| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1997: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1996: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Four unrelated japanese Patients with infantile Sandhoff disease (beta-hexosaminidase beta-subunit deficiency) have been studied for their molecular basis of their severe phenotype. Two patients had complex base substitutions ; one patient was homoallelic for a triple mutation (P417L,K121R and S255R) and the other was a compound heterozygote of a double (P417L and K121R) mutation and the triple mutation. K121R has been known to be a functional polymorphisms, while P417L (exon 11, +8 C*T) generates predominantly an abnormally spliced mRNA at +112 base of exon 11 and has been described in two patients with a juvenile form of the disease. The mild phenotype is attributed to the presence of a small amount of normally spliced mRNA.S255R is novel without prior description in the literature. 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
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activity of the already below-the threshold amount of normally spliced mRNA and accounts for the more severe phenotype in our patients. In the other two patients a novel disease-causing base transition was found within intron 10, away from the intron/exon junction (-17 a*g). This mutation caused abnormal 3' splicing at-37 of intron 10, and normally spliced product was detectable in RT-PCR analysis. We noted an unusually low splice site score (61.8) for the exon 10-intron 11 junction and suspected that this might be partially responsible for these abberant splicing in these mutations. To test this hypothesis, we constructed four chimeric cDNAs all with additinal intron 10 inserted and evaluated their splicing efficiency. They respectively had the normal sequence, P417L (exon 11, +8 C*T), the intronic mutation (-17 a*g), and an artificially engineered intron 10-exon 11 junction with a higher splice site score (85.1). Fifty-eight % and 31% of total transcript were correctly spliced in the normal chimeric construct and P417L,respectively, while no normally spliced product was generated either in the chimeric construct with -17 a*g or in that with a high splice site score. The sequence around the adenosine 17 residues upstream of the normal acceptor splice site in this report UUGCAAU (-22 to -16) matches the consensus branchpoint sequence YNYRAY reported in the literature. The mutation in this study is most likely to abolish the lariat formation because the artificial site of high splice acore did not improve splicing efficiency. Less
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