Dorschner Michael O, Barden Deborah, Stephens Karen
Department of Medicine, Medical Genetics 357720, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195-7720, USA.
J Mol Diagn. 2002 May;4(2):108-13. doi: 10.1016/S1525-1578(10)60689-7.
The autosomal-dominant spinocerebellar ataxias (ADCA) are a heterogeneous group of neurodegenerative disorders with variable expression and phenotypic overlap. An accurate diagnosis relies on detection of a mutation in a specific causative gene, which is typically an abnormal number of CAG trinucleotide repeats. To streamline testing in a clinical setting, we converted our current panel of tests for the spinocerebellar ataxias (SCA) types SCA1, SCA2, SCA3, SCA6, and SCA7 from five independent amplification reactions analyzed by polyacrylamide gel electrophoresis (PAGE) to a single multiplex amplification reaction analyzed by capillary electrophoresis (CE). Multiplex amplification was facilitated by the use of chimeric primers; different lengths and fluorochromes distinguished the amplicons. During CE with commercially available molecular weight standards, the SCA amplicons migrated faster than predicted, thereby underestimating their length compared to that determined previously by PAGE. This was observed to varying degrees for each of the five loci, with the greatest size differential occurring in amplicons with greater (CAG)(n). To determine accurate amplicon length, and therefore an accurate number of CAG repeats, a size correction formula was calculated for each locus. This multiplex semi-automated assay has been reliable during 1 year of use in a clinical setting during which 57 samples were tested and five positive samples were detected.
常染色体显性遗传性脊髓小脑共济失调（ADCA）是一组异质性神经退行性疾病，具有可变表达和表型重叠。准确的诊断依赖于检测特定致病基因中的突变，该突变通常是CAG三核苷酸重复序列数量异常。为了简化临床检测流程，我们将目前用于检测脊髓小脑共济失调（SCA）1型、2型、3型、6型和7型的检测方法，从通过聚丙烯酰胺凝胶电泳（PAGE）分析的五个独立扩增反应，转换为通过毛细管电泳（CE）分析的单个多重扩增反应。使用嵌合引物促进了多重扩增；不同的长度和荧光染料区分扩增子。在使用市售分子量标准品进行CE分析时，SCA扩增子迁移速度比预期快，因此与之前通过PAGE测定的长度相比，其长度被低估。在五个位点中的每个位点都观察到了不同程度的这种情况，在具有更多（CAG）（n）的扩增子中大小差异最大。为了确定准确的扩增子长度，从而确定准确的CAG重复次数，为每个位点计算了一个大小校正公式。这种多重半自动检测方法在临床应用的1年中一直可靠，在此期间共检测了57个样本，发现了5个阳性样本。
