Department of Neurology, Houston Methodist Research Institute, Houston, Texas, United States of America.
Movement Disorders Unit, Neurology Service, Department of Internal Medicine, Hospital de Clinicas, Federal University of Paraná, Curitiba, PR, Brazil.
PLoS One. 2020 Mar 11;15(3):e0228789. doi: 10.1371/journal.pone.0228789. eCollection 2020.
Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.
大片段的微卫星 DNA 扩张会引起多种神经疾病。在脊髓小脑共济失调 10 型（SCA10）中，重复中断会改变疾病表型；（ATTCC）n 或（ATCCT）n/（ATCCC）n 中断位于（ATTCT）n 重复内，与共济失调和癫痫的明显表型相关，而主要是纯（ATTCT）n 可能会降低外显率。SCA10 及许多其他微卫星扩张的大片段重复可以超过 10000 个碱基对（bp）。常规的下一代测序（NGS）技术无法确定这些扩展重复的内部序列内容或大小。我们使用重复引物 PCR（RP-PCR）与高灵敏度脉冲场毛细管电泳片段分析仪（FEMTO-Pulse，Agilent，Santa Clara，CA）（简称 RP-FEMTO）相结合，成功地确定了 SCA10 患者基因组 DNA 中大片段扩展重复的序列内容以及含有 SCA10 重复的酵母人工染色体。这种 RP-FEMTO 是一种简单经济的方法，可以补充新兴的 NGS 技术，用于非常长的序列读取，如单分子实时（SMRT）和纳米孔测序技术。
