BMJ Open: first published as 10.1136/bmjopen-2016-013279 on 23 August 2017. Downloaded from

PEER REVIEW HISTORY

BMJ Open publishes all reviews undertaken for accepted manuscripts. Reviewers are asked to complete a checklist review form (http://bmjopen.bmj.com/site/about/resources/checklist.pdf) and are provided with free text boxes to elaborate on their assessment. These free text comments are reproduced below.

ARTICLE DETAILS

TITLE (PROVISIONAL) Interaction effects among IFN-γ+874, IL-2-330, IL-10-1082, IL-10- 592, and IL-4-589 polymorphisms on the clinical progression of subjects infected with hepatitis B virus and/or hepatitis C virus: a retrospective nested case control study AUTHORS Gao, Qiu-jv; Xie, Jia-xin; Wang, Li-min; Zhou, Qiang; Zhang, Shiyong

VERSION 1 - REVIEW

REVIEWER Hend ibrahim Shousha endemic and hepatogastroenterology department, Faculty of medicine, Cairo University REVIEW RETURNED 28-Jul-2016

GENERAL COMMENTS thank you for your effort and research idea

How did you diagnose the controls who spontaneously cleared HCV or HBV? is there patients who had combined HCV and HBV infection and cleared both??

How many patients were infected with HCV, HBV or had combined http://bmjopen.bmj.com/ HBV and HCV within each group?? what is the treatment status of the patients??

Saxena R, Kaur J. Th1/Th2 cytokines and their genotypes as predictors of hepatitis B virus related hepatocellular carcinoma. World J Hepatol. 2015 Jun 18;7(11):1572-80. mentioned in their review (On associating the IL-2 (-330 T > G) genotypes with HCC

progression in HBV infected individuals, we showed that both the TG on September 29, 2021 by guest. Protected copyright. and GG genotypes remained largely non-significant in HBV chronicity, among controls and carriers [54]. Similarly, the IL-2 (-330 T > G) polymorphism did not appear to modify HBV-HCC risk in the Chinese and American populations[50,77]. On the contrary, a study by Gao et al[78] reported, IL-2-330 TT genotype to be associated with an increased risk of chronic hepatitis, in case of either HBV or HCV or HBV-HCV coinfection in Chinese population (your study)) Thus it is only your study which documented the association; could this be due to the synergism or the combined population of the study with HBV and HCV or combined HBV and HCV

BMJ Open: first published as 10.1136/bmjopen-2016-013279 on 23 August 2017. Downloaded from

REVIEWER Sara Tatiana Moreira Human Molecular Genetics Laboratory, Parana Federal University of Technology, UTFPR, Santa Helena, Parana, Brazil. REVIEW RETURNED 15-Aug-2016

GENERAL COMMENTS 1. This research not evaluated the effect of polymorphisms on the clinical progression of the diseases because they have not studied the same individuals over time. It was studied the influence of SNPs in different clinical outcomes. The objective needs to be corrected. 2. Correct the objective of the abstract and add the correct nomenclature for the studied SNPs, including the ID (rs) of each. 5. The study protocol have been performed according to the World Medical Association Declaration of Helsinki? 8. The number of references in the introduction must be reduced. Add references to discussion, when compare with results of similar researchs. 12. Add the limitation discuss to the text.

Title: Effect of interactions among IFN-γ+874, IL-2-330, IL-101082, IL-10-592, and IL-4-589 polymorphisms on the clinical progression of subjects infected with hepatitis B virus and/or hepatitis C virus

Manuscript ID: bmjopen-2016-013279

In this work, the authors found that IL-10-592AC and IL-4- http://bmjopen.bmj.com/ 589CC/CT showed synergistic effect on liver inflammatory injury (P <0.01). While IFN-γ+874AA and IL-2-330TT had synergistic impact (P<0.05), IFN-γ+874AA and IL-10-1082AA had antagonistic effect (P<0.01) on the clinical progression, including asymptomatic HBV and HCV carriers, and chronic hepatitis. IL-2-330TT and IL-10- on September 29, 2021 by guest. Protected copyright. 1082AA synergistically influenced the clinical outcome (P <0.05). IFN-γ+874AA, IL-2-330TT, and IL-10-1082AA interactively affected the clinical outcome as asymptomatic HBV and HCV carriers, chronic hepatitis (P<0.05).

What's more valuable in this work is the statistical method used because, as the authors said, “interleukins and the SNPs of cytokines may not play their roles alone, but interact with each other in establishing the clinical consequences of such patients”. However, the work has a large limitation also reported by the authors themselves, which corresponds to the engagement of patients infected with HBV and HCV. BMJ Open: first published as 10.1136/bmjopen-2016-013279 on 23 August 2017. Downloaded from

Comments:  Pg 2, line 11 - 12 “The mechanism still remains unknown”. The mechanism is not unknown, just is not fully understood.  The number of references in the introduction must be reduced.  Correct the objective: “evaluate the effect of interactions among the polymorphisms of IFN…on the clinical progression.” This research not evaluated the effect of polymorphisms on the clinical progression of the diseases because they have not studied the same individuals over time. It was studied the influence of SNPs in different clinical outcomes.  Add the correct nomenclature for the studied SNPs, including the ID (rs) of each.  The pathogenesis resulting of HCV infection is different from that derived from HBV infection. The lack of homogeneity of the sample originates vacant results, non-specific for HCV neither for HBV infection.  The study protocol have been performed according to the World Medical Association Declaration of Helsinki?  The sample stratification resulted in subgroups with individuals infected by HCV AND HBV, except for a subgroup, composed of individuals infected by HBV http://bmjopen.bmj.com/ AND/OR HCV. Because not follow a rule, the presence of this subgroup may have biased the results.  Pg 13, line 7: herein?  Eleven of the 13 references cited in the discussion refers to

the biological effects of cytokines. Only two references on September 29, 2021 by guest. Protected copyright. involve comparison with other results in the literature. The authors barely discussed their results compared with others of literature, but there are several studies of polymorphisms in cytokine genes x HCV or HBV that should be cited in the discussion.  Include: Discuss limitations of the study, taking into account sources of potential bias or imprecision. Discuss both direction and magnitude of any potential bias.

BMJ Open: first published as 10.1136/bmjopen-2016-013279 on 23 August 2017. Downloaded from

REVIEWER Sanak, Marek Department of Medicine, Medical College REVIEW RETURNED 21-Dec-2016

GENERAL COMMENTS Authors genotyped common variants in IL2, IFNG, IL10 and IL4 genes looking for their interactions with a chronic viral hepatitis B or C outcome. The comparator group were subjects who cleared the infection, chronic infection subjects were categorized according to the presence of active hepatitis symptoms. Most of my comments concern results section since numerous variants of SNP genotypes are not well presented. The main general comment is that the manuscript is disorganized.

Detailed comments: 1. Study groups. What is a difference between group 1 (controls) and group (2) chronic carriers of the virus without hepatitis, if no presence of HCV genome was tested. 2. Methods. All SNPs typed should be referred to using a standard SNP database entries (rs numbers), 3. Results. It is sufficient to discuss only the risk genotype affecting elevated AST. Actually, Authors do not explain if these are the same subjects as named chronic hepatitis group 3. 4. Association with “abnormal AST level” or “abnormal amounts of ALT” does not make much sense because group 2 subjects had also intermittent elevations of transaminases. Moreover, transaminases levels tend to fluctuate between their consecutive measuruments. 5. Gene-gene interactions and the clinical outcome. Certain combinations of IFN, IL10 and IL2 variants cross-verified predicting group 2 or group 3 membership. But the number of individuals with these predicted outcomes represented only a fraction of the total group. No corrections were used for multiple comparisons. http://bmjopen.bmj.com/ 6. Results. Effect of gene-gene interactions on the liver inflammatory injury. The table 4 with results refers to all the study group, however, only in group 3 a liver biopsy was done to evaluate the inflammation. 7. Discussion. Conclusions suggest that the outcome of viral hepatitis depends on the interactions of the genetic variants studied. In fact, all these genetic variants ought to manifest by altered expression of the respective cytokines and eventual changes in the

adaptive immunity response. No attempt to correlate these findings on September 29, 2021 by guest. Protected copyright. functionally with cytokine levels, lymphocyte populations or at least serological response was made.

VERSION 1 – AUTHOR RESPONSE

Reviewer: 1

How did you diagnose the controls who spontaneously cleared HCV or HBV? is there patients who had combined HCV and HBV infection and cleared both?? Answer: there should be under-represent. there are controls who spontaneously cleared HCV or HBV. Because there were data showing the controls were infected with HBV or HCV in plasm-donation in the late 1980s, and were tested negative to HCV or HBV in 2007.

How many patients were infected with HCV, HBV or had combined HBV and HCV within each group?? what is the treatment status of the patients?? BMJ Open: first published as 10.1136/bmjopen-2016-013279 on 23 August 2017. Downloaded from

Answer: There are 37(30.3%)HCV infection, 43(35.2%) HBV infection and 42(34.4%) HBV&HCV infection in ASC group, there are 18(22.2%) HCV infection, 36(44.4%) HBV infection and 27(33.3%) HBV&HCV infection in chronic hepatitis group. (2=2.256, P=0.324). This part was added in the results. The treatment status of the patients was hepatoprotective.

Saxena R, Kaur J. Th1/Th2 cytokines and their genotypes as predictors of hepatitis B virus related hepatocellular carcinoma. World J Hepatol. 2015 Jun 18;7(11):1572-80. mentioned in their review (On associating the IL-2 (-330 T > G) genotypes with HCC progression in HBV infected individuals, we showed that both the TG and GG genotypes remained largely non-significant in HBV chronicity, among controls and carriers [54]. Similarly, the IL-2 (-330 T > G) polymorphism did not appear to modify HBV-HCC risk in the Chinese and American populations[50,77]. On the contrary, a study by Gao et al[78] reported, IL-2-330 TT genotype to be associated with an increased risk of chronic hepatitis, in case of either HBV or HCV or HBV-HCV coinfection in Chinese population (your study)) Thus it is only your study which documented the association; could this be due to the synergism or the combined population of the study with HBV and HCV or combined HBV and HCV Answer: This association could be due to the synergism. This was reported by Bei et al that the interaction between IL-2-330 and IFN-γ-1615 was associated with an increased HCC risk (OR=1.078, 95% CI=1.022-1.136). Asian Pac J Cancer Prev. 2014;15(16):6961-7. The correlation cotent have been added in the discussion.

Reviewer: 2

1. This research not evaluated the effect of polymorphisms on the clinical progression of the diseases because they have not studied the same individuals over time. It was studied the influence of SNPs in different clinical outcomes. The objective needs to be corrected. Answer: the objective of the article has been changed according to the editor in colored text.

2. Correct the objective of the abstract and add the correct nomenclature for the studied SNPs, including the ID (rs) of each. http://bmjopen.bmj.com/ Answer: the nomenclature for the studied SNPs, including the ID (rs) of each are added using colored text in the paper.

5. The study protocol have been performed according to the World Medical Association Declaration of Helsinki? Answer: The study protocol has been performed according to the World Medical Association

Declaration of Helsinki. This content has been added in the Study subjects of MATERIALS AND on September 29, 2021 by guest. Protected copyright. METHODS.

8. The number of references in the introduction must be reduced. Add references to discussion, when compare with results of similar researchs. Answer: the number of references in the introduction is reduced from 17 to 14. We added references to discussion and compare with results of similar research.

12. Add the limitation discuss to the text. Answer: we add the limitation discuss to the text in the discussion.

Reviewer: 3

Authors genotyped common variants in IL2, IFNG, IL10 and IL4 genes looking for their interactions with a chronic viral hepatitis B or C outcome. The comparator group were subjects who cleared the infection, chronic infection subjects were categorized according to the presence of active hepatitis BMJ Open: first published as 10.1136/bmjopen-2016-013279 on 23 August 2017. Downloaded from symptoms. Most of my comments concern results section since numerous variants of SNP genotypes are not well presented. The main general comment is that the manuscript is disorganized.

Detailed comments: 1. Study groups. What is a difference between group 1 (controls) and group (2) chronic carriers of the virus without hepatitis, if no presence of HCV genome was tested. Answer: Group1 included some people as controls who infected with HBV or HCV in the plasm- donation in the late 1980s. and were tested negative to HCV or HBV in 2007. Group 2 were chronic carriers of the virus without hepatitis. In this study, we mainly analyze the interaction among the SNP- SNP, so HCV genome was not tested.

2. Methods. All SNPs typed should be referred to using a standard SNP database entries (rs numbers), Answer: the nomenclature for the studied SNPs, including the ID (rs) of each are added in red color.

3. Results. It is sufficient to discuss only the risk genotype affecting elevated AST. Actually, Authors do not explain if these are the same subjects as named chronic hepatitis group 3. Answer: We don‟t know how to answer this question, for it is not clear what the word “these” means in the question.

4. Association with “abnormal AST level” or “abnormal amounts of ALT” does not make much sense because group 2 subjects had also intermittent elevations of transaminases. Moreover, transaminases levels tend to fluctuate between their consecutive measurements. Answer: Considering of the fluctuation of the ALT and other influencial factors, we tested simultaneously the AST and ALT, and raised the standard of alanine aminotransferase (ALT) to an upper limit of 80U/L (Hsu HY, et al. Interferon-alpha treatment in children and young adults with chronic hepatitis B: a long-term follow-up study in Taiwan. Liver int, 2008,28(9):1288-1297 [PMID: 18397229])

5. Gene-gene interactions and the clinical outcome. Certain combinations of IFN, IL10 and IL2 http://bmjopen.bmj.com/ variants cross-verified predicting group 2 or group 3 membership. But the number of individuals with these predicted outcomes represented only a fraction of the total group. No corrections were used for multiple comparisons. Answer: We did not use multiple comparisons in the study.

6. Results. Effect of gene-gene interactions on the liver inflammatory injury. The table 4 with results refers to all the study group, however, only in group 3 a liver biopsy was done to evaluate the on September 29, 2021 by guest. Protected copyright. inflammation. Answer: Because in group 2, there were no evidence of liver cirrhosis based on the clinical criteria and ultrasound examination, and (iii) normal alanine aminotransferase (ALT), so in group 2 a liver biopsy was not done.

7. Discussion. Conclusions suggest that the outcome of viral hepatitis depends on the interactions of the genetic variants studied. In fact, all these genetic variants ought to manifest by altered expression of the respective cytokines and eventual changes in the adaptive immunity response. No attempt to correlate these findings functionally with cytokine levels, lymphocyte populations or at least serological response was made. Answer: we add the limitation discuss to the text in the discussion.

BMJ Open: first published as 10.1136/bmjopen-2016-013279 on 23 August 2017. Downloaded from

VERSION 2 – REVIEW

REVIEWER Marek Sanak Jagiellonian University Medical College, REVIEW RETURNED 02-Feb-2017

GENERAL COMMENTS Authors answered reviewers questions and introduced necessary changes into revised version of the manuscript. Page 13 line 52 - sentence ending "was homogeneity" requires grammar correction.

VERSION 2 – AUTHOR RESPONSE

Reviewer Name: Marek Sanak Institution and Country: Jagiellonian University Medical College, Poland Competing Interests: None declared Answer: The „Competing Interests‟ has been declared in the 17th page of the paper.

5.Authors answered reviewers questions and introduced necessary changes into revised version of the manuscript. Page 13 line 52 -sentence ending "was homogeneity" requires grammar correction. Answer5:Page 13 line 52-sentence ending “was homogeneity” was a grammar error, it has been corrected to “was homogenous”. http://bmjopen.bmj.com/ on September 29, 2021 by guest. Protected copyright.