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

丙酸血症是由丙酰辅酶A羧化酶（PCC）活性缺乏引起的常染色体隐性遗传代谢疾病。天然PCC可能是由六个含生物素的α-亚基和六个β-亚基组成的十二聚体。酶缺乏可由α或β亚基的突变引起。我们研究了从6名α-亚单位缺陷的日本患者（细胞编号91、168、295、330、409、419）中获得的成纤维细胞培养物。将mRNA逆转录并PCR扩增。将PCR产物亚克隆入pGEM-blue载体后进行测序。序列分析显示5个错义突变（R52 W，Q272 R，R374 Q，P398 L，W534 L），2个缺失（111 bp缺失，nt.1353-1463 ; 103 bp缺失，nt.1464-1566）和一个插入（84 bp插入，在患者330中介于NT.1209和1210之间）。使用包含84 bp插入的正常cDNA片段中的300个核苷酸的引物扩增来自6名患者和对照受试者的cDNA。令人惊讶的是，除了正常产物（300 bp）之外，在包括对照受试者的所有细胞系中检测到痕量水平的较大产物（384 bp）。基因组DNA序列分析表明，该片段为两个外显子之间的一个未剪接的内含子，其5'和3'端有类似于共有剪接位点的序列。84 bp片段周围的这些序列可能是RNA加工过程中的可变剪接位点。我们推测，这84 bp的插入不是一种致病突变，而是正常剪接的隐蔽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.