MOLECULAR GENETICS OF HUMAN ARPKD

常染色体隐性遗传性多囊肾病（ARPKD）是先天性肾脏疾病之一，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的临床谱变化很大，大多数病例在婴儿期出现。 这种疾病的遗传缺陷是未知的，表型的分子机制仍有待阐明。该疾病似乎是遗传同质性的，严重的围产期和轻度形式映射到6p 21 -12上的相同遗传区间。该提案是一个竞争性的更新申请，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，并招募了来自遗传同质背景的家庭参与。 与此同时，我们用多态性标记使遗传间隔饱和，并使用这些标记将PKHD 1的位置细化到小于1cM的间隔。 我们已经将基于YAC的物理地图替换为由PAC和BAC组成的物理地图。 新的序列就绪图具有1/20 kb的平均STS密度，并预测最近的侧翼遗传标记相距约400 kb。 我们也取得了重大进展，在产生一个转录图的时间间隔。 使用各种方法，我们已经确定了19套非重叠表达序列的最小间隔，其中大部分似乎是新基因的产物。目前正在进行研究，以完成其分离和表征。 在这次更新中，我们寻求资金来完成PKHD 1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体中使用新的多态性标记，鉴定新的重组体，并分析遗传同质群体来完善遗传图谱。 目标2提出了完成关键区间的转录图谱，并使用它来指导我们选择候选基因进行突变分析。 一旦PKHD 1被确定，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（目标编号3）。 与此同时，我们将生产PKHD 1基因产物的抗体，并表征目标4中蛋白质的表达模式。 最后，我们将使用基因靶向方法建立PKHD 1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.