The molecular basis for an animal model of inherited hyperuricosuria

遗传性高尿酸尿动物模型的分子基础

• 批准号: 7084992
• 负责人: DANIKA L BANNASCH
• 金额: $ 18.18万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7084992
• 关键词: animal genetic material tag; autosomal recessive trait; complementary DNA; dogs; functional /structural genomics; gene expression; gene mutation; genetic models; hyperuricemia; linkage disequilibriums; liver metabolism; northern blottings; nucleic acid sequence; polymerase chain reaction; purine /pyrimidine metabolism; purine /pyrimidine metabolism disorder; single nucleotide polymorphism; southern blotting; urate

DESCRIPTION (provided by applicant): The Dalmatian is unique among dogs in that it excretes uric acid rather than allantoin, the normal byproduct of purine metabolism. Thus, the Dalmatian is a natural model for the human conditions hyperuricosuria and hyperuricemia (i.e., elevated levels of uric acid in the urine and blood, respectively). Tissue transplantation experiments have demonstrated that the defect is intrinsic to the liver, consistent with an inborn error in purine metabolism. Although the defect is fixed within the Dalmatian population, a multigenerational Pointer x Dalmatian backcross family segregates a single locus responsible for the autosomal recessive disorder. The proposed research will identify the gene responsible for hyperuricosuria/hyperuricemia (huu) in the Dalmatian breed. A genome scan has been performed which demonstrated linkage of huu to CFA 3 with a LOD score of 6.5. Further fine structure mapping using microsatellites derived from the canine genome sequence localized huu to a 3.3 Mb interval flanked by recombination breakpoints. Based on the recent availability of the canine genome sequence, there are twenty four candidate genes in this interval. Interestingly, none of these genes have previously identified functions in purine metabolism or urate transportation, confirming that this work will yield novel insights into hyperuricosuria. A three tiered approach will be taken to identify the mutation responsible for hyperuricosuria in the Dalmatian. First, SNPs obtained from the canine genome sequence will be evaluated for linkage disequilibrium in the Dalmatian breed. The area of LD may exclude additional candidate genes as well as establish our expectations for potential disease-causing alleles. Second, candidate genes from the critical interval that are expressed in the canine liver will be determined using RT-PCR. At the same time, expression levels will be compared between Dalmatians and non-Dalmatians using Northern Blots to identify any qualitative or quantitative differences in expression. Based on the results of the three approaches, we will begin sequencing cDNA from the remaining candidates as well as performing Southern blot analysis to identify any large rearrangements. Putative mutation-causing alleles will be evaluated by verification of homozygosity in the Dalmatian breed, lack of homozygosity in other dog breeds, as well as appropriate functional studies of the mutation. Identification of the gene and mutation that cause high levels of uric acid in the Dalmatian dog will further establish it as a model system for the human condition. Uric acid levels are a contributing factor for many human diseases including gout, kidney stones, cardiovascular and renal disease. The elucidation of a novel gene function should provide new avenues of investigation for the treatment of the human condition.

描述(申请人提供)：斑点狗在狗中是独一无二的，因为它排泄尿酸而不是尿囊素，尿囊素是嘌呤代谢的正常副产品。因此，斑点狗是人类高尿酸尿症和高尿酸血症(即尿液和血液中尿酸水平分别升高)的自然模型。组织移植实验证明，这种缺陷是肝脏固有的，与嘌呤代谢的先天错误一致。虽然这种缺陷在斑点狗群体中是固定的，但一个多世代的指针x斑点狗回交家族分离了导致常染色体隐性遗传疾病的单个基因座。这项拟议的研究将确定斑点狗品种中导致高尿酸尿/高尿酸血症(HUU)的基因。基因组扫描显示Huu与CFA 3连锁，LOD分数为6.5。进一步使用从犬类基因组序列中获得的微卫星进行精细结构作图，将Huu定位到3.3Mb的区间，两侧是重组断裂点。根据最近获得的犬类基因组序列，在这个区间内有24个候选基因。有趣的是，这些基因之前都没有确定在嘌呤代谢或尿酸盐运输中的功能，这证实了这项工作将对高尿酸尿症产生新的见解。将采取三级方法来确定导致斑点狗高尿酸尿症的突变。首先，从犬类基因组序列中获得的SNPs将被评估在斑点狗品种中是否存在连锁不平衡。LD区域可能排除额外的候选基因，并建立我们对潜在致病等位基因的预期。其次，将使用RT-PCR确定在犬肝脏中表达的关键区间内的候选基因。同时，将使用Northern blotts比较斑点狗和非斑点狗之间的表达水平，以确定表达的质量或数量上的差异。根据这三种方法的结果，我们将开始对其余候选基因进行cDNA测序，并进行Southern杂交分析，以确定是否有大的重排。可能导致突变的等位基因将通过验证达尔马提亚犬品种的纯合性、其他犬种缺乏纯合性以及对突变进行适当的功能研究来评估。识别导致斑点狗尿酸水平高的基因和突变将进一步确立它作为人类状况模型系统的地位。尿酸水平是许多人类疾病的促成因素，包括痛风、肾结石、心血管疾病和肾脏疾病。阐明一种新的基因功能将为人类疾病的治疗提供新的研究途径。