key: cord-0969195-ri0oxo3k
authors: FREITAS, A. T.; Calhau, C.; Antunes, G.; Araujo, B.; Bandeira, M.; Barreira, S.; Bazenga, F.; Braz, S.; Caldeira, D.; Santos, S. C. R.; Faria, A.; Faria, D.; Fraga, M.; Nogueira-Garcia, B.; Goncalves, L.; Kovalchuk, P.; Lacerda, L.; Lopes, H.; Luis, D.; Medeiros, F.; Melo, A. M.; Melo-Cristino, J.; Miranda, A.; Pereira, C.; Pinto, A. T.; Pinto, J.; Proenca, H.; Ramos, A.; Rato, J. P.; Rocha, F.; Rocha, J. C.; Moreira-Rosario, A.; Vazao, H.; Volovetska, Y.; Guimaraes, J.-T.; Pinto, F.
title: Vitamin D-related polymorphisms and vitamin D levels as risk biomarkers of COVID-19 infection severity
date: 2021-03-26
journal: nan
DOI: 10.1101/2021.03.22.21254032
sha: 11fc28aec7e1f0de2d646b90c301fc73702d0871
doc_id: 969195
cord_uid: ri0oxo3k

Background: Vitamin D is a fundamental regulator of host defences by activating genes related to innate and adaptive immunity. Previous research shows a correlation between the levels of vitamin D in patients infected with SARS-CoV-2 and the degree of disease severity. This work investigates the impact of the genetic background related to vitamin D pathways on COVID-19 severity. For the first time, the Portuguese population was characterized regarding the prevalence of high impact variants in genes associated with the vitamin D pathways. Methods: This study enrolled 517 patients admitted to two tertiary Portuguese hospitals. The serum concentration of 25 (OH)D, was measured in the hospital at the time of patient admission. Genetic variants, 18 variants, in the genes AMDHD1, CYP2R1, CYP24A1, DHCR7, GC, SEC23A, and VDR were analysed. Results: The results show that polymorphisms in the vitamin D binding protein encoded by the GC gene are related to the infection severity (p = 0.005). There is an association between vitamin D polygenic risk score and the serum concentration of 25 (OH)D (p = 0.042). There is an association between 25 (OH)D levels and the survival and fatal outcomes (p = 1.5e-4). The Portuguese population has a higher prevalence of the DHCR7 RS12785878 variant when compared with its prevalence in the European population (19% versus 10%). Conclusion: This study shows a genetic susceptibility for vitamin D deficiency that might explain higher severity degrees in COVID-19 patients. These results reinforce the relevance of personalized strategies in the context of viral diseases.

From the end of 2019 until now, a new coronavirus named SARS-CoV-2 has been causing a worldwide pandemic leading to many deaths due to an acute respiratory infection named COVID-19. The lack of knowledge about the pathogenesis of this disease and the reasons underlying the different clinical outcomes have been pushing the society as an all to take preventive measures and to accelerate research. During the last year, several research groups and healthcare institutions have made available evidence that indicates that an uncontrolled inflammatory response is a major responsible for the occurrence of an acute respiratory distress syndrome (1) .

. CC-BY-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.22.21254032 doi: medRxiv preprint A statistical analysis of COVID-19 pandemic data from healthcare centers across China, France, Germany, Italy, Iran, South Korea, Spain, Switzerland, the United Kingdom and the United States, performed by Northwestern University, shows a strong correlation between severe vitamin D deficiency and mortality rates (2) .

Vitamin D has many relevant and documented roles in general health maintenance, being its deficiency particularly associated with severe impacts on the functional integrity of the immune system, such as influencing cytokine production (3) . Vitamin D deficiency has also been linked to hypertension, autoimmune, infectious and cardiovascular diseases, also known risk factors for severe COVID-19 (4) .

Previous research shows that genetics contribute to up to 28% of inter-individual variability in serum 25(OH)D concentrations, while season and vitamin D intake explain another 24% of the variability (5) . Large-scale genome-wide association studies (GWAS), considering 79,366 individuals with European ancestry (6), or considering imputed genotypes from 401,460 white British UK Biobank participants (7), have identified relatively common single nucleotide polymorphisms which play an important biological role in vitamin D metabolism, transport, degradation and downstream pathways, to evaluate their impact on circulating 25(OH) D concentrations. Vitamin D polymorphisms research shows that not all genomes will respond to vitamin D supplementation due to the loss of function of different genes (8) .

In a normal scenario, it is known that vitamin D deficiency is common in Europe and the Middle East. It occurs in <20% of the population in Northern Europe, in 30-60% in Western, Southern and Eastern Europe and up to 80% in Middle East countries (9) . Data from Portugal shows that 66% of adults present vitamin D insufficiency/deficiency (10) . This high percentage in a population living in a sunny country, motivated the characterization that was performed regarding the prevalence of high impact genetic variants in genes associated with the vitamin D pathways in the Portuguese population when compared with the European population.

The main objective of this study was to understand if an association exists between polymorphisms in vitamin D-related genes and vitamin D levels, and between these variables and the COVID-19 severity. Secondarily, we aimed to compare the frequency of the genetic variants under analysis with the observed frequency for the European population. To achieve this objective, we have assessed the genetic variants in vitamin D-related genes in hospitalized patients and the vitamin D serum levels, and evaluated the association between these data and the severity of the disease. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Health Organization (WHO) discrete clinical progression scale from 0 to 10 (11). In this study patients' severity levels were between 4 and 10.

From previous large-scale GWAS, several single nucleotide polymorphisms that play an important biological role in vitamin D metabolism, transport, degradation, and downstream pathways, have been identified as having an impact on circulating 25(OH) D concentrations (6, 7) . To understand if an association exists between the polymorphisms in the vitamin D-related genes and the disease severity, four polygenic risk scores (PRSs) were defined. These scores considered contributions from different genes and were identified as: Synthesis (DHCR7; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Regarding the association of the GC RS2282679 polymorphism with the disease severity, it might be explained by an additional role played by the vitamin D binding protein other than the transport of vitamin D in the bloodstream. Available data indicates that this protein may also act as a neutrophil chemotactic factor and a macrophage activator, therefore actively participating in the inflammation process (12, 13, 14) . Also, vitamin D binding protein is an extracellular scavenger for actin released from damaged/dead cells. When in excess, actin can cause intravascular coagulation resulting in multi-organ dysfunction and cardiac arrest (15) .

The functional consequence of this polymorphism in this pathway needs to be further explored in future studies.

It was observed a trend towards an increased proportion of patients with deficient levels of vitamin D (< 20 ng/ml) and an increase of severity from moderate to severe, and dead. The proportion of patients with deficient levels of vitamin D was higher in the group that died (76%) when compared with the two other groups (moderate -59% and severe -64% disease). The combined proportion of patients with insufficient and sufficient vitamin D levels was 40% in the group with moderate severity, compared with 24% in the group of deceased patients (see supplemental material). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint In this study, and for the first time, it is shown that the Portuguese population presents a genetic makeup for a higher predisposition to vitamin D deficiency when compared to the European population, therefore increasing the potential severity of the COVID-19 response, consequently impacting on patient outcomes. Furthermore, these results also explain, in part, the higher vitamin D deficiency in Portuguese population in different previous publications (10) . It is important to know that this metabolic vulnerability must be considered for a vitamin D supplementation clinical decision. This data proves that it is wrong to assume that sunny countries would not have an issue with vitamin D deficiency, showing that genetic characterization and vitamin D monitoring at a population level should be put in place in order to define guidelines for vitamin D intake.

One year after this pandemic scenario, it has been described that vitamin D deficiency may be a risk factor for mortality in COVID-19 infected patients (9) . On top of this, evidence revealed that supplementation with high-dose vitamin D3 booster therapy and reduced the risk of mortality (16) . Whereas vitamin D intake, sun exposure, demographics, and, specially, genes have been identified as being crucial determinants of vitamin D status, the impact of these factors is expected to be different across populations. To improve current prevention and treatment strategies, it is essential to propose novel diagnostic tools for personalization.

These results reinforce the role of vitamin D polymorphisms, in particular the GC RS2282679 polymorphism, and vitamin D levels as biomarkers for COVID-19 disease severity and emphasize is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint (17) . Thus, vitamin D adequacy was classified according to the following 25(OH) D cut-off levels: deficiency, <20 ng/mL; insufficiency, 20-29 ng/mL; and sufficiency, ≥30 ng/mL (18) . The season of blood sample collection was also considered, as it may affect the patient's vitamin D concentrations (19) .

Genotyping. All patients signed an informed consent to perform a genetic test for the analysis of is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Regarding the methodological approach, the following steps were undertaken: is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

The PRSs did not model other genetic variants that have been tested, since their impact has not been obtained by the same GWAS studies, which could introduce a bias in its relative impact.

Different statistical tests were employed, namely Mann-Whitney and Kruskal-Wallis

Tests, depending on the type of categorization under analysis. Spearman rank correlation coefficient was also calculated in order to analysed not only an eventual association but also to quantify it and observe its direction. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.22.21254032 doi: medRxiv preprint conceived and supervised the study, and analysed the results. All authors revised and approved the manuscript.

The cytokine storm in COVID-19: An overview of the involvement of the chemokine/chemokine-receptor system

The Possible Role of Vitamin D in Suppressing Cytokine Storm and Associated Mortality in COVID-19 Patients

Vitamin D and the immune system

Association of vitamin D status with arterial blood pressure and hypertension risk: a mendelian randomisation study

Current vitamin D status in European and Middle East countries and strategies to prevent vitamin D deficiency: a position statement of the European Calcified Tissue Society

Genome-wide association study in 79,366 European-ancestry individuals informs the genetic architecture of 25-hydroxyvitamin D levels

Genome-wide Association Study for Vitamin D Levels Reveals 69

Effects of Genetic and Nongenetic Factors on Total and Bioavailable 25(OH)D Responses to Vitamin D Supplementation

Editorial: low population mortality from COVID-19 in countries south of latitude 35 degrees North supports vitamin D as a factor determining severity

WHO Working Group on the Clinical Characterisation and Management of COVID-19 infection. A minimal common outcome measure set for COVID-19 clinical research

Vitamin D-binding protein contributes to COPD by activation of alveolar macrophages

New perspectives on the vitamin D binding protein

Behind the scenes of vitamin D binding protein: more than vitamin D binding

The Vitamin D Binding Protein and Inflammatory Injury: A Mediator or Sentinel of Tissue Damage?

The importance of vitamin d metabolism as a potential prophylactic, immunoregulatory and neuroprotective treatment for COVID-19

The vitamin D deficiency pandemic: Approaches for diagnosis, treatment and prevention

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Vitamin D levels and effects of vitamin D replacement in children with periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome

The REDCap consortium: Building an international community of software platform partners

We thank all the study participants, who donated blood and authorized the genetic analysis and the collection of the clinical and phenotypic personal data. This project was supported by the "Fundação para a Ciência e Tecnologia", program "Research 4 Covid-19 Apoio especial a projetos de implementação rápida para soluções inovadoras de resposta à pandemia de COVID-19".