Prodromal Elevation of DNA Damage and Cell Cycle Biomarkers HRAS and CHEK2 in Amyotrophic Lateral Sclerosis

Prodromal Elevation of DNA Damage and Cell Cycle Biomarkers HRAS and CHEK2 in Amyotrophic Lateral Sclerosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prodromal Elevation of DNA Damage and Cell Cycle Biomarkers HRAS and CHEK2 in Amyotrophic Lateral Sclerosis Steven Lehrer, Peter Rheinstein This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8328706/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Pre-symptomatic biomarkers that reflect upstream pathogenic processes in amyotrophic lateral sclerosis (ALS) are urgently needed to enable earlier diagnosis and trial enrichment. Methods: Using longitudinal plasma proteomics from the UK Biobank Pharma Proteomics Project, we analyzed pre-diagnostic samples from 23 individuals who later developed motor neuron disease (MND). Mixed-effects regression models evaluated protein trajectories relative to years before diagnosis. Results: As expected, neurofilament light (NfL) demonstrated a progressive rise approaching clinical onset. Exploratory proteome-wide analysis identified two additional markers—CHEK2, a DNA damage–response kinase, and HRAS, a regulator of MAPK/ERK cell-cycle signaling—that showed linear elevation beginning up to 10 years before diagnosis. Genetic analyses revealed no germline association between CHEK2 or HRAS variants and ALS risk, suggesting state-dependent biological activation rather than inherited susceptibility. Conclusion: These findings provide early human evidence that DNA damage–response and cell-cycle pathways are systemically activated during the ALS prodrome; they precede structural neuroaxonal injury marked by NfL, CHEK2 and HRAS, and merit further evaluation as components of multi-marker risk models for early disease detection and therapeutic stratification. Molecular Epidemiology ALS biomarkers prodromal neurodegeneration DNA damage response CHEK2 HRAS signaling plasma proteomics Figures Figure 1 Introduction Therapeutic intervention in amyotrophic lateral sclerosis (ALS) remains profoundly constrained by the prolonged and clinically silent interval between molecular disease initiation and symptomatic diagnosis. By the time weakness and upper or lower motor neuron signs become apparent, a substantial fraction of vulnerable motor neurons has already undergone irreversible degeneration. This temporal disconnect between disease onset and clinical recognition has contributed to the repeated failure of otherwise rational therapeutic strategies when deployed too late in the disease course. Consequently, there is intense interest in identifying biomarkers that reflect early pathogenic activity, rather than late-stage structural injury. Neurofilament light (NfL) has emerged as the most robust and reproducible circulating biomarker of ALS to date and is now widely used for diagnosis, prognosis, and pharmacodynamic monitoring (1-3). However, NfL reflects established neuroaxonal destruction, rising sharply only after the neurodegenerative cascade is well underway. While invaluable for tracking disease progression, NfL provides limited insight into the upstream molecular events that initiate neuronal vulnerability or drive early disease propagation. The lack of validated biomarkers that capture these earliest pathogenic processes remains a critical barrier to early intervention, preventive trial design, and biologically informed patient stratification. Converging experimental evidence indicates that DNA damage accumulation, impaired DNA repair, and aberrant cell-cycle re-entry are central features of motor neuron degeneration (4). Motor neurons exhibit unusually high metabolic demand, transcriptional burden, and oxidative stress, rendering them particularly susceptible to genotoxic injury (5). In familial ALS models, including C9orf72, SOD1, and TDP-43 systems, activation of the DNA damage response (DDR) precedes overt neuronal loss, and failure of checkpoint signaling accelerates neurodegeneration (6, 7). Similarly, inappropriate reactivation of cell-cycle signaling in post-mitotic motor neurons is a well-established trigger of apoptotic death, representing a maladaptive attempt at proliferation in terminally differentiated cells. Despite the compelling mechanistic evidence linking DDR and cell-cycle dysregulation to ALS pathogenesis, it remains unknown whether these processes are systemically detectable in humans during the clinically silent prodromal phase. Most biomarker studies have focused on cross-sectional sampling after diagnosis, thereby capturing the consequences rather than the causes of neurodegeneration. Whether circulating markers of genomic stress and proliferative signaling are altered years before symptom onset has remained largely unexplored. Here, we address this gap using unbiased high-throughput plasma proteomics applied to pre-diagnostic samples from the UK Biobank. We report evidence that systemic markers of DNA damage response and cell-cycle regulation—specifically CHEK2 and HRAS—exhibit progressive elevation up to a decade before clinical diagnosis of ALS. These findings suggest that molecular stress signaling is detectable long before structural axonal breakdown, providing early insight into the biological trajectory of human ALS and identifying candidate targets for prodromal risk stratification and early therapeutic intervention. Methods Data were derived from the UK Biobank resource under Application 57245 (SL and PHR). We performed high-throughput proteomic profiling using Olink data to measure the relative concentrations of 2,925 proteins across major biological pathways. We identified 23 incident motor neuron disease cases (ICD-10 G12.2) with samples collected 0–10 years prior to diagnosis (Mean age at recruitment: 64 years; 60% Male). Given that Amyotrophic Lateral Sclerosis (ALS) accounts for >85% of MND cases in this population (8), this cohort is referred to herein as ALS (Motor Neuron Disease Association. MND Statistics . Available at: https://www.mndassociation.org/about-mnd/statistics. Accessed December 2025). We performed linear regression modeling of normalized protein expression (NPX) against time-to-diagnosis to identify markers with significant prodromal trajectories. Statistics were done with R v4.5.2. Results As expected, NfL levels exhibited a characteristic upward trajectory approaching diagnosis (Fig. 1 , right panel), confirming the neurodegenerative phenotype of our cohort. Unbiased screening identified significant prodromal upregulation of HRAS (Fig. 1 , middle panel, a central regulator of cell division ( 9 )) and CHEK2 (Fig. 1 , left panel, a checkpoint kinase activated by DNA damage ( 10 )). Strikingly, both novel markers demonstrated elevation trajectories in the early prodromal window (5–10 years pre-diagnosis). HRAS exhibited the steepest rate of change, rising from a baseline of -0.5 NPX at 8 years pre-diagnosis to > 0.5 NPX at clinical onset. CHEK2 showed a similar, highly linear elevation, suggesting that systemic cellular stress responses are detectable years before overt motor symptom onset. Critically, we queried the UK Biobank Phenome-Wide Association Study (PheWAS) ( 11 ) data and found no significant germline association between HRAS or CHEK2 variants and ALS risk (SAIGE-GENE burden tests). This dissociation between genetics and proteomics suggests that the prodromal elevation of these proteins is not driven by germline risk alleles, but rather represents a functional, state-dependent response to early neurodegenerative stress. Unlike static genetic markers, these functional biomarkers may serve as dynamic readouts of disease activity amenable to therapeutic monitoring. Discussion Our identification of CHEK2 and HRAS is mechanistically consistent with emerging models of ALS pathogenesis. CHEK2 is a pivotal effector of the DNA Damage Response (DDR) ( 10 ). DDR activation has been observed in iPSC-derived motor neurons carrying C9orf72 expansions, where it precedes overt neurotoxicity. The systemic elevation of CHEK2 we observed may reflect a compensatory response to accumulating genotoxic stress in the prodromal window ( 12 ). Similarly, HRAS is a canonical driver of MAPK/ERK signaling and cell proliferation. While typically associated with tumorigenesis, aberrant cell-cycle reentry is a documented driver of motor neuron apoptosis ( 9 ). Our data imply that systemic dysregulation of these proliferative and repair pathways constitutes an early, detectable feature of the ALS prodrome, distinct from the later-stage cytoskeletal breakdown marked by NfL. Moreover, our findings suggest that cell-cycle reentry and DNA damage accumulation—mechanisms long implicated in motor neuron death—are reflected in the plasma proteome during the prodromal phase. While this pilot cohort is small (N = 23), the identification of HRAS and CHEK2 as prodromal biomarkers supports the development of multi-marker risk scores to enrich clinical trials for participants with active, early-stage disease pathology ( 13 ). Several important limitations should be considered when interpreting these findings. First, the sample size of pre-diagnostic ALS cases was modest, reflecting both the low incidence of ALS and the restricted availability of longitudinal pre-symptomatic plasma samples. Although this limitation is inherent to prodromal human ALS research, it reduces statistical power and increases susceptibility to false discovery. Accordingly, these findings should be viewed as exploratory and hypothesis-generating rather than definitive. Second, case identification relied on ICD-based motor neuron disease coding, which introduces phenotypic heterogeneity within the MND spectrum. While most of such cases in UK Biobank represent ALS, diagnostic misclassification cannot be fully excluded. Future studies incorporating detailed clinical subtyping, neurophysiology, and ALS-specific registries will be essential for diagnostic refinement. Third, plasma-based proteomic measurements do not permit direct inference regarding the cellular or tissue origin of CHEK2 or HRAS. Although both proteins are mechanistically implicated in neuronal DNA damage response and aberrant cell-cycle signaling, their circulating levels may also reflect systemic immune activation, endothelial stress, or peripheral tissue turnover. Direct linkage to motor neuron pathology will require integration with cerebrospinal fluid proteomics, neuroimaging, and postmortem tissue studies. Fourth, despite multivariable adjustment, residual confounding cannot be fully excluded, including unmeasured influences related to metabolic status, subclinical malignancy, chronic inflammation, medication use, or sample handling. Additionally, storage duration effects inherent to biobank plasma samples may introduce subtle bias that is difficult to fully eliminate. Fifth, this study remains observational in nature, precluding causal inference. The observed elevations in CHEK2 and HRAS may represent compensatory responses to early neurodegenerative stress rather than primary drivers of ALS pathogenesis. Finally, independent replication in larger, longitudinal cohorts is essential before these markers can be considered for clinical translation. Validation across diverse populations, proteomic platforms, and disease stages will be required to establish robustness, specificity, and predictive utility. In summary, this exploratory longitudinal proteomic analysis provides initial evidence that DNA damage–response and cell-cycle signaling pathways are systemically activated years before the clinical onset of ALS, well in advance of the rise in NfL that marks structural neuroaxonal injury. The prodromal elevation of CHEK2 and HRAS suggests that upstream molecular stress responses may be detectable during a long, previously inaccessible pre-symptomatic window. Although limited by small sample size and the observational nature of the dataset, these findings support a model in which molecular dysfunction precedes, and likely contributes to, later cytoskeletal breakdown and neuronal loss. If replicated in larger cohorts, CHEK2 and HRAS could form part of multi-marker biomarker panels that enable earlier biological stratification, facilitate prodromal therapeutic interventions, and improve the design of ALS platform trials. Further mechanistic and longitudinal studies are warranted to determine the specificity, predictive value, and translational utility of these early-stage biomarkers. Declarations Funding sources: none Conflicts of interest: The authors declare that they have no competing interests. This research was conducted using data from the UK Biobank under application number 57245. UK Biobank has received ethical approval from the North West Multi-Centre Research Ethics Committee (MREC) (Ref: 11/NW/0382). All participants provided written informed consent. The present study was conducted in accordance with the Declaration of Helsinki and approved under the material transfer agreement of UK Biobank. 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Nat Neurosci 20(9):1225–1235. 10.1038/nn.4604 Paganoni S, Berry JD, Quintana M, Macklin E, Saville BR, Detry MA, Chase M, Sherman AV, Yu H, Drake K, Andrews J, Shefner J, Chibnik LB, Vestrucci M, Cudkowicz ME, Healey ALSPTSG (2022) Adaptive platform trials to transform amyotrophic lateral sclerosis therapy development. Ann Neurol 91(2):165–175. 10.1002/ana.26285 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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16:31:21","extension":"html","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":56413,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8328706/v1/8bea61f508aacfba6f25b2af.html"},{"id":97951056,"identity":"c7040016-6901-4a74-b0a0-3d19c6a7aea5","added_by":"auto","created_at":"2025-12-11 07:05:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32463,"visible":true,"origin":"","legend":"\u003cp\u003eMulti-Omic Stratification of Pre-Symptomatic ALS. Longitudinal trajectories of plasma biomarkers in N=23 ALS patients sampled 0–10 years prior to clinical diagnosis. Lines represent linear regression models of Normalized Protein Expression (NPX) versus years remaining until diagnosis; shaded regions indicate 95% confidence intervals. Left: CHEK2 (DNA damage response) shows significant linear elevation (p\u0026lt;0.01). Middle: HRAS (Cell cycle regulation) exhibits a steep upward trajectory, indicating early systemic dysregulation. Right: NfL (Neurofilament Light) serves as the benchmark for neuroaxonal injury. Note the consistent elevation of all three markers as the time to diagnosis approaches zero (right side of x-axis).\u003c/p\u003e","description":"","filename":"46613b7e2fb14da7b7d41ae5457a125c.png","url":"https://assets-eu.researchsquare.com/files/rs-8328706/v1/7faa64b7d36085235c78aa5e.png"},{"id":98443775,"identity":"1b6abcf9-25f2-44fc-8c48-bde2bb7af051","added_by":"auto","created_at":"2025-12-17 17:14:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":275162,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8328706/v1/819850ea-d4e7-4f72-bcb4-add1d7c1300d.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eProdromal Elevation of DNA Damage and Cell Cycle Biomarkers HRAS and CHEK2 in Amyotrophic Lateral Sclerosis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTherapeutic intervention in amyotrophic lateral sclerosis (ALS) remains profoundly constrained by the prolonged and clinically silent interval between molecular disease initiation and symptomatic diagnosis. By the time weakness and upper or lower motor neuron signs become apparent, a substantial fraction of vulnerable motor neurons has already undergone irreversible degeneration. This temporal disconnect between disease onset and clinical recognition has contributed to the repeated failure of otherwise rational therapeutic strategies when deployed too late in the disease course. Consequently, there is intense interest in identifying biomarkers that reflect early pathogenic activity, rather than late-stage structural injury.\u003c/p\u003e\n\u003cp\u003eNeurofilament light (NfL) has emerged as the most robust and reproducible circulating biomarker of ALS to date and is now widely used for diagnosis, prognosis, and pharmacodynamic monitoring (1-3). However, NfL reflects established neuroaxonal destruction, rising sharply only after the neurodegenerative cascade is well underway. While invaluable for tracking disease progression, NfL provides limited insight into the upstream molecular events that initiate neuronal vulnerability or drive early disease propagation. The lack of validated biomarkers that capture these earliest pathogenic processes remains a critical barrier to early intervention, preventive trial design, and biologically informed patient stratification.\u003c/p\u003e\n\u003cp\u003eConverging experimental evidence indicates that DNA damage accumulation, impaired DNA repair, and aberrant cell-cycle re-entry are central features of motor neuron degeneration (4). Motor neurons exhibit unusually high metabolic demand, transcriptional burden, and oxidative stress, rendering them particularly susceptible to genotoxic injury (5). In familial ALS models, including C9orf72, SOD1, and TDP-43 systems, activation of the DNA damage response (DDR) precedes overt neuronal loss, and failure of checkpoint signaling accelerates neurodegeneration (6, 7). Similarly, inappropriate reactivation of cell-cycle signaling in post-mitotic motor neurons is a well-established trigger of apoptotic death, representing a maladaptive attempt at proliferation in terminally differentiated cells.\u003c/p\u003e\n\u003cp\u003eDespite the compelling mechanistic evidence linking DDR and cell-cycle dysregulation to ALS pathogenesis, it remains unknown whether these processes are systemically detectable in humans during the clinically silent prodromal phase. Most biomarker studies have focused on cross-sectional sampling after diagnosis, thereby capturing the consequences rather than the causes of neurodegeneration. Whether circulating markers of genomic stress and proliferative signaling are altered years before symptom onset has remained largely unexplored.\u003c/p\u003e\n\u003cp\u003eHere, we address this gap using unbiased high-throughput plasma proteomics applied to pre-diagnostic samples from the UK Biobank. We report evidence that systemic markers of DNA damage response and cell-cycle regulation—specifically CHEK2 and HRAS—exhibit progressive elevation up to a decade before clinical diagnosis of ALS. These findings suggest that molecular stress signaling is detectable long before structural axonal breakdown, providing early insight into the biological trajectory of human ALS and identifying candidate targets for prodromal risk stratification and early therapeutic intervention.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eData were derived from the UK Biobank resource under Application 57245 (SL and PHR). We performed high-throughput proteomic profiling using Olink data to measure the relative concentrations of 2,925 proteins across major biological pathways. We identified 23 incident motor neuron disease cases (ICD-10 G12.2) with samples collected 0\u0026ndash;10 years prior to diagnosis (Mean age at recruitment: 64 years; 60% Male). Given that Amyotrophic Lateral Sclerosis (ALS) accounts for \u0026gt;85% of MND cases in this population (8), this cohort is referred to herein as ALS (Motor Neuron Disease Association. \u003cem\u003eMND Statistics\u003c/em\u003e. Available at: https://www.mndassociation.org/about-mnd/statistics. Accessed December 2025). We performed linear regression modeling of normalized protein expression (NPX) against time-to-diagnosis to identify markers with significant prodromal trajectories. Statistics were done with R v4.5.2.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAs expected, NfL levels exhibited a characteristic upward trajectory approaching diagnosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, right panel), confirming the neurodegenerative phenotype of our cohort. Unbiased screening identified significant prodromal upregulation of HRAS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, middle panel, a central regulator of cell division (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)) and CHEK2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, left panel, a checkpoint kinase activated by DNA damage (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003eStrikingly, both novel markers demonstrated elevation trajectories in the early prodromal window (5\u0026ndash;10 years pre-diagnosis). HRAS exhibited the steepest rate of change, rising from a baseline of -0.5 NPX at 8 years pre-diagnosis to \u0026gt;\u0026thinsp;0.5 NPX at clinical onset. CHEK2 showed a similar, highly linear elevation, suggesting that systemic cellular stress responses are detectable years before overt motor symptom onset.\u003c/p\u003e \u003cp\u003eCritically, we queried the UK Biobank Phenome-Wide Association Study (PheWAS) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) data and found no significant germline association between \u003cem\u003eHRAS\u003c/em\u003e or \u003cem\u003eCHEK2\u003c/em\u003e variants and ALS risk (SAIGE-GENE burden tests). This dissociation between genetics and proteomics suggests that the prodromal elevation of these proteins is not driven by germline risk alleles, but rather represents a functional, state-dependent response to early neurodegenerative stress. Unlike static genetic markers, these functional biomarkers may serve as dynamic readouts of disease activity amenable to therapeutic monitoring.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur identification of CHEK2 and HRAS is mechanistically consistent with emerging models of ALS pathogenesis. CHEK2 is a pivotal effector of the DNA Damage Response (DDR) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). DDR activation has been observed in iPSC-derived motor neurons carrying C9orf72 expansions, where it precedes overt neurotoxicity. The systemic elevation of CHEK2 we observed may reflect a compensatory response to accumulating genotoxic stress in the prodromal window (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, HRAS is a canonical driver of MAPK/ERK signaling and cell proliferation. While typically associated with tumorigenesis, aberrant cell-cycle reentry is a documented driver of motor neuron apoptosis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Our data imply that systemic dysregulation of these proliferative and repair pathways constitutes an early, detectable feature of the ALS prodrome, distinct from the later-stage cytoskeletal breakdown marked by NfL.\u003c/p\u003e \u003cp\u003eMoreover, our findings suggest that cell-cycle reentry and DNA damage accumulation\u0026mdash;mechanisms long implicated in motor neuron death\u0026mdash;are reflected in the plasma proteome during the prodromal phase. While this pilot cohort is small (N\u0026thinsp;=\u0026thinsp;23), the identification of HRAS and CHEK2 as prodromal biomarkers supports the development of multi-marker risk scores to enrich clinical trials for participants with active, early-stage disease pathology (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral important limitations should be considered when interpreting these findings. First, the sample size of pre-diagnostic ALS cases was modest, reflecting both the low incidence of ALS and the restricted availability of longitudinal pre-symptomatic plasma samples. Although this limitation is inherent to prodromal human ALS research, it reduces statistical power and increases susceptibility to false discovery. Accordingly, these findings should be viewed as exploratory and hypothesis-generating rather than definitive.\u003c/p\u003e \u003cp\u003eSecond, case identification relied on ICD-based motor neuron disease coding, which introduces phenotypic heterogeneity within the MND spectrum. While most of such cases in UK Biobank represent ALS, diagnostic misclassification cannot be fully excluded. Future studies incorporating detailed clinical subtyping, neurophysiology, and ALS-specific registries will be essential for diagnostic refinement.\u003c/p\u003e \u003cp\u003eThird, plasma-based proteomic measurements do not permit direct inference regarding the cellular or tissue origin of CHEK2 or HRAS. Although both proteins are mechanistically implicated in neuronal DNA damage response and aberrant cell-cycle signaling, their circulating levels may also reflect systemic immune activation, endothelial stress, or peripheral tissue turnover. Direct linkage to motor neuron pathology will require integration with cerebrospinal fluid proteomics, neuroimaging, and postmortem tissue studies.\u003c/p\u003e \u003cp\u003eFourth, despite multivariable adjustment, residual confounding cannot be fully excluded, including unmeasured influences related to metabolic status, subclinical malignancy, chronic inflammation, medication use, or sample handling. Additionally, storage duration effects inherent to biobank plasma samples may introduce subtle bias that is difficult to fully eliminate.\u003c/p\u003e \u003cp\u003eFifth, this study remains observational in nature, precluding causal inference. The observed elevations in CHEK2 and HRAS may represent compensatory responses to early neurodegenerative stress rather than primary drivers of ALS pathogenesis.\u003c/p\u003e \u003cp\u003eFinally, independent replication in larger, longitudinal cohorts is essential before these markers can be considered for clinical translation. Validation across diverse populations, proteomic platforms, and disease stages will be required to establish robustness, specificity, and predictive utility.\u003c/p\u003e \u003cp\u003eIn summary, this exploratory longitudinal proteomic analysis provides initial evidence that DNA damage\u0026ndash;response and cell-cycle signaling pathways are systemically activated years before the clinical onset of ALS, well in advance of the rise in NfL that marks structural neuroaxonal injury. The prodromal elevation of CHEK2 and HRAS suggests that upstream molecular stress responses may be detectable during a long, previously inaccessible pre-symptomatic window. Although limited by small sample size and the observational nature of the dataset, these findings support a model in which molecular dysfunction precedes, and likely contributes to, later cytoskeletal breakdown and neuronal loss. If replicated in larger cohorts, CHEK2 and HRAS could form part of multi-marker biomarker panels that enable earlier biological stratification, facilitate prodromal therapeutic interventions, and improve the design of ALS platform trials. Further mechanistic and longitudinal studies are warranted to determine the specificity, predictive value, and translational utility of these early-stage biomarkers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding sources:\u003c/h2\u003e \u003cp\u003enone\u003c/p\u003e \u003cp\u003eConflicts of interest: The authors declare that they have no competing interests.\u003c/p\u003e \u003cp\u003eThis research was conducted using data from the UK Biobank under application number 57245. UK Biobank has received ethical approval from the North West Multi-Centre Research Ethics Committee (MREC) (Ref: 11/NW/0382). All participants provided written informed consent. The present study was conducted in accordance with the Declaration of Helsinki and approved under the material transfer agreement of UK Biobank.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun BB, Chiou J, Traylor M, Benner C, Hsu YH, Richardson TG, Surendran P, Mahajan A, Robins C, Vasquez-Grinnell SG, Hou L, Kvikstad EM, Burren OS, Davitte J, Ferber KL, Gillies CE, Hedman AK, Hu S, Lin T, Mikkilineni R, Pendergrass RK, Pickering C, Prins B, Baird D, Chen CY, Ward LD, Deaton AM, Welsh S, Willis CM, Lehner N, Arnold M, Worheide MA, Suhre K, Kastenmuller G, Sethi A, Cule M, Raj A, Alnylam Human G, AstraZeneca Genomics I, Biogen Biobank T, Bristol Myers S, Genentech Human G, GlaxoSmithKline Genomic S, Pfizer Integrative B, Regeneron Genetics S, Burkitt-Gray C, Melamud L, Black E, Fauman MH, Howson EB, Kang JMM, McCarthy HM, Nioi MI, Petrovski P, Scott S, Smith RA, Szalma EN, Waterworth S, Mitnaul DM, Szustakowski LJ, Gibson JD, Miller BW, Whelan MR (2023) Plasma proteomic associations with genetics and health in the uk biobank. 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Ann Neurol 91(2):165\u0026ndash;175. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ana.26285\u003c/span\u003e\u003cspan address=\"10.1002/ana.26285\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Icahn School of Medicine at Mount Sinai","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ALS biomarkers, prodromal neurodegeneration, DNA damage response, CHEK2 HRAS signaling, plasma proteomics","lastPublishedDoi":"10.21203/rs.3.rs-8328706/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8328706/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Pre-symptomatic biomarkers that reflect upstream pathogenic processes in amyotrophic lateral sclerosis (ALS) are urgently needed to enable earlier diagnosis and trial enrichment.\u003c/p\u003e\n\u003cp\u003eMethods: Using longitudinal plasma proteomics from the UK Biobank Pharma Proteomics Project, we analyzed pre-diagnostic samples from 23 individuals who later developed motor neuron disease (MND). Mixed-effects regression models evaluated protein trajectories relative to years before diagnosis.\u003c/p\u003e\n\u003cp\u003eResults: As expected, neurofilament light (NfL) demonstrated a progressive rise approaching clinical onset. Exploratory proteome-wide analysis identified two additional markers—CHEK2, a DNA damage–response kinase, and HRAS, a regulator of MAPK/ERK cell-cycle signaling—that showed linear elevation beginning up to 10 years before diagnosis. Genetic analyses revealed no germline association between CHEK2 or HRAS variants and ALS risk, suggesting state-dependent biological activation rather than inherited susceptibility.\u003c/p\u003e\n\u003cp\u003eConclusion: These findings provide early human evidence that DNA damage–response and cell-cycle pathways are systemically activated during the ALS prodrome; they precede structural neuroaxonal injury marked by NfL, CHEK2 and HRAS, and merit further evaluation as components of multi-marker risk models for early disease detection and therapeutic stratification.\u003c/p\u003e","manuscriptTitle":"Prodromal Elevation of DNA Damage and Cell Cycle Biomarkers HRAS and CHEK2 in Amyotrophic Lateral Sclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 07:05:38","doi":"10.21203/rs.3.rs-8328706/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d051c4c-616e-440c-a8d6-32fb81451b2f","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59429869,"name":"Molecular Epidemiology"}],"tags":[],"updatedAt":"2025-12-11T07:05:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 07:05:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8328706","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8328706","identity":"rs-8328706","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}