞争性更新申请，寻求资金来完成基因的分离和初步表征。在获得资助后的3年里，我们大大增加了ARPKD家庭数据库的规模，并招募了基因同质背景的家庭参与。与此同时，我们用多态性标记饱和了遗传区间，并利用这些标记将PKHD1的位置细化到小于1cM的区间。我们已经用由pac和bac组成的物理地图取代了基于yac的物理地图。新的序列准备图谱的平均STS密度为1/20kb，并预测最接近的侧翼遗传标记相距约400kb。我们在生成区间转录图方面也取得了重大进展。使用多种方法，我们在最小间隔内鉴定出19组非重叠表达序列，其中大多数似乎是新基因的产物。目前正在进行研究，以完成它们的分离和表征。在这次更新中，我们寻求资金来完成对PKHD1的搜索并开始表征其基因产物。在目标1中，我们建议通过在我们的关键重组中使用新的多态性标记来完善遗传图谱，识别新的重组，并分析遗传同质群体。目标2提出完成临界区间的转录图谱，并以此来指导我们选择候选基因进行突变分析。一旦确定了PKHD1，我们将描述突变的性质，并研究ARPKD基因型和表型之间的关系（目的3）。同时，我们将生产针对PKHD1基因产物的抗体，并在Aim 4中表征该蛋白的表达模式。最后，我们将利用基因靶向方法建立小鼠PKHD1模型。

Autosomal recessive polycystic kidney disease (ARPKD) is, among congenital renal disorders; a significant cause of pediatric morbidity and mortality. Affected children suffer from hypertension, progressive renal insufficiency and portal tract fibrosis. The clinical spectrum of ARPKD is widely variable with most cases presenting in infancy. The genetic defect in this disease is unknown and the molecular mechanisms underlying the phenotype remain to be elucidated. The disease appears to be genetically homogeneous, with both the severe perinatal and milder forms mapping to an identical genetic interval on 6p21-12. This proposal is a competitive renewal application that seeks funds to complete the gene's isolation and initial characterization. In the 3 years since the funding was awarded, we have greatly increased the size of our database of ARPKD families and recruited participation of families from genetically homogeneous backgrounds. In parallel, we have saturated the genetic interval with polymorphic markers and used these to refine the position of PKHD1 to an interval of less than 1cM. We have replaced our YAC-based physical map with one comprised of PACs and BACs. The new sequence-ready map has an average STS density of 1/20kb and predicts that the closest flanking genetic markers are approximately 400kb apart. We have also made significant progress in generating a transcription map of the interval. Using a variety of methods, we have identified 19 sets of non-overlapping expressed sequences in the minimal interval, the majority of which appear to be products of novel genes. Studies are presently underway to complete their isolation and characterization. In this renewal, we seek funds to complete the search for PKHD1 and begin characterization of its gene product. In Aim number 1, we propose to refine the genetic map by using new polymorphic markers in our critical recombinants, identifying new recombinants, and analyzing genetically homogenous populations. Aim number 2 proposes to complete the transcription map of the critical interval and use this to guide our selection of candidate genes for mutation analysis. Once PKHD1 is identified, we will characterize the nature of mutations and investigate the relationship between genotype and phenotype in ARPKD (Aim number 3). In parallel, we will produce antibodies to the PKHD1 gene product and characterize the protein's pattern of expression in Aim number 4. Finally, we will create a murine model of PKHD1 using gene-targeting methods.