Cloning of a type 2 diabetes modifier in obese mice

描述（由申请人提供）：2型糖尿病（T2 DM）影响超过5%的美国人口，造成巨大的痛苦，每年的直接医疗费用超过1000亿美元。在人类和啮齿类动物中，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。在人类中识别这些易感基因的固有复杂性使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，然后可以在人类中进行测试。在Lep-ob的B6/DBA鼠交叉分离的F2子代中，我们将T2 DM基因座定位到鼠Chr 1的区域（p<10[-8]），与人lq 23同线。该小鼠间隔位于人类lq 23间隔的中间，并且是其物理大小的约1/10，在人类连锁研究中反复鉴定为含有糖尿病易感基因。该杂交的肥胖F2后代的糖尿病内表型保持在B6.DBA N12同源系的遗传肥胖成员中，该同源系从小鼠区域1at 86分离约5 Mb的DBA DNA。同类动物中的相关表型为：低胰岛素血症性高血糖、HbA 1c升高和β细胞发育不良/营养不良。我们建议系统地识别和分析DBA同源间隔中的所有基因，使用我们已经开发的信息学和分子生物学技术。步骤包括：1.）登录和分析B6.DBA同源间隔的所有DNA序列，其将通过精细作图进一步减少。2.）的情况。通过计算技术鉴定所有转录物，通过合并的器官RNA中的表达分析确认。3.）第三章B6和DBA之间的序列比较以鉴定这些转录物中的所有非同义编码变体。1500 bp 5'端编码序列中典型启动子元件的鉴定和序列比较。4.）计算评估编码和非编码变体对二级蛋白质结构/功能和转录的影响。5.）通过间隔DBA剂量在同类动物中在30和60天时的体内/体外β细胞功能和骨骼肌胰岛素应答的分析。6.）基于体外体内研究，使用定量PCR对选定器官中选定转录物进行定量基因表达分析（B6 v. DBA）。7.）候选编码和调节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体，制备DBA等位基因的转基因B6敲入并评估后代中的相关糖尿病表型。9.）在参与与1 q23相关的T2 DM研究的5000多名个体中，检查正向候选基因。如果成功，该项目可以确定肥胖背景下糖尿病易感性的主要基因（可能是一种新途径）。这种基因可用于预测性诊断和预防，以及治疗剂的设计。

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.