The vitamin B12 processing enzyme, mmachc, is essential for zebrafish survival, growth and retinal morphology

Cobalamin C deficiency (cblC), the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia, and hypomethioninemia caused by impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study loss of mmachc function. mmachc homozygotes survived the embryonic period but perished in early juvenile life. The mutants displayed metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation, and lethality. Clinical and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine, and betaine. Furthermore, mmachc mutants, some bred to express rod and or cone fluorescent reporters, manifested a retinopathy and thin optic nerves. Expression analysis using whole eye mRNA revealed dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the phenotypic and biochemical features of the human disorder, including ocular pathology, and a show a response to established treatments mmachc heterozygous and mutant zebrafish whole eyes N=3 pools were studied by microarray at two timepoints 7 and 28 days post fertilization.

钴胺素C缺陷症（Cobalamin C deficiency, cblC）是最常见的细胞内钴胺素代谢先天异常，由负责维生素B12加工及细胞内转运的MMACHC基因突变所致。该常染色体隐性遗传病的核心特征为无法将钴胺素转化为腺苷钴胺与甲基钴胺，进而导致下游酶——甲基丙二酰辅酶A变位酶（methylmalonyl-CoA mutase）与甲硫氨酸合酶（methionine synthase）活性受损，引发甲基丙二酸血症、高同型半胱氨酸血症及低甲硫氨酸血症等生化紊乱。

钴胺素C缺陷症的临床表现谱跨度极广，可出现进行性失明；在小鼠模型中则表现为极早期胚胎致死。由于斑马鱼具备完整的钴胺素代谢酶系统，本研究通过基因组编辑（genome editing）技术探究mmachc的功能缺失效应。mmachc纯合子可顺利渡过胚胎发育期，但于幼年早期死亡。该突变体呈现出cblC缺陷的代谢与临床特征，包括甲基丙二酸血症、严重生长迟缓及致死性。当将突变体饲养于添加临床治疗用小分子（羟钴胺（hydroxocobalamin）、甲基钴胺（methylcobalamin）、甲硫氨酸（methionine）与甜菜碱（betaine））的水环境中时，其临床与代谢参数均得到改善。

此外，部分经杂交以表达视杆（rod）及/或视锥（cone）荧光报告基因（fluorescent reporters）的mmachc突变体，表现出视网膜病变（retinopathy）与视神经变薄。全眼mRNA表达分析显示，光转导（phototransduction）与胆固醇代谢（cholesterol metabolism）相关基因存在表达失调现象。罹患mmachc缺陷的斑马鱼重现了人类疾病的表型与生化特征，包括眼部病理改变，并对现有治疗方案产生响应。本研究在受精后7天与28天两个时间点，对3组mmachc杂合子及突变型斑马鱼的全眼混合样本进行了微阵列（microarray）分析。