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.