key: cord-0882826-r8c96vww authors: Ertosun, Mustafa Gökhan; Özkan, Özlenen; Darbaş, Şule; Özel, Deniz; BİLGE, Uğur; Sayin Ekinci, Nurten; Yilmaz, Vural Taner; Uçar, Fahri; Koçak, Hüseyin; Özkan, Ömer title: The relationship between COVID‐19 and HLA in kidney transplant recipients, an evaluation of predictive and prognostic factors date: 2021-11-24 journal: Clin Transplant DOI: 10.1111/ctr.14525 sha: 598fb01f5d42bfef82668f351b7b4ef8e9b239ca doc_id: 882826 cord_uid: r8c96vww INTRODUCTION: The purpose of this study was to determine the predictive and prognostic factors for COVID‐19 infection and its relationship with human leukocyte antigen (HLA) in kidney transplant recipients. MATERIAL AND METHOD: Three hundred fifty kidney transplant recipients were included in the study. Recipients were divided into two groups: COVID‐19(+) (n = 100) and control (n = 250). The relationships between HLA frequencies, COVID‐19 infection, and prognostic factors (age, donor type, immunosuppression protocol, etc.) were then evaluated. Logistic regression analysis, heatmap, and decision tree methods were used to determine predictive and prognostic factors. The study was performed retrospectively. RESULTS: Advanced age and deceased transplantation emerged as predictive of SARS‐CoV‐2 infection, while the presence of HLA‐A*11, the HLA match ratio, and high‐dose tacrolimus were identified as prognostic factors in kidney transplant recipients. HLA‐A10, HLA‐B*13, HLA‐B22, and HLA‐B*55 were shown to be associated with SARS‐CoV‐2 infection at univariate analysis, and HLA‐B*57, HLA‐DRB1*11, and HLA‐DRB1*13 at logistic regression analysis. CONCLUSION: HLA‐A10, HLA‐B*13, HLA‐B*55, HLA‐B*57, HLA‐DRB1*11, and HLA‐DRB1*13 were identified for the first time in the literature associated with SARS‐CoV‐2 infection in kidney transplant recipients. kidney transplant recipients based on genetic, clinical, and demographic factors. The continuing COVID-19 pandemic has had a significant impact on solid organ transplantation (SOT) worldwide and has become a threat to the lives of SOT recipients, as to other members of the community. 3 Since emerging as a specialty field in the 1980s, SOT has progressed rapidly due to advances in surgical techniques, immunosuppression, and genetics. The number of transplant operations performed and the number of SOT transplant recipients in society have therefore both increased. 4, 5 As the number of cases increases, the probability of encountering the SARS-CoV-2 virus also rises. Organ transplant recipients are thought to be more susceptible to COVID-19 infection due to the immunosuppressive medications they use. This makes it even more important to investigate the genetic and environmental factors that predispose to COVID-19 infection. One of the most important issues requiring investigation is human leukocyte antigens (HLAs), which are necessary for the immune system to distinguish between self and nonself (foreign) cells, and which are associated with family and population studies, transplantation, infectious diseases, autoimmune diseases, and many types of cancer. 6 The first study of the relationship between SARS and HLA was performed in 2003. 7 HLA-B*46:01 was found to be related to SARS disease. With the emergence of the SARS-CoV-2 virus, studies investigating the relationship between viral infection and HLA have acquired renewed importance. [8] [9] [10] Although common, possibly related HLAs have been described in studies from several countries, regional differences have also been observed. [11] [12] [13] Due to the regional variation in the frequency of HLA types, studies investigating HLAs and their relationship with COVID-19 in different regions are essential. The investigation of susceptibility factors for SARS-CoV-2 infection in organ transplant recipients is critical since these have to use immunosuppression agents throughout their lives. The relationship between COVID-19 and HLAs, which play a critical role in the immunological response against RNA viruses as well as in SOTs (especially kidney transplants), has previously been investigated. 14 The aim of the present study was to investigate the HLA type of the renal transplant recipient, donor-recipient tissue compatibility, and the effects of these genetic factors on COVID-19. In the study, clinical significance was accepted when the difference between the incidence of certain HLAs in kidney transplant recipients with and without COVID-19 diagnosis was at least 10%. Under these conditions (p1 = 0.15; p2 = 0.05), assuming 80% power, .05 margin of error and allocation ratio (N2 / N1) ratio is 2.5, the minimum number of samples to be included in the study according to the two-ratio difference test is group 1 (COVID-19(+)) for n = 90 and for group 2 (COVID-19(-)) n = 224. The sample number estimation of the study was made by using G*Power 3.1.9.2 program. 15 One hundred kidney transplant recipients with COVID-19 diagnosis and 250 kidney transplant recipients without COVID-19 diagnosis were included in the study in order to obtain the minimum number of volunteers against the loss in follow-up and the possibility of not having access to patient data. Despite this, the data of all patients included in the project were accessed; has been included in the analysis. One hundred kidney transplant recipients diagnosed with COVID-19 by means of PCR tests and presenting to our center between April 2020 and February 2021 were included in the study. Genomic DNA of healthy controls and patients infected with SARS-CoV-2 were isolated from 200 ul peripheral blood samples using a Bio-robot EZ1 advanced XL magnetic bead-based workstation (Qiagen, Germany). HLA-A, HLA-B, and HLA-DRB1 genotyping were performed on all subjects by the low-resolution polymerase chain reaction with sequence-specific oligonucleotide probe (PCR-eRES,SSO) hybridization method using Luminex technology (IMMUCOR-Lifecodes, Georgia). Whole blood was collected on citrate phosphate dextrose, and lymphocytes were isolated by centrifugation on Ficoll-Hypaque. The isolated cells were counted and adjusted to 3 × 10 6 cells/mL. Serological typing was performed on lymphocyte suspensions using the microlymphocytotoxicity technique (standard NIH) and local set of sera. Data for two HLA alleles were analyzed as serological typing, HLA-A10 and HLA-B22. Descriptive statistics are presented as frequency, percentage, mean, standard deviation, median, minimum, maximum, 25%-75% percentile (Q1-Q3), or IQR values. Normality assumptions were checked by examining the histogram, q-q plots, skewness, and kurtosis values with the Shapiro Wilk test. The independent two-sample t test was used to analyze the difference in numerical data between the two groups when the data conformed to normal distribution, and the Mann-Whitney U test was used when the data were not normally distributed. In addition to the classical statistical analyses, we applied nonlinear techniques to cluster, classify, and visualize data to provide insights. Presence of SARS-CoV-2 infection, etiology of end-stage renal disease, postoperative periods after transplantation, drugs used in the transplant process, and blood tacrolimus levels are shown in Table 3 . A statistically significant relationship was found between HLA match and presence of COVID-19 infection for all HLAs (p < 0.001). In pairwise comparisons using the Bonferroni correction, the mismatch rate for all alleles was higher among the COVID-19 patients than in the control group, while the match ratio to one allele was higher in the control group than in the COVID-19 patients. No difference in the proportions of patients with two alleles matched was observed between the COVID-19 and control groups. When examining how many of the six alleles had a match, full-mismatch and 1/6 match were higher in the COVID-19(+) group, while 3/6 match was higher in the control group. In addition, 2/6, 4/6, 5/6, and 6/6 matches exhibited no difference between the two groups. No statistically significant relationship was determined between compliance variables and hospitalization for all HLAs. Multivariate analysis was applied using logistic regression after adjusting for age and blood tacrolimus levels in order to examine which HLA alleles are associated with COVID-19(+) ( Table 4 The heatmap in Figure 1 shows . In the decision tree, the incidence of COVID-19 disease is higher in four out of 11 terminal nodes compared to the control group Our study aimed to investigate the relationship between HLA genotyping and COVID-19 infection in kidney transplant recipients. In addition to HLA, the relationship between COVID-19 and parameters such as age, gender, etiology of CKD, donor type, rejection, and drugs were also investigated. Our study includes one of the most comprehensive kidney transplant series on COVID-19 disease in the literature. 18 A statistically significant difference was observed in this study between the patient and control groups HLA-match rates. In terms of the HLA-A match, the one allele match rate was higher among living donor recipients than in the deceased group. In contrast, more than 50% of deceased donor recipients had 2/6 or less of the total match rate ( Supplementary Information 1) . In addition, cold-ischemia time and other immunological factors which were not evaluated within the scope of this study should also be investigated. As expected, the mean age of the COVID-19(+) patient group was higher than that of the control group, consistent with the previous literature. 8, 19 However, in contrast to previous studies, the donor type was statistically significant in terms of COVID-19 infection in the present study. 20 In seeking to explain this based on the data obtained, incidence rate. 9 On the other hand, the HLA-A10 group is no longer defined since this was a method previously used in our serological typing laboratory. Due to the age of this method, the average age of the recipients using it is high, considering the time elapsed. 27 The same gene and gene products (chemokines) are also involved in COVID-19 disease, suggesting the possibility of common molecular pathways between HLA-mismatches and SARS-CoV-2 virus infection. 28 Finally, studies have shown that multiple-dose influenza vaccine increases anti-HLA antibodies. These findings strongly suggest a potential relationship between viral infection immunity and anti-HLA antibodies. 29 The relationship between the anti-HLA antibody and the inflammation response created by the SARS-CoV-2 virus needs to be investigated in future studies. Evaluation of HLA frequencies and COVID-19-associated hospitalization status revealed that HLA-A*11 was statistically significant. In-silico analysis reported in the previous literature showed that the HLA-A*11 alleles may be associated with hospitalization in COVID-19 infection. 30 The analyses performed in the present study showed that this retrospective in silico analysis is clinically accurate. A study from Spain found that the HLA-A*11 alleles were associated with high mortality. 11 Considering that the patients were hospitalized due to poor prognosis of COVID-19 disease (WHO scores 6-9), this seems to be consistent with our findings concerning the HLA-A*11 allele. The present study shows, for the first time in the literature, that HLA-A*13, HLA-B*57, HLA-DRB1*11, and HLA-DRB1*13 are associated with SARS-CoV-2 infection. Previous studies have shown that these alleles are associated with viral and bacterial infection. [31] [32] [33] [34] Additionally, the conclusions are based on a single center study and further investigation in other populations is needed to confirm these associations. It is very important to analyze the SARS-CoV-2 peptide binding capacities of the HLA subtypes that we found associated with the SARS-CoV-2 viral infection in this study. In the literature, the binding capacity of HLA-A*26, which is the genetic subgroup of HLA-A10, to SARS-CoV-2 peptide has been investigated. 23 In addition, it has been stated in the literature that different HLA-B correlations may show different affinity for the SARS-CoV-2 peptide, which may be associated with daily deaths related to COVID-19. 35 Investigation of peptide binding capacities of other subgroups is also very important in the fight against COVID-19 infection. In addition, in the study conducted with the in silico analysis method in the literature, the relationship between Covid and HLA in our country was determined. In that study, the subtype with the highest HLA allele frequency is HLA A*02. Although there are other studies, including Turkey, examining the HLA-COVID19 relationship, 36 as is known, our country has a heterogeneous structure. This study has the feature of having the largest cohort showing the Covid-19&HLA relationship covering kidney transplant individuals covering the Mediterranean region in Turkey. One of the limitations of the present study is that we were unable to analyze the anti-HLA antibody since the genetic data were obtained retrospectively, and anti-HLA antibody data were not available for all patients. For the same reason, we were unable to analysis SARS-CoV-2 IgM and IgG antibody levels. Additionally, since the tissue typing results were evaluated retrospectively, this enabled us to access the serological typing results of some HLA alleles, HLA-A10 and HLA-B22. Lack of genotypic typing of statistically significant HLA alleles is a further lim-itation of this study. Finally, this study involved only kidney transplant recipients, and it is important that similar studies be performed in other SOTs. In our study, we can say that more statistical tests were performed than should be due to the large number of variables. This situation can lead to multiple testing (multiplicity) problem and this problem may highly increase random variation and type-I error. 37 According to ICH E9 guideline, to avoid this problem, we specified in the protocol the precise definition of the primary variable. 38 We did not change the statistical methods following finalization of the protocol and statistical analysis were pre-planned. On the other hand, our primary outcomes consisted of only COVID-19(+) and hospitalization. In subgroup testing through a post hoc evaluation, we use Bonferroni correction to avoid type I error. Finally, as this study is retrospective in nature, all results are associative and should be validated in future studies. 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Data available on request from the authors. Mustafa Gökhan Ertosun https://orcid.org/0000-0002-2557-7346Vural Taner Yilmaz https://orcid.org/0000-0002-1313-8856Ömer Özkan https://orcid.org/0000-0002-9031-5596 Additional supporting information may be found in the online version of the article at the publisher's website.