Gene Discovery in Heritable Renal Hypodysplasia

遗传性肾发育不良的基因发现

• 批准号: 7078580
• 负责人: SCOTT LOZANOFF
• 金额: $ 12.74万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7078580
• 关键词: chronic renal failure; congenital kidney disorder; gene expression; gene mutation; genetic disorder; immunocytochemistry; in situ hybridization; laboratory mouse; linkage mapping; molecular biology information system; nucleic acid sequence; single strand conformation polymorphism

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.

描述（由申请人提供）：肾发育不全（RHD）是一种先天性疾病，导致肾脏异常小和发育不良。RHD与慢性肾衰竭相关，因为排泄部分不能正确分化，肾小管由于滤过效率降低而进行性扩张。在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3 H1小鼠中称为Brachyrrhine（Br）的辐射诱导突变，该突变遗传了RHD作为常染色体半显性性状。该项目的总体目标是确定RHD的遗传基础，使用Br作为模型。以前，Br被映射到小鼠染色体17（chr 17）的远端部分。该项目的第一个具体目标是利用大的回交小鼠DNA样品进行远侧chr 17的高分辨率微卫星连锁分析以分离100 - 500个酶区域，随后使用Celera和桑格Gene Discovery数据库对候选基因区域进行计算机分析。第二个目的是使用北方印迹分析筛选在妊娠第13天和第14天的突变和正常胚胎的肾组织中的候选基因表达。预期将鉴定出基因缺失，因为突变体表型和遗传模式与辐射诱导的双链断裂和片段缺失一致。如果指示缺失，则使用RT-PCR和原位杂交将突变体中的肾mRNA表达模式与相应的正常肾组织进行比较，而相关蛋白质分析将使用免疫组织化学方法。具体目标3将使用3 H1 +/'+和Br/Br DNA样品对候选区域中的每个外显子进行测序。因此，即使在特异性目的2中未发现缺失，也将确定3 H1 Br中的突变。将使用其他方法，包括SSCP和异源双链体分析，以识别可能缺失的外显子突变。作为该项目的结果，将鉴定3 H1 Br/Br小鼠中导致RHD的基因。将对该序列进行小鼠-人同源性搜索，并且很可能将鉴定出相应的人基因，因为远端鼠chr 17与人chr2p21.1具有高度的序列亲和力。