HLA haplotypes and differential regional mortality caused by COVID-19 in Brazil: an ecological study based on a large bone marrow donor bank dataset

Abstract The coronavirus disease 2019 (COVID-19) mortality rates varied among the states of Brazil during the course of the pandemics. The human leukocyte antigen (HLA) is a critical component of the antigen presentation pathway. Individuals with different HLA genotypes may trigger different immune responses against pathogens, which could culminate in different COVID-19 responses. HLA genotypes are variable, especially in the highly admixed Brazilian population. In this ecological study, we aimed to investigate the correlation between HLA haplotypes and the different regional distribution of COVID-19 mortality in Brazil. HLA data was obtained from 4,148,713 individuals registered in The Brazilian Voluntary Bone Marrow Donors Registry. COVID-19 data was retrieved from epidemiological bulletins issued by State Health Secretariats via Brazil’s Ministry of Health from February/2020 to July/2022. We found a positive significant correlation between the HLA-A*01~B*08~DRB1*03 haplotype and COVID-19 mortality rates when we analyzed data from 26 states and the Federal District. This result indicates that the HLA-A*01~B*08~DRB1*03 haplotype may represent an additional risk factor for dying due to COVID-19. This haplotype should be further studied in other populations for a better understanding of the variation in COVID-19 outcomes across the world.


INTRODUCTION
Brazil surpassed 670,000 deaths due to coronavirus disease 2019 , caused by SARS-CoV-2 (WHO 2022).The mortality rates varied considerably among the Brazilian regions during the course of pandemics (CORONAVIRUS BRASIL 2020).This variability could be partially explained by the patient clinical profile and unequal access to the health system in different regions (Li et al. 2020, Zhou et al. 2020).In addition to the social and environmental conditions, genetic factors may also influence susceptibility and immune response to SARS-CoV-2 infection.Many studies are investigating genetic factors that could influence on COVID-19 susceptibility and severity worldwide.Several of them have focused on the variability of the genetic complex of the classic human leukocyte antigen (HLA), a critical component of the antigen presentation pathway.Individuals with different HLA genotypes may trigger different immune responses against SARS-CoV-2 (Dendrou et al. 2018, Saghazadeh 2020).The HLA variability observed worldwide JULIANO ANDRÉ BOQUETT et al.
In a recent review, Migliorini et al. (2021) showed that a few HLA alleles or haplotypes may have a protective effect or an increased susceptibility to SARS-CoV-2 infection.However, these studies had a limited number of patients and the associated alleles varied depending on the population under investigation.Additionally, in silico studies have analyzed the binding affinity between the SARS-CoV-2 peptides and different HLA class I genotypes (Barquera et al. 2020, Kiyotani et al. 2020, Nguyen et al. 2020, Tomita et al. 2020).In Italy, two studies compared HLA allele prevalence retrieved through the Italian Bone-Marrow Donor Registry with the incidence of SARS-CoV-2 infections in the different geographical regions of the country.This strategy increases the number of analyzed individuals and could be very informative, even though the HLA genotypes are not obtained directly from COVID-19 patients (Correale et al. 2020, Pisanti et al. 2020).
The diverse results obtained from HLA and COVID-19 studies highlight the need for additional analyses in different populations.
In the present study, we investigated a large sample of HLA haplotypes from all Brazilian states retrieved from The Brazilian Voluntary Bone Marrow Donors Registry (REDOME, Registro de Doadores de Medula Óssea), the third largest bone marrow donors bank in the world, and compared to COVID-19 data in the correspondent region.(Boquett et al. 2017, Buhler et al. 2012, Nunes 2015).Maps of COVID-19 mortality rates were generated in ArcGis v10.3.Spearman's correlation test between the five most frequent haplotypes in Brazil (Torres et al. 2017) and COVID-19 mortality rates in the 26 states and the Federal District was performed using IBM SPSS software, Version 20.0 (IBM Corp., Armonk, NY).P-values below 0.05 were considered statistically significant.Bonferroni's correction for multiple tests was applied for haplotype correlations (α Bonf = 0.01).

RESULTS
The geographical distribution of the COVID-19 epidemic in Brazil in our analysis was as follows: the states with the highest incidence of COVID-19 cases registered until July 6 th , 2022 were Espírito Santo, the Federal District and Roraima, respectively; while the states with the lowest incidence were Maranhão, Pará and Alagoas (Table I).Regarding COVID-19 mortality, Rio de Janeiro, Mato Grosso and Rondônia had the highest rates, respectively, while the lowest mortality rates were observed in Maranhão, Bahia and Alagoas, respectively (Table I, Figure 1).
HLA allele frequencies did not deviate from HWE expectations and were used to estimate the haplotypes.The five most frequent HLA-A~B~DRB1 haplotypes found in the analyzed population are presented in Table II.The most frequent haplotype was HLA-A*01~B*08~DRB1*03, with a frequency ranging from 1.2% in Pará, Maranhão and Amapá, states located in the North (Pará, Amapá) and Northeast (Maranhão) of the country, to 3.1% in Rio Grande do Sul and Santa Catarina, both states located in the South.Rio Grande do Sul and Santa Catarina had a high outlier frequency for haplotypes #1 (HLA-A*01~B*08~DRB1*03) and #3 (HLA-A*03~B*07~DRB1*15) (Figure 2).Spearman correlation coefficient was calculated to test if the regional COVID-19 mortality correlated with any of the five most frequent haplotypes in Brazilian population.We found a strong and significant correlation (rho = 0.687, P < 0.001) between the haplotype HLA-A*01~B*08~DRB1*03 frequency and COVID-19 mortality rates for the 26 states and the Federal District (Table III).No significant correlation was found in the remaining haplotypes analyzed.

DISCUSSION
Many studies have employed different approaches to investigate the role of the HLA system in COVID-19, such as in silico (Barquera et al. 2020, Kiyotani et al. 2020, Nguyen et al. 2020, Tomita et al. 2020), case-control (Kousathanas et al. 2022, Langton et al. 2021, Wang et al. 2020) and ecological (Correale et al. 2020, Pisanti et al. 2020).In this ecological study, we found a significant positive correlation between the HLA-A*01~B*08~DRB1*03 haplotype and COVID-19 mortality rates when analyzing data from the 26 states and the Federal District (Table III).Pisanti et al. ( 2020) also found a significant correlation between the two most frequent haplotypes in the Italian population with both COVID-19 incidence and mortality using a similar ecological approach.In their study, the haplotype HLA-A*01:01g~B*08:01g~C*07:01g~DRB1 *03:01g showed a positive correlation with both COVID-19 incidence and mortality.(2023) 95(3) e20220801 5 | 9 In Brazil, we observed that the four most common HLA haplotypes had higher frequencies in the South region and lower frequencies in the North (Table II).These regional differences reflect the different contributions of Native American, European, and African populations across the country after a long history of colonization and immigration (IBGE 2020).This is the first study in the country evaluating HLA haplotype frequencies and COVID-19 mortality rates.A previous descriptive study in Brazil focused on HLA alleles retrieved from Brazilian exome databases and compared the frequency of these HLA alleles with populations that  Together with genetic and immune system variation, environmental and social disparities among Brazilian regions may contribute to the differential burden of COVID-19, disproportionately affecting individuals carrying genetic factors of susceptibility and/or the most vulnerable people regarding social assistance.As the number of reported cases depends on the number and type of diagnostic tests performed by each city and state, and with limited testing it is unlikely that asymptomatic subjects would have been diagnosed for SARS-CoV-2 infection, these numbers should be taken with caution.In this sense, the number of COVID-19 related deaths used in this study could be more reliable.
Distinguishing among genetic, social and environmental variability, though challenging, is essential for building an efficient way to prevent, control and understand the disease (Zhao et al. 2019).In this sense, our study is an important initial step in the understanding of COVID-19 dispersion and behavior in Brazil.Overall, our analysis provided support for the association of HLA haplotype and a severe outcome from COVID-19.An important limitation of our study is that we used HLA data from bone Table III.Bivariate correlation analysis among regional haplotypes estimated frequency in the population and COVID-19 and mortality (N° deaths/100,000 inhabitants) in Brazilian states until July 6 th , 2022.marrow donors instead of directly genotyping individuals affected by COVID-19.However, this approach has some advantages.Bone marrow donor registries usually include very large sample sizes and a wide geographic coverage, as illustrated by the REDOME registry.Ecological studies based on databases such as this one are a cost-effective and useful alternative for raising hypotheses, which could be tested in further studies with patients.Similar studies have been performed with REDOME and other diseases databases in the Brazilian population (Boquett et al. 2018).In addition, because several countries maintain such large banks, statistically significant associations could reveal regions or populations in higher genetic risk for COVID-19, thus representing an additional tool for health policymakers in the fight against COVID-19.
In conclusion, in this ecological approach, we found a significant positive correlation between the HLA-A*01~B*08~DRB1*03 haplotype and COVID-19 mortality rates when analyzing data from the Brazilian 26 states and the Federal District.The probable association of the HLA haplotype with a severe outcome from COVID-19 in Brazil could be useful to identify more vulnerable populations and guide public policies and vaccination strategies in case of novel disease waves.
occupy the top 10 positions for most cases of COVID-19 and the five populations less affected by the disease (Secolin et al. 2021).They found that the HLA alleles HLA-DQB1*06:02 and HLA-DRB1*15:01 were frequent in Brazil and in less affected populations.

Figure 2 .
Figure 2. Box-and-whisker plots with the five most frequent HLA-A~B~DRB1 haplotypes in Brazil.AP: Amapá; RN: Rio Grande do Norte; RS: Rio Grande do Sul; SC Santa Catarina.

Table I .
Regional data relative to the impact of COVID-19 on the Brazilian population until July 6th, 2022.

Table II .
Frequencies (%) of the five most common haplotypes observed in the Brazilian population.