GENETIC AND BIOCHEMICAL STUDIES ON KRABBE'S DISEASE

克拉伯病（Krabbe disease， KD）是一种常染色体隐性溶酶体疾病

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.