The Utility of Long-Read Sequencing in Diagnosing Genetic Autosomal Recessive Parkinson's Disease: a genetic screening study.

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.

Genetic and clinical study of PARK7 in Japanese Parkinson's disease.

Background: Biallelic variants in PARK7, which encodes protein-nucleic acid deglycase DJ-1, can cause early-onset Parkinson's disease (PD). Although many patients with PARK7 variants have been identified from European and Middle Eastern ethnic groups, there have been no reports in the Japanese population. Objectives: To determine the prevalence and clinical features of patients with PD harboring PARK7 variants in Japan. Methods: We performed a molecular genetic analysis of PD patients with PARK7 variants identified using comprehensive panel sequencing, to explore the details of variants. Moreover, clinical neurological features were investigated, including neuroimaging analyses. This study followed STROBE guidelines. Results: Four patients with biallelic rare variants of PARK7 were identified in the cohort. All four patients presented with levodopa-responsive parkinsonism, with an age at onset in the early 30s. Furthermore, two of the four patients had psychiatric complications. Dopamine transporter imaging revealed nigrostriatal pathway dysfunction. Conclusions: To our knowledge, this is the first report of Japanese patients with PARK7 variants. We identified a relatively low frequency of PARK7 variants in patients in Japan. As opposed to typical patients with sporadic PD, the identified patients developed the disease in their 30s and presented with a variety of non-motor symptoms and complications. Further studies are needed to identify the clinical features related to PARK7 variants in Japanese patients with PD, and to analyze the pathophysiology of how the variants identified in the present study might affect DJ-1 function.

CHCHD2 P14L, found in amyotrophic lateral sclerosis, exhibits cytoplasmic mislocalization and alters Ca2+ homeostasis.

CHCHD2 and CHCHD10, linked to Parkinson's disease and amyotrophic lateral sclerosis-frontotemporal dementia (ALS), respectively, are mitochondrial intermembrane proteins that form a heterodimer. This study aimed to investigate the impact of the CHCHD2 P14L variant, implicated in ALS, on mitochondrial function and its subsequent effects on cellular homeostasis. The missense variant of CHCHD2, P14L, found in a cohort of patients with ALS, mislocalized CHCHD2 to the cytoplasm, leaving CHCHD10 in the mitochondria. Drosophila lacking the CHCHD2 ortholog exhibited mitochondrial degeneration. In contrast, human CHCHD2 P14L, but not wild-type human CHCHD2, failed to suppress this degeneration, suggesting that P14L is a pathogenic variant. The mitochondrial Ca2+ buffering capacity was reduced in Drosophila neurons expressing human CHCHD2 P14L. The altered Ca2+-buffering phenotype was also observed in cultured human neuroblastoma SH-SY5Y cells expressing CHCHD2 P14L. In these cells, transient elevation of cytoplasmic Ca2+ facilitated the activation of calpain and caspase-3, accompanied by the processing and insolubilization of TDP-43. These observations suggest that CHCHD2 P14L causes abnormal Ca2+ dynamics and TDP-43 aggregation, reflecting the pathophysiology of ALS.

Clinical characteristics and pathophysiological properties of newly discovered LRRK2 variants associated with Parkinson's disease.

Leucine-rich repeat kinase 2 (LRRK2) is the most common gene responsible for familial Parkinson's disease (PD). The gene product of LRRK2 contains multiple protein domains, including armadillo repeat, ankyrin repeat, leucine-rich repeat (LRR), Ras-of-complex (ROC), C-terminal of ROC (COR), kinase, and WD40 domains. In this study, we performed genetic screening of LRRK2 in our PD cohort, detecting sixteen LRRK2 rare variants. Among them, we selected seven variants that are likely to be familial and characterized them in terms of LRRK2 protein function, along with clinical information and one pathological analysis. The seven variants were S1120P and N1221K in the LRR domain; I1339M, S1403R, and V1447M in the ROC domain; and I1658F and D1873H in the COR domain. The kinase activity of the LRRK2 variants N1221K, S1403R, V1447M, and I1658F toward Rab10, a well-known phosphorylation substrate, was higher than that of wild-type LRRK2. LRRK2 D1873H showed enhanced self-association activity, whereas LRRK2 S1403R and D1873H showed reduced microtubule-binding activity. Pathological analysis of a patient with the LRRK2 V1447M variant was also performed, which revealed Lewy pathology in the brainstem. No functional alterations in terms of kinase activity, self-association activity, and microtubule-binding activity were detected in LRRK2 S1120P and I1339M variants. However, the patient with PD carrying LRRK2 S1120P variant also had a heterozygous Glucosylceramidase beta 1 (GBA1) L444P variant. In conclusion, we characterized seven LRRK2 variants potentially associated with PD. Five of the seven variants in different LRRK2 domains exhibited altered properties in kinase activity, self-association, and microtubule-binding activity, suggesting that each domain variant may contribute to disease progression in different ways.

Generation of hiPSCs (JUCGRMi003-A) from a patient with Parkinson's disease with PARK2 mutation.

PARK2 is the most common autosomal recessive form of Parkinson's disease and is caused by mutations in parkin that result in early-onset loss of dopaminergic neurons in the substantia nigra. In this study, we established an induced pluripotent stem cell (iPSC) line from a patient harboring a homozygous exon 3 deletion in PARK2. The established iPSCs showed pluripotency, the capacity to differentiate into the three germ layers, and normal karyotypes.

Generation of three clones (JUCGRMi002-A, B, C) of induced pluripotent stem cells from a Parkinson's disease patient with SNCA duplication.

Parkinson's disease is the second most common neurodegenerative disorder and is pathologically characterized by synuclein-rich aggregations (Lewy bodies) in neurons. Multiplication of the synuclein gene (SNCA) increases the mRNA and protein levels of synuclein, resulting in autosomal dominant hereditary Parkinson's disease. In the present study, we established three isogenic induced pluripotent stem cells (iPSCs) from a patient harboring SNCA duplication, which showed pluripotency, three-germ layer differentiation capacity, and normal karyotypes.

Long-Read Sequencing Resolves a Complex Structural Variant in PRKN Parkinson's Disease.

BACKGROUND: Parkin RBR E3 ubiquitin-protein ligase (PRKN) mutations are the most common cause of young onset and autosomal recessive Parkinson's disease (PD). PRKN is located in FRA6E, which is one of the common fragile sites in the human genome, making this region prone to structural variants. However, complex structural variants such as inversions of PRKN are seldom reported, suggesting that there are potentially unrevealed complex pathogenic PRKN structural variants. OBJECTIVES: To identify complex structural variants in PRKN using long-read sequencing. METHODS: We investigated the genetic cause of monozygotic twins presenting with a young onset dystonia-parkinsonism using targeted sequencing, whole exome sequencing, multiple ligation probe amplification, and long-read sequencing. We assessed the presence and frequency of complex inversions overlapping PRKN using whole-genome sequencing data of Accelerating Medicines Partnership Parkinson's disease (AMP-PD) and United Kingdom (UK)-Biobank datasets. RESULTS: Multiple ligation probe amplification identified a heterozygous exon three deletion in PRKN and long-read sequencing identified a large novel inversion spanning over 7 Mb, including a large part of the coding DNA sequence of PRKN. We could diagnose the affected subjects as compound heterozygous carriers of PRKN. We analyzed whole genome sequencing data of 43,538 participants of the UK-Biobank and 4941 participants of the AMP-PD datasets. Nine inversions in the UK-Biobank and two in AMP PD were identified and were considered potentially damaging and likely to affect PRKN expression. CONCLUSIONS: This is the first report describing a large 7 Mb inversion involving breakpoints outside of PRKN. This study highlights the importance of using long-read sequencing for structural variant analysis in unresolved young-onset PD cases. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Long-read sequencing resolves a complex structural variant in PRKN Parkinson's disease.

Background: PRKN mutations are the most common cause of young onset and autosomal recessive Parkinson's disease (PD). PRKN is located in FRA6E which is one of the common fragile sites in the human genome, making this region prone to structural variants. However, complex structural variants such as inversions of PRKN are seldom reported, suggesting that there are potentially unrevealed complex pathogenic PRKN structural variants. Objectives: To identify complex structural variants in PRKN using long-read sequencing. Methods: We investigated the genetic cause of monozygotic twins presenting with a young onset dystonia-parkinsonism using targeted sequencing, whole exome sequencing, multiple ligation probe amplification, and long-read. We assessed the presence and frequency of complex inversions overlapping PRKN using whole-genome sequencing data of AMP-PD and UK-Biobank datasets. Results: Multiple ligation probe amplification identified a heterozygous exon 3 deletion in PRKN and long-read sequencing identified a large novel inversion spanning over 7Mb, including a large part of the coding DNA sequence of PRKN. We could diagnose the affected subjects as compound heterozygous carriers of PRKN. We analyzed whole genome sequencing data of 43,538 participants of the UK-Biobank and 4,941 participants of the AMP-PD datasets. Nine inversions in the UK-Biobank and two in AMP PD were identified and were considered potentially damaging and likely to affect PRKN isoforms. Conclusions: This is the first report describing a large 7Mb inversion involving breakpoints outside of PRKN. This study highlights the importance of using long-read whole genome sequencing for structural variant analysis in unresolved young-onset PD cases.

Analysis of LIN28A variants in patients with Parkinson's disease.

A heterozygous loss-of-function variant in lin-28 homolog A (LIN28A) was recently reported as a novel pathogenic gene in patients with PD from Korea. Two patients harboring LIN28A variants had early- or middle-aged-onset PD with good responses to levodopa. In the current study, we aimed to identify the prevalence of LIN28A variants among PD patients of Japanese origin. We performed genetic sequencing of 284 patients with early-onset PD. We then estimated the frequency and functional effect of each variant using prediction tools. We identified three different rare variants in LIN28A (rs4623750, c.228 + 49 C > T; rs199541048, c.*7 A > G; and rs4659441, c.*43 C > T). The frequency of each variant in the PD patients did not differ from that of the general population. No variants were identified in the amino acid-coding regions. Our results do not support a strong association of LIN28A with early-onset PD among Japanese patients.

Pathophysiological evaluation of the LRRK2 G2385R risk variant for Parkinson's disease.

Missense variants in leucine-rich repeat kinase 2 (LRRK2) lead to familial and sporadic Parkinson's disease (PD). The pathological features of PD patients with LRRK2 variants differ. Here, we report an autopsy case harboring the LRRK2 G2385R, a risk variant for PD occurring mainly in Asian populations. The patient exhibited levodopa-responsive parkinsonism at the early stage and visual hallucinations at the advanced stage. The pathological study revealed diffuse Lewy bodies with neurofibrillary tangles, amyloid plaques, and mild signs of neuroinflammation. Biochemically, detergent-insoluble phospho-α-synuclein was accumulated in the frontal, temporal, entorhinal cortexes, and putamen, consistent with the pathological observations. Elevated phosphorylation of Rab10, a substrate of LRRK2, was also prominent in various brain regions. In conclusion, G2385R appears to increase LRRK2 kinase activity in the human brain, inducing a deleterious brain environment that causes Lewy body pathology.

α-Synuclein V15A Variant in Familial Parkinson's Disease Exhibits a Weaker Lipid-Binding Property.

BACKGROUND: The α-Synuclein (α-Syn) V15A variant has been found in two Caucasian families with Parkinson's disease (PD). However, the significance of this missense variant remained unclear. OBJECTIVE: We sought to elucidate whether V15A could increase aggregation or change phospholipid affinity. METHODS: A sequencing analysis for the SNCA encoding α-Syn from 875 patients with PD and 324 control subjects was performed. Comparing with known pathogenic missense variants of α-Syn, A30P, and A53T, we analyzed the effects of V15A on binding to phospholipid membrane, self-aggregation, and seed-dependent aggregation in cultured cells. RESULTS: Genetic screening identified SNCA c.44 T>C (p.V15A) from two Japanese PD families. The missense variant V15A was extremely rare in several public databases and predicted as pathogenic using in silico tools. The amplification activity of α-Syn V15A fibrils was stronger than that of wild-type α-Syn fibrils. CONCLUSIONS: The discovery of the V15A variant from Japanese families reinforces the possibility that the V15A variant may be a causative variant for developing PD. V15A had a reduced affinity for phospholipids and increased propagation activity compared with wild-type. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Clinical Manifestations and Molecular Backgrounds of Parkinson's Disease Regarding Genes Identified From Familial and Population Studies.

Over the past 20 years, numerous robust analyses have identified over 20 genes related to familial Parkinson's disease (PD), thereby uncovering its molecular underpinnings and giving rise to more sophisticated approaches to investigate its pathogenesis. α-Synuclein is a major component of Lewy bodies (LBs) and behaves in a prion-like manner. The discovery of α-Synuclein enables an in-depth understanding of the pathology behind the generation of LBs and dopaminergic neuronal loss. Understanding the pathophysiological roles of genes identified from PD families is uncovering the molecular mechanisms, such as defects in dopamine biosynthesis and metabolism, excessive oxidative stress, dysfunction of mitochondrial maintenance, and abnormalities in the autophagy-lysosome pathway, involved in PD pathogenesis. This review summarizes the current knowledge on familial PD genes detected by both single-gene analyses obeying the Mendelian inheritance and meta-analyses of genome-wide association studies (GWAS) from genome libraries of PD. Studying the functional role of these genes might potentially elucidate the pathological mechanisms underlying familial PD and sporadic PD and stimulate future investigations to decipher the common pathways between the diseases.

Genotype-phenotype correlation of Parkinson's disease with PRKN variants.

To investigate the prevalence and genotype-phenotype correlations of parkin RBR E3 ubiquitin protein ligase (PRKN) variants in Parkinson's disease (PD), we first included 2,527 patients with PD. Through the defined selection, we enrolled 2,322 patients, including 1,204 with familial and 1,118 with sporadic PD. We identified 242 patients harboring PRKN variants, which were thought to be susceptibility factors, comprising 137 patients with familial and 105 with sporadic PD; among the 26 identified variants, 13 were novel. We divided our cohort into 2 groups: heterozygote (hereafter called one-allele) and homozygote or compound heterozygote (hereafter called two-allele). The patients with two-allele were significantly younger at onset than those with one-allele. Six families harbored the complex forms of one- and two-allele in different individuals of the same family. The presence of two-allele reflected more frequent normal values of [123I] metaiodobenzylguanidine myocardial scintigraphy. The log-rank test revealed an exacerbation associated with two-allele over 15 years of the disease course. The patients with PRKN variants showed specific symptoms dependent on the number of mutated alleles.

A complex form of hereditary spastic paraplegia harboring a novel variant, p.W1515*, in the SPG11 gene.

Individuals with hereditary spastic paraplegia (HSP) are known to present with a variety of symptoms, including intellectual disability, cognitive decline, parkinsonism, and epilepsy. We report here our experience of treating a family with consanguinity, including three patients with HSP-related symptoms. We performed whole-exome sequencing and identified a novel pathogenic nonsense variant, c.4544G > A, p.W1515*, in the SPG11 gene. Proband and her affected sister showed the same course of gait disturbance due to spastic paraplegia from childhood and progressive cognitive decline from early adulthood. Brain MRI depicted a thinning of the corpus callosum, severe atrophic changes in the frontotemporal lobes, and ears of the lynx sign. Patients with SPG11 variants clinically present with distinctive symptoms.

Genetic analysis of ATP10B for Parkinson's disease in Japan.

Compound heterozygosity of ATP10B is thought to be a risk factor for young-onset Parkinson's disease (PD). We genetically screened 245 patients with young-onset sporadic PD and 33 patients with autosomal recessive PD for ATP10B. All 13 identified gene variants were heterozygous with little evidence of the pathogenicity.

A Novel LRRK2 Variant p.G2294R in the WD40 Domain Identified in Familial Parkinson's Disease Affects LRRK2 Protein Levels.

Leucine-rich repeat kinase 2 (LRRK2) is a major causative gene of late-onset familial Parkinson's disease (PD). The suppression of kinase activity is believed to confer neuroprotection, as most pathogenic variants of LRRK2 associated with PD exhibit increased kinase activity. We herein report a novel LRRK2 variant-p.G2294R-located in the WD40 domain, detected through targeted gene-panel screening in a patient with familial PD. The proband showed late-onset Parkinsonism with dysautonomia and a good response to levodopa, without cognitive decline or psychosis. Cultured cell experiments revealed that p.G2294R is highly destabilized at the protein level. The LRRK2 p.G2294R protein expression was upregulated in the patient's peripheral blood lymphocytes. However, macrophages differentiated from the same peripheral blood showed decreased LRRK2 protein levels. Moreover, our experiment indicated reduced phagocytic activity in the pathogenic yeasts and α-synuclein fibrils. This PD case presents an example wherein the decrease in LRRK2 activity did not act in a neuroprotective manner. Further investigations are needed in order to elucidate the relationship between LRRK2 expression in the central nervous system and the pathogenesis caused by altered LRRK2 activity.

Clinical manifestations of Parkinson's disease harboring VPS35 retromer complex component p.D620N with long-term follow-up.

INTRODUCTION: To identify and investigate patients with Parkinson's disease (PD) harboring VPS35 variants in Japan. METHODS: Using targeted gene panel screening, we analyzed 393 familial, 294 young-onset, and 52 late-onset sporadic PD patients derived from the Juntendo PD DNA bank, and obtained clinical information from the medical records on each patient in whom we found VPS35 p.D620N variants. RESULTS: We identified VPS35 p.D620N in three new patients: two patients with familial PD and one patient with sporadic PD. Additionally, we newly confirmed p.D620 from a patient of a family reported previously. The prevalence of familial PD was 0.7% (2/307), young-onset sporadic PD was 0.3% (1/294), and late-onset sporadic PD was 0% (0/52) in our cohort. Combining four patients with p.D620N from our previous reports, haplotype analysis indicated at least two founders in our cohort. Patients commonly showed a slow progression of parkinsonism with onset in middle or late age and mild parkinsonism with good response to levodopa and little cognitive decline even for more than 10 years of disease duration. Psychosis was occurred in two patients. One-half of patients required device-aided therapies such as deep brain stimulation or levodopa-carbidopa intestinal gel. Brain magnetic resonance imaging mostly showed normal findings, even at more than 10 years after onset. 123I-metaiodobenzylguanidine myocardial scintigraphy indicated normal heart-to-mediastinum ratio values among three of four patients. CONCLUSIONS: Patients with VPS35 p.D620N showed distinctive symptoms and neuroimaging. Our findings expand the clinical findings of patients with VPS35 variants.

PLA2G6 variants associated with the number of affected alleles in Parkinson's disease in Japan.

This study aimed to evaluate genotype-phenotype correlations of Parkinson's disease (PD) patients with phospholipase A2 group V (PLA2G6) variants. We analyzed the DNA of 798 patients with PD, including 78 PD patients reported previously, and 336 in-house controls. We screened the exons and exon-intron boundaries of PLA2G6 using the Ion Torrent system and Sanger method. We identified 21 patients with 18 rare variants, such that 1, 9, and 11 patients were homozygous, heterozygous, and compound heterozygous, respectively, with respect to PLA2G6 variants. The allele frequency was approximately equal between patients with familial PD and those with sporadic PD. The PLA2G6 variants detected frequently were identified in the early-onset sporadic PD group. Patients who were homozygous for a variant showed more severe symptoms than those who were heterozygous for the variant. The most common variant was p.R635Q in our cohort, which was considered a risk variant for PD. Thus, the variants of PLA2G6 may play a role in familial PD and early-onset sporadic PD.