Phenotypic characterization of a new EPM2A mutation (N163D)

Abstract

Lafora disease (LD, OMIM 254780) is a fatal rare disorder characterized by epilepsy and neurodegeneration. In the vast majority of cases LD is related to mutations in either the EPM2A gene (encoding the glucan phosphatase laforin) or the EPM2B gene (encoding the E3-ubiquitin ligase malin). Alterations in these genes consist of deletions or missense and nonsense mutations. These alterations are spread all over the laforin and malin protein sequences and it remains to be shown whether they correlate with the severity of the disease. We have recently gained access to a primary fibroblast sample form a compound heterozygous EPM2A patient (Y112X/N163D), who shows a slow progression of the disease. As the Y112X mutation is related to regular progression of the disease and to our knowledge the laforin N 163D mutation is novel, here we have carried out the phenotypic characterization of the laforin N163D mutation. We have expressed the laforin-N 163D mutant in bacteria and purified the corresponding protein. Using OMFP as substrate we have observed no major changes in phosphatase activity. The mutant protein was also as stable as wild type when expressed either in bacteria or in mammalian cells. However, it showed a severe impairment in the interaction with regular laforin partners, as laforin itself, malin, R5/PTG and R6 (by yeast two-hybrid assays). Probably, this lack of interaction is the cause of the pathogenic profile of this novel mutation. We have recently reported that human primary fibroblasts from LD patients have an impairment of mitochondrial function with increased production of reactive oxygen species (ROS). In agreement with these observations we found that primary fibroblasts from EPM2A Y112X/N 163D patient presented higher levels of superoxide and lower levels of superoxide dismutase activity. The levels of thioredoxin1 (Trx1) were also decreased in the LD samples. All these results indicate that primary fibroblasts from EPM2A Y112X/N163D patient suffer from oxidative stress.