Daida Kensuke, Yoshino Hiroyo, Malik Laksh, Baker Breeana, Ishiguro Mayu, Genner Rylee, Paquette Kimberly, Li Yuanzhe, Nishioka Kenya, Masuzugawa Satoshi, Hirano Makito, Takahashi Kenta, Kolmogolv Mikhail, Billingsley Kimberley J, Funayama Manabu, Blauwendraat Cornelis, Hattori Nobutaka
Integrative Neurogenomics Unit, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA.
Center for Alzheimer's and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
medRxiv. 2024 Jun 15:2024.06.14.24308784. doi: 10.1101/2024.06.14.24308784.
Mutations within the genes and are the leading cause of early onset autosomal recessive Parkinson's disease (PD). However, the genetic cause of most early-onset PD (EOPD) cases still remains unresolved. Long-read sequencing has successfully identified many pathogenic structural variants that cause disease, but this technology has not been widely applied to PD. We recently identified the genetic cause of EOPD in a pair of monozygotic twins by uncovering a complex structural variant that spans over 7 Mb, utilizing Oxford Nanopore Technologies (ONT) long-read sequencing. In this study, we aimed to expand on this and assess whether a second variant could be detected with ONT long-read sequencing in other unresolved EOPD cases reported to carry one heterozygous variant in or .
ONT long-read sequencing was performed on patients with one reported pathogenic variant. EOPD patients with an age at onset younger than 50 were included in this study. As a positive control, we also included EOPD patients who had already been identified to carry two known pathogenic variants. Initial genetic testing was performed using either short-read targeted panel sequencing for single nucleotide variants and multiplex ligation-dependent probe amplification (MLPA) for copy number variants.
48 patients were included in this study ( "one-variant" n = 24, "one-variant" n = 12, "two-variants" n = 12). Using ONT long-read sequencing, we detected a second pathogenic variant in six "one-variant" patients (26%, 6/23) but none in the "one-variant" patients (0%, 0/12). Long-read sequencing identified one case with a complex inversion, two instances of structural variant overlap, and three cases of duplication. In addition, in the positive control "two-variants" group, we were able to identify both pathogenic variants in in all the patients (100%, 12/12).
This data highlights that ONT long-read sequencing is a powerful tool to identify a pathogenic structural variant at the locus that is often missed by conventional methods. Therefore, for cases where conventional methods fail to detect a second variant for EOPD, long-read sequencing should be considered as an alternative and complementary approach.
基因 和 内的突变是早发性常染色体隐性帕金森病（PD）的主要病因。然而，大多数早发性PD（EOPD）病例的遗传原因仍未得到解决。长读长测序已成功鉴定出许多导致疾病的致病性结构变异，但该技术尚未广泛应用于PD研究。我们最近利用牛津纳米孔技术（ONT）长读长测序，通过发现一个跨越7 Mb以上的复杂结构变异，确定了一对同卵双胞胎中EOPD的遗传原因。在本研究中，我们旨在在此基础上进行拓展，评估在其他据报道在 或 中携带一个杂合变异的未解决EOPD病例中，ONT长读长测序是否能检测到第二个变异。
对报告有一个 致病变异的患者进行ONT长读长测序。本研究纳入发病年龄小于50岁的EOPD患者。作为阳性对照，我们还纳入了已被确定携带两个已知 致病变异的EOPD患者。最初的基因检测使用针对单核苷酸变异的短读长靶向panel测序和针对拷贝数变异的多重连接依赖探针扩增（MLPA）。
本研究共纳入48例患者（“一个变异”组n = 24，“一个变异”组n = 12，“两个变异”组n = 12）。使用ONT长读长测序，我们在6例“一个变异”患者（26%，6/23）中检测到第二个致病变异，但在“一个变异”患者中未检测到（0%，0/12）。长读长测序确定了1例复杂倒位、2例结构变异重叠和3例重复。此外，在阳性对照“两个变异”组中，我们能够在所有患者中鉴定出两个 致病变异（100%，12/12）。
这些数据表明，ONT长读长测序是一种强大的工具，可用于识别在 位点上的致病性结构变异，而这种变异通常会被传统方法遗漏。因此，对于传统方法未能检测到EOPD第二个变异的病例，长读长测序应被视为一种替代和补充方法。
