Department of Molecular Diagnosis and Graduate School of Medicine, Chiba University, Chiba, Japan.
J Hum Genet. 2012 Dec;57(12):807-8. doi: 10.1038/jhg.2012.112. Epub 2012 Sep 20.
Spinocerebellar ataxia type 31 (SCA31) is defined by the presence of an insertion mutation containing a TGGAA repeat within the intron of the brain-expressed, associated with NEDD4 (BEAN) gene. Detecting this mutation is conventionally done by southern blotting or DNA sequencing, but these methods are technically demanding and not easily implemented in clinical diagnosis. Here, we adapted repeat-primed PCR (RP-PCR) to develop a clinical genetic test for SCA31 using only the PCR process to detect the TGGAA repeat within the insertion mutation. Pentanucleotide RP-PCR and subsequent DNA fragment analysis demonstrated characteristic ladder peaks with a 5-bp periodicity, originating from the TGGAA repeat, in 100% of samples (n=14) from SCA31 patients in whom the presence of the TGGAA repeat had been verified by DNA sequencing. No peaks were observed in a normal control and two non-SCA31 patients, in whom the TGGAA repeat was absent. This method is valuable for genetic diagnosis of SCA31 in clinical practice.
脊髓小脑性共济失调 31 型（SCA31）的定义是存在 TGGAA 重复插入突变，该突变位于脑表达相关蛋白 NEDD4（BEAN）基因的内含子中。通常通过 Southern 印迹或 DNA 测序来检测这种突变，但这些方法技术要求高，在临床诊断中不易实施。在这里，我们通过重复引物 PCR（RP-PCR）进行了适应性研究，仅通过 PCR 过程即可检测插入突变中的 TGGAA 重复，从而开发了一种用于 SCA31 的临床基因检测方法。五核苷酸 RP-PCR 和随后的 DNA 片段分析显示，在通过 DNA 测序证实存在 TGGAA 重复的 100%（n=14）SCA31 患者样本中，存在具有 5bp 周期性的特征梯状峰，起源于 TGGAA 重复。在正常对照和两个不存在 TGGAA 重复的非 SCA31 患者中未观察到峰。该方法在临床实践中对 SCA31 的遗传诊断具有重要价值。
