PLTP Variants rs6065904 and rs378114 Are Associated with Acute Coronary Syndrome Risk and Altered Glucose and Triglyceride Levels in a Mexican Population

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors investigated whether the rs6065904 and rs378114 polymorphisms of the PLTP gene are associated with the risk of acute coronary syndrome, as well as with glucose and triglyceride levels, in a Mexican Mestizo population. Overall, this is an interesting study with a relatively large sample size; however, the concerns regarding group comparability, possible treatment-related confounding, and overinterpretation should be addressed before the manuscript can be considered for publication.

1.The title should be revised for precision. “Triglycerides Levels” should be corrected to “Triglyceride Levels.” The title may also be improved by specifying the study population more clearly.

2.The comparability of the case and control groups is a major concern. The ACS group and the control group were recruited during substantially different periods, which may introduce temporal bias. In addition, the groups differ significantly in baseline characteristics such as sex, hypertension, and smoking status. These issues should be discussed more explicitly as potential sources of residual confounding.

3.The lipid-related findings should be interpreted more cautiously. Since the authors note that lower cholesterol levels in ACS patients may be related to acute statin administration, the measured lipid values may not reflect baseline metabolic status. The timing of blood sampling and the effect of lipid-lowering treatment should therefore be clarified.

4.The statistical reporting should be clarified. The use of logistic regression and Bonferroni correction is appropriate; however, the manuscript should clarify whether multiple-testing correction was consistently applied across all genotype–phenotype analyses, particularly for glucose and triglyceride comparisons.

5.The conclusions should be slightly toned down. The data support a statistical association between the PLTP polymorphisms and ACS risk, but the mechanistic interpretation appears stronger than the results can fully justify. It would be more appropriate to state that the underlying biological pathways remain uncertain.

6.The presentation of tables, figures, and abbreviations should be improved. Baseline data in Table 1 should be presented more clearly, and the number of subjects in each genotype subgroup should be provided in the relevant figures or legends. In addition, all abbreviations should be defined at first mention and used consistently throughout the manuscript.

Author Response

Thank you very much for your comments and suggestions to Manuscript ID biomedicines-4513105

 

 

We would like to thank the Reviewer for their comments; they have helped to improve the manuscript

 

 

Comments to the Author:

 

 

Reviewer #1: Comments and Suggestions for Authors

 

The authors investigated whether the rs6065904 and rs378114 polymorphisms of the PLTP gene are associated with the risk of acute coronary syndrome, as well as with glucose and triglyceride levels, in a Mexican Mestizo population. Overall, this is an interesting study with a relatively large sample size; however, the concerns regarding group comparability, possible treatment-related confounding, and overinterpretation should be addressed before the manuscript can be considered for publication.

 

1. The title should be revised for precision. “Triglycerides Levels” should be corrected to “Triglyceride Levels.” The title may also be improved by specifying the study population more clearly.

 

Answer: We have corrected the grammatical mistake and refined the title specifying the specific population included in the study. We substitute the former title “The rs6065904 and rs378114 Polymorphisms of PLTP Gene Are Associated with Risk of Acute Coronary Syndrome, as well as with Glucose and Triglycerides Levels.” BY “PLTP Variants rs6065904 and rs378114 Are Associated with Acute Coronary Syndrome Risk and Altered Glucose and Triglyceride Levels in a Mexican Population.”

 

2.The comparability of the case and control groups is a major concern. The ACS group and the control group were recruited during substantially different periods, which may introduce temporal bias. In addition, the groups differ significantly in baseline characteristics such as sex, hypertension, and smoking status. These issues should be discussed more explicitly as potential sources of residual confounding.

 

Answer: We understand the Reviewer’s concern, biological samples were in fact collected asynchronously and in different periods. Despite the gap between the collection of biological samples from patients and controls, quality control program at our institution's laboratories ensure the accuracy of results over time. It is important to emphasize that plasma samples were analyzed in the clinical laboratory on the day of collection; frozen samples were not used for routine chemical chemistry determinations. In contrast, DNA analysis from white blood cells was performed on frozen samples as indicated in the manuscript; to minimize the likelihood of genotyping mistakes, DNA integrity was assessed prior to determining the polymorphisms. We included in the new version of the manuscript the following paragraphs to highlight the comparability of the patient and control samples: “Plasma samples were analyzed in the clinical laboratory on the day of collection for both, ACS patients and healthy controls; frozen samples were not used for routine chemical chemistry determinations.”  and “To minimize the likelihood of genotyping mistakes, DNA integrity was assessed prior to determining the polymorphisms.” In 2.2 Laboratory analysis and 2.3 Genetic Analysis subsections, respectively.

We have also included this issue in the Discussion section as follows:

First, samples from ACS patients and controls were independently collected in different periods that may introduce temporal bias; laboratory quality controls and DNA integrity analysis helped reduce this temporal factor, but it cannot be assumed that it was completely excluded. Lines 264 to 267

     

On the other hand, we are aware of the baseline differences between patients and controls. Consequently, we treated these variables as confounding variables when necessary, and accounted for the association analyses of the polymorphisms. In this context, the association of the rs6065904 A/G, and rs378114 T/C SNPs with susceptibility to development ACS were performed using the inheritance models indicated in the manuscript by multivariable logistic regression adjusted for sex, age, BMI, hy-pertension, smoking status, and T2DM.

In order to emphasize this point, we modified the following sentence: “To evaluate the association between PLTP gene polymorphisms and ACS susceptibility, logistic regression analysis was conducted under multiple inheritance models (additive, codominant, dominant, overdominant/heterozygous, and recessive) [22,23]” BY “To evaluate the association between rs6065904 A/G, and rs378114 T/C SNPs of the PLTP gene with the susceptibility to development ACS, we used the following inheritance models: additive (major allele homozygotes versus heterozygotes versus minor allele homozygotes), codominant (major allele homozygotes versus minor allele homozygotes), dominant (major allele homozygotes versus heterozygotes + minor allele homozygotes), over-dominant (heterozygotes versus major allele homozygotes + minor allele homozygotes), and recessive (major allele homozygotes + heterozygotes versus minor allele homozygotes) using logistic regression [22,23]. All models were evaluated using multivariable logistic regression adjusted for sex, age, BMI, hypertension, smoking status, and type 2 diabetes mellitus.” In 3.2. Association of PLTP SNPs with ACS subsection.

 

3.The lipid-related findings should be interpreted more cautiously. Since the authors note that lower cholesterol levels in ACS patients may be related to acute statin administration, the measured lipid values may not reflect baseline metabolic status. The timing of blood sampling and the effect of lipid-lowering treatment should therefore be clarified.

 

Answer: We agree with the Reviewer’s point of view, statins and the moment of blood sampling deeply influences cholesterol levels. For this reason, the potential relationship between PLTP gene polymorphisms and plasma lipid levels were analyzed only in the ACS patient group; we avoided to including the control group for these analyses or the comparisons between groups. Of course, we assumed that the lipid effect of statins or the acute state had the same effect in all ACS patients. Therefore, we also recognized this assumption as a weakness of the study in the discussion section as follows:

Also, PLTP polymorphisms associations with lipid levels have some limitations; lower levels of cholesterol in ACS patients than in controls clearly indicate an effect of the ACS-associated acute state and the use of statins as stated before. For this reason, the analysis of the PLTP gene polymorphisms on plasma lipid levels was performed only in ACS patients, assuming that the effect of statins and acute state is homogeneous in all the included individuals, but such assumption cannot be verified. Lines 269 to 275.

 

4.The statistical reporting should be clarified. The use of logistic regression and Bonferroni correction is appropriate; however, the manuscript should clarify whether multiple-testing correction was consistently applied across all genotype–phenotype analyses, particularly for glucose and triglyceride comparisons.

 

Answer: We apologize for this omission. We performed Kruskal–Wallis test, followed by post hoc Mann–Whitney U tests for the analyses of the PLTP SNPs associations with plasma lipid and glucose levels always corrected by Bonferroni test.

To be clearer on this point, we modified the following sentence “…the Kruskal–Wallis test, followed by post hoc Mann–Whitney U tests.”  BY “…the Kruskal–Wallis test, followed by post hoc Mann–Whitney U tests. p-values were corrected by Bonferroni test (pC).” In subsection 2.5 Association of PLTP SNPs with Plasma Lipid and Glucose Levels.

Furthermore, in the footnotes of Figures 1 and 2, we substituted the phrase "p-values ​​< 0.05 were considered statistically significant" to "p-values ​​were corrected using the Bonferroni test (pC)."

In addition, the “p” is changed by “pC”, in the Figures 1 and 2.

 

5. The conclusions should be slightly toned down. The data support a statistical association between the PLTP polymorphisms and ACS risk, but the mechanistic interpretation appears stronger than the results can fully justify. It would be more appropriate to state that the underlying biological pathways remain uncertain.

 

Answer: We agree with the reviewer´s comment, our data only supports a statical association of the PLTP SNPs with the susceptibility to development ACS, and with plasma glucose and triglyceride levels. In the discussion section, we recognized that this study did not demonstrate the molecular mechanisms that explain the PLTP contribution to the ACS physiopathology as follows:

“…, this study only showed a statistical relationship between genetic background and cardiovascular risk of developing ACS but did not establish the molecular mechanisms connecting PLTP variants to lipid and glucose shifts and ACS risk, that remain to be elucidated.” Lines 275 to 278

  

We also modified the sentence “The rs6065904 A/G and rs378114 T/C polymorphisms of the PLTP gene are significantly associated with ACS risk, as well as triglyceride and glucose levels in Mexican Mestizos..” BY “Our findings showed that minor allele frequencies of the rs6065904 A/G and rs378114 T/C polymorphisms of the PLTP gene are associated with susceptibility to developing ACS, and with triglyceride and glucose levels in the Mexican population.” In the 5. Conclusion section.

 

6.The presentation of tables, figures, and abbreviations should be improved. Baseline data in Table 1 should be presented more clearly, and the number of subjects in each genotype subgroup should be provided in the relevant figures or legends. In addition, all abbreviations should be defined at first mention and used consistently throughout the manuscript.

Answer: Following Reviewer’s suggestion, in the new version of the manuscript, the Baseline data of Table 1 has been improved. Additionally, all abbreviations have been carefully defined throughout the revised manuscript. We would like to emphasize that the tables and figures format we used adhere to the journal's guidelines and there was a page break after the figure, so the figure titles and legends appeared at the beginning of the next page. We have organized the text in order to maintain the figure legends just below the figures to be clearer. Moreover, the number of individuals carrying each genotype is indicated in parentheses in the horizontal axis of the figures. The significant p-values for the comparison between genotypes are represented with the horizontal bars above the boxes in each plot.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Overall, the manuscript is well written and presents an interesting study. However, there are several comments and suggestions that should be addressed to further improve the clarity and quality of the manuscript. Please refer to the attached file for the detailed comments and suggestions. Thank you. 

 

Comments for author File: Comments.pdf

Author Response

Thank you very much for your comments and suggestions to Manuscript ID biomedicines-4513105

 

 

We would like to thank the Reviewer for their comments; they have helped to improve the manuscript

 

Comments to the Author:

 

Reviewer #2: Comments and Suggestions for Authors

 

1. Title should be revised for clarity and grammatical accuracy. The phrase “as well as with” seems awkward and does not clearly convey the two outcomes being investigated. Suggestion: The rs6065904 and rs378114 Polymorphisms of PLTP Gene Are Associated with Risk of Acute Coronary Syndrome, Glucose and Triglycerides Levels.

 

Answer: We agree with the reviewer´s comment. In the new version of the manuscript, we have modified and specified the study population to refine the title, as follows: “PLTP Variants rs6065904 and rs378114 Are Associated with Acute Coronary Syndrome Risk and Altered Glucose and Tri-glyceride Levels in a Mexican Population.”

 

 

2. Introduction.

Line 58–59: The references should be cited individually for each disease/condition rather than grouped at the end of the sentence. For example: “…contribute to susceptibility to coronary artery disease (CAD) [4], carotid artery disease [9], hypercholesterolemia [10], T2DM [11], and dyslipidemia [12–14].” Please ensure that each reference specifically supports the corresponding statement. This would improve the accuracy and clarity of the information presented.

 

Answer: Following the reviewer’s recommendation, we ensure that each reference specifically supports the corresponding statement. In this context, we changed the phrase “… contribute to susceptibility to coronary artery disease (CAD), carotid artery disease, hypercholesterolemia, T2DM, and dyslipidemia [4,9-14].” BY “…contribute to susceptibility to hypercholesterolemia [4], coronary artery disease (CAD) [9,10], T2DM [11], and with alterations in fatty acids and the plasma lipid profile [12-14].” Lines 60 to 62, In the introduction section.

 

3. Method:

- Line 73: Please state what is the international guideline. Is it European Society of Cardiology (ESC)?

 

Answer: The diagnosis of ACS was made according with European Society of Cardiology guidelines. We therefore changed the phrase “The diagnosis of ACS was made followed international guidelines [15,16]” BY “The diagnosis of ACS was made following European Society of Cardiology (ESC) guidelines [15,16];…” Lines 75 to 76 of the materials and methods section.

 

- Line 78: Please define GEA

 

Answer: The GEA Mexican study is defined as Genetics of Atherosclerosis Disease (GEA) Mexican study. The GEA study investigates the genetic factors associated with premature coronary artery disease (CAD), atherosclerosis, and other coronary risk factors in the Mexican population. In this context we changed the sentence “The comparison group included 999 healthy participants without personal or family history of cardiovascular diseases (CVD) or heart failure, recruited between June 2008 and January 2013 for the GEA Mexican study at the same institution [17].” BY “The comparison group included 999 healthy participants of the Genetics of Atherosclerosis Disease (GEA) Mexican study, without personal or family history of cardiovascular diseases (CVD) or heart failure, recruited between June 2008 and January 2013 [17].” Lines 79 to 82 of the Material and methods section.

 

- Line 79: You stated that the exclusion criteria for the control group (“Exclusion criteria for controls comprised renal…”). However, the exclusion criteria for the ACS patient group are not clearly described. Please specify whether any exclusion criteria were applied to the ACS patients. If so, please clearly state these criteria in the Methods section.

 

Answer: According with the reviewer’s comment, we specified the exclusion criteria of ACS patients as follows: “Exclusion criteria for patients with ACS comprised heart failure, liver disease, thyroid dysfunction, cancer, infectious diseases, or autoimmune disorders.” Lines 78 to 79 of the Material and methods section.

 

- Lines 111–114: You have cited references [22,23] after describing the use of multiple inheritance models. Please clarify the rationale for applying these genetic models and the references [22,23] to support the statistical methodology used.

 

Answer: The association of the rs6065904 A/G, and rs378114 T/C SNPs with susceptibility to development ACS are perform under the following inheritance model (additive, codominant, dominant, overdominant/heterozygous, and recessive). These models make it possible to determine and compare which genotype is associated with the pathology. In this context, the inheritance models are structured as follows: additive (major allele homozygotes versus heterozygotes versus minor allele homozygotes), codominant (major allele homozygotes versus minor allele homozygotes), dominant (major allele homozygotes versus heterozygotes + minor allele homozygotes), over-dominant (heterozygotes versus major allele homozygotes + minor allele homozygotes), and recessive (major allele homozygotes + heterozygotes versus minor allele homozygotes) using logistic regression, adjusting for cardiovascular risk factors. The usefulness of such models in statistical population analyses are described in the indicated chapters of the references 22 and 23.

In order to clarify this point, we changed the following sentence “To evaluate the association between PLTP gene polymorphisms and ACS susceptibility, logistic regression analysis was conducted under multiple inheritance models (additive, codominant, dominant, overdominant/heterozygous, and recessive) [22,23]” BY “To evaluate the association between rs6065904 A/G, and rs378114 T/C SNPs of the PLTP gene with the susceptibility to development ACS, we used the following inheritance models: additive (major allele homozygotes versus heterozygotes versus minor allele homozygotes), codominant (major allele homozygotes versus minor allele homozygotes), dominant (major allele homozygotes versus heterozygotes + minor allele homozygotes), over-dominant (heterozygotes versus major allele homozygotes + minor allele homozygotes), and recessive (major allele homozygotes + heterozygotes versus minor allele homozygotes) using logistic regression [22,23].”  Lines 118 to 127 of Statistical analysis section.

 

4. Results:

- Table 1: Please revise the presentation of the categorical variables. For hypertension, smoking, and diabetes mellitus, only the “Yes” category is reported. Please clarify whether the reported values represent the number (%) of participants with the respective condition and provide the corresponding “No” category where appropriate. Alternatively, please indicate in the table footnote that only the presence (“Yes”) of each condition is being reported and explain why the “No” category is not presented in the table.

 

Answer: Following the Reviewer’s recommendation, in the new version of the manuscript, the Baseline data of the Table 1 were improved, including both, the percentage and the number of subjects (n) that had the specific condition and those that did not.

 

- Figure 1: I suggest indicating the statistical significance using significance symbols (e.g., asterisk) rather than displaying the exact p-value (e.g., p < 0.0001) directly on the figure. The corresponding significance symbols can be clearly defined in the figure legend.

Answer: We thank the reviewer for her/his suggestion. Unfortunately, the format for tables and figures is stipulated in the journal's guidelines. According to these guidelines, the figure title and captions should appear below the figure, and the significant p-values for the comparison between genotypes should represented within the plot.

5. Discussions:

- Line 218-220: you mentioned that: “ Jarvik et al. observed that the rs6065904 A and rs378114 T alleles were protective against carotid artery disease in Caucasian populations [5,25].” However, the no 5 reference does not refer to Jarvik. Please check and amend it accordingly.

 

Answer: We apologize for this mistake; the correct reference is the number 25. The corresponding modification was included in Line 249 of the Discussion section.

 

- Line 225-227: “Genome-wide association and meta-analysis studies also link the minor alleles of the rs6065904 and rs378114 PLTP SNPs with altered lipid profiles and risk of cardiovascular diseases” appears to be broader than what is directly supported by the cited references [13,14,27,28]. Please verify that each cited study specifically reports associations involving rs6065904 and/or rs378114 and clearly distinguish evidence relating to lipid traits from evidence relating to cardiovascular disease risk. Please cite appropriate references individually for each specific association.

 

Answer: To clarify this point, we verified each reference and ensured that each one specifically supports the corresponding claim. In this context we changed the following phrase “Genome-wide association and meta-analysis studies also link the minor alleles of the rs6065904 and rs378114 PLTP SNPs with altered lipid profiles and risk of cardiovascular diseases [13,14,27,28].” BY “Genome-wide association and meta-analysis studies also link the rs6065904 and rs378114 PLTP SNPs with altered lipid profiles [12-14] and risk of cardiovascular diseases [10,27,28].” Lines 254 to 256 of the Discussion section.

 

- Line 241: You have acknowledged that the molecular mechanisms linking PLTP variants to lipid/glucose alterations and ACS risk were not established. Please briefly discuss potential future studies connecting with relevant inflammatory or vascular signaling pathways.

 

Answer: We sincerely thank the reviewer for this insightful comment. We fully agree that proposing concrete functional mechanisms strengthens the potential value of our findings. We have expanded the Discussion section to propose some specific experimental paradigms. We suggest future studies focusing on inflammatory cascades by assessing whether these PLTP variants alter cell membrane lipid raft composition, thereby modulating downstream inflammation signaling activation in macrophages. Concerning endothelial function, we propose investigating how variant-induced functional composition modifications in lipoproteins that in turn impair cell membrane fluidity and consequently, endothelial nitric oxide synthase (eNOS) activation.

We included these potential studies in the Discussion section, lines 231-238, as follows:

“To bridge the gap between the observed PLTP gene polymorphisms associations in this study and biological mechanisms, future functional studies are warranted. Given that PLTP plays a pivotal role in phospholipid transfer between lipoproteins, this fact may affect membrane lipid raft assembly. Then, macrophage and endothelial cell models could help to elucidate whether the rs6065904 and rs378114 variants alter membrane microdomain structure. Such structural changes could modify inflammation or nitric oxide synthetase signaling pathways, accelerating vascular inflammation in acute coronary events.”

Adde the following cite:

Muñoz-Vega M, Massó F, Páez A, Vargas-Alarcón G, Coral-Vázquez R, Mas-Oliva J, Carreón-Torres E, Pérez-Méndez Ó. HDL-Mediated Lipid Influx to Endothelial Cells Contributes to Regulating Intercellular Adhesion Molecule (ICAM)-1 Expression and eNOS Phosphorylation. Int J Mol Sci. 2018 Oct 30;19(11):3394.

 

In addition, we added other studies necessary to ensure the validity, and reliability of these polymorphisms in clinical practice, using the following phrase “Therefore, future experimental studies on mRNA expression such as luciferase assays, RT-qPCR (quantitative reverse transcription-polymerase chain reaction), or RNA sequencing (RNA-Seq) are required to ensure the validity, and reliability of these polymorphisms in clinical practice.” Lines 278 to 281

  

6. Conclusions:

The conclusion is appropriate and reflects the main findings of the study. However, please revise the wording in Line 244 by replacing/removing “as well as” for better grammatical flow and clarity. For example: “The rs6065904 A/G and rs378114 T/C polymorphisms of the PLTP gene are significantly associated with ACS risk and with triglyceride and glucose levels in Mexican Mestizos.”

 

Answer: We agree with the reviewer’s comment. In response, we have reworded the conclusion as follows: “Our findings showed that minor allele frequencies of the rs6065904 A/G and rs378114 T/C polymorphisms of the PLTP gene are associated with the susceptibility to developing ACS, and with triglyceride and glucose levels in the Mexican population…”