PONE-D-19-22071

Association of Single Nucleotide Polymorphisms with Dyslipidemia in Antiretroviral Exposed HIV Patients: A Case-Control Study in a Ghanaian population

PLOS ONE

Dear Mr Acheampong,

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. We would appreciate receiving your revised manuscript by Nov 02 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.

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Kind regards,

Yong-Gang Yao

Academic Editor

PLOS ONE

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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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: No

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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: No

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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: Though obviously suppressing HIV transmission and extending the life expectancy of HIV-infected patients, antiretroviral therapy (ART) also shows some side effects on human health. For example, long-term use of ART is associated with metabolic disturbances, such as dyslipidemia. In this study, the author studied the relationship of gene variants with dyslipidemia in HIV-infected patients receiving ART. Their data suggested that protease inhibitor treatment might be associated with higher levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides [TG], compared with other ART treatment. In addition, four SNPs from four genes (ABCA1, LDLR, APOA5, and DSCAML1) are associated with dyslipidemia in HIV-infected patients receiving ART.

Major issues:

1. In the introduction, to easily make the reader understand the whole paper, the author had better explain why they selected these four SNPs to be studied.

2. The author said “Significantly increased levels of total cholesterol (TC) low-density lipoprotein cholesterol (LDL-C) and triglycerides [TG] were observed in protease inhibitor-based (PI) treated case subjects compared to non-PI-based case subjects” in the abstract. However, the similar presentation is deficient in the result section. Please add the corresponding content in the result section.

3. To make the reader better understand the relationship of different ART with dyslipidemia in HIV-infected patients, the author may as well describe the number of HIV-infected patients who receive PI or NNRTI.

Minor issues:

1. In the first paragraph of the result section, “All but HDL-cholesterol levels were significant levels in the control subjects compared to case subjects [ 1.44±0.33 vs. 0.99±0.53, p<0.0001].” should be “All but HDL-cholesterol levels were significantly lower levels in the control subjects compared to case subjects [ 1.44±0.33 vs. 0.99±0.53, p<0.0001].”

2. In Table 5, the author should clarify the definition of PRR in the sentence “DSCAML1 (T/T) [PRR=11.46(3.65-43.56), p<0.0001)”.

Reviewer #2: In the present study, Acheampong et al analyze the potential association of genetic variation in four different genes with the existence of dyslipidemia in HIV-infected patients on stable cART from Ghana.

Overall Major concern

The main concern of the study is that the design is very confusing. The authors state that the study is a case-control study. However there seems to be some confusion in the groups of selected individuals and their assignment to either cases or controls. In a case-control study, cases are those individual presenting the disease (in this case dyslipidemia) and controls are those not presenting the disease. The objective of the study (as stated by the authors) is to “..investigate the distribution of SNPs in four candidate genes and resultant association with plasma lipid levels in Ghanian HIV-infected patients on HAART”. For this purpose cases should be HIV patients with dyislipidemia and controls HIV patients without dyslipidemia. However, the authors recruit a population of HIV-seronegative adults without dyislipidemia as controls (in the author’s own words: “…alongside age-matched control subjects with no history of HIV, dyslipidemia, hypertension, and diabetes..”). In my opinion this is not the ideal control population as I have explained above. The inclusion of a population of individuals seronegative for HIV (with and without dyslipidemia) would serve to answer the question: Are the SNPs associated with dyslipidemia in the HIV population the same as those in the non-HIV population? However this is not the question that the objective addresses as the authors state (see above). Moreover, thereafter in the results section, the authors report that 18,3% of controls (ie. HIV-seronegative individuals) presented dyslipidemia. Thus there is a contradiction between the results and the inclusion criteria for the control population (“age-matched control subjects with no history of HIV, dyslipidemia, hypertension, and diabetes”). Also, the only data given for the control population is the prevalence of dyslipidemia and the distribution of allelic and genotypes frequencies for the different SNPs, but no data are given about the association of SNPs and dyslipidemia in this control population. Lastly, there is no discussion at all about the findings in the HIV-seronegative (“control”) population. Thus what is the reason to include this “control” population?.

In summary the authors must clarify this and explain the reason to include the “control” (HIV-seronegative individuals) population.

Specific concerns

1.- Introduction:

.- Some references are not the most adequate, for example references #3-5, reference #1 (this reference is very old), reference #19, references #21-22. The authors should change these references for other more appropriate references.

.- The last sentence of the second paragraph (“Specifically, a direct association has been observed between…”) should not be included here. This sentence is about the association between lipid levels and clinical endpoints, but in the previous sentence the authors are commenting about the association between genetic variation and metabolic dysfunction.

-. In the last paragraph the authors state that “…numerous SNP-associated dyslipidemia studies among HIV patients have come from non-African countries [18, 24, 25]”. However this is not true since the reference #24 is a study performed in a cohort of HIV patients from Zimbabwe.

2.- Materials and methods

.- In the section of sample size determination the authors assume an expected proportion of 0.1. Are there any previous study supporting this figure?.

.- In the data collection and biochemical analysis section the authors state that patients with triglycerides levels above 4.52 nmol/L were excluded from the study. The authors should explain to what extent this fact may impose a selection biass on the study population.

3.- Results

.- The proportion of females is much higher that the proportion of males. Is there any specific reason for this? Is this representative of the genre distribution among the HIV population in Ghana?. Moreover, the females proportion in the HIV-infected population is so high that the results obtained regarding the association between SNPs and dyslipidemia do really apply to only the female genre. The authors should discuss this fact and include it as one of the limitations of the study.

.- In table 1, immunovirological data of HIV population should be included (CD4 counts, time on HAART, pre-HAART HIV plasma viremia, etc..).

.- In the paragraph commenting the results given in table 2 the authors state that there was a significant difference (p=0.021) between cases and controls for rs662799 G/G genotype. However this p-value (as it appears on the table) is for the comparison of the frequency of allelic distribution and not of C/C genotype distribution. This is very confusing. Moreover, the authors must also compare the genotypes distribution and not only the allelic distribution and include this data in the table 2. The authors do not explain the reason to compare the allelic and genotypes distribution between HIV-infected and HIV-seronegative population. They do not comment anything about this comparison in the discussion section of the manuscript.

.-Table 3: data for these parameters in the control (HIV-seronegative) population should be given.

.- The way data are given on table 4 is somewhat confusing and not easy to quickly understand. A more understandable way to give the data would be to report the proportion of dyslipidemia in each group of patients according to the different SNPs genotypes. Moreover, when commenting the results given in this table, the authors state that “subjects with T/T genotype for LDLR polymorphism had a prevalence of dyslipidemia 3.46 times greater than non-dyslipidemic subjects.” Clearly this sentence makes no sense. Authors should pay more attention to the writing, since there are several errors either because a word is missing or because the word is not appropriate.

.- Data given in table 4 for HIV population should also be given for HIV-seronegative subjects.

.- Table 5: the authors report results from 3 different models. In each model the number of covariates differs, increasing from model 1 to model 3. Model 3 is the that includes the highest number of covariates and thus this is the only model that should be shown in table 5. In this table, the authors should include the definition of the terms: “Hypercholesterolemia”, “Hypertriglyceridemia”, “Low HDL-C”, and “High LDL-C”.

.- A table with the results of the multivariate logistic regression using dyslipidemia as the outcome variable should be included in the manuscript.

.-Supplementary data: a table comparing different characteristics between PI-base and NNRTI-based HAART regimens is shown as supplementary data. However there is no mention to this data in the text of the manuscript. The authors should briefly report in the text the findings of this supplementary data.

4.- Discussion

.- In the first sentence of the second paragraph the authors state that “the presence of….. among our study participants were very low..”Do the authors refer to the overall population of subjects included (HIV and HIV-seronegative together) of specifically to the HIV population. It is interesting that the prevalence of homozygous genotypes for the minor alleles (for ABCA1, DSCAML1 and APOA5 SNPs) is higher in HIV patients than in HIV-seronegative subjects, but the authors do not comment anything about this. They should discuss about this finding.

.- In the second sentence of the fifth paragraph the authors state that “This observation could be explained by the observation that almost half of the subjects carried the ABCA1 (rs2066714) G/G or A/G mutant……”. This is interpretation is incorrect since G/G and A/G carriers represented 36.6% of the population and this figure is far from “almost half”.

.- In the fourth sentence of the fifth paragraph the authors state that “In addition, Pollin et al..”In my opinion the sentence should start with “In contrast..” because the results of Pollin et al are different from the results reported in this manuscript.

.- In the first sentence of the sixth paragraph the word “homologous” has no sense at all. It must be changed to “homozygous”. Also in this same paragraph the sentence “The rs662799 is a SNP in the APOA5 gene” is not necessary since this has been already explained before throughout the text.

English grammar comment: I strongly recommend the manuscript to be revised by a native English-speaker.

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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". If this link does not appear, there are no attachment files to be viewed.]

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PONE-D-19-22071R1

Association of Single Nucleotide Polymorphisms with Dyslipidemia in Antiretroviral Exposed HIV Patients in a Ghanaian population

PLOS ONE

Dear Mr Acheampong,

Thank you for submitting your revised 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 second revised version of the manuscript that addresses the points raised during the review process. Please pay attention to the issues raised by the Reviewer #2, and properly answer her/his comments and revise the main text.

We would appreciate receiving your revised manuscript by Jan 13 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,

Yong-Gang Yao

Academic Editor

PLOS ONE

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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)

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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: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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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: No

Reviewer #2: Yes

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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: No

**********

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: The author has addressed all my questions. I don't have other major concerns. However, there is still a few minor issues. For example, in the introduction, "Low-density lipoprotein receptor (LDLR), positioned on chromosome 19p13.2 plays a significant role lipoprotein metabolism by mediating the uptake of cholesterol through

the binding and subsequent cellular uptake of apolipoprotein-E and B- constituting lipoproteins." should be "Low-density lipoprotein receptor (LDLR), positioned on chromosome 19p13.2 plays a significant role in lipoprotein metabolism by mediating the uptake of cholesterol through the binding and subsequent cellular uptake of apolipoprotein-E and B- constituting lipoproteins." So the author needs to proofread the whole manuscript carefully.

Reviewer #2: The revised version of the manuscript by Acheampong et al is still very far from being suitable for publication, for the next reasons:

1.- The authors did not satisfactorily answer the overall major concern regarding the design of the study and the rationale to include HIV-seronegative individuals. This is a study about the influence of several SNPs in the presence of dyslipidemia in HIV-patients on HAART. So, unless there is a very clear and strong reason as I explained in my first revision, HIV-seronegative individuals must be excluded from the study.

I strongly recommend the authors to exclude the 104 HIV-seronegative individuals from the study and reanalyze the data again only with the 289 HIV-patients.

2.- The design of the study is not clear at all and authors must clarify this. Is this a case-control study? (in my opinion it is). I already explained in my first revision who are the cases and who are the controls in a case/control study. If the authors do not consider that this is a case/control study, then the use of the terms “study groups”; “HIV+Dys+”; “HIV+Dys-“ is OK but all mention to “cases” and “controls” must be deleted from the manuscript.

I strongly recommend the help of a statistician.

3.- The way data are presented in tables 4 and 5 (logistic regression with diyslipidemia and with individual lipid abnormalities) and in figures 2 and 3, is incomprehensible. As the authors explain, the odds ratios shown in these tables and figures are referred to the “control” HIV-seronegative population. This is very difficult to understand. The clearest way to show evidence for association between the genotype of the different SNPs and the existence of dyslipidemia, is to calculate the odds ratios of having dyslipidemia in carriers of the minor homozygous genotype for each of the SNPs, taking the other individuals (carriers of the major homozygous and of the heterozygote genoypes) as reference (OR=1).

4.- The manuscript is still plagued with grammatical errors, sentences difficult to understand and words not appropriate, etc…

I strongly recommend that the whole manuscript is revised by a native English speaker

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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: 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". 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.

Association of single nucleotide polymorphisms with dyslipidemia in antiretroviral exposed HIV patients in a Ghanaian population:  a case-control study

PONE-D-19-22071R2

Dear Dr. Acheampong,

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.

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.

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With kind regards,

Yong-Gang Yao

Academic Editor

PLOS ONE