Cloning of a type 2 diabetes modifier in obese mice

描述(申请人提供)：2型糖尿病(T2 DM)影响超过5%的美国人口，造成巨大的痛苦，每年直接医疗费用超过1000亿美元。在人类和啮齿动物中，肥胖背景下对T2 DM的易感性受到尚未确定的基因的强烈影响。在人类中识别此类易感基因的固有复杂性导致我们在遗传性肥胖小鼠中使用双等位基因系统来识别T2 DM的候选基因和途径，然后可以在人类身上进行测试。在B6/DBA小鼠Lep-ob交叉分离的F2后代中，我们将T2 DM基因座定位到小鼠ChR1(p&lt；10[-8])的一个区域，该区域与人类Lq23同线。这个小鼠区间位于Lq23的人类区间的中间，大约是人类区间的十分之一，在人类连锁研究中反复被确认为含有糖尿病易感基因。这个杂交的肥胖F2后代的糖尿病内表型保持在B6的遗传肥胖成员中。DBA N12同源系从小鼠区域186分离出约5Mb的DBA DNA。同源基因动物的相关表型有：低胰岛素血症、糖化血红蛋白升高和发育不良/营养不良的β细胞。我们建议利用我们开发的信息学和分子生物学技术，系统地识别和分析DBA同源区间内的所有基因。这些步骤将包括：1)获得并分析B6.DBA同源区间的所有DNA序列，这将通过精细作图进一步减少。2.)通过计算技术鉴定所有转录本，通过在汇集的器官RNA中的表达分析证实这一点。3.)B6和DBA之间的序列比较，以确定这些转录本中的所有非同义编码变体。1500bp5‘端编码序列中典型启动子元件的鉴定和序列比较。4.)编码和非编码变体对蛋白质二级结构/功能和转录的影响的计算评估。5.)DBA间歇给药对同基因动物体内/体外β细胞功能和骨骼肌胰岛素反应的影响6.)基于体外活体研究，使用定量聚合酶链式反应对选定器官中选定的转录本进行定量基因表达分析(B6v.DBA)。7.)候选编码和调控序列变体的功能结果的体外分析。8.)对于最引人注目的序列变异，准备DBA等位基因的转基因B6敲入，并评估后代中相关的糖尿病表型。9.)在参与了与1q23相关的T2 DM研究的5000多名个体中，检查同源候选基因。如果成功，该项目可以确定肥胖背景下糖尿病易感性的一个主要基因(可能还有一条新的途径)。这种基因可用于预见性诊断和预防，以及治疗药物的设计。

DESCRIPTION (provided by applicant): Type 2 diabetes mellitus (T2DM) affects over 5% of the US population, causing tremendous suffering with annual direct medical costs of over $100 billion. In both humans and rodents, susceptibility to T2DM in the context of obesity is powerfully influenced by genes that have not been identified. The inherent complexity of identifying such susceptibility genes in humans led us to use a biallelic system in enetically obese mice to identify candidate genes and pathways for T2DM that can then be tested in humans. In F2 progeny of a B6/DBA murine cross segregating for Lep-ob, we mapped a T2DM locus to a region of murine Chr1 (p<10[-8]), syntenic to human lq23. This mouse interval lies in the middle of, and is about 1/10th the physical size of, the human interval at lq23 repeatedly identified as containing diabetes susceptibility genes in human linkage studies. The diabetic endophenotypes of the obese F2 progeny of this cross are maintained in the genetically obese members of a B6.DBA N12 congenic line segregating about 5 Mb of DBA DNA from the mouse region 1at 86. The associated phenotypes in the congenic animals are: hypoinsulinemic hyperglycemia, elevated HbA1c, and hypoplastic/hypotrophic beta cells. We propose to systematically identify and analyze all genes in the DBA congenic interval, using informatics and molecular biological techniques that we have developed. The steps will include: 1.) accession and analysis of all DNA sequence for the B6.DBA congenic interval that will be further reduced by fine mapping. 2.) identification of all transcripts by computational techniques, confirmed by expression analysis in pooled organ RNAs. 3.) sequence comparisons between B6 and DBA to identify all non-synonymous coding variants in these transcripts. Identification and sequence comparison of canonical promoter elements in 1500 bp 5' of coding sequences. 4.) computational assessment of effects of coding and non-coding variants on secondary protein structure/function and transcription. 5.) analysis of in vivo/in vitro beta cell function and skeletal muscle insulin response at 30 and 60 days in congenic animals by interval DBA dose. 6.) based upon in vitro in vivo studies, quantitative gene expression analysis (B6 v. DBA) of selected transcripts in selected organs using quantitative PCR. 7.) in vitro analysis of the functional consequences of candidate coding and regulatory sequence variants. 8.) for the most compelling sequence variants, preparation of transgenic B6 knock-ins of the DBA alleles and assessment of the relevant diabetes phenotypes in the progeny. 9.) in over 5000 individuals who have participated in studies of T2DM linked to 1q23, examine orthologous candidate genes. If successful, this project could identify a major gene (and possibly a novel pathway) for diabetes susceptibility in the context of obesity. Such a gene could be used for anticipatory diagnosis and prevention, and the design of therapeutic agents.