Molecular characterization of variant alpha-subunit of electron transfer flavoprotein in three patients with glutaric acidemia type II--and identification of glycine substitution for valine-157 in the sequence of the precursor, producing an unstable mature protein in a patient.
Indo, Yasuhiro et al., Molecular characterization of variant alpha-subunit of electron transfer flavoprotein in three patients with glutaric acidemia type II--and identification of glycine substitution for valine-157 in the sequence of the precursor, producing an unstable mature protein in a patient., American journal of human genetics , 49-I(3), 1991, pp. 575-580
In our previous study of eight glutaric acidemia type II (GAII) fibroblast lines by using [35S]methionine labeling and immunoprecipitation, three of them had a defect in the synthesis of the alpha-subunit of electron transfer flavoprotein (alpha-ETF) (Ikeda et al. 1986).In one of them (YH1313) the labeling of the mature alpha-ETF was barely detectable, while that of the precursor (p) was stronger. In another (YH605) no synthesis of immunoreactive p alpha-ETF was detectable.In the third cell line (YH1391) the rate of variant p alpha-ETF synthesis was comparable to normal, but its electrophoretic mobility was slightly faster than normal.In the present study, the northern blot analysis revealed that all three mutant cell lines contained p alpha-ETF mRNA and that their size and amount were comparable to normal.In immunoblot analysis, both alpha- and beta-ETF bands were barely detectable in YH1313 and YH605 but were detectable in YH1391 in amounts comparable to normal.Sequencing of YH1313 p alpha-ETF cDNA via PCR identified a transversion of T-470 to G.We then devised a simple PCR method for the 119-bp section (T-443/G-561) for detecting this mutation.In the upstream primer, A-466 was artificially replaced with C, to introduce a BstNI site into the amplified copies in the presence of G-470 from the variant sequence.The genomic DNA analysis using this method demonstrated that YH1313 was homozygous for T----G-470 transversion.It was not detected either in two other alpha-ETF-deficient GAII or in seven control cell lines.The alpha-ETF cDNA sequence in YH605 was identical to normal.