Propionic Acidemia : Mutation Analysis of the alpha-subunit of Propionyl-CoA Carboxylase.

丙酸血症是一种常染色体隐性代谢性疾病，由丙酰辅酶a羧化酶（PCC）活性缺乏引起。天然PCC可能是由6个含生物素的α -亚基和6个β -亚基组成的十二聚体。酶缺乏症可由a或β亚基突变引起。我们研究了从6名α -亚单位缺乏的日本患者(细胞号：no.；[91][168],[295,330],[409,419]。逆转录mrna并进行pcr扩增。PCR产物亚克隆至pGEM-blue载体后测序。序列分析显示5个错义突变（R52W、Q272R、R374Q、P398L、W534L）， 2个缺失（11 lbp del）。， nt.1353-1463；l03bp德尔。， nt.1464-1566)和一个插入(84bp英寸。（330例患者在nt.1209 - 1210之间）。在包含84bp插入的正常cDNA片段中，引物跨越300个核苷酸，用于扩增来自6名患者和对照组的cDNA。令人惊讶的是，除了正常产物（300bp）外，在包括对照组在内的所有细胞系中都检测到较大的产物（384bp）。基因组DNA序列分析显示，该84bp片段为位于两个外显子之间的未剪接内含子，在该片段的5′和3′端附近存在与一致剪接位点相似的序列。这些围绕84bp片段的序列可能是RNA加工过程中的备选剪接位点。我们推测这个84 bp的插入不是致病突变，而是正常剪接的隐型mRNA的产物。

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.