Somatic mutation of the glucose-6-phosphate dehydrogenase (g6pd) gene in colonic stem cells and crypt restricted loss of G6PD activity

Abstract

The study of somatic mutation frequency, particularly stem cell somatic mutation, is important to the understanding of mechanisms of carcinogenesis. The models currently in use for studies in stem cell tissues such as the colon infer the presence of stem cell somatic mutation from alteration in enzyme function, when this has shown to be mutagen dose dependent, restricted to the unit of clonal architecture, and persistent. The present study identifies and characterises somatic mutations in the g6pd gene in individual mouse colonic crypts showing histochemically demonstrable loss of G6PD activity. Microdissection of single crypts, showing either normal or low G6PD activity by histochemistry was performed in mice treated with ethylnitrosourea (ENU), and the presence of point mutations sought by PCR and direct sequencing. Because of the limitation of the small amount of partially degraded (due to fixation) DNA available from each crypt, only about 20% of the coding region of the g6pd gene could be sequenced. Despite this, somatic mutations were identified in 3 of the 9 crypts analysed which showed loss of G6PD activity, but in none of the crypts with normal activity. Each of the mutations identified would be predicted to lead to a decrease in enzyme activity. We conclude that we have confirmed that the crypt restricted loss of G6PD activity is indeed due to stem cell somatic mutation in the g6pd gene, and suggest that the G6PD model can be used as a paradigm for other models where somatic mutation is inferred from a change in histochemically identifiable gene expression.