Causal relationships between lipid and glycemic levels in an Indian population: A bidirectional Mendelian randomization approach

Tripti Agarwal; Tanica Lyngdoh; Frank Dudbridge; Giriraj Ratan Chandak; Sanjay Kinra; Dorairaj Prabhakaran; K. Srinath Reddy; Caroline L. Relton; George Davey Smith; Shah Ebrahim; Vipin Gupta; Gagandeep Kaur Walia

doi:10.1371/journal.pone.0228269

Peer Review History

Original SubmissionAugust 6, 2019
30 Oct 2019 Decision Letter - Paolo Magni, Editor PONE-D-19-22133 Causal relationships between lipid and glycemic levels in an Indian population: A bidirectional Mendelian randomization approach PLOS ONE Dear Dr Walia, 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 Dec 14 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. 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Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: 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 ****** 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: This interesting study by Agarwal et al. examined the causal relationships between plasma lipids and glycemic traits in 4,900 participants of the Indian Migration Study using bidirectional Mendelian randomization. The authors identified 3 gene variants associated with circulating triglycerides and developed a weighted genetic risk score for triglycerides that correlated with fasting plasma insulin and with markers of insulin sensitivity and beta cell function. These findings support a causal impact of hypertriglyceridemia in the early alterations of glucose metabolism that characterize the pathogenesis of type 2 diabetes. The question addressed by this trial is relevant from a clinical and physiological perspective. The effort to avoid potential confounders and limit the biases typical of MR studies is laudable and is a strength of the study. The manuscript is overall well written. Some issues and limitations listed below should be carefully considered, along with those already mentioned by the authors in the Discussion. Major comments: 1. The lack of more accurate indexes of glucose tolerance, insulin sensitivity and beta cell function provided by dynamic metabolic tests is a major limitation of the study. The HOMA-b in particular is a poor marker of beta cell function for at least two reasons: it does not provide information on glucose-stimulated insulin secretion and it does not take into account potential differences in insulin clearance, which is known to be associated with plasma lipids and significantly affected by the ethnic background. The second issue might be circumvented using fasting levels of C peptide instead of insulin, if available. 2. The choice of the different confounders for lipids and glycemic traits is somehow questionable. It is not clear why the BMI was included in mixed linear models only for glycemic outcomes and not for lipids, since there is a clear correlation between body weight and plasma lipid profile. Moreover, triglyceride levels seem to be influenced more by the carbohydrate intake than by the fat intake (Samaha FF et al, NEJM 2003, doi: 10.1056/NEJMoa022637). Triglyceride and cholesterol levels are also influenced by the alcohol intake, which was available in the Indian Migration Study (Table 1). 3. The main finding of this study, which is the impact of triglycerides on glycemic traits and especially on beta cell function, has been extensively examined in recent years (Seghieri M et al., Diabetes Metab 2017, doi: 10.1016/j.diabet.2017.04.010). The authors should carefully revise the literature and discuss concordant and conflicting results. The genetic susceptibility for higher triglycerides has been associated with a paradoxically lower risk for type 2 diabetes (Klimentidis YC et al, PLoS Genet 2015, doi: 10.1371/journal.pgen.1005204; White et al. JAMA Cardiol 2016, doi: 10.1001/jamacardio.2016.1884; Ahmad S et al, Arterioscler Thromb Vasc Biol 2019, doi: 10.1161/ATVBAHA.118.311562). With regards to insulin secretion, circulating triglycerides have been reported to have a positive effect on beta cell function, regardless of the ethnic background, in some (Tricò D et al, Diabetes Obes Metab 2018, doi: 10.1111/dom.13467) but not all studies (Bedogni G et al, Endocr Connect 2019, 10.1530/EC-19-0333). 4. The number of SNPs available and eventually selected for the development of each genetic instrument was consistently lower compared with similar Mendelian randomization analyses (see for instance the above-mentioned studies). 5. At the end of the Discussion it is stated that “an increase in triglyceride levels can be a factor for […] pancreatic beta cell dysfunction”, which is apparently in contrast with the current study findings. Although I personally agree that, in the long term, insulin hypersecretion may lead to beta cell dysfunction, this study shows a positive rather than negative effect of triglyceride on the HOMA-b. 6. The Introduction is rather long and might benefit from being shortened and more focused. Appropriate references should be provided in the second paragraph. 7. The statement “The study was adequately powered…” is too vague. The authors should provide the actual power of the study. Minor comment 1. I would change “p=0.000” with “p<0.001” in supplementary tables ****** 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 [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. 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Revision 1
14 Dec 2019 Author Response Dear Editor Thank you for informing your decision and sending the comments and suggestions from the reviewers which have helped in improving the manuscript. We have responded to each comment below in blue coloured text and have revised the manuscript accordingly. Reviewer #1: This interesting study by Agarwal et al. examined the causal relationships between plasma lipids and glycemic traits in 4,900 participants of the Indian Migration Study using bidirectional Mendelian randomization. The authors identified 3 gene variants associated with circulating triglycerides and developed a weighted genetic risk score for triglycerides that correlated with fasting plasma insulin and with markers of insulin sensitivity and beta cell function. These findings support a causal impact of hypertriglyceridemia in the early alterations of glucose metabolism that characterize the pathogenesis of type 2 diabetes. The question addressed by this trial is relevant from a clinical and physiological perspective. The effort to avoid potential confounders and limit the biases typical of MR studies is laudable and is a strength of the study. The manuscript is overall well written. Some issues and limitations listed below should be carefully considered, along with those already mentioned by the authors in the Discussion. Response: We are thankful to the reviewer for recognizing the importance of examining causal relationships between cardiometabolic traits and dour efforts to address the possible limitations of Mendelian randomization (MR) design. We have now revised the manuscript according to the below mentioned comments and suggestions. Major comments: 1. The lack of more accurate indexes of glucose tolerance, insulin sensitivity and beta cell function provided by dynamic metabolic tests is a major limitation of the study. The HOMA-b in particular is a poor marker of beta cell function for at least two reasons: it does not provide information on glucose-stimulated insulin secretion and it does not take into account potential differences in insulin clearance, which is known to be associated with plasma lipids and significantly affected by the ethnic background. The second issue might be circumvented using fasting levels of C peptide instead of insulin, if available. Response: We agree with the reviewer that glycemic indices have evolved over time and currently more precise and clinically relevant markers are available. However, the present analyses were done utilizing the available de-identified data from Indian Migration Study which was conducted during 2005-2008 when only these glycemic levels (Fasting glucose, fasting insulin, HOMA-IR and HOMA-β) were generally collected in large epidemiological surveys. 2. The choice of the different confounders for lipids and glycemic traits is somehow questionable. It is not clear why the BMI was included in mixed linear models only for glycemic outcomes and not for lipids, since there is a clear correlation between body weight and plasma lipid profile. Moreover, triglyceride levels seem to be influenced more by the carbohydrate intake than by the fat intake (Samaha FF et al, NEJM 2003, doi: 10.1056/NEJMoa022637). Triglyceride and cholesterol levels are also influenced by the alcohol intake, which was available in the Indian Migration Study (Table 1). Response: We would like to thank the reviewers to point out the other potential confounders that we missed in our analyses. As per the suggestion, we examined all the possible confounders that were available in the study and have now revised our analyses after including BMI uniformly in model-1 for both glycemic and lipid outcomes along with age, gender, location and site; and additional confounders like carbohydrates, alcohol use and tobacco smoking in model-2 along with previously included confounders for dietary factors and physical activity. 3. The main finding of this study, which is the impact of triglycerides on glycemic traits and especially on beta cell function, has been extensively examined in recent years (Seghieri M et al., Diabetes Metab 2017, doi: 10.1016/j.diabet.2017.04.010). The authors should carefully revise the literature and discuss concordant and conflicting results. The genetic susceptibility for higher triglycerides has been associated with a paradoxically lower risk for type 2 diabetes (Klimentidis YC et al, PLoS Genet 2015, doi: 10.1371/journal.pgen.1005204; White et al. JAMA Cardiol 2016, doi: 10.1001/jamacardio.2016.1884; Ahmad S et al, Arterioscler Thromb Vasc Biol 2019, doi: 10.1161/ATVBAHA.118.311562). With regards to insulin secretion, circulating triglycerides have been reported to have a positive effect on beta cell function, regardless of the ethnic background, in some (Tricò D et al, Diabetes Obes Metab 2018, doi: 10.1111/dom.13467) but not all studies (Bedogni G et al, Endocr Connect 2019, 10.1530/EC-19-0333). Response: We would like to thank the reviewer for sharing the above studies. We have now included these studies along with other related recent literature in the introduction and discussion sections. 4. The number of SNPs available and eventually selected for the development of each genetic instrument was consistently lower compared with similar Mendelian randomization analyses (see for instance the above-mentioned studies). Response: We agree with the reviewer that the recent MR studies utilize the allelic scores based on more number of variants compared to the present study. The present study was a small Masters thesis work based on available de-identified genotype and phenotype data. The genotype data on only 9 lipids and 35 glycemic loci was available in the study and out of these only those variants were included in the instrument that fulfilled Hardy-Weinberg Equilibrium and the three MR assumptions. We hope that the reviewers will agree that despite the lower number of variants, the genetic instruments were very robust and did provide very insightful findings. We would also like to state that there are very few MR studies from low resource settings like India and even those are based on smaller sample size and weaker instruments. However, we do understand that despite the various attempts, the present study has some limitations which have been discussed in the manuscript. Based on these preliminary results, our team is now involved in larger studied for examining causal relationships between various cardiometabolic traits utilizing more robust genetic instruments. 5. At the end of the Discussion it is stated that “an increase in triglyceride levels can be a factor for […] pancreatic beta cell dysfunction”, which is apparently in contrast with the current study findings. Although I personally agree that, in the long term, insulin hypersecretion may lead to beta cell dysfunction, this study shows a positive rather than negative effect of triglyceride on the HOMA-b. Response: We would like to thank the reviewer for pointing this mistake. We have now revised the text accordingly. 6. The Introduction is rather long and might benefit from being shortened and more focused. Appropriate references should be provided in the second paragraph. Response: As per reviewer suggestions, we have revised the introduction section and have provided relevant references. 7. The statement “The study was adequately powered…” is too vague. The authors should provide the actual power of the study. Response: We have now provided the actual power of the study to examine the causal association using instrument variable regression in the methods section. Minor comment 1. I would change “p=0.000” with “p<0.001” in supplementary tables Response: We have revised the tables as suggested by the reviewer. Attachments Attachment Submitted filename: Responses to Reviewers.docx https://doi.org/10.1371/journal.pone.0228269.r002
13 Jan 2020 Decision Letter - Paolo Magni, Editor Causal relationships between lipid and glycemic levels in an Indian population: A bidirectional Mendelian randomization approach PONE-D-19-22133R1 Dear Dr. Walia, 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. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. 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With kind regards, Paolo Magni 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 authors have addressed my comments in a satisfactory manner. I appreciate particularly the inclusion of additional potential confounders in the analyses, and the work put by the authors in their rebuttal and revisions. Please note that authors' first and last names are inverted in some of the new references. In my opinion, this issue may be addressed by the authors or the Editorial office after acceptance, without further revisions. ****** 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: Yes: Domenico Trico https://doi.org/10.1371/journal.pone.0228269.r003
Formally Accepted
21 Jan 2020 Acceptance Letter - Paolo Magni, Editor PONE-D-19-22133R1 Causal relationships between lipid and glycemic levels in an Indian population: A bidirectional Mendelian randomization approach Dear Dr. Walia: 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. Paolo Magni Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0228269.r004

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