|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1999 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1998 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Three distinct mutant alleles of β-hexosaminidase β-subunit gene in Japanese patients with infantile Sandhoff disease (GM2-gangliosidosis type O) have been found for their molecular basis of their severe phenotype. They were the allele with a tripe mutation (P417L, K121R and S255R), a double (P417L and K121R) mutation, and an intronic mutation away from the intron 10/exon11 junction (-17, a→g). S255R is novel without prior description in the literature. Both P417L and the intronal muatation lVS 10, 17 a→g caused splicing abnormalities, although they did not abolish the consensus sequence for splicing. An expression study of the normally spliced cDNA with the double and the triple mutations gave 78% and 28% of normal activity, respectively. This finding suggested that S255R further reduces the catalytic activity of the already below-the-threshold amount of normally spliced mRNA and accounts for the severe phenotype.
Number of missence mutations in β-hexosaminidase β-subunit gene that cause GM2-gangliosidosis type O with various phenotypes have been reported. To determine the active site for the enzyme activity or the binding site for the dimerization, we constructed four kinds of mutant cDNAs with l207V, S255R, Y456S, or C533Y by in vitro mutagenesis. The human cultured fibroblasts which do not produce α-subunit protein but produce mal β-subunit protein were transformed with those mutant cDNAs and analysed hexosaminidase B activity. When the β-subunit protein derived from mutant cDNA could bind with normal β-subunit protein, the activity of hexosaminidase B would decrease by a dominant negative like effect.