Molecular basis of neonatal-onset multiple carboxylase deficiency

已知新生儿发病的多发性羧化酶缺乏症是由全羧化酶合成酶（HCS）缺乏引起的。HCS在细胞中生物素利用中起重要作用。我们从牛肝细胞质中纯化了HCS，发现它由一个分子量为64，000 Da的单亚基组成，通过对该多肽的鉴定，我们确定了真核生物HCS的部分氨基酸序列，并克隆了人HCS cDNA，定位于染色体21q22.1。在日本HCS缺乏症患者的HCS基因中已经鉴定出两种突变：从T到C的转变，其导致在位置237（L237 P）处的氨基酸由脯氨酸替换为亮氨酸，以及单个鸟嘌呤碱基缺失（DELTAG 1067），随后过早终止。在培养的成纤维细胞中的瞬时表达分析，从一个病人后，定点突变表明，L237 P突变体构建产生显着降低HCS活性，尽管存在正常量的免疫反应蛋白的Western印迹。使用等位基因特异性寡核苷酸探针的杂交分析表明，突变L237P和DELTAG1067的患病率分别为50%和30%，在日本HCS缺乏症患者。

Neonatal-onset multiple carboxylase deficiency is known to be caused by deficiency of holocarboxylase synthetase (HCS). HCS plays an essential role in biotin utilization in cells. We purified HCS from bovine liver cytosol and found that it consists of a single subunit with a molecular mass of 64,000 Da. Identification of this polypeptide from bovine liver enabled us to determine the partial amino acid sequence of eukaryotic HCS.We have cloned the human HCS cDNA,which maps to chromosome 21q22.1. Two mutations in the HCS genes of Japanese patients with HCS deficiency have been identified : a transition from T to C which causes an amino acid substitution of proline for leucine at position 237 (L237P) and A single guanine base deletion (DELTAG1067) followed by premature termination. Transient expression analysis in cultured fibroblasts from a patient after site-directed mutagenesis demonstrated that the L237P mutant construct produce markedly decreased HCS activity, despite the presence of normal amounts ofimmunoreactive protein by Western blotting. Hybridization analysis using allele-specific oligonucleotide probes demonstrated that the prevalance of the mutations-L237P and DELTAG1067-was 50% and 30%, respectively, among Japanese patients with HCS deficiency.