key: cord-1049677-s51e32bx
authors: Mongelli, A.; gaetano, c.; gottardi zamperla, m.; barbi, v.; atlante, s.; la rovere, m. t.; bachetti, t.; catalano, o.; bussotti, m.; della vecchia, l.; nanni, s.; farsetti, a.; martelli, f.
title: EVIDENCE FOR BIOLOGICAL AGE ACCELERATION AND TELOMERE2 SHORTENING IN COVID-19 SURVIVORS
date: 2021-04-27
journal: nan
DOI: 10.1101/2021.04.23.21255973
sha: 7764502b8b834cbaaaa4697ccd37421caa7bc80b
doc_id: 1049677
cord_uid: s51e32bx

Introduction & Background: the SARS-CoV-2 infection determines the COVID-19 syndrome characterized, in the worst cases, by severe respiratory distress, pulmonary and cardiac fibrosis,inflammatory cytokines release, and immunodepression. This condition has led to the death of about 2.15% of the total infected world population so far. Among survivors, the presence of the so-called post-COVID19 syndrome (PPCS) is a common finding. In patients who survived the SARS-CoV-2 infection, overt PPCS presents one or more symptoms such as fatigue, dyspnea, memory loss, sleep disorders, and difficulty concentrating. The pathophysiology of PPCS is currently poorly understood, and whether epigenetic mechanisms are involved in this process is unexplored. Methods & Results: In this study, a cohort of 117 COVID19 survivors (post-COVID19) and 144 non-infected volunteers (COVID19-free) were analyzed using pyrosequencing of defined CpG islands previously identified as suitable for biological age determination. Besides, telomere length (TL) and ACE2 and DPP4 receptor expression were determined. The results show a consistent biological age increase in the post-covid population (mean 58,44 DS 14,66 ChronoAge Vs. mean 67,18 DS 10,86 BioAge, P<0,0001), determining a DeltaAge acceleration of 10,45 DS 7,29 years (+5.25 years above range of normality) compared to 3,68 DS 8,17 years for the COVID19-free population (P<0,0001). A significant telomere shortening parallels this finding in the post-COVID19 cohort compared to COVID19-free subjects (post-COVID19 TL: 3,03 DS 2,39 Kb vs. COVID19-free: 10,67 DS 11,69 Kb; P<0,0001). Additionally, ACE2 expression was decreased in post-COVID19 patients compare to COVID19-free, while DPP-4 did not change. Conclusion: In light of these observations, we hypothesize that some epigenetic alterations are associated with the post-COVID19 condition, particularly in the younger (<60 years). Although the consequences of such modifications on the long-term clinical outcome remain unclear, they might 46 indicate a direction to investigate the pathophysiological basis of the post-COVID19 syndrome

(1), which recently contributed to defining the so-called persistent post-COVID19 syndrome (PPCS) 60 (1). 61 It has been observed that SARS-CoV-2 infected people who developed the adult respiratory 62 distress syndrome (ARDS) also accumulated an excessive deposition of extracellular matrix in the 63 alveolar compartment, causing pulmonary function's worsening (2). Fibrosis accumulation has also 64 been observed in the cardiovascular and nervous systems. The infection frequently caused increased 65 circulating Troponin T and Brain Natriuretic Peptides, suggesting the presence of myocardium 66 damage with possible activation of a remodeling process (3). Besides, a compromised heart function 67 with reduced contractility has often been observed (4). Other effects were on the vascular system and 68 included altering the coagulation cascade and clotting (5). In this case, the fibrinogen pathway's fast 69 activation enhanced the risk of thrombotic events, leading to acute stroke and pulmonary embolism 70 (6)(5). Interestingly, coagulation problems have been seen in post-COVID survivors and in PPCS 71 patients, in which anticoagulants are routinely prescribed. 72 In some COVID19 survivors, also neurological symptoms are present. They diversify going 73 from the most common and light, such as headache, dizziness, anosmia, and dysgeusia, to the most 74 severe ones, including ataxia and epilepsy. This clinical picture has often been associated with a 75 disrupted blood-brain barrier, possibly due to an intense cytokine storm (7)(8). Chronic headaches 76 and neuropathic pain, often localized in the back and neck, have been frequently reported as part of 77 the PPCS and resistant to standard analgesics (9). 78 Similar to what was observed in 2003 SARS and the Middle East respiratory syndrome 79 (MERS) epidemics, in COVID19 survivors, an increase of psychological disorders that include 80 depression, posttraumatic stress, and anxiety has been observed (10). Similar disorders are often 81 present in the general population exposed to stress determined by sociological factors often linked to 82 economic damage or the sudden loss of some beloved ones (10). Remarkably, during the SARS or 83 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In humans, telomere shortening is associated in vivo with the aging process, and, in vitro, it 114 characterizes the cellular replicative senescence (23). Telomeres possess properties that make them 115 suitable as biomarkers in several diseases or conditions, including cancer, CVDs, or aging (24) (25).

The inverse correlation between telomere length (TL) and chronological age has been used for age 117 prediction (26). Interestingly, among individuals infected by Sars-CoV-2, a reduced TL has been 118 associated with the risk of developing more severe symptoms suggesting that TL at the moment of In recent years, several studies aimed to identify biological or molecular markers of aging that 123 correlate with chronological age and could be helpful to estimate the biological vs. chronological age 124 (28). Some of these parameters have been defined based on modifications of the DNA methylome 125 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. partially overlapping with the post-COVID19 patients (see Table I ) were recruited among the hospital 173 workers and non-COVID patients. Genomic DNA was extracted from the whole blood by a robotized (Table II) (Table II) . Interestingly, the DeltaAge distribution within the two groups showed that (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Telomere length quantification. 226 TL shortening has been reported as a risk factor for developing more severe COVID19 syndrome 227 (27). We investigated this parameter, which is also associated with the progression of the aging (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. were taken, longer than four weeks from the end of the viral infection (see Table I ), ACE2 expression 251 was significantly reduced (Fig. 5A) . The expression level of DPP4 was unchanged (Fig. 5B) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Interestingly, we found that the increased DeltaAge of the post-COVID19 group correlated well with 268 the lowest ACE2 expression level ( Fig. 5C; P<0 .01). No differences were observed in the distribution 269 of DPP4 by DeltaAge (Fig. 5D) .

Discussion. 272 Nowadays, the global vaccination program against the SARS-CoV-2 is actively ongoing, and 273 the incidence of COVID19 will soon decrease sensibly worldwide. Nevertheless, among the millions 274 of COVID19 survivors, many will require long-term assistance due to increased post-COVID19 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The two groups considered in this study were not significantly different in terms of age, sex, 337 and known clinical conditions before SARS-CoV-2 infection except for a relatively higher incidence 338 of BMI>30 (15.3% vs. 9%) in the post-COVID19 population compared to controls as well as for a 339 record of more frequent lung diseases (20.2% vs. 1.6%; see Table I ). The origin of the persistent (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 27, 2021. ; https://doi.org/10.1101/2021.04.23.21255973 doi: medRxiv preprint

A Review of 442 Persistent Post-COVID Syndrome (PPCS)

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No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted

Renin-angiotensin 485 system and cardiovascular functions

COVID-19 and chronological aging: Senolytics and 487 other anti-aging drugs for the treatment or prevention of corona virus infection

Structure of MERS-CoV spike 490 receptor-binding domain complexed with human receptor DPP4

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Recent progress, methods and perspectives in forensic epigenetics

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aging of blood can be 530 tracked by DNA methylation changes at just three CpG sites

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Interferon-Stimulated Genes Correlate to COVID-19 Susceptibility in Vertebrates

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Telomere length quantification. 396 The chromosome end has been quantified by PCR Real-Time of Absolute Human Telomere Length 397 Quantification qPCR Assay Kit (ScienCell, cat. 8918) following the manufacturer's instructions. According to manufacturer instruction, the total RNA has been isolated from whole blood using 401 QIAmp RNA blood mini (Qiagen, cat. 52304) and automatized extractor QIACube. The RNA has 402 been quantified with QIAxpert. 403 cDNA synthesis and qPCR Real-Time.

Omniscript RT kit (Qiagen, cat. 205113) has been used to convert total RNA into cDNA following 405 the manufacturer's instructions.

The qPCR RealTime has been performed on RotorGene 2plex HRM using the RT2 SYBR Green (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted April 27, 2021. ; https://doi.org/10.1101/2021.04.23.21255973 doi: medRxiv preprint