The sphingolipidoses are a group of severe disorders caused by inherited defects in the degradation of sphingolipids. In order to study pathogenesis and new treatment approaches we developed models of some of these disorders by total gene knockout technology in the mouse. We have applied this technology to establish models of Tay-Sachs, Sandhoff disease and the GM2 activator deficiency. In some of sphingolipidoses, such as Gaucher disease, effective modeling in the mouse requires the introduction of subtle mutational changes. Gaucher disease is caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GC). Three clinical types of Gaucher disease have been defined according to the presence (type 2 and 3) or absence (type 1) of central nervous system disease and severity of clinical manifestations. The clinical course of the disease correlates with the type of mutation carried by the GC gene. In order to produce mice with point mutations that correspond to the clinical types of Gaucher disease, we have devised a highly efficient one-step mutagenesis method -- the Single Insertion Mutagenesis Procedure (SIMP) -- to insert human disease mutations into the mouse GC gene. Using SIMP, mice have been generated carrying either the very severe RecNciI mutation that can cause type 2 disease or the less severe L444P mutation associated with type 3 disease. Mice homozygous for the RecNciI mutation had little GC enzyme activity and accumulated glucosylceramide in brain and liver. In contrast, the mice homozygous for the L444P mutation had higher levels of GC activity and no detectable accumulation of glucosylceramide in brain and liver. Both the type 2 and 3 mice died within 48 hrs of birth of a compromised epidermal permeability barrier caused by defective glucosylceramide metabolism in the epidermis. Current work is aimed at introducing genetic modifications that will allow the Gaucher mice to survive past the neonatal period.
