MOLECULAR GENETICS OF HUMAN ARPKD

常染色体隐性遗传性多囊肾病(ARPKD)是先天性肾脏疾病中的一种，是儿科发病率和死亡率的重要原因。受影响的儿童患有高血压、进行性肾功能不全和门脉纤维化。ARPKD的临床谱系差异很大，大多数病例出现在婴儿期。该病的遗传缺陷尚不清楚，表型的分子机制仍有待阐明。这种疾病似乎在遗传上是同质的，严重的围产期和较轻的形式都映射到6p21-12上的相同遗传区间。这项提议是一项竞争性的更新申请，旨在寻求资金来完成基因的分离和初步鉴定。在资金发放后的3年里，我们大大增加了ARPKD家庭数据库的规模，并招募了遗传背景相同的家庭参加。同时，我们用多态标记饱和了遗传区间，并用这些标记将PKHD1的位置细化到不到1 cM的区间。我们已经将基于YAC的物理地图替换为由PAC和BAC组成的地图。新的序列就绪图谱的平均STS密度为1/20kb，并预测最近的侧翼遗传标记大约相距400kb。我们在生成该间隔的转录图谱方面也取得了重大进展。使用各种方法，我们已经在最小间隔内鉴定了19组不重叠的表达序列，其中大部分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，并开始对其基因产物进行鉴定。在目标1中，我们建议通过在关键重组子中使用新的多态标记、鉴定新的重组子和分析遗传同源群体来完善遗传图谱。目标2建议完成关键区间的转录图谱，并用它来指导我们选择候选基因进行突变分析。一旦鉴定出PKHD1，我们将确定突变的性质，并调查ARPKD(目标编号3)的基因型和表型之间的关系。同时，我们将生产针对PKHD1基因产物的抗体，并在目标编号4中表征蛋白质的表达模式。最后，我们将使用基因靶向方法创建PKHD1的小鼠模型。

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.