Using zebrafish to assess novel therapeutics and model the eye disease of cblC disease. N. P. Achilly, J. L. Sloan, K. Bishop, M. S. Jones, R. Sood, C. P. Venditti National Human Genome Research Institute, National Institutes of Health, Bethesda, MD.

   Cobalamin C disease (cblC) is the most common inborn error of intracellular cobalamin metabolism and is caused by mutations in MMACHC, a gene responsible for processing and trafficking intracellular cobalamin. Disease manifestations include growth failure, anemia, heart defects, and progressive blindness. At present, the pathological basis of these symptoms remains unknown, as no animal viable model exists. Using zinc-finger nucleases, we generated two independent lines with mutations in the zebrafish homolog of MMACHC. mmachchg12/hg12 and mmachchg13/hg13 fish display many of the classic phenotypes of cblC disease, including growth retardation, impaired survival, anemia, and metabolite perturbations. Hydroxocobalamin (OH-cbl) injections typically administered to the patients to ameliorate some of the disease-related complications. Although hypothetical, the effectiveness of other potential therapeutics, such as methylcobalamin (Me-cbl) and methionine, has not been evaluated. Growth parameters improved significantly when mmachchg13/hg13 fish were maintained in water supplemented with Me-cbl (200 g/ml) or methionine (10 mM), Standard length (SL) increased by 21% and 16% (p<0.0001), respectively, and height at anterior of anal fin (HAA) increased by 63% and 61% (p<0.0001), respectively, compared to the untreated group. These changes represent a marked improvement compared to OH-cbl or betaine treatment (SL: 14% and 13%; HAA: 31% and 38%; p<0.0001). To visualize the cblC-related eye disease, we bred heterozygote mmachc fish with Tg(rho:EGFP,gnat2:tdTomato) fish, a double transgenic line expressing GFP in the rods and RFP in the cones. Using confocal microscopy, we observed a thinner photoreceptor layer in mmachchg13/hg13;Tg(rho:EGFP,gnat2:tdTomato) mutants as well as a reduction in outer segment material present in the retinal pigment epithelium. To understand the underlying gene expression changes associated with the eye phenotype, we performed microarray analysis on eyes dissected from mmachc+/hg13 and mmachchg13/hg13 fish. 327 genes involved in cholesterol metabolism, phototransduction, cAMP signaling, and oxidant stress emerged as differentially regulated (>2-fold change and p<0.05). Our zebrafish model recapitulates the phenotypic and biochemical features of cblC disease and demonstrates a response to conventional and novel therapeutics, and will be important to further delineate the pathophysiological mechanism and assess additional therapies.