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羧基酶(PCC)活性缺陷引起。天然PCC可能是由6个含有生物素的α-亚基和6个β-亚基组成的十二聚体。酶缺乏症可能是由a或β亚基突变引起的。我们研究了从6名阿尔法亚单位缺陷的日本患者(细胞编号91,168,295,330,409,419)获得的成纤维细胞培养。逆转录和聚合酶链式反应扩增mRNAs。将扩增产物亚克隆至pGEM-BLUE载体后测序。序列分析发现5个错义突变(R52W、Q272R、R374Q、P398L、W534L)，2个缺失(11个LBP del.，nt.1353-1463；103bpdel.，nt.1464-1566)和1个插入(84bpIns，在患者330的nt.1209和1210之间)。用包含84bp插入片段的300个核苷酸的引物扩增6例患者和对照组的cDNAs。令人惊讶的是，除了正常的产物(300bp)外，在所有细胞系中都检测到了更大的产物(384bp)，包括对照组的痕量水平。基因组DNA序列分析表明，该84bp片段为两个外显子之间的非剪接内含子，与共同剪接位点相似的序列位于该片段的5‘和3’端附近。在RNA加工过程中，84bp片段周围的这些序列可能是替代剪接位点。我们推测这个84个碱基的插入不是一种引起突变的疾病，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.