Plasma proteome-wide analysis of cerebral small vessel disease identifies novel biomarkers and disease pathways

Abstract Cerebral small vessel disease (SVD), as defined by neuroimaging characteristics such as white matter hyperintensities (WMHs), cerebral microhemorrhages (CMHs), and lacunar infarcts, is highly prevalent and has been associated with dementia risk and other clinical sequelae. Although conditions such as hypertension are known to contribute to SVD, little is known about the diverse set of subclinical biological processes and molecular mediators that may also influence the development and progression of SVD. To better understand the mechanisms underlying SVD and to identify novel SVD biomarkers, we used a large-scale proteomic platform to relate 4,877 plasma proteins to MRI-defined SVD characteristics within 1,508 participants of the Atherosclerosis Risk in Communities (ARIC) Study cohort. Our proteome-wide analysis of older adults (mean age: 76) identified 13 WMH-associated plasma proteins involved in synaptic function, endothelial integrity, and angiogenesis, two of which remained associated with late-life WMH volume when measured nearly 20 years earlier, during midlife. We replicated the relationship between 9 candidate proteins and WMH volume in one or more external cohorts; we found that 11 of the 13 proteins were associated with risk for future dementia; and we leveraged publicly available proteomic data from brain tissue to demonstrate that a subset of WMH-associated proteins was differentially expressed in the context of cerebral atherosclerosis, pathologically-defined Alzheimer’s disease, and cognitive decline. Bidirectional two-sample Mendelian randomization analyses examined the causal relationships between candidate proteins and WMH volume, while pathway and network analyses identified discrete biological processes (lipid/cholesterol metabolism, NF-kB signaling, hemostasis) associated with distinct forms of SVD. Finally, we synthesized these findings to identify two plasma proteins, oligodendrocyte myelin glycoprotein (OMG) and neuronal pentraxin receptor (NPTXR), as top candidate biomarkers for elevated WMH volume and its clinical manifestations. Competing Interest Statement B.M.P. serves on the Steering Committee of the Yale Open Data Access Project funded by Johnson & Johnson. C.R.J. serves on an independent data monitoring board for Roche, has served as a speaker for Eisai, and consulted for Biogen, but he receives no personal compensation from any commercial entity; he receives research support from NIH, the GHR Foundation and the Alexander Family Alzheimers Disease Research Professorship of the Mayo Clinic. A.J.N. receives research funding from Janssen Pharmaceuticals, Ono Pharma, and GlaxoSmithKline. J.C. (Josef Coresh) is a scientific advisor with personal fees to SomaLogic and Healthy.io. Proteomic assays in ARIC were conducted free of charge as part of a data exchange agreement with Soma Logic. The remaining authors declare no competing interests. Funding Statement The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201700001I, HHSN268201700002I, HHSN268201700003I, HHSN268201700004I, HHSN268201700005I). Neurocognitive data is collected by U01 2U01HL096812, 2U01HL096814, 2U01HL096899, 2U01HL096902, 2U01HL096917 from the NIH (NHLBI, NINDS, NIA and NIDCD), and with previous brain MRI examinations funded by R01HL70825 from the NHLBI. The authors thank the staff and participants of the ARIC study for their important contributions. Funding was also supported by R01HL087641, R01HL059367 and R01HL086694; National Human Genome Research Institute contract U01HG004402; and National Institutes of Health contract HHSN268200625226C. Infrastructure was partly supported by Grant Number UL1RR025005, a component of the National Institutes of Health and NIH Roadmap for Medical Research. SomaLogic Inc. conducted the SomaScan assays in exchange for use of ARIC data. This work was supported in part by NIH/NHLBI grant R01 HL134320. Reagents for GDF15 assays were donated by the Roche Diagnostics Corporation. The Cardiovascular Health Study was supported by contracts HHSN268201200036C, HHSN268200800007C, HHSN268201800001C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, N01HC85086, 75N92021D00006, and grants U01HL080295, U01HL130114, R01HL144483, and R01HL105756 from the National Heart, Lung, and Blood Institute (NHLBI), with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS); additional support was provided by N01HC15103 and R01AG023629 from the National Institute on Aging (NIA). In addition, this work was supported, in part, by the Intramural Research Program of the National Institute on Aging, NIH. M.R.D., G.N.D., Z.P., and K.A.W. are supported by the National Institute on Aging Intramural Research Program. R.F.G. is supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program. A.E.F. is supported by K01AG071689. P.S. was supported by the German Research Foundation (DFG) Project-ID 431984000 CRC 1453 NephGen, Project-ID 192904750 CRC 992 Medical Epigenetics, and by the DFG grant SCHL 2292/1 (Walter Benjamin Fellowship). T.M.H. is supported by the following grants from the National Institutes of Health: P30AG072947, R01AG054069, R01AG058969, RF1NS110043, U01HL096812, R01HL153191, R01AG070867, R01AG071032, R01AG072634, R01AG068629, R01HL158622, R01AG070881, R01AG067557, R21AG075291, and R01AG074971. P.L.L. is supported by NIH/NHLBI K24 HL159246. M.F. was supported in part by NIA grants U01AG052409 and U01AG058589. L.S. is funded by the Virtual Brain Cloud from European commission (grant no. H2020 SC1 DTH 2018 1). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Atherosclerosis Risk in Communities (ARIC) Study: Study protocol were approved by Institutional Review Boards at each participating center: University of North Carolina at Chapel Hill, Chapel Hill, NC; Wake Forest University, Winston-Salem, NC; Johns Hopkins University, Baltimore, MD; University of Minnesota, Minneapolis, MN; and University of Mississippi Medical Center, Jackson, MS. All participants gave written informed consent at each study visit, and proxies provided consent for participants who were judged to lack capacity. Cardiovascular Health Study (CHS): CHS was approved by institutional review committees at each field center and individuals in the present analysis gave informed consent including consent for the study of cardiovascular disease. Baltimore Longitudinal Study of Aging (BLSA): The BLSA protocol has been approved by the Institutional Review Board of the National Institute of Environmental Health Science, National Institutes of Health (03AG0325). Informed consent was obtained from all participants. De-identified data were used for all BLSA analyses. Generation Scotland (GS): Written informed consent was provided by all participants; ethical approval for STRADL was formally obtained from the NHS Tayside committee. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data availability All data generated in this study are either included in this article (and its Supplementary Information), available on reasonable request, or are available in an online public database. Pre-existing data access policies for each of the parent cohort studies (ARIC, CHS, BLSA, GS) specify that research data requests can be submitted to each steering committee; these will be reviewed for confidentiality or intellectual property restrictions and will not unreasonably be refused. Individual level patient or protein data may further be restricted by consent, confidentiality or privacy laws/considerations. These policies apply to both clinical and proteomic data. Tissue-specific gene expression data is available at https://www.gtexportal.org/home/. Tissue-specific protein expression data is available at https://www.proteinatlas.org/. Brain cell-specific gene expression data is available at http://www.brainrnaseq.org/. Brain gene expression date were derived from the Brain eQTL Almanac (BRAINEAC; http://www.braineac.org/) and the Functional Mapping and Annotation of Genome-Wide Association Studies105 (FUMA) platform (https://fuma.ctglab.nl/). eQTL gene enrichment was performed using data from the Molecular signatures (MsigDB; http://www.broadinstitute.org/msigdb) and the NHGRI-EBI Catalog of published genome-wide association studies (GWAS Catalog; https://www.ebi.ac.uk/gwas/). Functional enrichment of protein networks was conducted using the g:Profiler web tool https://biit.cs.ut.ee/gprofiler/gost.