Gene Discovery in Heritable Renal Hypodysplasia

描述(申请人提供)：肾发育不良(RHD)是一种先天性疾病，导致肾脏异常小和发育不良。风湿性心脏病与慢性肾功能衰竭有关，因为排泄物部分分化不正确，由于过滤效率降低，肾小管逐渐扩张。风湿性心脏病中指导畸形发生事件的基因仍然不清楚。该项目利用了3H1小鼠中一种名为Brachyrrhine(BR)的辐射诱导突变，该突变遗传了RHD作为常染色体的半显性特征。这个项目的总体目标是用BR作为模型来确定风湿性心脏病的遗传基础。在此之前，BR被定位于小鼠17号染色体(Chr17)的末端。该项目的第一个具体目标是进行远端chrl 7的高分辨率微卫星连锁分析，以利用大的回交小鼠DNA样本分离出100-500千碱基的区域，然后使用Celera和Sanger基因发现数据库对候选基因区域进行电子分析。第二个目的是利用Northern印迹分析筛选妊娠第13天和第14天突变和正常胚胎的肾组织中候选基因的表达。由于突变的表型和遗传模式与辐射诱导的双链断裂和片段缺失一致，因此有望鉴定出基因缺失。如果有缺失，突变的肾脏组织中的mRNA表达模式将通过RT-PCR和原位杂交与相应的正常肾组织进行比较，而相关蛋白的分析将利用免疫组织化学方法。特定目标3将使用3H1+/‘+和BR/BR DNA样本对候选区中的每个外显子进行测序。因此，即使在特定的目的2中没有发现缺失，3H1 BR中的突变也将被确定。将利用包括SSCP和异源双链分析在内的其他方法来鉴定可能丢失的外显子突变。作为这个项目的结果，3H1BR/BR小鼠中导致RHD的基因将被识别出来。该序列将进行鼠-人同源搜索，很可能会鉴定出相应的人类基因，因为远端小鼠的chr17与人的chr 2p21.1具有高度的序列相似性。

DESCRIPTION (provided by applicant): Renal hypodysplasia (RHD) is a congenital disease that results in abnormally small and dysplastic kidneys. RHD is associated with chronic renal failure since the excretory portion does not differentiate properly and renal tubules progressively distend due to decreased filtration efficiency. Genes directing dysmorphogenetic events in RHD remain obscure. This project utilizes a radiation induced mutation in the 3H1 mouse called Brachyrrhine (Br) that inherits RHD as an autosomal semidominant trait. The overall goal of this project is to determine the genetic basis of RHD using Br as a model. Previously, Br was mapped to the distal portion of murine chromosome 17 (chr17). The first specific aim of this project is to undertake a high resolutioin microsatellite linkage analysis of distal chrl 7 to isolate a 100 - 500 kilobase region utilizing a large back cross mouse DNA sample followed by an in silico analysis of the candidate gene region using Celera and Sanger Gene Discovery databases. The second aim is to screen renal tissues of mutant and normal embyos at gestational days 13 and 14 for candidate gene expression using Northern blot analysis. It is expected that a gene deletion will be identified since the mutant phenotype and inheritance pattern is consistent with a radiation induced doubled stranded break and segment deletion. If a deletion is indicated, renal mRNA expression patterns in the mutant will be compared to corresponding normal renal tissue using RT-PCR and in situ hybridization, while associated protein analysis will utilize immunohistochemical methods. Specific aim 3 will sequence each exon in the candidate region using 3H1+/'+ and Br/Br DNA samples. Thus, the mutation in 3H1 Br will be determined even if a deletion is not found in Specific Aim 2. Additional methods will be utilized including SSCP and heteroduplex analysis to identify mutations in exons that may have been missed. As a result of this project, the gene responsible for RHD in 3H1 Br/Br mice will be identified. This sequence will be subjected to a mouse-human homology search and it is likely that a corresponding human gene will be identified since distal murine chr17 shares a high degree of sequence affinity with human chr 2p21.1.