Studies of genetic analysis and gene therapy for myelin-associated disorders.

我们研究了日本戈谢病（GD）偏色差性脑白质营养不良（MLD）的基因型，以评估基因型与表型的相关性，并对其族群特征进行表征。在7例II型GD患者中，3例在异等位基因上发现了新的754A错义突变。在2例日本MLD患者中发现了一个新的异等位1070A错义突变。这两种突变在犹太和高加索人群中未见报道，这表明GD和MLD基因型在日本是独特的。为了评估MLD的潜在基因治疗效果，我们构建了芳基磺化酶cDNA的逆转录病毒载体，并在人MLD成纤维细胞中进行了转染和表达。基因转染后，MLD成纤维细胞的酶活性比正常成纤维细胞水平提高约70%。这些观察结果证明了基因治疗MLD的可行性。为了解克拉布病（GLD）脱髓鞘的原因，研究了神经细胞培养中精神碱的神经毒性。抗神经丝抗体免疫荧光染色法显示，精神素处理的细胞出现细胞骨架破坏和细胞内病变。电镜观察显示线粒体肿胀，线粒体嵴破坏，微管和神经丝消失。这些数据表明，精神病影响线粒体功能，可能是通过抑制细胞成分的破坏。

We investigated the genotype of Japanese Gaucher disease (GD), metachromatic leukodystrophy (MLD) to evaluate the correlation between genotype and phenotype, and to characterize the ethnicity. In 7 patients with type II GD, a new 754A missense mutation was identified in 3 cases heteroallelically.A new heteroalleic 1070A missense mutation was found in 2 cases of Japanese patients with MLD.These two mutations have not been reported mong Jewish and Caucasian population, which suggests that the genotypes of GD and MLD in Japan is unique.In order to evaluate the potential gene therapy for MLD, we constructed a retroviral vector for the transfer of the arylsulfatase cDNA, and transfected and expressed the gene in human MLD fibroblasts. As a result of gene transfer, the enzyme activity of MLD fibroblasts was increased about 70% of normal fibroblast level. Those observation demonstrates the feasibility of gene therapy for MLD.To understand the cause of demyelination in Krabbe's disease (GLD), the neurotoxicity of psychosine in neural cell culture was investigated. By immunofluorescence staining method using an anti-neurofilament antibody, psychosine treated cells showed destruction of cytoskelton and pathy intracellular changes. An electron microscopic study showed swelling of mitochondria, desruption of cristae in mitochondria, and disappearance of microtubules and neuro-filaments. These data suggest that psychosine influences mitochondrial function, possibly through inhibition in the destruction of cellular components.