Tachikawa Keiji, Shimizu Takahiro, Imai Takeshi, Ko Riyoko, Kawai Yosuke, Omae Yosuke, Tokunaga Katsushi, Frith Martin C, Yamano Yoshihisa, Mitsuhashi Satomi
Department of Neurology, St. Marianna University School of Medicine, Kawasaki, Japan.
Genome Medical Science Project, National Center for Global Health and Medicine, Tokyo, Japan.
J Mol Diagn. 2024 Feb;26(2):85-95. doi: 10.1016/j.jmoldx.2023.10.004. Epub 2023 Nov 24.
Hereditary repeat diseases are caused by an abnormal expansion of short tandem repeats in the genome. Among them, spinocerebellar ataxia (SCA) is a heterogeneous disease, and currently, 16 responsible repeats are known. Genetic diagnosis is obtained by analyzing the number of repeats through separate testing of each repeat. Although simultaneous detection of candidate repeats using current massively parallel sequencing technologies has been developed to avoid complicated multiple experiments, these methods are generally expensive. This study developed a cost-effective SCA repeat panel [Flongle SCA repeat panel sequencing (FLO-SCAp)] using Cas9-mediated targeted long-read sequencing and the smallest long-read sequencing apparatus, Flongle. This panel enabled the detection of repeat copy number changes, internal repeat sequences, and DNA methylation in seven patients with different repeat expansion diseases. The median (interquartile range) values of coverage and on-target rate were 39.5 (12 to 72) and 11.6% (7.5% to 16.5%), respectively. This approach was validated by comparing repeat copy number changes measured by FLO-SCAp and short-read whole-genome sequencing. A high correlation was observed between FLO-SCAp and short-read whole-genome sequencing when the repeat length was ≤250 bp (r = 0.98; P < 0.001). Thus, FLO-SCAp represents the most cost-effective method for conducting multiplex testing of repeats and can serve as the first-line diagnostic tool for SCA.
遗传性重复疾病是由基因组中短串联重复序列的异常扩增引起的。其中，脊髓小脑共济失调（SCA）是一种异质性疾病，目前已知有16种相关的重复序列。通过对每个重复序列进行单独检测来分析重复序列的数量，从而进行基因诊断。尽管已经开发出利用当前的大规模平行测序技术同时检测候选重复序列的方法，以避免复杂的多次实验，但这些方法通常成本高昂。本研究利用Cas9介导的靶向长读长测序和最小的长读长测序设备Flongle，开发了一种具有成本效益的SCA重复序列检测面板[Flongle SCA重复序列检测面板测序（FLO-SCAp）]。该检测面板能够检测7例患有不同重复序列扩增疾病患者的重复序列拷贝数变化、内部重复序列和DNA甲基化情况。覆盖度和靶向率的中位数（四分位间距）分别为39.5（12至72）和11.6%（7.5%至16.5%）。通过比较FLO-SCAp和短读长全基因组测序测得的重复序列拷贝数变化，验证了该方法。当重复序列长度≤250 bp时，FLO-SCAp与短读长全基因组测序之间观察到高度相关性（r = 0.98；P < 0.001）。因此，FLO-SCAp是进行重复序列多重检测最具成本效益的方法，可作为SCA的一线诊断工具。
