| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
|
|---|
| Research Abstract |
Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl-CoA carboxylase (PCC) activity. Native PCC is probably a dodecamer composed of six biotin - containing alpha-subunits and six BETA-subunits. Enzyme deficiency can result from mutations in the either a or beta subunit. We investigated fibroblast cultures obtained from six alpha-subunit deficient Japanese patients (cell no.91, 168, 295,330, 409,419). mRNAs were reverse-transcribed and PCR-amplified. The PCR products were sequenced after subcloning into pGEM-blue vector. Sequence analysis revealed five missense mutations (R52W, Q272R, R374Q, P398L, W534L), two deletions (11 lbp del., nt.1353-1463 ; l03bp del., nt.1464-1566) and one insertion (84bp ins., between nt.1209 and 1210 in patient 330). The primers spanning 300 nucleotides in the section of normal cDNA that encompassed the 84bp insertion were used to amplify cDNAs from six patients and control subjects. Surprisingly, in addition to normal products (300bp), larger products (384bp) were detected in all cell lines including control subjects at trace level. Sequence analysis of genomic DNA)revealed that the 84bp fragment was an unspliced intron between two exons and that sequences similar to consensus splicing sites were located adjacent to the 5' and 3' end of this fragment. Such sequences surrounding the 84bp fragment could be alternative splice sites during RNA processing. We speculate that this 84 bp insertion was not a disease causing mutation but the products of normally spliced cryptic mRNA.
|
