Association between HLA-C alleles and COVID-19 severity in a pilot study with a Spanish Mediterranean Caucasian cohort

Lorena Vigón; Miguel Galán; Montserrat Torres; Antonio J. Martín-Galiano; Sara Rodríguez-Mora; Elena Mateos; Magdalena Corona; Rosa Malo; Cristina Navarro; María Aránzazu Murciano-Antón; Valentín García-Gutiérrez; Vicente Planelles; Jorge Martínez-Laso; María Rosa López-Huertas; Mayte Coiras; on behalf of the Multidisciplinary Group of Study of COVID-19 (MGS-COVID)

doi:10.1371/journal.pone.0272867

Peer Review History

Original SubmissionApril 21, 2022
16 May 2022 Decision Letter - Aftab A. Ansari, Editor PONE-D-22-11703Association between HLA-C alleles and COVID-19 severity in a pilot study with a Spanish Mediterranean Caucasian cohortPLOS ONE Dear Dr. Vigon, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. As you may note reviewr one had some serious concerns and along with reviewer 2 has made a major issue with regards to the small sample size and the requirment for using appropriate statistics for the analysis of data. Kindly, please pay attention to each of the issues raised by reviewer one and the issue raised by reviewer two. Please submit your revised manuscript by Jun 30 2022 11:59PM. 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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ******** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Reviewer comments The authors conducted HLA and KIR association studies in 72 individuals with Spanish Mediterranean Caucasian ethnicity 1. There are a couple of previously published articles focusing on the same population group, please kindly describe these studies results in comparison to the authors results in both the introduction and the discussion part of the paper. 2. Please describe the clinical protocols that the authors used for the defining of mild, severe and critical COVID-19 patients, did the authors follow the WHO guideline for example SpO2 level, etcs? 3. Please perform a multiple correction adjustment for all the p-values in the HLA association analysis, for example the HLA-B14:01 would not be significant after multiple correction; P=0.02312, thus there are no HLA alleles significantly associate with the severity of COVID-19 and the title and the writings of the paper should reflect as such. 4. The obvious disadvantage of this paper are the tiny sample size used in this study, the lack of healthy control, the lack of replication sample set, the lack of appropriate adjustment for multiple testing. 5. The authors performed extra HLA association test by HLA carrier frequencies, but most of the significant alleles are either very rare (example: less than 5), these results should be gathered and put in a combined group such as the rare HLA group. Minor comments 1. “Most of these alleles concentrate the highest rate of mutations in the exon that encodes the peptide-binding pocket, likely due to the selection pressure exerted by infectious diseases during pandemics. “ the reason why mutations have been reported in the exon are mainly caused by the fact that conventional HLA genotyping method such as PCR-SSOP often only targets exon2.3 of the HLA genes, causing a bias in the reporting of exonic variants in the IMGT/HLA database, in fact, the recent database is now full of intronic/3’UTR/5’UTR variants. Reviewer #2: Overall this is a very interesting study. I would have one major comment, before any interpretation of the data is possible. I could not find the p-values adjusted for multiple comparisons. Please add ******** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". 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Revision 1
23 Jun 2022 Author Response Answers to Reviewer#1: Q1. The authors conducted HLA and KIR association studies in 72 individuals with Spanish Mediterranean Caucasian ethnicity 1. There are a couple of previously published articles focusing on the same population group, please kindly describe these studies results in comparison to the authors results in both the introduction and the discussion part of the paper. A1. We thank the Reviewer#1 for this comment. We had already included two references (44 and 45) published by Iturrieta-Zuazo I, et al. 2020 and Lorente L, et al. 2021 that describe the frequency of HLA alleles in the Spanish population. The sentence “Other HLA-C alleles from HLA-C1 group have been described to show a higher degree of recognition of SARS-CoV-2 peptides (Iturrieta-Zuazo I, et al. 2020), and HLA-C01 has also been correlated with a higher mortality during COVID-19 (Lorente L, et al. 2021)” had been included in line 154, page 8 (line 149, page 7 in the Revised version). We also discussed these references in lines 643-646, and we have added one sentence highlighted in red: “…it is similar to other studies previously published (44, 45, 82, 83) that also validate a correlation between HLA-C and higher COVID-19 severity in the Spanish population (44, 45).” Q2. Please describe the clinical protocols that the authors used for the defining of mild, severe and critical COVID-19 patients, did the authors follow the WHO guideline for example SpO2 level, etcs? A2. We agree with Reviewer#1 that this is an essential question. Patients were classified depending on the medical assistance they received and according to the progression of the disease. Mild COVID-19 received primary health assistance from Primary Healthcare Centers, whereas severe and critical COVID-19 were hospitalized. Critical COVID-19 were admitted to the Intensive Care Unit (ICU). This information was included in lines 279-289. This classification was also based on the WHO’s recommendations (World Health Organization. (2021). Living guidance for clinical management of COVID-19 [Ebook]. Retrieved from https://www.who.int/publications/i/item/WHO/2019-nCoV/clinical/2021.2). We also include Tables S1 and S2 to clarify the data. Q3. Please perform a multiple correction adjustment for all the p-values in the HLA association analysis, for example the HLA-B14:01 would not be significant after multiple correction; P=0.02312, thus there are no HLA alleles significantly associate with the severity of COVID-19 and the title and the writings of the paper should reflect as such. A3. We thank Reviewer#1 for this critical observation. We agree with the necessity of performing this analysis and therefore, P values have been corrected using Bonferroni correction after alleles with an expected value <5 in each group had been grouped as “rare alleles”. For each HLA haplotype the corrected P value (Pc) was calculated by multiplying the P value obtained with the two-tailed Chi-square or Fisher’s exact test by the number of tested allelic combinations. Only Pc values lower than 0.05 were considered to be statistically significant. Significant differences regarding HLA-C08:02 are maintained between Mild and Critical COVID-19 (pc=0.0240) (Table 2). Similarly, HLA-C08:02 or HLA-C12:03 or HLA-C16:01 are significantly more frequent in Mild group than in Severe (pc=0.033) and Critical (pc=0.0154) groups (Table 3). In addition, HLA-C06:02 or HLA-C07:01 are significantly more prevalent in the Severe group regarding Mild COVID-19 (pc=0.0132) (Table 3). After Bonferroni correction, no statistical significance was observed in HLA haplotypes (Table 4) or in allele HLA-B14:01. We have modified the manuscript accordingly, adding several sentences highlighted in red throughout the text. Q4. The obvious disadvantage of this paper are the tiny sample size used in this study, the lack of healthy control, the lack of replication sample set, the lack of appropriate adjustment for multiple testing. A4. We understand the concern raised by Reviewer#1 about this limitation of our study. We previously discuss this issue in lines 645-652. We would like to indicate, first, that we attained statistical significance in some comparison, despite the reduced sample size. And second, that other studies that evaluate the association of HLA with COVID-19 severity is a small sample have been published previously (Iturrieta-Zuazo I, et al. 2020; Lorente L, et al. 2021; Wang W, et al 2020; Abdelhafiz AS, et al.2021). Besides, these cohorts of individuals with COVID-19 pre-vaccination era are very precious as this situation is unlikely to be repeated again, due to more than 95% of the Spanish population and 65% of world population have at least received one dose of the authorized vaccines. Finally, we also indicated in the manuscript that this observational study is consider a pilot study, and the results would need to be confirmed in a larger cohort. The aim of this study was to find a possible association between HLA alleles and COVID-19 severity. Because of that, a healthy group was not considered, similarly to previous reports with a similar objective (Iturrieta-Zuazo I, et al. 2020). Moreover, the frequency of the different HLA alleles in the Spanish population have been mentioned in the introduction (Montero-Martín G, et al.2019) (Lines 123-124) and discussed (Montero-Martín G, et al.2019; Arnaiz-Villena A, et al. 1997. Martinez-Laso J, et al. 1995) lines 521-524; 529-530; 538-539; 546-552; 553-556). We also agree with Reviewer#1 that the lack of multiple adjustment was necessary to obtain significant results. According to Reviewer#1 suggestion, we applied Bonferroni correction to adjust p values, as explained above. Q5. The authors performed extra HLA association test by HLA carrier frequencies, but most of the significant alleles are either very rare (example: less than 5), these results should be gathered and put in a combined group such as the rare HLA group. A5. We agree with Reviewer#1 that this suggestion is also necessary to clarify data expressed in Table 2 and also contributes to get a better understanding of the results. Consequently, those alleles with an expected value <5 in each group have been grouped as “rare alleles” for every HLA class I and class II genes tested. Next, Bonferroni correction was performed, considering this new group of “rare alleles” as one determination. Minor comments Q1. “Most of these alleles concentrate the highest rate of mutations in the exon that encodes the peptide-binding pocket, likely due to the selection pressure exerted by infectious diseases during pandemics“. The reason why mutations have been reported in the exon are mainly caused by the fact that conventional HLA genotyping method such as PCR-SSOP often only targets exon2.3 of the HLA genes, causing a bias in the reporting of exonic variants in the IMGT/HLA database, in fact, the recent database is now full of intronic/3’UTR/5’UTR variants. A1. We thank Reviewer#1 for this suggestion. In order to avoid misunderstandings, we believe that is better to delete this sentence. Answers to Reviewer#2: Q1. I would have one major comment, before any interpretation of the data is possible. I could not find the p-values adjusted for multiple comparisons. Please add. A1. We thank Reviewer#1 for this suggestion. We agree with the necessity of performing this analysis and therefore, P values have been corrected using Bonferroni correction after alleles with an expected value <5 in each group had been grouped as “rare alleles”. For each HLA haplotype the corrected P value (Pc) was calculated by multiplying the P value obtained with the two-tailed Chi-square or Fisher´s exact test by the number of tested allelic combinations. Only Pc values lower than 0.05 were considered to be statistically significant. Significant differences regarding HLA-C08:02 are maintained between Mild and Critical COVID-19 (pc=0.0240) (Table 2). Similarly, HLA-C08:02 or HLA-C12:03 or HLA-C16:01 are significantly more frequent in Mild group than in Severe (pc=0.033) and Critical (pc=0.0154) groups (Table 3). In addition, HLA-C06:02 or HLA-C07:01 are significantly more prevalent in the Severe group regarding Mild COVID-19 (pc=0.0132) (Table 3). After Bonferroni correction, no statistical significance was observed in HLA haplotypes (Table 4) or in allele HLA-B14:01. We have modified the manuscript accordingly, adding several sentences highlighted in red throughout the text. Attachments Attachment Submitted filename: Response to Reviewers.pdf* https://doi.org/10.1371/journal.pone.0272867.r002
28 Jul 2022 Decision Letter - Gualtiero I. Colombo, Editor Association between HLA-C alleles and COVID-19 severity in a pilot study with a Spanish Mediterranean Caucasian cohort PONE-D-22-11703R1 Dear Dr. Coiras, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Gualtiero I. Colombo, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0272867.r003
Formally Accepted
3 Aug 2022 Acceptance Letter - Gualtiero I. Colombo, Editor PONE-D-22-11703R1 Association between HLA-C alleles and COVID-19 severity in a pilot study with a Spanish Mediterranean Caucasian cohort Dear Dr. Coiras: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Gualtiero I. Colombo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0272867.r004

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