Genetic polymorphism in C3 is associated with progression in chronic kidney disease (CKD) patients with IgA nephropathy but not in other causes of CKD

Sara T. Ibrahim; Rajkumar Chinnadurai; Ibrahim Ali; Debbie Payne; Gillian I. Rice; William G. Newman; Eman Algohary; Ahmed G. Adam; Philip A. Kalra

doi:10.1371/journal.pone.0228101

Peer Review History

Original SubmissionSeptember 8, 2019
13 Nov 2019 Decision Letter - Giuseppe Remuzzi, Editor PONE-D-19-25255 Genetic polymorphism in C3 is associated with progression in chronic kidney disease (CKD) patients with IgA nephropathy but not in other causes of CKD PLOS ONE Dear Dr Ibrahim, 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. The manuscript focuses on a topic of potential interest. However, the study has several shortcomings that should be addressed. To mention few of them, i) concern about the novelty regarding the association of the R102G and CKD; ii) need to analyze the results of the study considering the already available GWAS data on CKD; iii) concern about the sample size of the 37 patients with IgA nephropathy too small to be convincing of a different distribution of the polymorphism on the RP and SP groups; iv) need to evaluate the pathophysiological link between the R102G polymorphism and complement activation; v) need to include in the multivariate Cox regression model the evaluation of the IgAN subgroup CRP; vi) concern about the fact that the study featured a cohort that was primarily Caucasian which limits the generalization of the results; vii) concern about the significant differences in age between RP ad SP likely contributing to findings; viii) need to indicate how common the C3F variant is in the general population; ix) concern about the fact that Table 6 shows a regression analysis of the IgAN group in which the multivariate regression analysis did not include the C3SS genotype as a variant. We would appreciate receiving your revised manuscript by Dec 28 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Giuseppe Remuzzi Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records used in your retrospective study. Specifically, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. 3. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? 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: No 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: Yes 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: In this paper the authors provide evidence for the association of a genetic variant with CKD in a clinical study. Their goal was to identify novel genetic risk factors that lead to progression of kidney diseases. The target of interest was a variant in the human complement component C3. The study shows that within the cohort, there was a statistical overrepresentation of the C3FF variant in patients with fast progressing CKD and IgAN. The study featured a cohort that was primarily caucasian which limits the impact of the results. Significant differences in age between RP and SP likely contributes to findings, given likely strong survival bias. More clarification on how age when adjusted for affects the results. The data in tables 1, 2, and 3 features multiple observations on various measurements, with a p-value determined by a Mann-Whitney U test or chi-square analysis. In the figure there are 15 observations made, and there should be some sort of adjustment for the false discovery rate as it is misleading to mark observations as significant otherwise. The paper does not state how common the C3F variant is in the general population, which would affect the interpretation of the observed frequency of the C3FF variant in the patients. Table 6 shows a regression analysis of the IgAN group in which the multivariate regression analysis did not include the C3SS genotype as a variant, although the p-value for the univariate model seems to be most statistically significant. Overall, the manuscript presents interesting findings for which the methods are clearly written out and presented. However, the statistical analysis of the findings need further explanation and clarification, with the issues presented above. Reviewer #2: In this study Sara T Ibrahim and collaborators evaluated the role of the R102G variant in complement 3 (C3) in a coohort of CKD patients. They analyzed the distribution of the polymorphism compared to a healthy control group, in the CKD rapid progressive group (RP) against the CKD stable function subjects (SP). Finally, they evaluated the role of the polymorphism in the sub group of the biopsy proven glomerulonephritis affected patients. C3F allele frequency was found to be significantly higher in the CKD cohort compared with the healthy control group. There was no significant difference in C3F allele frequency between the RP and SP groups. In the glomerulonephritis subgroup Cox regression showed an association between C3F and progression only in those with IgA nephropathy. Major concerns The role of complement in many renal conditions and in particular in many glomerulonephrites (IgAN, C3 glomerulonephritis, HUS, membranous nephropathy, MPGN IgG mediated and others) is well known. The evaluation of a C3 polymorphism can be interesting. The authors recognize the small size of their cohort, but justify the importance of their study in consideration of the better clinical characterization of their sample compared to much larger GWAS studies. However the results of the study are not novel regarding the association of the R102G and CKD: in particular the same evaluation could be better analyzed considering the already available GWAS data on CKD. The sample size of the 37 patients with IgA nephropathy is too small to be convincing of a different distribution of the polymorphism in the RP and SP groups. Before publication it is necessary to increase the IgAN sample size of a factor of 10 times at least. Most important the authors did not try to make any evaluation of the pathophysiological link between the R102G polymorphism and complement activation. I understand that the C3F and C3S are two variants with different electrophoretic migration characteristic. What is the difference in C3 activity of the two variants. Minor concern In the multivariate Cox regression evaluation of the IgAN subgroup CRP has not been included in the model. Because it was highly colinear with R102G polymorphism in the analysis of the outcome of risk of death it should be maintained even in the other analyses. ****** 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: Yes: RICCARDO MAGISTRONI [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". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0228101.r001
Revision 1
27 Nov 2019 Author Response Response to reviewers Dear editor and reviewers, Thank you so much for reviewing our manuscript and providing us with your comments and queries, aiming to improve our work. The following is our reply to your comments and queries. 1- Concern about the novelty regarding the association of the R102G and CKD The association between the R102G polymorphism of C3 and CKD or GN is not a novel one and it has been addressed by some studies before. This is the main point we have relied upon in the rationale of our study that the R102G may be associated with progression of all-cause CKD or in specific sub-types of GN (hence study 2 which included all the biopsy proven GN patients in our cohort). The novel point in our study is the association between the R102G and progression in IgA nephropathy patients. This association has not been addressed by any of the previous studies. -We have now illustrated this point in a clearer way in the introduction and the discussion of the revised manuscript. 2- Need to analyze the results of the study considering the already available GWAS data on CKD Most available GWAS that have been conducted in CKD [1-5] or IgA nephropathy [6-8] patients aimed to investigate the genetic factors associated with prevalent CKD or IgAN; as these studies lacked the detailed clinical characteristics of the patients and follow up data they were unable to include progression as an end-point. The only 2 GWAS that attempted to search for genetic factors associated with progression rather than prevalence of CKD are Boger CA et al [9] and Parsa A et al [10].Boger et al`s GWAS tested only 16 SNPs and found that 11 of them associated with incident CKD (during a follow up period of 7 years). Our targeted SNP, the R102G, was not one of the 16 tested SNPs in this GWAS. Parsa et al`s GWAS tested a million SNPs in a large CKD cohort with 5 years follow up and found 12 SNPs that were associated with time to ESRD in black patients and 6 in white patients; the R102G SNP was not one of the latter 6 SNPs and this agrees with our finding that the R102G was not associated with progression in all-cause CKD. However in Pars et al`s study the specific causes of CKD in their cohort were not defined and so they could not relate primary renal disease-specific progression to specific SNPs. -We have added a paragraph about this in our discussion in the revised manuscript. 3- Concern about the sample size of the 37 patients with IgA nephropathy too small to be convincing of a different distribution of the polymorphism on the RP and SP groups This is correct and we mentioned this point in our manuscript`s discussion as a limitation. However in study 2 with larger number of IgAN patients (114) we found that C3F is strongly and independently associated with the progression in IgAN patients. We believe that our study could underpin future studies in larger cohorts of IgAN patients which could then replicate this association. 4- Need to evaluate the pathophysiological link between the R102G polymorphism and complement activation The pathophysiological link between the R102G and complement activation has been evaluated previously by Heurich M et al [11]. They used C3FF and C3SS from plasma of healthy individuals and added them separately to antibody coated sheep RBCs and then added factor B (FB) to them. They found that the samples containing C3SS needed larger amount of FB to lyse the RBCs. Demonstrating that plasma containing C3FF has more complement activity than that containing C3SS. They further investigated this issue and proved that factor H (one of the complement regulator proteins) bound less well to C3FF than C3SS. -We have added a paragraph about this issue in the introduction of the revised manuscript. 5- Need to include in the multivariate Cox regression model the evaluation of the IgAN subgroup CRP -We have added the CRP to the univariate Cox regression in table 6 in the revised manuscript. It showed no significant association with progression in IgAN patients so we have not added it to the multivariate Cox model. The variables which showed significant association in the univariate model are the only variables that were used in the multivariate model to be consistent with the statistical rule that one variable can be included in the Cox model for every 8-10 patients with the event of interest (we had 59 events). 6- Concern about the fact that the study featured a cohort that was primarily Caucasian which limits the generalization of the results All genetic studies should define the ethnicity of the studied cohort according to the prevalence of studied SNPs. If the studied SNP prevalence is ˂ 5% in a certain ethnicity the presence or the absence of the association between this SNP and the disease will not be accurate due to the rarity of the SNP in this ethnicity. The frequency of the C3F allele differs in different ethnicities: Caucasian (20%), black (5%) and Asian (1%) [12]. Hence the Caucasian cohort appears to be the optimal cohort to be used to investigate the R102G SNP in different diseases. 7- Concern about the significant differences in age between RP and SP likely contributing to findings. The difference between the age of patients in RP and SP groups was only significant in the whole CKD cohort but it was not significant in the sub group of IgAN patients (37 patients: 21 RP and 16 SP). Also the age was not significantly associated with progression in the IgAN patients in the Cox regression (table 6). The Cox regression analysis for death was undertaken in the whole cohort without separation into SP and RP; adjustment for age in the multivariate analysis was also performed (table 4). -We have added the table comparing the baseline characteristics of the IgAN subgroup to the supplementary tables in the revised manuscript (S4 table). 8- Need to indicate how common the C3F variant is in the general population As mentioned above the frequency of the C3F allele differs in different ethnicities (Caucasian (20%), black (5%) and Asian (1%)). We have previously referred to its frequency in Caucasian cohorts and in our healthy control group in the discussion section. -We have now illustrated this point in the introduction of the revised manuscript. 9- Concern about the fact that Table 6 shows a regression analysis of the IgAN group in which the multivariate regression analysis did not include the C3SS genotype as a variant. Statistically we cannot include C3SS in the same multivariate model with C3FF and C3FS as they are constant or linearly dependent covariates; C3SS = 1- (C3FF + C3FS). -We have added a second multivariate model to table 6 in the revised manuscript with C3SS (but without C3FF and C3FS) to show that it is still protective of progression in the multivariate model. All of the above modifications have been highlighted in yellow in the revised manuscript. Thank you so much, Sara T Ibrahim References 1. Kottgen A, Glazer NL, Dehghan A, Hwang SJ, Katz R, Li M, et al. Multiple loci associated with indices of renal function and chronic kidney disease. Nature Genetics. 2009;41:712-7. 2. Kottgen A, Pattaro C, Boger CA, Fuchsberger C, Olden M, Glazer NL, et al. New loci associated with kidney function and chronic kidney disease. Nature genetics. 2010;42(5):376-84. 3. Pattaro C, Kottgen A, Teumer A, Garnaas M, Boger CA, Fuchsberger C, et al. Genome-wide association and functional follow-up reveals new loci for kidney function. PLoS genetics. 2012;8(3):e1002584. 4. Pattaro C, Teumer A, Gorski M, Chu AY, Li M, Mijatovic V, et al. Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function. Nat Commun. 2016;7:10023. 5. Chambers J, Zhang W, Lord G, van der Harst P, Lawlor DA, Sehmi JS, et al: Genetic loci influencing kidney function and chronic kidney disease. Nat Genet. 2010;42:373-5. 6. Gharavi AG, Kiryluk K, Choi M, Li Y, Hou P, Xie J, et al: Genome-wide association study identifies susceptibility loci for IgA nephropathy. Nat Genet. 2011;43:321-7. 7. Yu XQ, Li M, Zhang H, Low HQ, Wei X, Wang JQ, et al: A genome-wide association study in Han Chinese identifies multiple susceptibility loci for IgA nephropathy. Nat Genet. 2011;44:178-82. 8. Kiryluk K, Li Y, Scolari F, Sanna-Cherchi S, Choi M, Verbitsky M, et al: Discovery of new risk loci for IgA nephropathy implicates genes involved in immunity against intestinal pathogens. Nat Genet. 2014;46:1187-96. 9. Böger CA, Gorski M, Li M, Hoffmann MM, Huang C, Yang Q, et al. Association of eGFR-Related Loci Identified by GWAS with Incident CKD and ESRD. PLoS Genet. 2011;7(9):e1002292. 10. Parsa A, Kanetsky PA, Xiao R, Gupta J, Mitra N, Limou S, et al. Genome-Wide Association of CKD Progression: The Chronic Renal Insufficiency Cohort Study. J Am Soc Nephrol. 2017;28(3):923-34. 11. Heurich M, Martínez-Barricarte R, Francis NJ, Roberts DL, Rodríguez de Córdoba S, Morgan BP, et al. Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk. Proc Natl Acad Sci U S A. 2011;108(21):8761–6. 12. Bazyar N, Azarpira N, Khatami RS, Galehdari H. The investigation of allele and genotype frequencies of human C3 (rs2230199). Mol Biol Rep. 2012;39(9):8919–24. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0228101.r002
18 Dec 2019 Decision Letter - Giuseppe Remuzzi, Editor PONE-D-19-25255R1 Genetic polymorphism in C3 is associated with progression in chronic kidney disease (CKD) patients with IgA nephropathy but not in other causes of CKD PLOS ONE Dear Dr Ibrahim, 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. The revised manuscript is definitely improved. It remains, however, a minor issue raised by Reviewer #2 that should be easily addressed. We would appreciate receiving your revised manuscript by Feb 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Giuseppe Remuzzi Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? 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 ****** 5. 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: Yes Reviewer #2: Yes ****** 6. 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: Appreciate the additional explanations in the introduction and discussion sections to address concerns. Reviewer #2: The authors have answered to the questions I raised. However the Parsa et al. study should not be reported as a GWAS (Genome Wide Association Study) but rather as a Candidate Genes Association Study. ****** 7. 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: Yes: RICCARDO MAGISTRONI [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". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0228101.r003
Revision 2
19 Dec 2019 Author Response Dear 2nd reviewer, Thank you so much for your comment (The Parsa et al. study should not be reported as a GWAS (Genome Wide Association Study) but rather as a Candidate Genes Association Study). You are right, but I think you meant the Böger et al study not the Parsa et al study. Parsa et al study is a large GWAS which tested million SNPs but Böger et al study is the one which tested only 16 SNPs that were identified by a previous GWAS. I have now corrected this and highlighted it in yellow in the revised manuscript. Thanks Sara T Ibrahim https://doi.org/10.1371/journal.pone.0228101.r004
8 Jan 2020 Decision Letter - Giuseppe Remuzzi, Editor Genetic polymorphism in C3 is associated with progression in chronic kidney disease (CKD) patients with IgA nephropathy but not in other causes of CKD PONE-D-19-25255R2 Dear Dr. Ibrahim, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. The re-revised version of the manuscript is definitely improved. The authors have adequately addressed all the reviewers’ comments. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Giuseppe Remuzzi Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? 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 ****** 5. 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: Yes Reviewer #2: Yes ****** 6. 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: As stated on prior review of submission, the authors have already addressed my comments. Although there are still some limitations, the authors were responsive. Reviewer #2: (No Response) ****** 7. 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: Yes: Riccardo Magistroni https://doi.org/10.1371/journal.pone.0228101.r005
Formally Accepted
17 Jan 2020 Acceptance Letter - Giuseppe Remuzzi, Editor PONE-D-19-25255R2 Genetic polymorphism in C3 is associated with progression in chronic kidney disease (CKD) patients with IgA nephropathy but not in other causes of CKD Dear Dr. Ibrahim: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Giuseppe Remuzzi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0228101.r006

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