A zebrafish model of cobalamin C deficiency displays development defects of the central nervous system. J. Sloan¹, T. Blake², R.J. Chandler¹, M.S. Tsai¹, B.P. Brooks³, C.P. Venditti¹. 1) GDRB/NHGRI/NIH, Bethesda, MD; 2) Zebrafish Core Facility/NHGRI/NIH, Bethesda, MD; 3) OSD/NEI/NIH,Bethesda, MD.
   Cobalamin C deficiency (cblC) features a combined impairment of the cobalamin dependent enzymes, methionine synthase and methylmalonyl-CoA mutase. It is caused by mutations in MMACHC, a gene of unknown function that is suspected to participate in intracellular cobalamin trafficking. The clinical spectrum of cblC is wide and can feature prenatal manifestations, such as congenital microcephaly and intrauterine growth retardation (IUGR). To examine the phenotype caused by loss of function of this gene, we have created and studied a zebrafish model of cobalamin C deficiency. The zebrafish MMACHC gene was identified using informatics and cDNA cloning. FITC-tagged morpholinos targeting the cognate ATG and an exonic junction were designed and used to knock-down the zebrafish homologue. Both morpholinos produced a similar phenotype and displayed a dose-dependent response. Injected fish began to display defects at 24 hours, including brain necrosis and diminished movement when compared to injected controls. The morphants had delayed hatching and by 48 hours, were significantly smaller in size, had less blood, smaller heads and eyes, and grossly abnormal swimming and behavior compared to controls. By 96 hours, some animals displayed pericardial edema. Additionally, histologic examination revealed a fatty liver. RT-PCR studies using total RNA from the exonic morphants harvested on day 6 showed a knock-down of the cognate message. Metabolic analysis using extracts derived from the morphants showed increased homocysteine, methylmalonic acid and cystathionine, a pattern similar to that seen in affected patients. The model presented here is the first animal model of cblC and faithfully replicates some of the more severe findings seen in humans. Furthermore, it demonstrates the utility of zebrafish to easily examine aspects of metabolic diseases that will be difficult to study in other organisms, such as embryonic manifestations, and should facilitate the testing of new therapies for cobalamin C as well as allow developmental mechanisms affected in this disorder to be explored.