New genetic tools to define the pathophysiology of inborn errors of cobalamin metabolism impacting mammalian...

The congenital, autosomal recessive disorder combined methylmalonic acidemia and homocystinuria - cblC type, is the most common inborn error of cobalamin (vitamin B₁₂) metabolism. In its early onset form, cblC profoundly impacts fetal development of the central nervous system, hematopoietic system, and other tissues. Previously, mutations in the MMACHC gene, which encodes a protein required for the intracellular trafficking and enzymatic processing of free cobalamin into active coenzyme forms, were found to cause cblC. These coenzymes are required in two metabolic pathways which produce either succinyl-CoA in the mitochondria or methionine in the cytosol. However, due to a lack of sufficient animal models, the exact pathophysiology of cblC remains unknown. Moreover, there is evidence to suggest that MMACHC may have roles outside of cobalamin metabolism and that cobalamin itself may be required for additional, unknown metabolic pathways. Here, we report the generation and characterization of three new mouse lines aimed at further defining the role of MMACHC and cobalamin in mammalian development. CRISPR/Cas9 genome editing was used to develop an HA-tagged version of Mmachc, which will aid in affinity purification and spatiotemporal localization of the MMACHC protein. To clarify which metabolic perturbations downstream of Mmachc loss give rise to tissue-specific developmental defects, we also created floxed alleles for both methionine synthase (Mtr) and methylmalonyl-CoA mutase (Mmut), which are the only known cobalamin dependent enzymes in mammals. In total, these new mouse models significantly expand upon the repertoire of genetic reagents to clarify the pathophysiology of cblC as well as define both the canonical and hypothesized noncanonical roles of MMACHC in mammalian development.