Background Mutations within the genes PRKN and PINK1 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 PRKN or PINK1. Methods ONT long-read sequencing was performed on patients with one reported PRKN/PINK1 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 PRKN 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. Results 48 patients were included in this study (PRKN 'one-variant' n = 24, PINK1 'one-variant' n = 12, PRKN 'two-variants' n = 12). Using ONT long-read sequencing, we detected a second pathogenic variant in six PRKN 'one-variant' patients (26%, 6/23) but none in the PINK1 '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 PRKN 'two-variants' group, we were able to identify both pathogenic variants in PRKN in all the patients (100%, 12/12). Conclusions This data highlights that ONT long-read sequencing is a powerful tool to identify a pathogenic structural variant at the PRKN 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.

The authors have declared no competing interest.

This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (NIA), National Institutes of Health, the Japan Society for the Promotion of Science (JSPS) KAKENHI [Grant Numbers: 24K02372 and 23K06958 for MF, 22K07542 for HY, 21K07283 for YL, 20K07893 for KN, 21H04820 and 24H00068 for NH], the Japan Science and Technology Agency (JST) Moonshot R&D Program [Grant Number: JPMJMS2024-5 for NH], AMED under Grant Number 23bm1423015h0001 for MF and NH, and 24ek0109677h0002 for NH, Subsidies for Current Expenditures to Private Institutions of Higher Education from the Promotion and Mutual Aid Corporation for Private Schools of Japan, through a subaward from Juntendo University (for MF and NH), and the Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine (for MF, YH, and NH).

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The ethics committee of Juntendo University, gave ethical approval for this work (M08-0477-M09).

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All the data publicly available is included in the manuscript. The authors declare that the genomics data in this study is not available to share due to the ethics.