Gene Discovery in Heritable Renal Hypodysplasia

遗传性肾发育不良的基因发现

• 批准号: 6770095
• 负责人: SCOTT LOZANOFF
• 金额: $ 17.15万
• 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/6770095
• 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）：肾发育不良（RHD）是一种先天性疾病，导致肾脏异常小和发育不良。RHD与慢性肾衰竭有关，因为排泄部分不能正常分化，肾小管因滤过效率下降而逐渐扩张。指导RHD畸形发生事件的基因仍然不清楚。该项目利用辐射诱导的3H1小鼠Brachyrrhine （Br）突变，将RHD遗传为常染色体半显性性状。该项目的总体目标是以Br为模型确定RHD的遗传基础。此前，Br被定位到小鼠17号染色体（chr17）的远端部分。该项目的第一个具体目标是利用大型背交小鼠DNA样本对远端chrl 7进行高分辨率微卫星连锁分析，以分离出100 - 500千碱基区域，然后使用Celera和Sanger基因发现数据库对候选基因区域进行计算机分析。第二个目的是使用Northern blot分析筛选妊娠13和14天突变和正常胚胎的肾组织中候选基因的表达。由于突变体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，预计将鉴定出基因缺失。如果存在缺失，将使用RT-PCR和原位杂交技术将突变体肾脏mRNA表达模式与相应的正常肾组织进行比较，同时使用免疫组织化学方法进行相关蛋白分析。Specific aim 3将使用3H1+/'+和Br/Br DNA样本对候选区域的每个外显子进行测序。因此，即使在Specific Aim 2中没有发现缺失，也可以确定3H1 Br中的突变。其他的方法将被利用，包括SSCP和异双工分析来识别可能被遗漏的外显子突变。作为这个项目的结果，在3H1 Br/Br小鼠中负责RHD的基因将被确定。该序列将进行小鼠-人类同源性搜索，由于远端小鼠chr17与人类chr 2p21.1具有高度的序列亲和性，因此很可能鉴定出相应的人类基因。