GENETIC AND BIOCHEMICAL STUDIES ON KRABBE'S DISEASE

克拉伯病的遗传学和生物化学研究

• 批准号: 3238301
• 负责人: DAVID A WENGER
• 金额: $ 20.58万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3238301
• 关键词: Krabbe's disease; alleles; alpha galactosidase; beta galactosidase; cerebrosides; complementary DNA; enzyme activity; enzyme biosynthesis; gene expression; gene mutation; genetic disorder diagnosis; genetic library; genetic regulatory element; genetically modified animals; human genetic material tag; laboratory mouse; laboratory rabbit; molecular cloning; molecular pathology; monoclonal antibody; nucleic acid hybridization; nucleic acid sequence; polymerase chain reaction; protein metabolism; protein purification; southern blotting; tissue /cell culture

Krabbe disease (KD) is an autosomal recessive lysosomal disorder caused by the deficiency of galactocerebrosidase (GALC) activity. this results in a severe, fatal disorder primarily affecting young children. While most patients die by 13 months of age, later onset forms are diagnosed. Five animal species (monkeys, mice, dogs, cats and sheep) have been identified with GALC deficiency. This laboratory has diagnosed 190 patients of all ages with KD. Until now GALC had not been purified to homogeneity, and nothing is known about the mutations causing KD in humans or animal models. This is a proposal to utilize information from purified human GALC to prepare monospecific antibodies, clone and sequence the human and mouse cDNAs and genes, identify the mutations causing low GALC activity in all clinical types of human KD and the twitcher mouse, study the biosynthesis and processing of GALC in cultured cells, and make transgenic mice (homozygous for twitcher mutation but with the normal human gene), and follow these mice clinically, biochemically, and pathologically. In addition, the gene coding for the activator of GALC will be cloned and sequenced. Very recently we succeeded in purifying GALC from human urine, and 19 amino acids at the N-terminal were determined. From this sequence, probes (degenerate and best guess sequence for human usage) for screening genomic and cDNA libraries, and primers for polymerase chain reaction amplification of cDNA and genomic DNA will be synthesized. Candidate clones will be purified, and tested for their ability to express GALC activity in COS-1 cells, hybridize to chromosome 14 DNA, and demonstrate a difference between digested DNA samples from patients with KD and normal people. The sequence of normal cDNA will be obtained, and mutations causing very low GALC activity in all clinical types of KD, and high and low GALC activity in normal people will be identified. The mouse GALC cDNA and gene will be cloned, and the mutation causing the twitcher phenotype will be found. The human GALC gene will be cloned, and the 5' regulatory elements and exon-intron boundaries will be sequenced. The GALC subunit and synthetic peptides coupled to KLH will be injected into rabbits, and the antibodies will be used to measure antigen levels and to study biosynthesis and processing in cultured cells after 35S-methionine labeling. In an attempt to prevent the consequences of GALC deficiency, the GALC gene with appropriate 5' regulatory elements will be introduced into twitcher mouse embryos, and the resulting embryos will be evaluated. Better method of identifying patients and carriers of KD, as well as a better understanding of the role of GALC in producing healthy myelin will come from this research.

Krabbe病（KD）是一种常染色体隐性遗传的溶酶体疾病， 半乳糖苷酶（GALC）活性缺乏。 这导致 一种严重致命的疾病主要影响幼儿 而 大多数患者在13个月大时死亡，诊断为晚发型。 五种动物物种（猴子，老鼠，狗，猫和羊）已经被 发现GALC缺乏症。 该实验室已诊断出190例 所有年龄段的KD患者。 到目前为止，GALC还没有被纯化， 同质性，并没有什么是已知的突变导致KD在 人或动物模型。 这是一个利用信息的建议， 纯化人GALC制备单特异性抗体、克隆和 对人类和小鼠的cDNA和基因进行测序， 在所有临床类型的人KD中引起低GALC活性， twitcher小鼠，研究培养的GALC的生物合成和加工 细胞，并使转基因小鼠（纯合子抽搐突变，但 与正常的人类基因），并在临床上跟踪这些小鼠， 从生化和病理学角度来看 此外，编码 GALC的激活因子将被克隆和测序。 最近，我们 成功地从人尿中纯化了GALC，并在 N-末端测定。 从该序列，探针（简并和 用于筛选基因组和cDNA的最佳猜测序列 文库和用于聚合酶链反应扩增的引物， 将合成cDNA和基因组DNA。 候选克隆将是 纯化，并测试它们在COS-1中表达GALC活性的能力， 细胞，与染色体14 DNA杂交，并显示出差异 KD患者和正常人消化DNA样本之间的差异。 将获得正常cDNA的序列，并且突变引起非常 在所有临床类型的KD中GALC活性低，以及高和低GALC 将识别正常人的活动。 小鼠GALC cDNA和 基因将被克隆，引起抽搐表型的突变将 将克隆人GALC基因，并将5'调控区与人GALC基因的5'调控区结合。 将对元件和外显子-内含子边界进行测序。 GALC亚基 将与KLH偶联的合成肽注射到兔子体内， 这些抗体将被用来测量抗原水平， 35 S-蛋氨酸后培养细胞的生物合成和加工 标签。 为了防止GALC缺乏的后果， 将引入具有适当5 ′调控元件的GALC基因 植入到抽搐小鼠胚胎中，并对所产生的胚胎进行评估。 更好的识别KD患者和携带者的方法，以及 更好地了解GALC在产生健康髓鞘中的作用， 来自这项研究。