key: cord-0683949-qoke03gq authors: Fallerini, C.; Daga, S.; Benetti, E.; Picchiotti, N.; Zguro, K.; Catapano, F.; Baroni, V.; Lanini, S.; Bucalossi, A.; Marotta, G.; Baldassarri, M.; Fava, F.; Beligni, G.; Di Sarno, L.; Alaverdian, D.; Palmieri, M.; Croci, S.; Isidori, A. M.; Furini, S.; Frullanti, E.; Multicenter Study, G.-C.; Renieri, A.; Mari, F. title: SELP Asp603Asn and severe thrombosis in COVID-19 males: implication for anti P-selectin monoclonal antibodies treatment date: 2021-05-27 journal: nan DOI: 10.1101/2021.05.25.21257803 sha: 4e45d56ae6e06533b736d303e7a46eb80b99f13f doc_id: 683949 cord_uid: qoke03gq Thromboembolism is a frequent cause of severity and mortality in COVID-19. However, the etiology of this phenomenon is not well understood. A cohort of 1,186 subjects, from the GEN-COVID consortium, infected by SARS-CoV-2 with different severity were stratified by sex and adjusted by age. Then, common coding variants from whole exome sequencing were mined by LASSO logistic regression. The homozygosity of the cell adhesion molecule P-selectin gene (SELP) rs6127 (c.1807G>A; p.Asp603Asn) which increases platelet activation is found to be associated with severity in the male subcohort of 513 subjects (Odds Ratio= 2.27, 95% Confidence Interval 1.54-3.36). As the SELP gene is downregulated by testosterone, the odd ratio is increased in males older than 50 (OR 2.42, 95% CI 1.53-3.82). Asn/Asn homozygotes have increased D-dimers values especially when associated with poly Q[≥]23 in the androgen receptor (AR) gene (OR 3.26, 95% CI 1.41-7.52). These results provide a rationale for the repurposing of antibodies against P-selectin as adjuvant therapy in rs6127 male homozygotes especially if older than 50 or with impaired AR gene. a rationale for the repurposing of antibodies against P-selectin as adjuvant therapy in rs6127 male homozygotes especially if older than 50 or with impaired AR gene. Since its onset in early 2020, the global pandemic of coronavirus disease 2019 , caused by SARS-CoV-2, has posed a multitude of challenges and triggered intensive research on the disease and the virus. However, a lot of questions still remain unanswered, especially those regarding its heterogeneity of clinical manifestations and the appropriate therapeutic strategies. It is now widely recognized that COVID-19 is a systemic disease 1 characterized by dysregulation of the immune system and presence of a hypercoagulable state 2 . COVID-19 severe patients show coagulation abnormalities with increased incidence of arterial thrombosis and mortality has been shown to correlate with elevated levels of both interleukin-6 (IL-6) and D-dimer 2, 3 . These values have often been reported accompanied by a relatively modest decrease in platelet count 4 , and prolongation of the prothrombin time 3 . Genetic bases of this prothrombotic susceptibility remain until now elusive, despite the fact that it is evident that phenotypic variability associated with the viral infection is obviously also due to host genetic factors. Some rare variants of genes involved in adaptive immunity have been identified by us and others in Mendelian forms of COVID-19 [5] [6] [7] . Among common genetic factors, the protective role of the zero blood group has been linked to destabilization of the von Willebrand factor and protection from thrombosis 8 . We have shown that longer polyQ repeats (> 23) in the androgen receptor gene (AR) predisposes to severe COVID-19 outcome due to reduced anti-inflammatory and anti-thrombotic effect of testosterone 9 . 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. The testosterone inhibited P-selectin (SELP) gene encodes a cell adhesion molecule that is stored in the alpha-granules of platelets and in Weibel-Palade bodies of endothelial cells. It mediates the interaction of activated platelets on endothelium with leukocytes playing a key role in the thrombotic process 10,11 . A significantly increased P-selectin concentration in plasma samples of severe COVID-19 patients compared to healthy controls, together with other prothrombotic biomarkers has been recently demonstrated 12,13 . Among SELP variants, the Asp603Asn functional polymorphism (rs6127; c.1807G>A) -previously reported as Asp562Asn or Asp541Asn -has been associated with risk of thrombotic disease, tissue damage in diabetes, abortion, as well as myocardial infarction 14-18 . The polymorphism is located within the consensus repeat domain of the SELP protein 19 , and it has been shown to affect the binding of P-selectin to its ligand (PSGL-1) on leukocytes, resulting in a protein that is more efficient at recruiting leukocytes to the endothelium 16 . Here, we applied Ordered Logistic Regression (OLR) analysis on clinical data and LASSO logistic regression on WES data within our prospectively recruited GEN-COVID cohort and identified SELP as a key player for severity and thromboembolism in severe COVID-19. A cohort of 1,186 SARS-CoV-2-infected subjects was recruited during the first pandemic wave within the Italian GEN-COVID Study (https://sites.google.com/dbm.unisi.it/gen-covid) 1 . ORL model was applied to the clinical WHO gradings, stratified by sex and adjusted by age, as described elsewhere 20 . The male subset consists of 513 COVID-19 male patients: 236 severe COVID-19 patients (cases) and 277 SARS-CoV-2 PCR-positive oligo-asymptomatic subjects (controls). WES with at least 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. Within the Italian GEN-COVID cohort, we tested different combination of coding polymorphisms of human genes at homozygous state and found that the Asp603Asn polymorphism of the SELP gene correlate with severity in the subcohort of males (Figure 1 , 1, 20 . The genotypic frequencies of the polymorphism in cases and controls were confirmed to be in Hardy-Weinberg equilibrium; the minor allele frequency in our cohort was similar to that reported in the European (non-Finnish) population in the gnomAD database (16.4% versus 15.9%) (https://gnomad.broadinstitute.org/). We reasoned that the hyper-inflammatory and hyper-thrombotic state, due to viral injury of the vascular endothelium, leads to the release of P-selectin by activated platelets, driving thrombosis and vascular inflammation especially in those individuals with enhanced P-selectin activities due a double copy of Asparagine in position 603 without any other additional coding polymorphisms 16 . These results are in line with the demonstration that SARS-CoV-2 induces thrombosis by binding to ACE2 on platelets and subsequent integrin α IIbβ3 activation and P-selectin expression 21 , and that P-selectin soluble isoform is increased in thrombosis 10,11 and severe COVID-19 12,13 . 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. The association between Asp603Asn polymorphism (rs6127) in homozygous state, in absence of other coding polymorphisms, and severity is quite strong with an OR 2.27 in males (95% CI, 1.54 to 3.36; p-value 2.8x10 -5 , Table 1a ). We reasoned that, since SELP transcription is inhibited by androgens 22 , the strength of the association should increase with age. Indeed, in males aged > 50 years we found an increased OR of 2.42 (95% CI, 1.53 to 3.82; p-value 1.19x10 -4 , Table 1b ). While the significance is, instead, lost in young males (<50 years), (data not shown p= 1.1x10 -1 ). In a subset of 52 severely affected hospitalised males, 4 main laboratory parameters indicating thrombosis (D-dimer, platelets and LDH) and severity (lymphocytes) were longitudinally followed (Figure 1, Panel B-E) . We observed that the maximum pick, over 10 times of the normal value, was exclusive of Asp/Asn and Asn/Asn genotypes and older patients (Figure 1, Panel B-E) . The pick timing was earlier in Asn/Asn (median 7.5 days from infection) than Asp/Asn (median 13.5 days from infection), (p-value = 3x10 -2 , Figure 1 , panel F). As the von Willebrand factor (vWF) is a downstream effector for clotting, the nonzero blood group, associating with more stable vWF, also correlate with higher D-dimer and LDH values (Figure 1, panel G-H) , in agreement with previous reports 8 . Given the higher association of the SELP polymorphism in older males we reasoned that the AR poly-Q status would impact on the SELP genotype 9 : the combination of poly-Q polymorphism ≥ 23 with homozygous SELP polymorphism versus D-dimer value reached an OR of 3.26 (95% CI 1.41-7.52; p-value 3.73x10 -3 ), (Table 1c) . This result indicates that the two polymorphisms enhance each other, and that the SELP Asp603Asn and the AR poly-Q are two factors belonging to the same puzzle contributing to thrombosis in COVID-19 in males. Anti-P-Selectin monoclonal antibodies have been developed for human use: the phase 3 Inclacumab and the FDA and EMA approved Crizanlizumab. The latter is indicated to 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. 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. The copyright holder for this preprint this version posted May 27, 2021. PMC7938922. 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. (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 May 27, 2021. ; https://doi.org/10.1101/2021.05.25.21257803 doi: medRxiv preprint Employing a systematic approach to biobanking and analyzing genetic and clinical data for advancing COVID-19 research Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China Inborn errors of type I IFN immunity in patients with life-threatening COVID-19 Presence of Genetic Variants Among Young Men With Severe COVID-19 Unit of Respiratory Diseases and Lung Transplantation, Department of Internal and 37. Department of Infectious and Tropical Diseases, University of Brescia and ASST Spedali Civili Hospital Infectious Diseases Clinic, Policlinico San Martino Hospital, IRCCS for Cancer Research Genova, Italy 49. Microbiology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS Member of the European Reference Network for Rare, Low Prevalence and Complex Diseases of the Heart-ERN GUARD-Heart 67 Istituti Clinici Scientifici Maugeri, IRCCS, Department of Cardiac Rehabilitation 81. IRCCS C. Mondino Foundation 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.