PLOS ONE RESEARCH ARTICLE DNA methylation analyses identify an intronic ZDHHC6 locus associated with time to recurrent stroke in the Vitamin Intervention for Stroke Prevention (VISP) clinical trial Nicole M. Davis Armstrong1, Wei-Min Chen2,3, Fang-Chi Hsu4, Michael S. Brewer1, Natalia Cullell5,6, Israel Fernández-Cadenas5,6, Stephen R. Williams7, Michèle M. Sale2,3, Bradford B. Worrall3,7, Keith L. Keene1,8* a1111111111 1 Department of Biology, East Carolina University, Greenville, NC, United States of America, 2 Center for a1111111111 Public Health Genomics, University of Virginia, Charlottesville, VA, United States of America, 3 Department a1111111111 of Public Health Sciences, University of Virginia, Charlottesville, VA, United States of America, 4 Department a1111111111 of Biostatistics and Data Science, Division of Public Health Sciences, Wake Forest School of Medicine, a1111111111 Winston-Salem, NC, United States of America, 5 Stroke Pharmacogenomics and Genetics, Fundació Docència i Recerca Mútua Terrassa, Hospital Universitari Mútua de Terrassa, Terrassa, Barcelona, Spain, 6 Stroke Pharmacogenomics and Genetics, Sant Pau Institute of Research, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 7 Department of Neurology, University of Virginia, Charlottesville, VA, United States of America, 8 Center for Health Disparities, Brody School of Medicine, East Carolina University, Greenville, NC, United States of America OPEN ACCESS Citation: Davis Armstrong NM, Chen W-M, Hsu F- * keenek@ecu.edu C, Brewer MS, Cullell N, Fernández-Cadenas I, et al. (2021) DNA methylation analyses identify an intronic ZDHHC6 locus associated with time to Abstract recurrent stroke in the Vitamin Intervention for Stroke Prevention (VISP) clinical trial. PLoS ONE Aberrant DNA methylation profiles have been implicated in numerous cardiovascular dis- 16(7): e0254562. https://doi.org/10.1371/journal. pone.0254562 eases; however, few studies have investigated how these epigenetic modifications contrib- ute to stroke recurrence. The aim of this study was to identify methylation loci associated Editor: Giulia Bivona, University of Palermo, ITALY with the time to recurrent cerebro- and cardiovascular events in individuals of European and Received: March 1, 2021 African descent. DNA methylation profiles were generated for 180 individuals from the Vita- Accepted: June 30, 2021 min Intervention for Stroke Prevention clinical trial using Illumina HumanMethylation 450K Published: July 12, 2021 BeadChip microarrays, resulting in beta values for 470,871 autosomal CpG sites. Ethnicity- stratified survival analyses were performed using Cox Proportional Hazards regression Peer Review History: PLOS recognizes the benefits of transparency in the peer review models for associations between each methylation locus and the time to recurrent stroke or process; therefore, we enable the publication of composite vascular event. Results were validated in the Vall d’Hebron University Hospital all of the content of peer review and author cohort from Barcelona, Spain. Network analyses of the methylation loci were generated responses alongside final, published articles. The editorial history of this article is available here: using weighted gene coexpression network analysis. Primary analysis identified four signifi- https://doi.org/10.1371/journal.pone.0254562 cant loci, cg04059318, ch.2.81927627R, cg03584380, and cg24875416, associated with Copyright: © 2021 Davis Armstrong et al. This is time to recurrent stroke. Secondary analysis identified three loci, cg00076998, cg16758041, an open access article distributed under the terms and cg02365967, associated with time to composite vascular endpoint. Locus cg03584380, of the Creative Commons Attribution License, which is located in an intron of ZDHHC6, was replicated in the Vall d’Hebron University Hos- which permits unrestricted use, distribution, and pital cohort. The results from this study implicate the degree of methylation at cg03584380 reproduction in any medium, provided the original author and source are credited. is associated with the time of recurrence for stroke or composite vascular events across two ethnically diverse groups. Furthermore, modules of loci were associated with clinical traits Data Availability Statement: Individual level genetics and epigenetics data is considered and blood biomarkers including previous number of strokes, prothrombin fragments 1 + 2, sensitive controlled data and cannot be shared thrombomodulin, thrombin-antithrombin complex, triglyceride levels, and tissue PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 1 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP publicly, as specified by the IRBs of Wake Forest plasminogen activator. Ultimately, these loci could serve as potential epigenetic biomarkers University School of Medicine, the University of that could identify at-risk individuals in recurrence-prone populations. North Carolina at Chapel Hill School of Medicine, and the University of Virginia School of Medicine, along with the NIH Data Access Committee. Controlled-access data can only be obtained if a user has been authorized by the appropriate Data Introduction Access Committee. The individual level Genomics and Randomized Trials Network (GARNET) VISP Ischemic strokes account for 87% of all strokes and are heterogeneous, multifactorial diseases data are available in the database of Genotypes and comprising genetic and environmental contributions. Of the approximately 795,000 incident Phenotypes (dbGaP) (Accession: phs000343.v3. stroke cases annually in the United States, approximately 25% are recurrent events [1]. Indi- p1) and can be requested through the dbGaP viduals experiencing an ischemic stroke are at high risk of having a recurrent stroke or devel- Authorized Access System (https://dbgap.ncbi.nlm. nih.gov/aa/wga.cgi?page=login). The authors will oping cardiovascular diseases including myocardial infarction (MI), coronary heart disease, or also share the data on request. vascular death. Only a proportion of the estimated 37.9% heritability for ischemic stroke [2] is accounted for from genetic variants identified in genome-wide association studies, suggesting Funding: This work was supported by the National Institutes of Health, grant U01HG005160 and other mechanisms, such as epigenetic modifications, could comprise some of the remaining Supplement U01HG005160-03S1 (MMS/BBW) heritability for ischemic stroke and stroke recurrence risk. from the National Human Genome Research Epigenetics, such as DNA methylation, refer to chemical modifications of DNA structure Institute, an American Heart Association Scientist that can be maintained over cellular generations [3] and serve to propagate cellular memory Development Award 12SDG9180012 (KLK), and [4]. Abnormal DNA methylation patterns have been implicated in a number of cardiovascular funds from the Walking for My Life 5K Stroke Walk diseases [5, 6]; however, few studies have investigated these epigenetic contributions to stroke sponsored by Faith Christian Center International (Charlottesville, VA). The original study recruitment recurrence [7–9]. The aim of this study was to elucidate DNA methylation loci associated with and datasets for the VISP clinical trial were the time to vascular events, including recurrent stroke, MI, and death. Single locus and com- supported by research grant R01 NS34447 (JT) prehensive loci networks were analyzed in 180 individuals from the Vitamin Intervention for from the National Institute of Neurological Stroke Prevention (VISP) clinical trial and further validated in the independent Vall d’Hebron Disorders and Stroke. The funders had no role in University Hospital cohort. Findings from this study support the utility of epigenetic marks as study design, data collection and analysis, decision to publish, or preparation of the manuscript. potential biomarkers and may lead to improved prognosis or prevention of recurrent stroke and cardiovascular events. Competing interests: The authors have declared that no competing interests exist. Methods Ethics statement The institutional review boards (IRBs) of Wake Forest University School of Medicine, the Uni- versity of North Carolina at Chapel Hill School of Medicine, and individual recruitment sites approved the VISP clinical trial study protocol. All VISP participants provided written, informed consent. A subset of 2,100 participants agreed to be included in subsequent genetic studies. IRB approval from the University of Virginia and East Carolina University was obtained for the genetic and epigenetic components. Discovery cohort: Vitamin Intervention for Stroke Prevention (VISP) clinical trial VISP was a multi-centered, double-blinded, randomized and controlled clinical trial that enrolled participants aged 35 years or older with baseline homocysteine levels at or above the 25th percentile and was designed to determine whether pyridoxine (B6), cyanocobalamin (B12), and folic acid (B9) supplementation reduced recurrent cerebral infarction, MI, or fatal coronary heart disease (CHD) [10]. Participants were enrolled within 120 days of suffering a non-disabling cerebral infarction, assigned a daily B vitamin high-dose or low-dose formula- tion, and followed for two years. While ischemic subtype was not adjudicated, based on inclu- sion/exclusion criteria [10], the VISP enrollment stroke was most likely a small-vessel (lacunar) infarct. PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 2 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP A subset of 2,100 VISP participants consented for inclusion in genetic studies of which, methylation data were generated for 204 participants. Upon quality control (QC), the methyla- tion profiles of 180 individuals were used in subsequent analyses, including 76 individuals of African descent (AFR) and 104 of European descent (EUR). Individual level genetics and epi- genetics data is considered sensitive controlled data and cannot be shared publicly, as specified by the IRBs of Wake Forest University School of Medicine, the University of North Carolina at Chapel Hill School of Medicine, and the University of Virginia School of Medicine, along with the NIH Data Access Committee. Controlled-access data can only be obtained if a user has been authorized by the appropriate Data Access Committee. The individual level Genomics and Randomized Trials Network (GARNET) VISP data are available in the database of Geno- types and Phenotypes (dbGaP) (Accession: phs000343.v3.p1) and can be requested through the dbGaP Authorized Access System (https://dbgap.ncbi.nlm.nih.gov/aa/wga.cgi?page= login). The authors will also share the data on request. Replication cohort: The Vall d’Hebron University Hospital cohort From a cohort of 1,900 patients with stroke from Vall d’Hebron University Hospital (Barce- lona, Spain), 28 subjects with composite vascular recurrence events were selected. Of these par- ticipants, 18 had a recurrent ischemic stroke. Composite vascular recurrence was described as new ischemic stroke, MI, peripheral vascular disease, or cardiovascular death and was detected through telephone interviews every three months or direct clinical visit. The epigenome-wide methylation profiles were generated using Illumina Infinium 450k BeadChip microarrays. The profiles were processed in a single working batch and preprocessing, correction, normalization steps, and QC was performed using R, as previously described [8]. The epigenetics and genet- ics summary data used for replication will be shared for research studies on approval of the principal investigators of the GRECOS (Genotyping Recurrence Risk of Stroke) cohort. Stroke recurrence and composite vascular event definition The primary vascular endpoint analyzed in the current study was an incident recurrent stroke during the VISP trial (VISP recurrent stroke). VISP recurrent stroke was defined as an acute neurological ischemic event, requiring a sudden onset of symptoms lasting at least 24 hours, as reported on the Follow-up Stroke Symptoms (FSS) form or as determined by the VISP End- point Review Committee [10]. A composite vascular event was examined as a secondary endpoint and was defined as fatal coronary heart disease, a non-fatal hospitalized MI and resuscitation for cardiac collapse, coro- nary bypass surgery, coronary angioplasty, or a VISP recurrent cerebrovascular event. Methylation data generation Genomic DNA was extracted from whole blood samples of the VISP participants, as previously described [9]. Briefly, the DNA was denatured and bisulfite-converted using Zymo EZ DNA Methylation Kits (Zymo Research Corp., Irvine, CA). Illumina Infinium Human Methyla- tion450k BeadChip microarrays were used to interrogate CpG sites across the genome and the resulting intensity files were analyzed using GenomeStudio. Beta scores were generated as the ratio of methylated intensities divided by the sum of the methylated and unmethylated intensi- ties (i.e. the proportion of total signal from the methylation-specific probe). Probes were removed if they failed to hybridize (detection p>5%) or if they were located on sex chromo- somes. The filtered beta scores underwent stratified quantile normalization with the minfi package in R [11–13]. A total of 470,871 autosomal CpGs were used in subsequent analyses. PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 3 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP Covariate generation To account for population substructure, a principal component analysis was performed using KING software [14] on VISP genotype data. The top four and ten principal components (PCs) were generated and used as covariates in the survival analysis for the EUR population and AFR population, respectively. Batch effect was adjusted for using factor variables indicative of meth- ylation data generation round, while cellular heterogeneity due to the variation in cell popula- tion proportions within whole blood samples was controlled for using cell proportion estimates generated by the estimateCellCounts function in minfi [12, 15–17]. This function generates cell counts for B-lymphocytes, CD4+ and CD8+ T-lymphocytes, natural killer cells, granulocytes, and monocytes. Statistical analysis Baseline characteristics of the study participants with and without recurrent stroke were com- pared using t-tests and χ2 tests for continuous and categorical traits, respectively, by each eth- nic stratum. Survival analyses utilizing Cox Proportional Hazards (PH) regression models were per- formed for the time in days to VISP recurrent stroke or composite vascular event for both AFR and EUR participants independently. The degree of methylation was the exposure and the models adjusted for age, sex, PCs, batch effect, treatment arm, and cellular proportions. The replication cohort adjusted for age, sex, the top two PCs, cell heterogeneity proportions, and batch effect. Statistical significance was calculated at p�1.06e-07 (= 0.05/470871 total number of loci) for the discovery cohort. Statistical power ranged from 0.5847 to 0.7466 based on analysis phenotype [18]. A significance threshold of p�7.14e-03 (= 0.05/7) was determined for the look-up analysis in the replication cohort. Weighted Gene Co-expression Network Analysis (WGCNA) [19] was used to identify net- works or modules of highly correlated DNA methylation loci and blood biomarkers or clinical traits (n = 27 traits; S1 Table). Modules were calculated using the blockwise module function in the WGCNA R package. Outlier samples were identified and removed to ensure reliable network construction. An appropriate soft threshold power was calculated for each stratum. A soft thresholding power of 20 in the AFR cohort and 26 in the EUR cohort was determined at a threshold �0.8. These parameters were used in a signed-hybrid network model with the min- imum number of loci set to 30 and a maximum block size set to 10,000. The loci comprising each module were represented by a weighted average, which is indicative of the first principle component in the analysis. Statistical significance was calculated as p�3.93e-09 (= 0.05/(470871 loci� 27 traits)), while the suggestive threshold was set at p<1.00e-03. Gene ontology (GO) term enrichment was performed using GOrilla (Gene Ontology enRIchment anaLysis and visuaLizAtion) [20, 21] for the seven significant loci identified in the survival analysis with p�1.06e-07. Statistical significance for GO term enrichment included terms with a false discovery rate (FDR) q-value � 0.05. To further evaluate the biological mechanisms of statistically and marginally significant (p<1.00e-06) CpG sites from the AFR VISP recurrent stroke and composite vascular event analyses, the Functional Mapping and Annotation of Genome-Wide Association Studies (FUMA GWAS) version 1.3.6a [22] online platform and the GENE2FUNC process was uti- lized. The EUR composite vascular event analysis resulted in two CpGs that met or exceeded the suggestive threshold and therefore, functional mapping was not performed. A total of 36 (AFR VISP recurrent stroke) and 30 (AFR VISP composite vascular event) gene names were combined, resulting in 57 unique gene names uploaded into FUMA for evaluation of gene PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 4 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP expression and enrichment of differentially expressed gene sets in tissues from the Genotype- Tissue Expression (GTEx) 8 RNA sequencing data [23]. Upon filtering by ENSEMBL identifi- ers, 53 genes were used in the GTEx analyses. A multiple testing correction was performed using a Benjamini-Hochberg adjustment. Statistical significance was calculated using a p- threshold of p<0.05. Results The baseline characteristics of 76 AFR and 104 EUR VISP study participants with and without recurrent stroke were compared using χ2 and t-tests for categorical and continuous variables, respectively, and are presented in Table 1. Of these individuals, 28 AFR and 32 EUR partici- pants had an incident VISP recurrent stroke. In the AFR individuals, the average baseline age of those experiencing a recurrent stroke was five years older compared to those not having a recurrent stroke (65 years versus 60 years; p = 0.047). Of particular interest, those individuals having a VISP recurrent stroke had a more severe enrollment stroke compared to the non- recurrent control group, as measured on the modified Rankin stroke scale (RSS). While the enrollment criteria for VISP included mild-to-moderate RSS scores of 0–3, 35.7% of those suf- fering a recurrent stroke during VISP follow-up experienced an enrollment stroke with a RSS of 3, which is indicative of moderate disability, while 10.4% of the non-recurrent individuals had a similar enrollment stroke severity. Although not as extreme, VISP EUR participants had similar baseline age differences (70 years versus 68 years for VISP recurrence and nonrecur- rence, respectively), as well as enrollment stroke severity compared with AFR participants. Approximately 44% of EUR VISP participants who experienced a recurrent stroke had RSS of 0 or 1, compared to nearly 71% of participants in the non-recurrent group having a similar score indicative of no significant disability (Table 1). Ethnicity-stratified Cox PH analyses identified a total of seven methylation loci associated with time to event for recurrent stroke or composite vascular event (Tables 2 and 3). Four sta- tistically significant loci were identified for days to VISP recurrent stroke. The most significant association was observed for cg04059318 (HR [95% CI] = 7.19 [3.55–14.57]; p = 4.52e-08), located on chromosome 10. Three additional loci, ch.2.81927627R (HR = 2.72 [1.89–3.93]; p = 9.11e-08), cg03584380 (HR = 5.41 [2.91–10.06]; p = 9.66e-08), and cg24875416 (HR = 2.43 [1.75–3.37]; p = 9.82e-08) were also implicated in the AFR recurrent stroke analysis (Table 2; Fig 1). There were no statistically significant loci in the days to VISP stroke recurrence analysis in EUR. cg03584380, an intronic locus of ZDHHC6, was the only locus to remain significant in the replication cohort from Vall d’ Hebron University in Barcelona (HR = 1.83(1.21–2.77); p = 4.00e-03; Table 2). The ENCODE annotation and gene position for cg03584380 is pre- sented in Fig 2. For the time to composite vascular event analysis, cg00076998 (HR = 5.58 [2.98–10.44]; p = 7.87e-08) and cg16758041 (HR = 3.44 [2.18–5.43]; p = 1.04e-07) were identi- fied in AFR (Fig 3; Table 3), while cg02365967 (HR: 0.42 [0.31–0.58]; p = 8.08e-08) was identi- fied in the EUR stratum (Fig 4; Table 3). Using Schoenfeld residual tests, we did not observe any evidence of violation to the proportional hazards assumptions for any of the base models indicated by a global p<0.05 (p = 0.084 for AFR VISP recurrent stroke, p = 0.059 for AFR composite vascular endpoint, p = 0.320 for EUR VISP recurrent stroke, and p = 0.121 for EUR composite vascular endpoint analyses). GO analysis was performed on the eight genes located closest to the significant loci identi- fied in the survival analyses (PTEN, KLLN, PIK3CB, HERC2, CTNNA2, ZDHHC6, STRIP1, and NDUFB6). Nine terms describing biological processes and molecular function met a mod- est significance threshold (p�1.00e-03), including prepulse inhibition (GO: 0060134; p = 8.22e-06), brain morphogenesis (GO: 0048854, p = 2.87e-05), and axonogenesis (GO: PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 5 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP Table 1. Baseline demographics for VISP participants. AFR EUR VISP Recurrent Stroke Nonrecurrent Stroke pa VISP Recurrent Stroke Nonrecurrent Stroke pa Cases Controls Cases Controls N 28 48 32 72 Age, yrsb 65.18 (11.54) 60.40 (8.94) 0.047 70.03 (11.48) 68.01 (10.81) 0.391 Sex Male (%) 17 (60.7) 29 (60.4) 1.000 18 (56.2) 39 (54.2) 1.000 Female (%) 11 (39.3) 19 (39.6) 14 (43.8) 33 (45.8) Treatment Arm High-dose (%) 14 (50.0) 22 (45.8) 0.910 14 (43.8) 40 (55.6) 0.368 Low-dose (%) 14 (50.0) 26 (54.2) 18 (56.2) 32 (44.4) Current Smoker (%) 8 (28.6) 13 (27.1) 1.000 3 (9.4) 9 (12.5) 0.898 Body Mass Index, kg/m2 29.01 (5.45) 30.35 (6.65) 0.370 29.64 (7.21) 28.52 (5.68) 0.403 Diabetes mellitus (%) 9 (32.1) 20 (41.7) 0.562 13 (40.6) 21 (29.2) 0.356 Myocardial infarction (%) 3 (10.7) 1 (2.1) 0.274 0 (0.0) 4 (5.6) 0.419 Recurrent stroke ever (%) 28 (100.0) 16 (33.3) <0.001 32 (100.0) 15 (20.8) <0.001 PNS 0 12 (44.4) 32 (66.7) 0.139 19 (61.3) 57 (79.2) 0.212 1 8 (29.6) 13 (27.1) 6 (19.4) 10 (13.9) 2 4 (14.8) 2 (4.2) 5 (16.1) 4 (5.6) 3 2 (7.4) 1 (2.1) 1 (3.2) 1 (1.4) 4 1 (3.7) 0 (0.0) 0 (0.0) 0 (0.0) RSS 0 2 (7.1) 7 (14.6) 0.001 4 (12.5) 17 (23.6) 0.071 1 16 (57.1) 20 (41.7) 10 (31.2) 34 (47.2) 2 0 (0.0) 16 (33.3) 14 (43.8) 16 (22.2) 3 10 (35.7) 5 (10.4) 4 (12.5) 5 (6.9) Hypertension (%) 24 (85.7) 42 (87.5) 1.000 28 (87.5) 56 (77.8) 0.373 Systolic Blood Pressure, mmHg 144.34 (20.43) 143.65 (20.16) 0.886 146.77 (19.29) 140.11 (19.67) 0.112 Diastolic Blood Pressure, mmHg 80.09 (9.89) 82.05 (10.12) 0.414 81.34 (8.67) 78.14 (9.81) 0.115 Creatinine, mg/dL 1.16 (0.39) 1.41 (0.68) 0.076 1.26 (0.55) 1.11 (1.00) 0.44 Total cholesterol, mg/dL 190.09 (53.10) 212.85 (55.33) 0.107 206.48 (37.67) 201.93 (48.04) 0.641 High-density lipoprotein, mg/dL 42.17 (10.76) 51.76 (16.99) 0.016 45.17 (13.78) 46.59 (16.10) 0.676 Triglycerides, mg/dL 146.95 (105.06) 162.46 (138.70) 0.65 193.83 (110.43) 201.89 (193.05) 0.834 Total plasma homocysteine, 15.20 (5.16) 14.95 (6.62) 0.862 15.58 (6.85) 16.82 (12.82) 0.607 μmol/L B6, nmol/L 36.08 (33.36) 22.33 (20.91) 0.042 29.99 (19.26) 43.03 (50.34) 0.18 B12, pmol/L 421.63 (226.23) 418.30 (190.21) 0.947 354.97 (132.03) 313.50 (124.92) 0.144 Folate, ng/mL 31.92 (32.59) 21.85 (9.26) 0.057 20.84 (10.59) 24.65 (11.25) 0.122 C-reactive protein, mg/L 12.49 (8.92) 12.64 (10.26) 0.953 13.50 (8.41) 11.37 (8.92) 0.277 Prothrombin fragments 1+2, 0.99 (0.56) 1.25 (1.42) 0.386 1.14 (0.63) 1.14 (1.28) 0.996 nmol/L Thrombin-antithrombin 9.11 (12.52) 5.26 (3.49) 0.099 6.47 (8.94) 6.42 (6.73) 0.98 complex, μg/L Thrombomodulin, ng/mL 10.33 (18.47) 11.74 (25.00) 0.84 10.66 (22.63) 8.34 (12.82) 0.588 Tissue plasminogen activator, ng/ 15.42 (10.61) 15.78 (19.58) 0.933 14.32 (15.14) 18.07 (21.12) 0.419 mL (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 6 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP Table 1. (Continued) AFR EUR VISP Recurrent Stroke Nonrecurrent Stroke pa VISP Recurrent Stroke Nonrecurrent Stroke pa Cases Controls Cases Controls von Willebrand Factor, IU/L 1551.15 (747.11) 1222.32 (616.71) 0.053 1284.78 (652.09) 1315.66 (874.32) 0.871 a p-value calculated using t-tests and χ2 for continuous and categorical traits, respectively. b Continuous traits described as mean (SD). Categorical traits described as N (%). Abbreviations: AFR- African descent stratum; EUR- European descent stratum; PNS- number of strokes prior to VISP enrollment; RSS- modified Rankin stroke scale. https://doi.org/10.1371/journal.pone.0254562.t001 0007409, p = 6.94e-04) (S2 Table). To further elucidate any biological implications of our find- ings, we performed functional annotation and mapping of the 57 unique genes with suggestive (p<1.00E-06) CpG loci from the Cox PH results on AFR VISP recurrent stroke and composite vascular endpoint analyses. Tissue analysis on 53 specific types from the GTEx project, revealed statistically significant differential down-regulated expression in the pancreas (padj = 3.05e-08), putamen basal ganglia (padj = 2.42e-06), left ventricle of the heart (padj = 1.54e-05), liver (padj = 8.75e-05), amygdala (padj = 1.50e-04), caudate basal ganglia (padj = 2.30e-04), hip- pocampus (padj = 3.52e-04), nucleus accumbens basal ganglia (padj = 9.30e-04), anterior cingu- late cortex BA24 (padj = 5.93e-04), substantia nigra (padj = 6.02e-03), skeletal muscle (padj = 2.72e-02), whole blood (padj = 2.91e-02), hypothalamus (padj = 2.93e-02), and the cortex (padj = 3.89e-02) (Fig 5, Table 4), when compared to the background gene set from GTEx v8. To evaluate comprehensive networks of methylation loci associated with stroke related clin- ical traits, WGCNA was performed in the two ethnic strata upon outlier removal, which resulted in 100 EUR and 73 AFR participants included. Twenty-seven traits, including baseline biomarker levels, stroke risk factors, and outcome statuses were included in the analyses (Table 1; S1 Table). In the AFR stratum, 106 modules were produced, of which six were observed in significant module-clinical trait associations (p�3.93e-09; Table 5). The significant associations observed were between modules and the previous number of strokes experienced before VISP enrollment (r = -0.90; 5.00e-27), prothrombin fragments 1 + 2 (r = -0.79; p = 8.00e-17), thrombomodulin (r = -0.77; 1.00e-15), thrombin-antithrombin complex (r = -0.73; p = 3.00e-13), triglyceride levels (r = -0.71; p = 2.00e-12), and tissue plasminogen activa- tor (r = -0.68; p = 3.00e-11). Additional module associations in AFR were detected with traits and outcomes including MI, hypertension, thrombomodulin, and thrombin-antithrombin complex. In the EUR analysis, 27 modules were produced; however, no module-trait associa- tions observed met the significance threshold. Notable nominal associations in this stratum included modules correlated with composite vascular endpoint, VISP recurrent stroke, and Table 2. Significant methylation loci associated with time to VISP recurrent stroke in AFR (p�1.06e-07). a Discovery Cohort: VISP Replication Locus CHR:BP Locus Location Gene Mean Beta Value HR (95% CI) P P cg04059318 10:89622526 TSS1500; Exon 1 PTEN; KLLN 0.07 7.19 (3.55–14.57) 4.52E-08 0.665 ch.2.81927627R 2:82074116 Downstream CTNNA2 0.09 2.72 (1.89–3.93) 9.11E-08 0.178 cg03584380 10:114206433 Intron 1 ZDHHC6 0.06 5.41 (2.91–10.06) 9.66E-08 0.004 cg24875416 1:110577936 Intron 1 STRIP1 0.08 2.43 (1.75–3.37) 9.82E-08 0.784 a Discovery model adjusts for age, sex, top 10 genetic principal components, treatment arm, batch effect, and estimated cellular proportions. Abbreviations: CHR- chromosome; BP-base position (hg19); TSS1500- covers from 200 to 1500 nucleotides upstream of transcriptional start site (TSS); HR-hazard ratio; CI- confidence interval. https://doi.org/10.1371/journal.pone.0254562.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 7 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP Table 3. Significant methylation loci associated with time to VISP composite vascular endpoint (p�1.06e-07). a Discovery Cohort: VISP Replication Strata Locus CHR:BP Locus Location Gene Mean Beta Value HR (95% CI) P P AFR cg00076998 3:138553170 Upstream PIK3CB 0.06 5.58 (2.98–10.44) 7.87E-08 0.608 cg16758041 9:32573371 TSS200 NDUFB6 0.07 3.44 (2.18–5.43) 1.04E-07 0.738 EUR cg02365967 15:28473380 Exon 35 HERC2 0.94 0.42 (0.31–0.58) 8.08E-08 0.163 a Discovery model adjusts for age, sex, genetic principal components (first 4 in EUR; first 10 in AFR), treatment arm, batch effect, and estimated cellular proportions. Abbreviations: CHR- chromosome; BP-base position (hg19); TSS200- region from the transcriptional start site (TSS) to 200 nucleotides upstream of TSS; HR-hazard ratio; CI- confidence interval. https://doi.org/10.1371/journal.pone.0254562.t003 total plasma homocysteine. Only two loci identified in the Cox PH models were included in any of the modules. While identified in the AFR survival analyses, cg04059318 and cg24875416 were included in the turquoise module for EUR. Interestingly, this was one of two modules nominally associated with VISP recurrent stroke or composite vascular endpoint (r = 0.43, p = 8.00e-06 and r = 0.44, p = 5.00e-06, respectively) (Table 5). Discussion To the best of our knowledge, this study represents the first epigenome-wide association study evaluating association between time to recurrent stroke (or composite vascular event) and the degree of methylation. This study was performed in a subset of the VISP clinical trial Fig 1. Epigenome-wide Manhattan plot for time (days) to VISP recurrent stroke survival analysis in AFR. Each point corresponds to the -log10(P-value) for a CpG site at its specific chromosome location (y-axis). Horizontal line is indicative of epigenome-wide significance threshold (p� 1.06e-07). https://doi.org/10.1371/journal.pone.0254562.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 8 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP Fig 2. ENCODE annotation of cg03584380 on intron 1 of ZDHHC6. The annotation for cg03584380 includes CpG islands, cell line chromatin state (ChromHMM), H3K27Ac marks, and cell line methylation at CpG sites on the Methyl450 Bead Arrays from ENCODE/Hudson Alpha Institute for Biotechnology (ENCODE/HAIB; bright blue, purple, and orange CpGs correspond to unmethylation, partially methylated, and methylated states, respectively). https://doi.org/10.1371/journal.pone.0254562.g002 participants, providing a diverse cohort of individuals of both AFR and EUR descent. The inclusion of individuals of AFR descent is a strength of this study, since this population is 60% more likely to experience a recurrent stroke within two years compared to individuals of EUR descent, albeit likely mediated by stroke risk factors and comorbidities [24]. Seven loci reached Fig 3. Epigenome-wide Manhattan plot for AFR time to composite vascular event. Horizontal line is indicative of epigenome-wide significance threshold (p� 1.06e-07). https://doi.org/10.1371/journal.pone.0254562.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 9 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP Fig 4. Epigenome-wide Manhattan plot for EUR time to composite vascular event. Horizontal line is indicative of epigenome-wide significance threshold (p� 1.06e-07). https://doi.org/10.1371/journal.pone.0254562.g004 or exceeded a Bonferroni corrected significance threshold, with cg03584380 statistically signif- icant in AFR stroke recurrence and validated upon replication in the Vall d’Hebron University Hospital cohort. While similar trends were observed across phenotypes due to the strong cor- relation between the primary (VISP recurrent stroke) and secondary (composite vascular) out- comes of interest at these loci, results were not significant across separate cohorts, suggesting potential ethnic disparities, as demonstrated by the significant association between Fig 5. Differentially expressed down-regulated genes from FUMA. Red bars are indicative of statistically significant differentially expressed tissues based on Benjamini-Hochberg adjustment. Significance threshold padj<0.05. https://doi.org/10.1371/journal.pone.0254562.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 10 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP Table 4. Differentially expressed genes from FUMA using GTEx version 8 tissue expression. General Tissue Type Specific Tissue Type from GTEx N genesa N overlapb pc padjd Pancreas Pancreas 9668 37 5.64E-10 3.05E-08 Brain Putamen basal ganglia 7853 31 4.49E-08 2.42E-06 Heart Left ventricle 9531 33 2.85E-07 1.54E-05 Liver Liver 8059 29 1.62E-06 8.75E-05 Brain Amygdala 7751 28 2.77E-06 1.50E-04 Brain Caudate basal ganglia 6927 26 4.26E-06 2.30E-04 Brain Hippocampus 7571 27 6.53E-06 3.52E-04 Brain Nucleus accumbens basal ganglia 6479 24 1.72E-05 9.30E-04 Brain Anterior cingulate cortex BA24 6725 23 1.10E-04 5.93E-03 Brain Substantia nigra 7223 24 1.11E-04 6.02E-03 Muscle Skeletal 6908 22 5.03E-04 2.72E-02 Blood Whole blood 6940 22 5.38E-04 2.91E-02 Brain Hypothalamus 5967 20 5.43E-04 2.93E-02 a The number of background genes expressed in specific tissue from GTEx version 8. b The number of overlap occurring between input genes and background. c Unadjusted p-value from the hypergeometric test performed in FUMA. d Benjamini-Hochberg adjusted p-value. https://doi.org/10.1371/journal.pone.0254562.t004 cg03584380 and stroke recurrence in the AFR and Spanish cohorts, but not in EUR (p = 4.07e- 01). Within the AFR stratum, an increase in the beta values at the six significant loci were asso- ciated with shorter duration to recurrent stroke or composite vascular event, thus suggesting Table 5. Significant WGCNA module-trait associations. Strata Module Trait r pa AFR bisque4 Previous number of strokes -0.90 5.00E-27 AFR coral2 Prothrombin fragments 1 + 2 -0.79 8.00E-17 AFR pink4 Thrombomodulin -0.77 1.00E-15 AFR coral Thrombin-antithrombin complex -0.73 3.00E-13 AFR yellowgreen Triglycerides -0.71 2.00E-12 AFR lightsteelblue Tissue plasminogen activator -0.68 3.00E-11 AFR darkturquiose Myocardial infarction 0.51 4.00E-06 EUR turquoise Composite endpoint 0.44 5.00E-06 EUR turquoise VISP recurrent stroke 0.43 8.00E-06 AFR honeydew1 Myocardial infarction 0.49 9.00E-06 AFR blue2 Myocardial infarction -0.49 1.00E-05 AFR coral1 Myocardial infarction -0.48 2.00E-05 AFR lightcyan1 Myocardial infarction -0.48 2.00E-05 AFR lightslateblue Myocardial infarction 0.48 2.00E-05 AFR coral Thrombomodulin -0.43 1.00E-04 AFR mediumpurple2 Thrombin-antithrombin complex -0.44 1.00E-04 AFR royalblue Myocardial infarction -0.42 3.00E-04 EUR cyan Composite endpoint 0.34 5.00E-04 AFR thistle3 Hypertension 0.39 7.00E-04 EUR cyan Total plasma homocysteine 0.33 8.00E-04 a Statisitical significance (bold): p� 3.93e-09. https://doi.org/10.1371/journal.pone.0254562.t005 PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 11 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP that increased methylation at these loci could be indicative of earlier event recurrence. Three of these loci have implications in the phosphatidylinositol pathway, while the remaining three loci were located in genes or gene families linked to cardiovascular traits including the patho- genesis of cardiac disease and diabetes [25], regulation of hyperlipidemia and arteriosclerosis [26], and insulin sensitivity [27]. cg03584380 was the only locus replicated in our analyses. This methylation site is located in the first intron of ZDHHC6, the gene which encodes palmitoyltransferase ZDHHC6 [28] and mediates the palmitoylation of critical endoplasmic reticulum (ER) proteins, including cal- nexin and the inositol 1,4,5-trisphosphate receptor (ITPR1) [29]. Calnexin is an ER chaperone that has been implicated in cardiomyocyte viability and in ER stress, a prominent clinical fea- ture of cardiovascular disease [29], while ITPR1 mediates the influx and release of intracellular Ca2+ and is regulated by the palmitoylation cascade of ZDHHC16/ZDHHC6 [30, 31]. Addi- tionally, ITPR1 can be phosphorylated by Akt kinase and further regulated by phosphatidyli- nositol 3-kinase (PI3K) [32]. Results from the ENCODE annotation suggest this region around cg03584380 is an active regulatory site. There are several active promoters for both the ZDHHC6 and VTI1A genes as indicated by the red bars in Fig 2 in the chromatin state seg- mentation by the Hidden Markov Model (HMM) track, as well as a CpG island just down- stream of this locus. Furthermore, there is evidence of increased H3K27 (lysine 27 of the H3 histone) acetylation, which also indicates enhanced transcription. Therefore, it is plausible that cg03584380 could regulate chromatin and histone states related to transcription and transcrip- tion factor binding, although future gene expression and functional work is needed. Although the association between cg04059318 and AFR stroke recurrence was not validated in the EUR only analysis (p = 7.34e-02) or look-up efforts (p = 6.65e-01), it was the most statis- tically significant association detected in the VISP survival analysis. cg04059318 is located within the 5’ untranslated region of PTEN and within an intron of KLLN. KLLN encodes KILLIN, a DNA-binding protein that inhibits DNA synthesis and mediates p53/TP53-induced apoptosis [28]. PTEN encodes PTEN, a tumor suppressor that regulates angiogenesis [33] and has been associated with a number of stroke-related clinical traits including triglycerides [34] and type 2 diabetes [35]. PTEN negatively regulates the Akt signaling pathway through intra- cellular phosphatidylinositol 3-phosphate (PI3P), which is of interest due to the neuroprotec- tive properties of Akt against ischemia-induced damage [36, 37]. A third locus involved in the phosphatidylinositol pathway was identified in the composite vascular event analysis. cg00076998 is located upstream of PIK3CB, which encodes the catalytic subunit of phosphati- dylinositol 3-kinase, beta (PI3Kβ). PI3K activates cellular signaling cascades through the gen- eration of phosphatidylinositol (3,4,5)-trisphosphate and recruitment of Akt and phosphoinositide-dependent kinase-1 [38]. PI3K is involved in platelet activation signaling triggered by G-protein coupled receptors [28] and is required for platelet-induced aggregation induced by thrombin and thromboxane A2 (TxA2) [39]. PI3K/Akt signaling has been observed in regulation of vascular tone, or the degree of vasoconstriction experienced by a blood vessel, in both vascular endothelium and smooth muscle cells [40]. Exposure to homo- cysteine, a well-documented risk factor for atherosclerosis and stroke [41, 42], in endothelial cells can form the intermediate S-nitroso-homocysteine, a vasodilator implicated in reducing vascular tone and altering arteriole calcium levels [40] Collectively, these associations have identified a novel association with ZDHHC6 and implicate PI3K/Akt signaling in time to stroke and/or vascular event following stroke [43, 44]. These findings could in part, help explain the ethnic disparities in stroke severity and recovery seen in African American patients; however, further functional analyses are needed to confirm the biological significance of our results. PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 12 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP cg02365967 was the only locus identified in our EUR cohort (composite vascular event; AFR composite p = 2.54e-01), and was the sole locus that exhibited hypermethylated beta val- ues (mean β = 0.9411). Increased beta values at this locus were associated with longer duration to event, which differed from the other identified loci. cg02365967 is located in exon 35 of HERC2, which encodes the ubiquitin-protein ligase HERC2. This protein regulates ubiquitin- dependent retention of repair proteins and is a binding protein with SIRT1, a NAD-dependent protein deacetylase that plays a vasoprotective role in endothelial cells [45]. Increased levels of SIRT1 have been described to promote endothelial angiogenesis, enhance vasodilation, and suppress vascular inflammation [45]. Overexpression of SIRT1 in murine endothelial cells pre- vented hypertension and adverse arterial remodeling; however, a knockdown of HERC2 abol- ished any beneficial effects of SIRT1 [45], suggesting a neuroprotective regulatory role of HERC2. Network analyses using WGCNA, identified six statistically significant associations between modules and clinical traits in the AFR stratum, including a correlation between a module com- prised of 58 methylation loci within 38 genes and the previous number of strokes prior to VISP enrollment variable. Of these genes, A2ML1, AGGF1, CBS, ECE1, GABRB3, GALNT2, MRPS6/SLC5A3, and SPG7 had documented associations with hypertension, ischemic stroke and methionine metabolism, atherosclerosis, and early-onset MI [46–48]. GO term enrich- ment of the genes identified in survival analyses, resulted in terms associated with prepulse inhibition (GO: 0060134), brain morphogenesis (GO: 0048854), axonogenesis (GO: 0007409), and postsynaptic assembly, organization, and regulation (GO: 0098698, GO: 2000463, GO: 0098815, and GO: 0099084). Prepulse inhibition (PPI) describes the regulated transmission of sensory information, while disrupted PPI has been linked to neurological disorders including Tourette’s syndrome and Schizophrenia [49]. Brain morphogenesis generates and organizes anatomical structures of the brain, which can be a crucial process after ischemia. Axonogen- esis, or the de novo generation of a neuron’s long process [50], requires the melatonin MT2 receptor [51]. Deficits in MT2 signaling have been observed in a number of neurological disor- ders, including Alzheimer’s disease, suggesting that axonogenesis may be beneficial in regard to the outcome of these disorders [51]. The GO results, in addition to the differential gene expression analysis in GTEx tissues, provide evidence of altered cerebro- and cardiovascular regulation. Recurrent stroke is a vastly understudied phenotype and its etiology is not well understood. The utilization of this phenotype is a strength of this study; however, one limitation is the lack of formally adjudicated ischemic stroke subtype. We can conclude that due to the inclusion and exclusion criteria of VISP, the enrollment strokes were most likely lacunar or small vessel infarctions. DNA methylation profiles were generated from whole blood samples. Although not optimal due to cellular heterogeneity, whole blood provides a valuable resource that is potentially available for replication studies and represents a minimally invasive source for potential biomarker testing. To overcome this limitation, cellular proportions were calculated in silico, and used as covariates in the Cox PH models. Although our analyses detected seven statistically significant loci and replicated cg03584380 in the Vall d’Hebron University Hospital cohort, one constraint of this study that could not be fully addressed was the limited statistical power due to the modest sample sizes of both the discovery cohort and replication cohort. Fur- thermore, previous studies have shown that global methylation patterns differ across ethnici- ties, as well as within ethnic subgroups. In a study of 573 individuals from diverse Latino ethnic sub-groups, genetic ancestry explained approximately 75% of the variation in methyla- tion between the sub-groups [52]. Therefore, the use of cohorts from different races or ethnici- ties should allow for the identification of methylation sites with global implications (affecting most ethnicities similarly) but will limit our ability to identify race/ethnicity-specific PLOS ONE | https://doi.org/10.1371/journal.pone.0254562 July 12, 2021 13 / 17 PLOS ONE DNA methylation analyses identify an intronic locus associated with time to recurrent stroke in VISP associations. In conclusion, findings from this study provide insight into the relationship between the degree of DNA methylation and the duration to recurrent stroke and vascular event following a stroke and lay the foundation for further studies investigating these out- comes in diverse populations. Supporting information S1 Table. List of blood biomarkers and clinical traits used in WGCNA. (DOCX) S2 Table. Gene ontology results from significant methylation loci. (DOCX) Acknowledgments The authors would like to thank the individuals who participated in the VISP study. Co-author Michele M. Sale passed away before the submission of the final version of this manuscript. The corresponding author (KLK) accepts responsibility for the integrity and validity of the data collected and analyzed. Author Contributions Conceptualization: Nicole M. Davis Armstrong, Stephen R. Williams, Michèle M. Sale, Brad- ford B. Worrall, Keith L. Keene. Data curation: Wei-Min Chen, Fang-Chi Hsu, Michèle M. Sale. Formal analysis: Nicole M. Davis Armstrong, Fang-Chi Hsu, Michael S. Brewer, Natalia Cul- lell, Keith L. Keene. Funding acquisition: Michèle M. Sale, Bradford B. Worrall, Keith L. Keene. Methodology: Nicole M. Davis Armstrong, Wei-Min Chen, Fang-Chi Hsu, Michael S. Brewer, Stephen R. Williams, Bradford B. Worrall, Keith L. Keene. Resources: Michèle M. Sale, Bradford B. Worrall, Keith L. Keene. Supervision: Keith L. Keene. 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