
==== Front
NPJ Parkinsons Dis
NPJ Parkinsons Dis
NPJ Parkinson's Disease
2373-8057
Nature Publishing Group UK London

784
10.1038/s41531-024-00784-1
Article
Parkinson’s disease is associated with clonal hematopoiesis with TET2 mutation
http://orcid.org/0000-0001-5457-7348
Woo Kyung Ah 1
http://orcid.org/0000-0001-8219-9663
Kim Han-Joon movement@snu.ac.kr

1
http://orcid.org/0000-0001-9559-5286
Lee Chan Young 2
http://orcid.org/0000-0003-4182-3612
Shin Jung Hwan 1
Sun Choonghyun 3
Im Hogune 3
An Hongyul 3
Lim Jiwoo 3
Choi Su-Yeon 4
http://orcid.org/0000-0002-4750-931X
Koh Youngil 35
Jeon Beomseok 1
1 grid.31501.36 0000 0004 0470 5905 Department of Neurology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
2 https://ror.org/053fp5c05 grid.255649.9 0000 0001 2171 7754 Department of Neurology, Ewha Womans University Mokdong Hospital, Ewha Womans University College of Medicine, Seoul, Republic of Korea
3 NOBO Medicine Inc, Seoul, Republic of Korea
4 grid.412484.f 0000 0001 0302 820X Department of Internal Medicine, Seoul National University College of Medicine, Seoul National University Hospital Healthcare System Gangnam Center, Seoul, Republic of Korea
5 grid.31501.36 0000 0004 0470 5905 Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea
6 9 2024
6 9 2024
2024
10 16818 10 2023
27 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Clonal hematopoiesis of indeterminate potential (CHIP), a premalignant expansion of mutated hematopoietic stem cells, is linked to immune alterations. Given the role of neuroinflammation and immune dysfunction in Parkinson’s disease (PD), we hypothesized a connection between CHIP and PD. We analyzed peripheral blood DNA from 341 PD, 92 isolated REM sleep behavior disorder (iRBD) patients, and 5003 controls using targeted sequencing of 24 genes associated with hematologic neoplasms. PD cases were classified by clinical progression mode: fast, slow, and typical. Using multivariable logistic regression models, CHIP prevalence was assessed against controls with a 1.0% variant allele fraction threshold. CHIP with TET2 mutations was more prevalent in PD than controls (aOR 1.75, 95% CI 1.11–2.77, p = 0.017), particularly in the fast motor progression subgroup (aOR 3.19, p = 0.004). No distinct associations were observed with iRBD. PD is linked to increased odds of CHIP with TET2 mutations, suggesting immune dysregulation in PD pathophysiology.

Subject terms

Parkinson's disease
Mutation
https://doi.org/10.13039/501100003725 National Research Foundation of Korea (NRF) 2022R1A2C2091254 Kim Han-Joon issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

The potential connection between “inflammaging”, i.e., chronic inflammation during aging1, and clonal hematopoiesis of indeterminate potential (CHIP) has emerged as an area of interest, as the link between CHIP and nonmalignant diseases, i.e., atherosclerotic cardiovascular disease, stroke, and most recently, Alzheimer’s disease (AD), have been reported2–4. CHIP refers to the nonmalignant overgrowth of specific hematopoietic stem cell progeny that carry a cancer-related mutation with a growth advantage5. Importantly, CHIP has been linked to an increased risk of cardiovascular death, and experimental evidence suggests that aberrant innate immune responses in CHIP may serve as a common underlying mechanism6–8.

The inflammatory immune responses play a critical role in the pathogenesis of Parkinson’s disease (PD)9–11. Alterations in the intestinal microbiota and inflammation, elevated levels of pro-inflammatory cytokines, activation of the innate and adaptive immune systems, and neuroinflammation in the central nervous system (CNS) with activated microglia mark the pro-inflammatory profiles of PD10. The presence of mutations in DNMT3A and TET2, the two most frequently mutated genes in CHIP5, in the whole-brain DNA of elderly individuals12 raises the possibility of CNS involvement in CHIP.

Based on these observations, we aimed to investigate the association between CHIP and PD, as well as iRBD as a prodromal entity for Lewy body disorders. We performed targeted sequencing of genes commonly associated with hematologic neoplasms on peripheral blood samples from patients with PD, iRBD, and healthy controls. This included 24 genes, notably TET2 and DNMT3A which together account for two-thirds of CHIP cases and are implicated in inflammation and immune dysfunction. Additionally, utilizing prospectively collected longitudinal data from the PD cohort, we examined whether specific clinical progression subtypes of PD—such as slow-progressing or fast-progressing with prominent motor or cognitive decline—had distinct associations with CHIP.

Results

Clinical characteristics

A total of 341 subjects with PD, 92 with iRBD, and 5003 controls were included in the analysis. Among patients with PD, 95 (27.8%) were classified as Slow PD, 98 (28.7%) as Fast PD, and 148 (43.4%) as Typical PD. The demographic characteristics of the subjects are summarized in Table 1. The patient groups exhibited a higher average age compared to the control groups, and there was a difference in sex distribution between the groups. To address these factors, all subsequent analyses were appropriately adjusted for both sex and age.Table 1 Demographic information of study subjects

	Group	P value	
Control	iRBD	
N	3676	92		
Age	57.0 [53.0; 63.0]	69.5 [61.5; 75.0]	<0.001	
Sex, M (%)	2578 (70.1)	50 (54.3)	0.002	
	Control	All PD		
N	5003	341		
Age	54.0 [49.0; 60.0]	66.0 [60.0; 73.0]	<0.001	
Sex, M	3644 (72.8)	185 (54.3)	<0.001	
	Control	Slow PD		
N	5002	95		
Age	54.0 [49.0; 60.0]	63.0 [57.0;70.0]	<0.001	
Sex, M	3643 (72.8)	44 (46.3)	<0.001	
	Control	Typical PD		
N	5001	148		
Age	54.0 [49.0; 60.0]	66.0 [61.0; 72.0]	<0.001	
Sex, M	3642 (72.8)	85 (57.4)	<0.001	
	Control	Fast PD		
N	4360	98		
Age	55.0 [51.0; 62.0]	69.5 [61.0; 74.0]	<0.001	
Sex, M	3112 (71.4)	56 (57.1)	0.003	
IQR interquartile range, iRBD isolated rapid eye movement behavior disorder, PD Parkinson’s disease.

Information at the time of sample collection is provided, with age presented as mean [IQR].

Association between CHIP, PD, and iRBD

There was no significant difference in the odds of harboring CHIP in any gene between the control group and either the iRBD or PD groups (Fig. 1). However, gene-specific analyses revealed a significant enrichment of CHIP with TET2 mutations in participants with PD compared to controls (adjusted odds ratio [aOR], 1.75; 95% CI, 1.11–2.77; p = 0.017; Fig. 1A). No specific genetic associations were found in iRBD (Fig. 1B).Fig. 1 Associations between clonal hematopoiesis of indeterminate potential (CHIP), Parkinson’s disease, and isolated rapid eye movement (REM) sleep behavior disorder.

The forest plots in the panels illustrate the odds ratios for the associations between CHIP and A PD and B iRBD, as determined by multivariate logistic regression models with age and sex as covariates. P values were not adjusted for multiple comparisons. Additional data for 2.0% VAF threshold is provided in Supplementary Fig. 2. vaf variant allele fraction, CI confidence interval.

Association between CHIP and mode of PD progression

For the 341 subjects with PD, the mean follow-up duration after disease onset was 7.42 (SD 3.48) years. Detailed characteristics the PD subgroups with different modes of clinical progression and the criteria can be found in Supplementary Table 1 and the Methods section.

In brief, during an average follow-up of 9.97 (SD 3.08) years after PD onset, 80% of the Slow PD subgroup maintained a Hoehn and Yahr (HY) stage < 3, and the remaining 20% reached HY stage 3 after an average of 10.11 (SD 3.45) years. In contrast, 70% of the Fast PD subgroup reached HY 3 within the follow-up period, with a mean interval of 3.63 (SD 1.92) years between PD onset to HY 3. These participants were subsequently followed for an additional average duration of 2.84 (SD 2.43) years after reaching HY 3, which confirmed the absence of atypical parkinsonism. Additionally, 65 patients met the criteria for fast cognitive decline, with 43 patients classified in the Fast Cognition subgroup and 22 in the Both (Motor & Cognition) subgroup. The average duration from onset to dementia for these patients was 3.07 years (SD 1.78). Of these, 37 patients developed dementia within 3 years. For these individuals, the mean period of additional clinical follow-up was 4.32 years (SD 3.04), during which no exclusion criteria or red flags for PD diagnosis were identified.

Regarding the odds of harboring CHIP, no significant differences were observed when comparing the Fast, Slow, or Typical subgroups of PD with the control group. However, gene-specific analysis revealed the Fast PD subgroup was 2.14 times more likely to harbor a TET2 clonal mutation (aOR, 2.14; 95% CI, 1.08 – 4.25; p = 0.030; Fig. 2A). Such associations were not seen in the Slow PD or Typical PD subgroups (Supplementary Fig. 1). The overall and gene-specific prevalence of CHIP in the Fast, Slow, and Typical PD subgroups are illustrated in Fig. 2B.Fig. 2 Association between CHIP and the mode of PD progression.

Panel A presents the association between CHIP and fast-progressing PD (Fast PD). P values were not adjusted for multiple comparisons. Panel B shows the prevalence of overall CHIP and CHIP related to specific genes in each PD subgroup.

To further investigate the clinical aspects of PD associated with CHIP, the Fast PD group was subdivided into three categories based on whether they met criteria related to motor and/or cognition (see Methods): ‘Fast Motor,’ ‘Fast Cognition,’ and ‘Both’ for those meeting both criteria. Those with fast motor decline (55 subjects in the ‘Fast Motor’ and ‘Both’ subgroups) reached HY 3 after an average of 3.05 (SD 1.47) years. Individuals with fast cognitive decline (65 subjects in the ‘Fast Cognition’ and ‘Both’ subgroups) developed dementia after an average of 3.02 (SD 1.81) years.

Within the Fast PD group, the subjects with fast motor progression had marked enrichment of CHIP with TET2 mutations compared to controls, with 10 out of 55 subjects being TET2-CHIP carriers (aOR, 3.19; 95% CI, 1.46–6.95; p = 0.004; Supplementary Table 2). The association remained significant with higher variant allele fraction (VAF) thresholds for CHIP: the adjusted odds ratio was 3.26 (95% CI, 1.38–7.70; p = 0.007) at a 1.5% threshold, and 3.63 (1.44–9.16, p = 0.006) at a 2.0% threshold. No significant associations were found between cognitive decline and CHIP.

Association of CHIP with TET2 mutation and PD after propensity score matching

To rigorously verify the association between TET2-CHIP and PD, we performed propensity score matching for age and sex and reassessed the significant results compared to the overall control group (Supplementary Tables 3–5). Even after propensity score matching, PD patients (n = 341) demonstrated higher odds of CHIP with TET2 mutations compared to 1:2-matched controls at a VAF ≥ 1.0% threshold (aOR, 1.94; 95% CI, 1.16 – 3.27; p = 0.012). The association between the Fast PD group (n = 98) and TET2-CHIP also remained significant (aOR, 2.41; 95% CI, 1.13–5.15; p = 0.023). Specifically, the subgroup of Fast PD patients with fast motor progression (n = 55) showed a marked enrichment of TET2-CHIP (aOR, 4.07; 95% CI, 1.61–10.29; p = 0.003). When applying a VAF ≥ 2.0% threshold, the results for the overall PD and Fast PD groups were not significant; however, the results for the PD with fast motor progression group remained significant (p = 0.004).

Discussion

In this cross-sectional cohort study encompassing patients with PD, iRBD, and controls, we observed a 75% higher odds of CHIP with TET2 mutations in patients with PD compared to controls. This association was particularly evident in the subgroup of PD patients with accelerated motor decline.

Our investigation reveals a novel association between PD and increased odds of TET2 clonal mutation in circulating blood cells. TET2 encodes the enzyme ten-eleven translocation 2 that catalyzes the conversion of DNA methylation to hydroxymethylation13. While mutations in this pivotal epigenetic regulator are frequent in CHIP and myeloid malignancies5,14, the pathways interconnecting these somatic mutations in the systemic circulation with CNS pathologies demand further exploration. One possible explanation involves microglia, the macrophage population of innate immune cells residing in the brain parenchyma. Evidence suggests that the overactivation of microglia by pathologic α-synuclein in PD contributes to dopaminergic neurodegeneration via phagocytosis, the spread of pathologic α-synuclein, and the initiation of immunologic cascades through antigen presentation and proinflammatory cytokine release15. However, it remains uncertain whether marrow-derived macrophages in the systemic circulation can reach the brain parenchyma to partake in these processes16. In a murine model, α-synuclein overexpression prompted pro-inflammatory peripheral monocyte infiltration into the substantia nigra, and intervention to block this entry prevented subsequent neurodegeneration17. Among CNS macrophage populations in humans, only choroid plexus macrophages are traditionally recognized to partially turnover with blood-borne monocytes18, and the interactions of these border-associated macrophages (BAM) with other CNS compartments remained unknown, although BAMs have been implicated in immunopathological processes, including antigen presentation to circulating lymphocytes18. Importantly, recent research on Alzheimer’s disease and CHIP provides preliminary evidence that marrow-derived mutant microglia-like cells substantially infiltrate the brain, which may imply a potentially similar role in CNS pathogenesis in PD4.

An alternate potential mechanism involves the systemic immune dysregulation observed in both TET2 mutant mice models and PD. Mounting evidence underscores the role of peripheral inflammatory conditions in PD initiation and progression, engaging cytokines and peripheral immune cells10. Meta-analyses in PD have reported elevated peripheral cytokine concentrations, including interleukin (IL)-1β, IL-2, IL-6, and IL-1019. Similarly, TET2 dysregulation triggers an immune-active, hyperinflammatory phenotype. Tet2-deficient mice display heightened expression of IL-1β, IL-6, and other inflammatory mediators, accompanied by more pronounced inflammatory responses to endotoxin shock6,20. Notably, TET2-mutant clonal hematopoiesis is associated with elevated IL-1β secretion in macrophages through nucleotide-binding oligomerization domain-leucine-rich repeat-pyrin domain-containing 3 (NLRP3) inflammasomes6, suggesting a role for this pathway in TET2-CHIP-PD. The NLRP3 inflammasome has a pivotal role in PD pathophysiology by amplifying local inflammation through IL-1β secretion triggered by α-synuclein aggregates15; it is also systemically upregulated in immune cells in the blood of PD patients21,22. Monocytes could potentially contribute to CHIP-related PD through systemic immune modifications. α-synuclein demonstrates antigenicity for both microglia and monocytes9. Activated monocytes are elevated in the periphery of PD patients23, and monocytes display distinct subpopulation changes and immune-related gene expression patterns in early stages of the disease9,24.

TET2 dysregulation in neurons has also been linked to PD pathomechanism in the brain via enhanced immune activation, although this may not be directly associated with TET2 somatic mutation in the peripheral circulation. TET2 transcript levels are significantly upregulated in PD neurons; conversely, in vivo Tet2 inactivation in a mouse model blocked inflammation-induced dopaminergic neurodegeneration. Interestingly, Tet2 knockout mice showed significantly attenuated microglial activation and transcriptional immune responses to inflammatory triggers, suggesting an immune-mediated contribution of neuronal TET2 to PD pathogenesis25. This diverges from the findings in the periphery, where Tet2 deficiency in bone marrow cells is linked to a hyperinflammatory state6. Nevertheless, taken together, these findings imply that TET2 could play a role in PD pathogenesis through a certain mechanism, despite the specific site and directionality of this contribution remaining to be clarified.

The link between TET2-CHIP and the accelerated motor decline observed in our PD cohort is intriguing. Plasma IL-1β levels, which are elevated in TET2-CHIP, exhibit a positive correlation with motor severity in PD patients21. TET2 is also engaged in the differentiation of CD4+ helper T cells26, and emerging evidence indicates that peripheral inflammation, marked by an elevated neutrophil-to-lymphocyte ratio due to reduced lymphocyte count, aligns with parkinsonian motor severity and striatal dopaminergic denervation27. Taken together, our findings may imply that TET2-CHIP and associated immune alterations primarily exert a detrimental effect on the central dopaminergic system, potentially resulting in early parkinsonian motor decline.

Our study found no significant association between CHIP and iRBD, despite potential immune implications in iRBD. For example, blood monocytes in iRBD show elevated expression of CD11b and CCR2 involved in tissue adhesion and infiltration, similar to changes in early PD28. Also, toll-like receptor 4 expression on iRBD monocytes correlates with nigral immune activation and dopaminergic degeneration28. Meanwhile, the peripheral pro-inflammatory cytokine profile in iRBD might differ from that assumed in TET2-CHIP-PD. In multiple iRBD studies, alterations in IL-1β have not been observed, while serum TNF-α is elevated and predicts earlier phenoconversion to synucleinopathies29,30. Microglia-secreted TNF-α contributes to CNS neuronal death31; TNF-α is also produced in the intestinal epithelium32; peripheral neutralization of soluble TNF alleviates dopaminergic neurodegeneration in rodents33. The link between chronic anti-TNF therapy and reduced PD odds in inflammatory bowel diseases suggests the role of TNF-α in the gut-brain inflammatory pathogenesis of PD34, and possibly, iRBD. In essence, TET2-CHIP and iRBD may connect to PD through distinct systemic immune mechanism changes. Nonetheless, the shared and synergistic biological activities of IL-1 and TNF-α35 may lead to a common inflammatory outcome. Additionally, a study reported that Tet2-mutant clonal hematopoiesis is favored in a chronic TNF-α exposure environment, which may imply a possible active cross-talk between the two mechanisms36.

Several limitations in the present study require attention. Firstly, the VAF cutoff for CHIP was empirically set at 1.0%. While multiple studies set the CHIP threshold at 2.0, varying thresholds such as 1.0%, 2.0%, and 5.0% are also utilized depending on the disease context, testing facility, and cohort characteristics5. In our analysis of the entire PD or iRBD participants, thresholds of 1.5% or 2.0% did not yield significant outcomes, possibly due to sample size constraints, as higher thresholds result in fewer CHIP-positive patients. Nevertheless, the results of the subgroups that exhibited a strong enrichment of TET2-CHIP, specifically the group with fast motor decline, remained significant with the 1.5% and 2.0% thresholds. One concern is that the statistics might be influenced by the smaller size of the PD subsets. We speculate that replication of the findings in a larger PD cohort in the future will be useful to confirm the association between TET2-CHIP and PD. Additionally, it is important to note that information on medical comorbidities, including coronary heart disease and type 2 diabetes, was not collected in the present study. Given the reported associations between these conditions and CHIP and the role of inflammatory mediators in these links2,5, as well as the suggested relationships between PD and these systemic diseases37,38, the possibility that they may have acted as potential confounding factors should be considered in future studies. Lastly, the mode of PD progression was determined based on the onset of dementia or postural instability, which are presentations of advanced-stage PD. Milder clinical features or quantitative scales, if available, could allow for a more sensitive evaluation of the association between CHIP and PD progression.

To summarize, our findings demonstrate the association between PD and increased odds of TET2-related CHIP, especially in PD patients with accelerated motor decline. Inflammatory dysregulation may play a critical role in this relationship.

Methods

Study participants

The DNA samples of patients diagnosed with PD or iRBD at the Movement Disorders Clinic of Seoul National University Hospital between 2013 and 2020 who provided informed consent for the DNA repository database were included. The diagnosis of PD was established by a movement disorders specialist (HJK) based on the Movement Disorders Society clinical diagnostic criteria for PD39. iRBD was diagnosed according to the third edition of the International Classification of Sleep Disorders with video polysomnography.

The control group comprised of an institutional cohort, called the “GENIE” cohort, of healthy subjects who voluntarily underwent a routine health screening program provided by Seoul National University Hospital Healthcare System Gangnam Center and provided informed consent to be included in the DNA repository database.

The demographic profiles of the subjects with PD, iRBD, and controls were collected. Additionally, for PD subjects, regular follow-up observations were conducted at 3- to 4-month intervals to ensure the reliability of PD diagnosis (i.e., to exclude the possibility of atypical parkinsonism) and to track changes in the HY stage and the occurrence of dementia after sample collection in the outpatient clinic. The criteria for diagnosing dementia were set as a Mini-Mental Status Examination (MMSE) score of 24 or below or a Montreal Cognitive Assessment (MoCA) score of 22 or below. Based on this, the PD subject group was categorized according to clinical progression patterns. Those progressing to HY stage 3 or higher, or developing dementia within 6 years of onset, were classified as fast-progressing PD (Fast PD). Patients who remained stable, showing unilateral motor involvement for 5 years after onset, or a HY stage of 2.0 or below for 10 years without dementia, were categorized as the Slow PD group. The Typical PD group included patients with onset in or before 2020, regardless of severity or rate of progression, to represent PD patients commonly encountered in outpatient settings.

To address the potential age differences between the control and patient groups, comparative analyses were performed by selecting control cohort samples within the age range of each patient group. Specifically, for the comparison with iRBD, subjects from the GENIE cohort aged between 50 and 85 years, matching the age range of the iRBD patient group, were chosen as the control group. Likewise, for the comparison with PD, control subjects aged between 40 and 86 years, corresponding to the age range of the PD patient group, were selected.

The study protocol was approved by the Institutional Review Board of the Seoul National University Hospital (IRB No. 2106-116-1227, H-1908-121-1056), and informed consent was waived by the board.

Sample preparation and analysis

Participants’ genomic DNA extracted from peripheral blood mononuclear cell was used for targeted sequencing for twenty-four genes frequently involved in CHIP (Supplementary Table 6). Twist target enrichment panel (Twist Bioscience, USA) and DNBSEQ-G400 Dx (MGI Tech, China) were used with 2 × 100 bp paired-end reads with a minimum coverage of 970x (average coverage 1,724x; 960x ~ 3100x). Detailed methods for CHIP variant calling are described below.

CHIP variant calling

Data preprocessing and quality control

Targeted sequencing reads for peripheral blood mononuclear cell samples were demultiplexed using Illumina’s bcl2fastq (v2.17.1.14) to generate FASTQ files. We used SeqPrep for adapter trimming (default settings) and Sickle (v1.33) for low BQV base trimming (minimum average base quality value [BQV] = 20). Subsequently, trimmed FASTQ files were submitted to GATK best practice pipeline, which includes alignment to the hg19 reference with BWA-MEM (v0.7.10). For all samples, duplicate marking and sorting were performed using PICARD (v1.94) MarkDuplicates, followed by indel realignment and base quality score recalibration using GATK Light (v2.3.9). This was followed by duplicate marking again, resulting in a final coordinate-sortedm analysis-ready BAM file for each sample. Duplication metrics and BAM quality metrics were computed using PICARD (v1.94; MarkDuplicates, CalculateHsMetrics, CollectGcBiasMetrics).

CHIP-Negative cohort

To filter out artifactual variants, we generated CHIP-negative cohort data (n = 209, age 20–30 years) for the CHIP variant calling filtering process. Putative CHIP variants with VAF ≥ 0.1% were collected and organized as variant frequency tables.

CHIP mutation calling and filtering

Analysis-ready BAM files for the analyzed cohort and CHIP-negative cohort were qualified based on depth of coverage (average DOC > 700x), ensuring a 2% VAF limit of detection. These BAM files were processed through a somatic variant calling pipeline, which includes VarDict40, Mutect2 (v4.1.4.1)41, SNVer (v0.4.1)42, for calling single nucleotide variants (SNVs), insertions, and deletions. To achieve comprehensive somatic variant calling, we enforced a union approach among the variant callers. The criteria for positive SNVs/Indels were as follows: total reads ≥ 20, alternative reads ≥ 10 (with at least 5 positive and 5 negative), and VAF between 1.5% and 30%. We further filtered out common germline variants with a minor allele frequency (MAF) > 0.1% in gnomAD. Additionally, we excluded a subset of artifactual calls with a MAF > 2% in somatic variants on the negative cohort data that do not exist in COSMIC hematological criteria. Final variants were curated by IGV review to filter out potential artifacts driven by PCR, highly homologous regions, and repeat regions.

CHIP threshold

While CHIP has been defined by VAF of at least 2.0%43, several studies have reported an association of hematopoietic clones with VAF < 2% with cardiovascular disease44,45. We sought to minimize false negatives by applying a more sensitive method with a cut-off of 1.0% VAF. Thus, all non-synonymous variants with a VAF of 1.0% to 30% were considered CHIP variants. Common germline variants with gnomAD allele frequency ≥ 0.1% were excluded unless previously reported to be somatic and involved in COSMIC hematologic malignancies. We filtered a subset of artifactual calls with MAF > 2% in somatic variants on 20–year CHIP-negative cohort data, which do not exist in COSMIC hematological criteria. Final variants were curated by IGV review to filter out potential artifacts driven by PCR, highly homologous regions, and repeat regions.

Among the twenty-five genes sequenced, individuals with mutations in the four most frequently mutated genes in CHIP, namely DNMT3A, TET2, ASXL1, and PPM1D, were additionally specified according to the mutated gene.

Variant annotation

All dependable non-synonymous variants associated with malignancy were annotated as CHIP-driving mutations in accordance with the criteria: (1) any truncating mutations; (2) any somatic variants previously reported on at least 20 occasions with solid malignancy or on 10 occasions with hematopoietic malignancy in the COSMIC database v83.

Statistical analysis

Continuous variables are represented as mean and interquartile range or standard deviation, or as the median with range. Categorical data is presented as percentages (%). For comparing continuous variables, we employed either an independent t-test or the Mann–Whitney U test. Categorical variables were compared using the χ2 test or Fisher’s exact test. Multivariate logistic regression, adjusted for age and sex, was utilized to identify independent risk factors for CHIP with a VAF threshold of 1.0% in both PD and iRBD. Statistical analyses were conducted using R statistical software version 4.0.2 (http://www.r-project.org).

Propensity score matching

To verify the significant findings by rigorously balancing age and sex between groups, we further performed propensity score matching using the MatchIt package in R. Age and sex was included in the propensity score model as covariates. A nearest-neighbor matching algorithm with a patient-to-control ratio of 1:2 or 1:4 was applied. The quality of matching was visually inspected using propensity score histograms to ensure a balanced distribution of propensity scores. The balance of covariates after matching was assessed using the independent t-test. The matching ratio that best eliminated statistically significant differences in age and sex was selected for each comparison. The matched dataset was then used for further multivariate logistic regression, adjusted for age and sex, to confirm the association between CHIP and PD or iRBD.

Supplementary information

Supplementary tables and figures

Supplementary information

The online version contains supplementary material available at 10.1038/s41531-024-00784-1.

Acknowledgements

This study was supported by a research grant from NOBO Medicine Inc. and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2022R1A2C2091254). The targeted NGS of CHIP was supported by NOBO Medicine Inc.

Author contributions

1. Research project: A. Conception, B. Organization, C. Execution; 2. Statistical Analysis: A. Design, B. Execution, C. Review and Critique; 3. Manuscript Preparation: A. Writing of the first draft, B. Review and Critique. KAW: 1B, 1C, 2A, 2B, 3A. HJK: 1A, 1B, 1C, 2C, 3A, 3B. CYL: 1B, 1C. JHS: 2C, 3B. CS, HI, HA, JL: 1B, 1C, 2A, 2B, 3B. SYC: 1C. YIK: 1A, 1B, 2C, 3B. BJ: 2C, 3B.

Data availability

The data is available from the corresponding author upon reasonable request.

Competing interests

CS, HI, HA, JL, and YIK are employed in NOBO Medicine Inc. CS, HI, HA, and YIK are stockholders of NOBO Medicine Inc.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Franceschi C Garagnani P Parini P Giuliani C Santoro A Inflammaging: a new immune-metabolic viewpoint for age-related diseases Nat. Rev. Endocrinol. 2018 14 576 590 10.1038/s41574-018-0059-4 30046148
Franceschi, C., Garagnani, P., Parini, P., Giuliani, C. & Santoro, A. Inflammaging: a new immune-metabolic viewpoint for age-related diseases. Nat. Rev. Endocrinol. 14, 576–590 (2018).30046148 10.1038/s41574-018-0059-4
2. Jaiswal S Clonal Hematopoiesis and risk of atherosclerotic cardiovascular disease N. Engl. J. Med. 2017 377 111 121 10.1056/NEJMoa1701719 28636844
Jaiswal, S. et al. Clonal Hematopoiesis and risk of atherosclerotic cardiovascular disease. N. Engl. J. Med. 377, 111–121 (2017).28636844 10.1056/NEJMoa1701719
3. Bhattacharya R Clonal Hematopoiesis is associated with higher risk of stroke Stroke 2022 53 788 797 10.1161/STROKEAHA.121.037388 34743536
Bhattacharya, R. et al. Clonal Hematopoiesis is associated with higher risk of stroke. Stroke 53, 788–797 (2022).34743536 10.1161/STROKEAHA.121.037388
4. Bouzid H Clonal hematopoiesis is associated with protection from Alzheimer’s disease Nat. Med. 2023 29 1662 1670 10.1038/s41591-023-02397-2 37322115
Bouzid, H. et al. Clonal hematopoiesis is associated with protection from Alzheimer’s disease. Nat. Med. 29, 1662–1670 (2023).37322115 10.1038/s41591-023-02397-2
5. Jaiswal S Ebert BL Clonal hematopoiesis in human aging and disease Science 2019 366 eaan4673 10.1126/science.aan4673 31672865
Jaiswal, S. & Ebert, B. L. Clonal hematopoiesis in human aging and disease. Science 366, eaan4673 (2019).31672865 10.1126/science.aan4673
6. Fuster JJ Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice Science 2017 355 842 847 10.1126/science.aag1381 28104796
Fuster, J. J. et al. Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice. Science 355, 842–847 (2017).28104796 10.1126/science.aag1381
7. Sano S CRISPR-mediated gene editing to assess the roles of Tet2 and Dnmt3a in clonal hematopoiesis and cardiovascular disease Circ. Res. 2018 123 335 341 10.1161/CIRCRESAHA.118.313225 29728415
Sano, S. et al. CRISPR-mediated gene editing to assess the roles of Tet2 and Dnmt3a in clonal hematopoiesis and cardiovascular disease. Circ. Res. 123, 335–341 (2018).29728415 10.1161/CIRCRESAHA.118.313225
8. Sano S Tet2-mediated clonal hematopoiesis accelerates heart failure through a mechanism involving the IL-1beta/NLRP3 inflammasome J. Am. Coll. Cardiol. 2018 71 875 886 10.1016/j.jacc.2017.12.037 29471939
Sano, S. et al. Tet2-mediated clonal hematopoiesis accelerates heart failure through a mechanism involving the IL-1beta/NLRP3 inflammasome. J. Am. Coll. Cardiol. 71, 875–886 (2018).29471939 10.1016/j.jacc.2017.12.037
9. Harms AS Ferreira SA Romero-Ramos M Periphery and brain, innate and adaptive immunity in Parkinson’s disease Acta Neuropathol. 2021 141 527 545 10.1007/s00401-021-02268-5 33555429
Harms, A. S., Ferreira, S. A. & Romero-Ramos, M. Periphery and brain, innate and adaptive immunity in Parkinson’s disease. Acta Neuropathol. 141, 527–545 (2021).33555429 10.1007/s00401-021-02268-5
10. Tansey MG Inflammation and immune dysfunction in Parkinson disease Nat. Rev. Immunol. 2022 22 657 673 10.1038/s41577-022-00684-6 35246670
Tansey, M. G. et al. Inflammation and immune dysfunction in Parkinson disease. Nat. Rev. Immunol. 22, 657–673 (2022).35246670 10.1038/s41577-022-00684-6
11. Kim R Peripheral blood inflammatory markers in early Parkinson’s disease J. Clin. Neurosci. 2018 58 30 33 10.1016/j.jocn.2018.10.079 30454693
Kim, R. et al. Peripheral blood inflammatory markers in early Parkinson’s disease. J. Clin. Neurosci. 58, 30–33 (2018).30454693 10.1016/j.jocn.2018.10.079
12. Keogh MJ High prevalence of focal and multi-focal somatic genetic variants in the human brain Nat. Commun. 2018 9 4257 10.1038/s41467-018-06331-w 30323172
Keogh, M. J. et al. High prevalence of focal and multi-focal somatic genetic variants in the human brain. Nat. Commun. 9, 4257 (2018).30323172 10.1038/s41467-018-06331-w
13. Ito S Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification Nature 2010 466 1129 1133 10.1038/nature09303 20639862
Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).20639862 10.1038/nature09303
14. Delhommeau F Mutation in TET2 in myeloid cancers N. Engl. J. Med. 2009 360 2289 2301 10.1056/NEJMoa0810069 19474426
Delhommeau, F. et al. Mutation in TET2 in myeloid cancers. N. Engl. J. Med. 360, 2289–2301 (2009).19474426 10.1056/NEJMoa0810069
15. Tan EK Parkinson disease and the immune system - associations, mechanisms and therapeutics Nat. Rev. Neurol. 2020 16 303 318 10.1038/s41582-020-0344-4 32332985
Tan, E. K. et al. Parkinson disease and the immune system - associations, mechanisms and therapeutics. Nat. Rev. Neurol. 16, 303–318 (2020).32332985 10.1038/s41582-020-0344-4
16. Jaiswal S Clonal hematopoiesis and nonhematologic disorders Blood 2020 136 1606 1614 32736379
Jaiswal, S. Clonal hematopoiesis and nonhematologic disorders. Blood 136, 1606–1614 (2020).32736379
17. Harms AS Peripheral monocyte entry is required for alpha-Synuclein induced inflammation and Neurodegeneration in a model of Parkinson disease Exp. Neurol. 2018 300 179 187 10.1016/j.expneurol.2017.11.010 29155051
Harms, A. S. et al. Peripheral monocyte entry is required for alpha-Synuclein induced inflammation and Neurodegeneration in a model of Parkinson disease. Exp. Neurol. 300, 179–187 (2018).29155051 10.1016/j.expneurol.2017.11.010
18. Goldmann T Origin, fate and dynamics of macrophages at central nervous system interfaces Nat. Immunol. 2016 17 797 805 10.1038/ni.3423 27135602
Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016).27135602 10.1038/ni.3423
19. Qin XY Zhang SP Cao C Loh YP Cheng Y Aberrations in peripheral inflammatory cytokine levels in Parkinson Disease: A systematic review and meta-analysis JAMA Neurol. 2016 73 1316 1324 10.1001/jamaneurol.2016.2742 27668667
Qin, X. Y., Zhang, S. P., Cao, C., Loh, Y. P. & Cheng, Y. Aberrations in peripheral inflammatory cytokine levels in Parkinson Disease: A systematic review and meta-analysis. JAMA Neurol. 73, 1316–1324 (2016).27668667 10.1001/jamaneurol.2016.2742
20. Zhang Q Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6 Nature 2015 525 389 393 10.1038/nature15252 26287468
Zhang, Q. et al. Tet2 is required to resolve inflammation by recruiting Hdac2 to specifically repress IL-6. Nature 525, 389–393 (2015).26287468 10.1038/nature15252
21. Fan Z Systemic activation of NLRP3 inflammasome and plasma alpha-synuclein levels are correlated with motor severity and progression in Parkinson’s disease J. Neuroinflamm. 2020 17 11 10.1186/s12974-019-1670-6
Fan, Z. et al. Systemic activation of NLRP3 inflammasome and plasma alpha-synuclein levels are correlated with motor severity and progression in Parkinson’s disease. J. Neuroinflamm. 17, 11 (2020).10.1186/s12974-019-1670-6
22. Lai TT Kim YJ Ma HI Kim YE Evidence of inflammation in Parkinson’s disease and its contribution to Synucleinopathy J. Mov. Disord. 2022 15 1 14 10.14802/jmd.21078 35124957
Lai, T. T., Kim, Y. J., Ma, H. I. & Kim, Y. E. Evidence of inflammation in Parkinson’s disease and its contribution to Synucleinopathy. J. Mov. Disord. 15, 1–14 (2022).35124957 10.14802/jmd.21078
23. Tian J Specific immune status in Parkinson’s disease at different ages of onset NPJ Parkinsons Dis. 2022 8 5 10.1038/s41531-021-00271-x 35013369
Tian, J. et al. Specific immune status in Parkinson’s disease at different ages of onset. NPJ Parkinsons Dis. 8, 5 (2022).35013369 10.1038/s41531-021-00271-x
24. Grozdanov V Inflammatory dysregulation of blood monocytes in Parkinson’s disease patients Acta Neuropathol. 2014 128 651 663 10.1007/s00401-014-1345-4 25284487
Grozdanov, V. et al. Inflammatory dysregulation of blood monocytes in Parkinson’s disease patients. Acta Neuropathol. 128, 651–663 (2014).25284487 10.1007/s00401-014-1345-4
25. Marshall LL Epigenomic analysis of Parkinson’s disease neurons identifies Tet2 loss as neuroprotective Nat. Neurosci. 2020 23 1203 1214 10.1038/s41593-020-0690-y 32807949
Marshall, L. L. et al. Epigenomic analysis of Parkinson’s disease neurons identifies Tet2 loss as neuroprotective. Nat. Neurosci. 23, 1203–1214 (2020).32807949 10.1038/s41593-020-0690-y
26. Ichiyama K The methylcytosine dioxygenase Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells Immunity 2015 42 613 626 10.1016/j.immuni.2015.03.005 25862091
Ichiyama, K. et al. The methylcytosine dioxygenase Tet2 promotes DNA demethylation and activation of cytokine gene expression in T cells. Immunity 42, 613–626 (2015).25862091 10.1016/j.immuni.2015.03.005
27. Munoz-Delgado L Peripheral inflammation is associated with dopaminergic degeneration in Parkinson’s disease Mov. Disord. 2023 38 755 763 10.1002/mds.29369 36912400
Munoz-Delgado, L. et al. Peripheral inflammation is associated with dopaminergic degeneration in Parkinson’s disease. Mov. Disord. 38, 755–763 (2023).36912400 10.1002/mds.29369
28. Farmen K Monocyte markers correlate with immune and neuronal brain changes in REM sleep behavior disorder Proc. Natl Acad. Sci. USA 2021 118 e2020858118 10.1073/pnas.2020858118 33658371
Farmen, K. et al. Monocyte markers correlate with immune and neuronal brain changes in REM sleep behavior disorder. Proc. Natl Acad. Sci. USA 118, e2020858118 (2021).33658371 10.1073/pnas.2020858118
29. Zhang H Plasma immune markers in an idiopathic REM sleep behavior disorder cohort Parkinsonism Relat. Disord. 2020 78 145 150 10.1016/j.parkreldis.2020.07.017 32835920
Zhang, H. et al. Plasma immune markers in an idiopathic REM sleep behavior disorder cohort. Parkinsonism Relat. Disord. 78, 145–150 (2020).32835920 10.1016/j.parkreldis.2020.07.017
30. Kim R Serum TNF-alpha and neurodegeneration in isolated REM sleep behavior disorder Parkinsonism Relat. Disord. 2020 81 1 7 10.1016/j.parkreldis.2020.09.041 33027749
Kim, R. et al. Serum TNF-alpha and neurodegeneration in isolated REM sleep behavior disorder. Parkinsonism Relat. Disord. 81, 1–7 (2020).33027749 10.1016/j.parkreldis.2020.09.041
31. Liddelow SA Neurotoxic reactive astrocytes are induced by activated microglia Nature 2017 541 481 487 10.1038/nature21029 28099414
Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481–487 (2017).28099414 10.1038/nature21029
32. Roulis M Armaka M Manoloukos M Apostolaki M Kollias G Intestinal epithelial cells as producers but not targets of chronic TNF suffice to cause murine Crohn-like pathology Proc. Natl Acad. Sci. USA 2011 108 5396 5401 10.1073/pnas.1007811108 21402942
Roulis, M., Armaka, M., Manoloukos, M., Apostolaki, M. & Kollias, G. Intestinal epithelial cells as producers but not targets of chronic TNF suffice to cause murine Crohn-like pathology. Proc. Natl Acad. Sci. USA 108, 5396–5401 (2011).21402942 10.1073/pnas.1007811108
33. Barnum CJ Peripheral administration of the selective inhibitor of soluble tumor necrosis factor (TNF) XPro(R)1595 attenuates nigral cell loss and glial activation in 6-OHDA hemiparkinsonian rats J. Parkinsons Dis. 2014 4 349 360 10.3233/JPD-140410 25061061
Barnum, C. J. et al. Peripheral administration of the selective inhibitor of soluble tumor necrosis factor (TNF) XPro(R)1595 attenuates nigral cell loss and glial activation in 6-OHDA hemiparkinsonian rats. J. Parkinsons Dis. 4, 349–360 (2014).25061061 10.3233/JPD-140410
34. Peter I Anti-Tumor Necrosis Factor Therapy and Incidence of Parkinson Disease Among Patients With Inflammatory Bowel Disease JAMA Neurol. 2018 75 939 946 10.1001/jamaneurol.2018.0605 29710331
Peter, I. et al. Anti-Tumor Necrosis Factor Therapy and Incidence of Parkinson Disease Among Patients With Inflammatory Bowel Disease. JAMA Neurol. 75, 939–946 (2018).29710331 10.1001/jamaneurol.2018.0605
35. Dayer JM Interleukin 1 or tumor necrosis factor-alpha: which is the real target in rheumatoid arthritis? J. Rheumatol. Suppl. 2002 65 10 15 12236616
Dayer, J. M. Interleukin 1 or tumor necrosis factor-alpha: which is the real target in rheumatoid arthritis? J. Rheumatol. Suppl. 65, 10–15 (2002).12236616
36. Abegunde SO Buckstein R Wells RA Rauh MJ An inflammatory environment containing TNFalpha favors Tet2-mutant clonal hematopoiesis Exp. Hematol. 2018 59 60 65 10.1016/j.exphem.2017.11.002 29195897
Abegunde, S. O., Buckstein, R., Wells, R. A. & Rauh, M. J. An inflammatory environment containing TNFalpha favors Tet2-mutant clonal hematopoiesis. Exp. Hematol. 59, 60–65 (2018).29195897 10.1016/j.exphem.2017.11.002
37. Chua SKK Saffari SE Lee SJY Tan EK Association between Parkinson’s disease and coronary artery disease: a systematic review and meta-analysis J. Parkinsons Dis. 2022 12 1737 1748 10.3233/JPD-223291 35694936
Chua, S. K. K., Saffari, S. E., Lee, S. J. Y. & Tan, E. K. Association between Parkinson’s disease and coronary artery disease: a systematic review and meta-analysis. J. Parkinsons Dis. 12, 1737–1748 (2022).35694936 10.3233/JPD-223291
38. De Pablo-Fernandez E Goldacre R Pakpoor J Noyce AJ Warner TT Association between diabetes and subsequent Parkinson disease: A record-linkage cohort study Neurology 2018 91 e139 e142 10.1212/WNL.0000000000005771 29898968
De Pablo-Fernandez, E., Goldacre, R., Pakpoor, J., Noyce, A. J. & Warner, T. T. Association between diabetes and subsequent Parkinson disease: A record-linkage cohort study. Neurology 91, e139–e142 (2018).29898968 10.1212/WNL.0000000000005771
39. Postuma RB MDS clinical diagnostic criteria for Parkinson’s disease Mov. Disord. 2015 30 1591 1601 10.1002/mds.26424 26474316
Postuma, R. B. et al. MDS clinical diagnostic criteria for Parkinson’s disease. Mov. Disord. 30, 1591–1601 (2015).26474316 10.1002/mds.26424
40. Lai Z VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research Nucleic Acids Res. 2016 44 e108 10.1093/nar/gkw227 27060149
Lai, Z. et al. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Res. 44, e108 (2016).27060149 10.1093/nar/gkw227
41. Benjamin D Calling Somatic SNVs and Indels with Mutect2 bioRxiv 2019 1 20
Benjamin, D. et al. Calling Somatic SNVs and Indels with Mutect2. bioRxiv 1, 20 (2019).
42. Wei Z Wang W Hu P Lyon GJ Hakonarson H SNVer: a statistical tool for variant calling in analysis of pooled or individual next-generation sequencing data Nucleic Acids Res. 2011 39 e132 10.1093/nar/gkr599 21813454
Wei, Z., Wang, W., Hu, P., Lyon, G. J. & Hakonarson, H. SNVer: a statistical tool for variant calling in analysis of pooled or individual next-generation sequencing data. Nucleic Acids Res. 39, e132 (2011).21813454 10.1093/nar/gkr599
43. Steensma DP Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes Blood J. Am. Soc. Hematol. 2015 126 9 16
Steensma, D. P. et al. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood J. Am. Soc. Hematol. 126, 9–16 (2015).
44. Dorsheimer L Association of mutations contributing to clonal hematopoiesis with prognosis in chronic ischemic heart failure JAMA Cardiol. 2019 4 25 33 10.1001/jamacardio.2018.3965 30566180
Dorsheimer, L. et al. Association of mutations contributing to clonal hematopoiesis with prognosis in chronic ischemic heart failure. JAMA Cardiol. 4, 25–33 (2019).30566180 10.1001/jamacardio.2018.3965
45. Mayerhofer E Prevalence and therapeutic implications of clonal hematopoiesis of indeterminate potential in young patients with stroke Stroke 2023 54 938 946 10.1161/STROKEAHA.122.041416 36789775
Mayerhofer, E. et al. Prevalence and therapeutic implications of clonal hematopoiesis of indeterminate potential in young patients with stroke. Stroke 54, 938–946 (2023).36789775 10.1161/STROKEAHA.122.041416
