Positional cloning of genes modifying susceptibility to type 2 diabetes in obese mice

Obese mice of the C57BL/6J (resistant) and DBA/2J (susceptible) inbred strains are discordant for type 2 diabetes (T2DM). In obese F2 and F3 progeny of a B61DBA cross segregating for Lepob, we mapped a quantitiative trait locus for T2DM (most strongly associated with fasting blood glucose and glycosylated hemoglobin in males) to a region of Chr1 (p < 10-8), 86cM from the centromere. Based upon metabolic and pancreatic/islet of Langerhans anatomic phenotypes of N12 B(B6).D(DBA) mouse congenic lines, the diabetes susceptibility gene(s) was predicted to reside in a 3.5 Mb interval on mouse Chr1.; In vivo phenotypes of diabetic (D/D for the region 1 86) congenic animals are hypoinsulinemic hyperglycemia (apparent as early as four weeks of age), and elevated glycosylated hemoglobin at 8 weeks of age. The primary endophenotype in D/D mice is a reduction in beta cell proliferation as reflected in 2-fold lower numbers of ki67-stained beta cells observed as early as p1 in D/D mice.; A systematic computational analysis of all genes and predicted transcripts in the congenic interval identified 18 genes of which 13 correspond to "known" genes, and 5 are computationally-predicted confirmed transcripts. Three coding SNPs were identified in transcripts encoding flavin monooxygenase-like proteins, and two cSNPs were identified in a newly identified transcript, Ll (Lisch-like).; To assess possible contributions of non-coding variants in mediating diabetes susceptibility, analysis of mRNA expression levels by real-time qRT-PCR was performed in tissues relevant to diabetes for each of the 18 genes in Lepob/ob D/D and B/B mice. This analysis showed that Ll was significantly down-regulated in all diabetes-relevant tissues/organs that we studied (liver, pancreatic islets, skeletal muscle, brain and adipose tissue) in 4-week old obese D/D (v. B/B) mice. Immunohistochemical staining for Ll appeared only in insulin positive cells in islets of B/B congenic mice, while Ll protein was undetectable by immunohistochemistry in islets of D/D congenics. Morpholino-mediated down-regulation of putative Ll ortholog in zebrafish resulted in an early scattered beta cell phenotype. Ll, a novel, putative transmembrane receptor of unknown function, is a prime candidate gene modifying T2DM susceptibility in obese mice.