MOLECULAR GENETICS OF HUMAN ARPKD

常染色体隐性遗传性多囊肾病（ARPKD）是先天性 肾脏疾病是儿科发病率和死亡率的重要原因。 受影响的儿童通常患有进行性肾性高血压 肝硬化门静脉高压症。ARPKD的临床谱是 变化很大，大多数病例在婴儿期出现。的遗传缺陷 在这种疾病中的作用尚不清楚，肾脏疾病的分子机制 囊肿形成、肝脏疾病和高血压的发展仍然存在 有待阐明。导致这种温和形式的基因 最近已将该疾病定位于染色体6p 21 -12。我们有 随后证实了该基因座与严重的围产期 疾病的形式，并已完善了ARPKD遗传区间使用 重组映射到等于或小于4的区间 厘摩根。 拟议的项目是一个互动的国际财团的一部分， 努力识别ARPKD基因。为了准备这项研究，我们 收集了最大的ARPKD家族和组织数据库， 受影响的个人。我们构建了一个重叠的基因组重叠群， 克隆在酵母人工染色体（YACs）中的片段， 最近的侧翼遗传标记之间的间隔，并已验证 它的真实性使用序列标记的网站（STS）在我们的 laboratories.收集家系、ARPKD肾脏和肝脏组织， 物理图谱和克隆的基因组片段代表了 用于分离人ARPKD基因的资源。 在本申请中，我们建议构建一个高密度的遗传图谱， ARPKD候选地区。我们将研究目前确定的 重组，并招募更多的家庭参与， 进一步完善重组图谱。我们将分离和表征新的 微卫星标记映射在由当前 最接近的侧翼遗传标记和高密度STS图谱（<50 kb 分辨率）将被产生。基于YAC 物理图将被转换成一组重叠的P1和/或粘粒 克隆后一种试剂将用于鉴定表达的序列 使用方法的组合（例如，cDNA文库筛选，外显子 捕获和cDNA选择方案）。这些数据将与 YAC的现有EST内容图，以产生以下转录图： ARPKD间期。我们将评估每个基因的潜在候选资格 通过测定其序列和表达模式， 组织.我们会.扫描主要候选人的致病序列 使用包括SSCA、异源双链体 分析、直接基因组测序和/或RNA酶保护。最终 方法的选择将取决于基因的大小和表达的速率。 在开发更新、更有效的技术方面取得进展。

Autosomal recessive polycystic kidney disease (ARPKD) is, among congenital renal disorders, a significant cause of pediatric morbidity and mortality. Affected children typically 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 involved in renal cyst formation, liver disease and the development of hypertension remain to be elucidated. The gene responsible for the milder form of this disorder has recently been mapped to chromosome 6p21-12. We have subsequently confirmed linkage between this locus and the severe perinatal form of the disease and have refined the ARPKD genetic interval using recombination mapping to an interval of equal to or less than 4 centiMorgan. The proposed project is part of an interactive, international consortium effort to identify the ARPKD gene. In preparation for this study, we have assembled the largest available database of ARPKD families and tissue from affected individuals. We have constructed a contig of overlapping genomic fragments cloned in yeast artificial chromosomes (YACs) that spans the interval between the closest flanking genetic markers and have verified its authenticity using sequence tagged sites (STSs) generated in our laboratories. The collection of pedigrees, ARPKD kidney and liver tissues, the physical map and the cloned genomic segments represent critical resources for the isolation of the human ARPKD gene. In this application, we propose to construct a high density genetic map of the ARPKD candidate region. We will study currently identified recombinants and recruit the participation of additional families to further refine the recombination map. We will isolate and characterize new microsatellite markers mapping within the interval defined by the current closest flanking genetic markers and a high density STS map (<50 kb resolution) of the refined interval will be produced. The YAC-based physical map will be converted into a set of overlapping P1 and/or cosmid clones. The latter reagents will be used to identify expressed sequences using a combination of methods (e.g., cDNA library screening, exon trapping and cDNA selection protocols). This data will be integrated with existing EST content maps of the YACs to produce a transcription map of the ARPKD interval. We will evaluate the potential candidacy of each gene by determining its sequence and expression pattern in normal and affected tissue. We will.scan leading candidates for pathogenic sequence differenceS using a combination of methods including SSCA, heteroduplex analysis, direct genomic sequencing and/or RNase protection. The ultimate selection of a method will depend on the size of the gene and the rate of progress in the development of newer, more efficient technologies.