key: cord-0928928-k21z3wij authors: De Marco, Renato; Faria, Tathyane C.; Mine, Karina L.; Cristelli, Marina; Medina‐Pestana, José O.; Tedesco‐Silva, Hélio; Gerbase‐DeLima, Maria title: HLA‐A homozygosis is associated with susceptibility to COVID‐19 date: 2021-06-29 journal: HLA DOI: 10.1111/tan.14349 sha: 0af5079e35b0cac60f4f61532487ea118922c32d doc_id: 928928 cord_uid: k21z3wij The purpose of this single center retrospective study was to investigate the relationship between HLA and ABO polymorphisms and COVID‐19 susceptibility and severity in kidney transplant recipients. It included 720 recipients who had COVID‐19 and 1680 controls composed by recipients in follow‐up who did not contact the transplantation center for COVID‐19 symptoms, up to the moment of their inclusion in the study. HLA‐A, ‐B, and ‐DRB1 allele groups and ABO frequencies were compared between recipients with COVID‐19 (all cases, or separately mild/moderate and severe disease) and controls. The HLA association study was conducted in two case–control series and only associations that showed a p‐value <0.05 in both series were considered. No HLA association regarding COVID‐19 occurrence or severity met this criterion. Homozygosity at HLA‐A locus was associated with COVID‐19 susceptibility (odds ratio 1.4) but not severity. Blood groups A and O were associated with susceptibility and resistance to COVID‐19, respectively. COVID‐19 severity was associated only with older age and cardiac disease, in a multivariate analysis. We conclude that an influence of HLA on COVID‐19 susceptibility is supported by the association with homozygosity at HLA‐A locus but that there is no evidence for a role of any particular HLA‐A, ‐B, or ‐DRB1 polymorphism. Thus, we suggest that what matters is the overall capability of an individual's HLA molecules to present SARS‐CoV‐2 peptides to T cells, a factor that might have a great influence on the breadth of the immune response. The coronavirus disease 19 (COVID-19) is a highly infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in Wuhan, China, in December, 2019, and was declared as a pandemic by the World Health Organization (WHO) in March 2020. [1] [2] [3] [4] The first case in Brazil was diagnosed in February 2020. 5 The main independent risk factor for COVID-19 severity and death is older age, besides other factors including diabetes, hypertension, obesity, chronic respiratory and cardiovascular diseases, smoking and chronic immunosuppression, male gender and black race. 2, [6] [7] [8] [9] [10] [11] Because COVID-19 susceptibility and severity varies among individuals with apparently similar environmental risk factors, it is conceivable that viral and host genetic variants may both influence outcomes. Several genetic factors have been suggested to be involved in susceptibility to COVID-19 and its outcomes including HLA and ABO polymorphisms as the most frequently investigated ones. [12] [13] [14] [15] [16] [17] The extensive polymorphism of HLA genes and the influence of HLA alleles on the adaptive immune response, at the level of selection of peptides for presentation to T cells, make HLA genes attractive candidates with impact on susceptibility to viral infections. Actually, there is evidence that the extensive HLA polymorphism results from pathogen-driven balancing selection or, in other words, from the necessity for a species to be immunologically diverse in order to maximize the probability that at least some individuals within the general population would survive an epidemic. The advantage of HLA heterozygosis over homozygosis in terms of fitness to survive infections is a mechanism that contributes to maintenance of HLA polymorphism. [18] [19] [20] [21] The demonstration, by in silico analysis, that HLA molecules vary on their binding affinities for SARS-CoV-2 peptides, have strengthened the interest in the investigation of the relationship between HLA and COVID-19 and there is preliminary evidence that the peptide binding capacity of HLA class molecules may influence the outcome of COVID-19. [22] [23] [24] [25] [26] [27] [28] [29] [30] Several associations between HLA polymorphisms and COVID-19 have been described in investigations that compared HLA frequencies between geographical regions with different COVID-19 prevalences, [31] [32] [33] or in case-control studies within the same population. [34] [35] [36] [37] [38] [39] However, confidence in these associations is limited, not only because the majority loose significance when the p-value is corrected for multiple comparisons, but also because there is no consistency of the associated polymorphisms among the different studies, not even in those conducted in populations with similar genetic backgrounds. ABO blood groups have been investigated in several studies and the great majority reported association of group A with susceptibility and of group O with resistance. [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] It has been suggested that the resistance of group O individuals is due to the presence of anti-A and anti-B isoagglutinins and, conversely, that the susceptibility conferred by group A is caused by the absence of anti-A isoagglutinins. This hypothesis is based on the fact that SARS-CoV-2 S proteins express A and/or B glycan antigens of the host and on the observation that in an experimental SARS cell model monoclonal or human polyclonal anti-A antibodies inhibited the interaction between viral S proteins carrying A antigens and cellular ACE2 proteins. 41, 51, 52 The purpose of the present study was to investigate the relationship between HLA and ABO polymorphisms and COVID-19 susceptibility and severity in kidney transplantation recipients (KTR), a population that is at increased risk for COVID-19 due to chronic immunosuppression and high prevalence of comorbidities. 53, 54 2 | METHODS AND MATERIALS This single-center, retrospective observational case-control study included 720 KTR that had COVID-19, between March and December, 2020, and 1680 KTR transplanted in the years of 2018 and 2019 who did not seek medical assistance at the transplant center, up to the moment of their inclusion in the present study. The transplants were from living or deceased donors and were performed and followed-up in Hospital do Rim, São Paulo, Brazil. For the diagnosis of COVID-19 it was required at least one positive test for SARS-CoV-2 by reverse transcription-polymerase chain reaction (RT-PCR) from nasopharyngeal swabs or bronchoalveolar lavage. All COVID-19 cases were diagnosed after transplantation, but the onset of the disease was considered the day of the first symptoms. COVID-19 was classified as mild/moderate (N = 501) when there was no need for hospitalization or, when hospitalization was required, there was no need for mechanical ventilation. COVID-19 was classified as severe (N = 219) in cases with intubation or death. Demographics, ABO blood type, comorbidities, and clinical data related to COVID-19 were collected from medical records of the hospital. Data on comorbidities were not collected from the control group. Concerning race, KTR were classified as white or non-white. Nonwhite comprised black and admixed (white/black/indigenous) individuals. HLA typing at allele group level (low resolution) and pre-transplant calculated panel reactive antibodies (cPRA) were retrieved from the database of our laboratory, where all the pre-transplant tests were performed. The study was approved by the local ethics committee and informed consent was obtained from all recipients diagnosed with COVID-19. Informed consent was waived for KTR without COVID-19 because the analyses used only anonymous data. ABO phenotypic frequencies were assessed by direct counting. The reference group consisted of all ABO groups, except the one that was being considered in the comparison. The study included the analysis of 21 HLA-A, 35 HLA-B, and 13 HLA-DRB1 allelic groups. HLA-A, -B, -DRB1 phenotypic frequencies were assessed by direct counting. The 720 KTR with and the 1680 KTR controls without COVID-19 were randomly subsampled in order to construct two case-control series. HLA allele groups frequencies were compared between KTR with COVID-19 (all cases, or separately, mild/moderate and severe disease) and controls in each of the two case-controls series and only the associations that showed a p-value <0.05 in both series were considered. An individual carrying only one HLA-A, -B, or -DRB1 allelic group was considered homozygous. The percentages of homozygous individuals were compared between the 720 KTR with COVID-19 (all cases, or separately, mild/moderate and severe disease) and the 1680 controls. Pre-transplant cPRA levels (0%-10%, 11%-49%, 50%-79%, and ≥ 80%) were compared between KTR with or without COVID-19 and between mild/moderate and severe cases. Categorical variables were compared with chi-square or Fisher's exact test, as appropriate. Continuous variables were analyzed with Mann-Whitney test. Logistic regression was used to test the independence of variables, which presented p-value < 0.05 in the univariate analyses. Values of p < 0.05 were reported as statistically significant. The analyses were performed with IBM SPSS Statistics for Windows Version 21.0 (IBM Corp, Armonk, NY). The median time from transplant to the first symptoms of COVID-19 was 5.6 (À0.01-21.2) years. In one KTR the symptoms began 3 days before transplantation. Among the severe cases, 83% died. The comparison between KTR with (N = 720) and without (N = 1680) COVID-19 T A B L E 2 HLA-A,-B, and -DRB1 phenotypic frequencies that differed, considering an uncorrected p-value <0.05, between kidney transplant recipients without and with COVID-19 (all cases and cases stratified by severity) in the analyses performed in any one of the two case-control series regarding gender, age, and race, as well the comparison between cases with mild/moderate (N = 501) and severe (N = 219) COVID-19 regarding gender, age, race, and comorbidities, are presented in Table 1 . Occurrence of COVID-19 was associated with age ≥ 60 years (p = 0.002), but not with gender or race. Severe disease was associated with male gender (p = 0.02), age ≥ 60 years (p < 0.0001), hypertension (p = 0.022), diabetes (p < 0.0001), heart disease (p < 0.0001), and other co-morbidities that included chronic lung and liver diseases and neoplasia (p = 0.01). White and non-white patients did not differ regarding the prevalence of severe disease. KTR with COVID-19 did not differ between case-control series 1 and 2, in respect to gender, age, race, time posttransplant to first COVID-19 symptoms, and the comorbidities considered in this study (Table S1) . Frequencies of HLA-A (N = 21), -B (N = 35), and -DRB1 (N = 13) allele group in KTR with any type of COVID-19, in mild/moderate cases and in severe cases were compared to those in KTR without COVID-19, in each of the two case-control series. The 12 HLA allele groups that showed significant differences in frequencies, considering a p < 0.05, in any of these comparisons are presented in Table 2 . No p-value remained < 0.05 after correction for the number of comparisons, even considering only the number of investigated HLA-A, -B and -DR polymorphisms (N = 69) and not all comparisons between the groups. Moreover, no association was present in both studies. The association with HLA-B*48, although statistically significant (p < 0.05) in both series, was negatively associated in series 1 and positively associated with COVID-19 in series 2. The comparison of HLA-A, -B, or -DRB1 homozygosity between all KTR with and without COVID-19 showed higher frequency of homozygosity at HLA-A locus (11.5% vs. 8.5%, p = 0.020), a tendency for higher frequency at HLA-DR locus (7.8% vs. 5.8%, p = 0.075), and no significant difference at HLA-B locus (6.7% vs. 6.0%, p = 0.54) ( Table 3) . No association was observed between HLA-A, -B, or -DRB1 homozygosity and severity of the disease. 46 .5%, p = 0.026) associated with COVID-19. No differences were observed concerning frequencies of B and AB blood groups. No association was observed between any blood group and severity of the disease. Pre-transplant cPRA levels (0%-10%, 11%-49%, 50%-79% and ≥ 80%) did not show any statistically significant differences between KTR with or without COVID-19 or between mild/moderate and severe cases (Table 5 ). All factors that showed association in the univariate analyses were considered in the multivariate analysis. Regarding COVID-19 susceptibility, the multivariate logistic regression analysis included age ≥ 60 years, blood group A and homozygosity at HLA-A locus and these three variables remained significantly associated with odds ratios and 95% CI of 1.4 (1.1-1.7), 1.2 (1.0-1.5), and 1.4 (1.0-1.8), respectively ( Table 6 ). Also when blood group O instead of group A was analyzed, the three variables remained significantly associated with odds ratios and 95% CI of 1.4 (1.1-1.7), 0.8 (0.7-1.0), and 1.4 (1.0-1.8), respectively (data not shown). Regarding COVID-19 severity, however, only advanced age ≥ 60 years (OR 4.2; 95% CI 2.9-6.1; p < 0.0001) and heart disease (OR 2.1; 95% CI 1.1-3.9; p = 0.022) were independently associated with severe disease, whereas blood groups, male gender, hypertension, diabetes and other comorbidities were not. The main purpose of this study was to evaluate association of HLA and ABO polymorphisms with susceptibility to COVID-19 in KTR. The investigation included 720 KTR with COVID-19 and 1680 KTR who did not report COVID-19 symptoms. Univariate analyses showed that older age, but not gender or race, was associated with susceptibility to COVID, while older age, male gender and co-morbidities were found to be associated with severe disease, defined as intubation or death. These findings are in agreement with previous reports. 2,6-9 On the other hand, we did not observe a higher prevalence of severe disease in nonwhites, as reported in UK and in the United States, [9] [10] [11] possibly because all KTR were from the same transplantation center, with equitable access to COVID-19 treatment. This interpretation is in line with authors of UK and United States that believe that environmental factors, such as poverty and limited health care, rather than genetic factors, are responsible for the higher SARS-CoV-2 infection morbidity and mortality in racial minority populations. [9] [10] [11] The main findings of our investigation were the association between homozygosity at HLA-A locus and susceptibility to COVID-19 and the lack of association between any HLA-A, -B, or -DRB1 polymorphism and COVID-19. A study of class I HLA polymorphism and COVID-19 in the Spanish population 22 found higher proportion of homozygosity at HLA-A and -C loci in moderate and severe as compared to mild COVID-19. Interestingly, as in our study, there was no association with HLA-B homozygosis. Furthermore, the study in the Spanish population showed that patients with moderate and severe disease presented an overall lower theoretical capacity of their HLA class I molecules to bind SARS-CoV-2 peptides. We therefore suggest that susceptibility to COVID-19 is not affected by any particular HLA-A, -B, or -DRB1 polymorphism, but rather by the overall limitation of the possibilities of HLA molecules to present SARS-CoV-2 peptides to T cells. There are several examples of infectious, especially viral, diseases in animals that are influenced by homozygosity at genes of the major histocompatibility complex (MHC). 18, [55] [56] [57] [58] [59] [60] The clearest example in humans is AIDS, where HIV-positive individuals who are homozygous at one or more HLA class I loci progress to AIDS faster than the heterozygous patients. [58] [59] [60] The lack of association between any particular HLA-A, -B, or -DRB1 polymorphism and COVID-19 susceptibility or severity observed in our study was not surprising, considering that it was designed with the idea of avoiding false-positive discoveries. The inconsistencies of associations across different reports concerning COVID-19, 28,31-39 as well as in studies with diseases caused by other Coronavirus, namely Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome (MERS) (MERS-CoV) [61] [62] [63] [64] [65] [66] suggest that the majority, if not all, of the reported associations are falsepositive. False-positive results, in fact, are a common statistical pitfall in HLA and disease association studies where cases and controls are subjected to multiple comparisons. 67 Concerning false-negative associations, the size of the samples reduced very much this possibility. A sensitivity power analysis 68 was conducted to define the detectable differences in proportions between KTR without COVID-19 and KTR with COVID-19, in each one of the casecontrol series. Considering a statistical power of 80%, the observed sample sizes and the observed proportions as references, the results (not shown) indicated that for HLA allelic groups with frequencies <10% and ≥10%, the detectable percentage points differences ranged from 0.04 to 5.52, and from 3.86 to 8.84, respectively. Our study confirmed the association of blood group A with susceptibility and of blood group O with resistance to COVID-19. These results are in accordance with findings of several previous studies. 28, [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] On the other hand, we did not find any association between ABO blood groups and severity of COVID-19, which is agreement with some studies, 28, [48] [49] [50] and at variance with other ones. 42, 46, 47 Multivariate analyses of variables associated with COVID-19 susceptibility in univariate analyses showed that advanced age ≥ 60 years, blood group A and HLA-A homozygosity were independently associated with higher susceptibility, while blood group O was associated with resistance. Concerning severity of COVID-19, only age ≥ 60 years and cardiac disease were independently associated with the more severe form of disease. As a high cPRA is sometimes considered to reflect an individual's ''high responder'' immune status not only to HLA but also to other antigens, we tested whether there was a relationship between the level of cPRA and susceptibility or severity of COVID-19. No associations were disclosed, in accordance with an observation in another study of KTR. 28 This study has strengths as well as limitations. Among the strengths are the high number of KTR with (720) and without (1680) COVID-19 from the same transplantation center, and the design of the study of association between HLA and COVID-19 that aimed to avoid false-positive discoveries. The limitations include those inherent to a single center retrospective study and the impossibility to rule out the presence of KTR with symptomatic SARS-CoV-2 infection in the control group, since mild to moderate symptoms may not always have been reported to the hospital. In summary the association with homozygosity at HLA-A locus supports an influence of HLA on COVID-19 susceptibility but, on the other hand, our study does not indicate an influence of any particular HLA-A, -B, or -DRB1 polymorphism on COVID-susceptibility or severity. Thus, we suggest that what matters is the overall capability of an individual's HLA molecules to present SARS-CoV-2 peptides to T cells, a factor that might have a great influence on the breadth of the immune response. We are grateful to Professor Caner Süsal for the critically revision of the manuscript and suggestions. The authors declare no potential conflict of interest. Renato De Marco designed the study, performed research, analyzed the data and revised the manuscript; Tathyane C Faria performed research, analyzed the data and revised the manuscript; Karina L Mine designed the study, analyzed the data and revised the manuscript; Marina Cristelli designed the study, performed research and revised the manuscript; José O Medina-Pestana revised the manuscript; Hélio Tedesco-Silva designed the study and revised the manuscript, Maria Gerbase-DeLima designed the study, analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Renato De Marco https://orcid.org/0000-0002-8388-6403 A novel coronavirus from patients with pneumonia in China The SARS-CoV-2 outbreak: what we know COVID-19 infection: origin, transmission, and characteristics of human coronaviruses WHO declares COVID-19 a pandemic Painel de casos de doença pelo coronavírus 2019 (COVID-19) no Brasil pelo Ministério da Saúde. Coronavirus Brasil Clinical features of patients infected with 2019 novel coronavirus in Wuhan Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Coronavirus COV-19/SARS-CoV-2 affects women less than men: clinical response to viral infection Clinical, regional, and genetic characteristics of Covid-19 patients from UKbiobank The COVID-19 pandemic: a call to action to identify and address racial and ethnic disparities COVID-19 and African Americans Genetic gateways to COVID-19 infection: implications for risk, severity, and outcomes The ABO blood group locus and a chromosome 3 gene cluster associate with SARS-CoV2 respiratory failure in an Italian-Spanish genomewide association analysis The role of host genetic factors in coronavirus susceptibility: review of animal and systematic review of human literature The role of host genetics in the immune response to SARS-CoV-2 and COVID-19 susceptibility and severity Angiotensin-converting enzymes (ACE, ACE2) gene variants and COVID-19 outcome Initial whole-genome sequencing and analysis of the host genetic contribution to COVID-19 severity and susceptibility Enhanced immunological surveillance in mice heterozygous at the H-2 gene complex Pathogen-driven selection and worldwide HLA class I diversity HLA and infectious diseases How pathogens drive genetic diversity: MHC, mechanisms and misunderstandings Prediction of SARS-CoV-2epitopesacross9360HLAclassIalleles Possible role of HLA class-I genotype in SARS-CoV-2 infection and progression: a pilot study in a cohort of Covid-19 Spanish patients Immunoinformatic approach to assess SARS-CoV-2 protein S epitopes recognised by the most frequent MHC-I alleles in the Brazilian population Human leukocyte antigen susceptibility map for severe acute respiratory syndrome coronavirus 2 Class I HLA allele predicted restricted antigenic coverages for spike and nucleocapsid proteins are associated with deaths related to COVID-19 Retrospective in silico HLA predictions from COVID-19 patients reveal alleles associated with disease prognosis Italian network of regional transplant coordinating centers. HLA and AB0 polymorphisms may influence SARS-CoV-2 infection and COVID-19 severity A bioinformatic prediction of antigen presentation from SARS-CoV-2 spike protein revealed a theoretical correlation of HLA-DRB1*01 with COVID-19 fatality in Mexican population: an ecological approach Association of HLA class I genotypes with severity of coronavirus Disease-19 Population difference in allele frequency of HLA-C*05 and its correlation with COVID-19 mortality Correlation of the two most frequent HLA haplotypes in the Italian population to the differential regional incidence of Covid-19 Predictive immunogenetic markers in COVID-19 Human leukocyte antigen complex and other Immunogenetic and clinical factors influence susceptibility or protection to SARS-CoV-2 infection and severity of the disease course Members of the BIOMEPOC group. HLA genetic polymorphisms and prognosis of patients with COVID-19 HLA allele frequencies and susceptibility to COVID-19 in a group of 99 Italian patients A role for human leucocyte antigens in the susceptibility to SARS-Cov-2 infection observed in transplant patients Distribution of HLA allele frequencies in 82 Chinese individuals with coronavirus disease-2019 (COVID-19) Association of HLA-B22 serotype with SARS-CoV-2 susceptibility in Hong Kong Chinese patients Association between ABO blood group system and COVID-19 susceptibility in Wuhan Blood group ABO polymorphism inhibits SARS-CoV-2 infection and affects COVID-19 progression Relationship between the ABO blood group and the COVID-19 susceptibility ABO blood group and COVID-19: a review on behalf of the ISBT COVID-19 working group Association between ABO blood groups and risk of SARS-CoV-2 pneumonia Association between ABO and Rh blood groups and SARS-CoV-2 infection or severe COVID-19 illness: a population-based cohort study ABO blood groups in COVID-19 patients Association between ABO blood groups and COVID-19 infection, severity and demise: a systematic review and meta-analysis ABO blood group predisposes to COVID-19 severity and cardiovascular diseases The association of ABO blood group with indices of disease severity and multiorgan dysfunction in COVID-19 ABO phenotype and death in critically ill patients with COVID-19 COVID-19 and ABO blood group: another viewpoint Inhibition of the interaction between the SARS-CoV spike protein and its cellular receptor by anti-histo-blood group antibodies Results from the TANGO international transplant consortium COVID-19 infection in kidney transplant recipients at the epicenter of pandemics MHC heterozygosity confers a selective advantage against multiple-strain infections Why did they die? MHC heterozygote advantage in simian immunodeficiency virus-infected Mauritian cynomolgus macaques HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage The influence of HLA genotype on AIDS HLA heterozygote advantage against HIV-1 is driven by quantitative and qualitative differences in HLA allelespecific peptide presentation Association of HLA class I with severe acute respiratory syndrome coronavirus infection Association of human-leukocyteantigen class I (B*0703) and class II (DRB1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome Lack of association between HLA-A, -B and -DRB1 alleles and the development of SARS: a cohort of 95 SARS-recovered individuals in a population of Guangdong, southern China Association of human leukocyte antigen class II alleles with severe acute respiratory syndrome in the Vietnamese population Human-leukocyte antigen class I Cw 1502 and class II DR 0301 genotypes are associated with resistance to severe acute respiratory syndrome (SARS) infection Association of human leukocyte antigen class II alleles with severe Middle East respiratory syndrome-coronavirus infection HLA and disease 1982-a survey Sample Size Calculations in Clinical Research Additional supporting information may be found online in the Supporting Information section at the end of this article.