Mapping, Positional Cloning, and Characterization of Mammalian Diabetes Genes Using N-ethyl-N-nitrosourea (ENU)-Mutagenesis

Abstract

Type 2 diabetes mellitus (T2DM) is the leading disorder of metabolism worldwide, and it is predicted to affect more than 330 million people over the next quarter century, generating increasing social and economic burdens. At the pathophysiological level, T2DM arises from a combination of resistance to the hormone insulin and insufficient production of insulin by the endocrine pancreas. T2DM is a polygenic disorder, and many of the genes involved are still unknown. N-ethyl-N-nitrosourea (ENU)-mutagenesis is a forward genetic approach that can be used to identify genes that can be mutated to cause a phenotype of interest. In an effort to identify genes that play a role in regulating blood glucose levels we performed an ENU screen in mice. Recessive and dominant screens were performed, and we identified fourteen lines of mice with evidence for heritable hyperglycemia.

Glucokinase (Gck) was a candidate gene, and so we sequenced for Gck mutations in most of the fourteen lines. We identified two lines of mice that had Gck mutations. One had a K140E coding mutation, and the other had a P417R coding mutation. Further characterization of the mutations in live mice and in recombinant proteins suggested that the mutations caused thermolability of the glucokinase protein, and this thermolability was responsible for the hyperglycemic phenotype in mice.

The remaining lines were sequenced for other candidate genes known to play a role in monogenic diabetes. A few of the remaining lines were put into mapping matings in order to map, and potentially positionally clone, the ENU-induced mutations. No novel genes have yet been identified through this screen. ENU-mutagenesis and phenotypic screening can unquestionably be used to identify lines that carry mutations that cause hyperglycemia. However, the majority of the mutations from this screen had small effect, which may make identification of the mutation more difficult.