ALOX5, LPA, MMP9 and TPO gene polymorphisms increase atherothrombosis susceptibility in middle-aged Mexicans

Atherothrombosis is the cornerstone of cardiovascular diseases and the primary cause of death worldwide. Genetic contribution to disturbances in lipid metabolism, coagulation, inflammation and oxidative stress increase the susceptibility to its development and progression. Given its multifactorial nature, the multiloci studies have been proposed as potential predictors of susceptibility. A cross-sectional study was conducted to explore the contribution of nine genes involved in oxidative stress, inflammatory and thrombotic processes in 204 subjects with atherothrombosis matched by age and gender with a healthy group (n = 204). To evaluate the possibility of spurious associations owing to the Mexican population genetic heterogeneity as well as its ancestral origins, 300 unrelated mestizo individuals and 329 Native Americans were also included. ALOX5, LPA, MMP9 and TPO gene polymorphisms, as well as their multiallelic combinations, were twice to four times more frequent in those individuals with clinical manifestations of atherothrombosis than in the healthy group. Once adjusting for population stratification was done, these differences remained. Our results add further evidence on the contribution of ALOX5, LPA, MMP9 and TPO polymorphisms to atherothrombosis development in the middle-aged group, emphasizing the multiethnic studies in search of gene risk polymorphisms.


Major comments
1. rs numbers of analyzed variants should given, names of all alleles, location within the gene, their functional effect should be given in introduction and association with diseases. Clearly give in tables allele and all genotypes. Methods applied should be described clearly, with references. Results, emphasize important revelations clearly, and give concrete not descriptive results. 2. The authors should avoid using confusing structure, and explain methods and protocols used clearly and simply. genotyping should be given more clearly, regardless the process in which they are involved in. one term should be used in the whole manuscript polymorphism or variant Association test should be written more clearly 3. Results should not be descriptive, than clearly emphasis the difference with statistical parameters Table 1, family history, positive (%) 4. genome control-give explanation of the term and reference 5. Give results of HW disequilibrium in CME group, what is observed and what expected freq 6. multi loci comparison, how it was calculated by cumulative effect, gene-gene interaction, should be explained It is not clear 7. >0.05 should be changed to ns-non significant, keep consistency in presenting results through manuscript 8. instead loci, authors could use a term variants in the whole manuscript, they use different terms for the same thing and make confusion with that 9. Titles of the tables should be clear, concise and without sufficient information 10. Sentence Epidemiological studies, cell cultures, and animal models have positively related the low numbers of tandem repeats on the promoter region of ALOX5 to an increased risk of CMA, reinforcing our findings (24)(25)(26)30) is very confusing Smaller and larger alleles than the LPA*8 have been associated with the pro thrombotic activity, reinforcing the our findings of the contribution of LPA to atherothrombosis development (40). It is not clear what is smaller or larger alleles, 11. instead of reinforcing authors should say which is in accordance with our results 12. MMP9 is mainly regulated at transcriptional level; polymorphisms located on the promoter region have been associated with MI, ischemic stroke, and coronary artery disease (38,42), lung disorders and carcinoma should be added: References, e.g. Gene-environment interaction between the MMP9 C-1562T promoter variant and cigarette smoke in the pathogenesis of chronic obstructive pulmonary disease. 13. collective gene participation, could be multifactorial nature of disease 14. results of genotypes should be given also for vWA,eNOS,HMOX1, 15. reinforce, cornerston-too many repeats in the manuscript 16. Weaknesses were the modest sample size, sample size is quite satisfying and should not be mentioned as weekness Authors could negelect limitations of the study mentioned.
17. title table 1. characteristics of the groups  table 2 first columns give data of the groups than p-values of comparisons, title should be short  and concise  table 3 give OR (95% CI) format per column 18. Authors should Proof read the manuscript with native English editor etc. no need for dashes 2. Biological pathways, biochemical pathways 3. Page4 morbid-, population-, ancestral-? 4. evaluated starting with the questions do you smoke or drink alcohol? evaluated by questionnaire 5. (units of alcohol consumed) * (standard drink unit) * (frequency of drink habits by week) this is confusing 6.The genetic structure for the nine polymorphism, this is confusing 7. Titles Genotyping assessment, should be Genotyping or Determination of genotypes Differences between the groups according to cardiovascular diseases could be Characteristics of (analzyed) groups Allele and genotype disparities among the studied groups, could be distribution of alleles and genotypes among groups Statistical population genetic parameters, very confusing (define group or population in paper) Multi-loci comparisons among the evaluated groups, could be Multi-loci comparisons 8. what is drastic decrease, avoid descriptive terms in the results 9. peoples, should be singular peopling ? The editors assigned to your paper ("ALOX5, LPA, MMP9 and TPO genetic variants increase atherothrombosis susceptibility in middle-aged Mexican population.") have now received comments from reviewers.
While both reviewers are relatively positive about publication of the paper, they both raise a substantive number of major comments regarding the presentation and analysis of the data. These will need careful consideration. We would like you to revise your paper in accordance with the referee suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) The researchers looked for genetic associations with atherothrombosis development (clinical manifestations) in the protein-coding genomes of 408 unrelated Mexican individuals. 204 Individuals having clinical manifestations of atherothrombosis (n=204) were paired by age and gender to an equal number of blood donors without clinical manifestations. They then replicated the study in an independent group of 300 unrelated mestizo individuals, the so-called genome control, and in a group of 329 native subjects belonging to different ethnic groups. From the nine gene studied, the researchers discovered frequency differences in 4 of them: ALOX5, LPA, MMP9, and TPO. These were significantly more frequent in individuals with clinical atherothrombosis even after being adjusted by family history of cardiovascular disease, smoking, alcohol intake, and physical activity Main concerns 1. In the abstract the authors included both patients and control groups in a collective of "1037 unrelated Mexican individuals". The real number of patients is 204, which had clinical atherothrombosis. There is an equal number of blood donors without clinical manifestations of atherothrombosis. There are also a genome control group of 300 unrelated individuals (equality in gender) and an ancestral control group of 329 from different ethnic origin. These differences should be clearly indicated not only in the Abstract but also sufficiently detailed in section 3.1 Population studied. 2. I would like to understand which is the difference between the 204 asymptomatic blood donor controls and the so-called genome control group of 300 unrelated individuals (beside their geographical origin). I suppose that residents of Mexico City and those from the Central Valley of Mexico are not different in terms of ethnic origin, i.e. mestizos. 3. Classical risk factors of atherothrombosis could be indicated under the form of Tables to avoid extensive description in the text including the way they were collected. 4. Lines 49-51, p.5, "one gene was involved in coagulation mechanism" vWA is a STR of intron 40 of the von Willebrand factor gene (Table S2) and not "the gene involved in coagulation mechanism" This tetranucleotide repeat is an excellent marker for genetic studies. 5. Page 10, lines 13-17. "Lipoprotein(a) [Lp(a)], an enhancer of foam cell production and a fibrinolytic inhibitor, is encoded by LPA, which genetic variants control both its isoforms size variation as plasma Lp(a) levels (10)" This sentence needs clarification. First, the main enhancer of foam cell production is LDL. Only when Lp(a) is considerably increased it could contribute to foam cell production via its LDL component. Second Lp(a) is a mix of LDL and apolipoprotein apo(a); LPA gene is the code for apo(a). Genetic variants of the LPA gene control isoforms size of apo(a) and thereby variation as plasma Lp(a) levels. Association of high Lp(a) levels and small isoform size is a well recognized factor of atherothrombosis. Was Lp(a) dosage and apo(a) isoform identification considered by authors? If not, it could be an important asset for further investigation in these populations.

Minor corrections
Page 8 The sentence "The higher consumption of tobacco (> two pack/year) and alcohol (> 20 g/week) was presented within the CMA individuals." needs correction.

Page 10
Lines 19-31 The whole paragraph should be revised and corrected. The sentence "The atherogenic component of Lp(a) -apoprotein (a)-, is structurally homologous to plasminogen and competes for bind to its receptor as well as fibrin and fibrinogen," Please correct as Binding of apo(a) to fibrin does not promote free radicals generation (36-38).
Lp(a) stimulates the endothelial permeability and adhesion molecules production as well as proinflammatory cytokines release. "besides to be an independent risk factor for cerebral and CV atherothrombosis (4, 39). Smaller and larger alleles than the LPA*8 have been associated with the prothrombotic activity, reinforcing the our findings of the contribution of LPA to atherothrombosis development (40 Reviewer: 2 Comments to the Author(s) The authors of the study entitled: ALOX5, LPA , MMP9 and TPO genetic variants increase atherothrombosis susceptibility in middle-aged Mexican population, aimed to examine the role of genetic variants of ALOX5, IL6, LPA, MMP9, vWA, eNOS, HMOX1, NOX4, and TPO in atherothrombosis in Mexican population and found association of ALOX5, LPA, MMP9 and TPO variants. The study is interesting and worth of publishing.
Major comments 1. rs numbers of analyzed variants should given, names of all alleles, location within the gene, their functional effect should be given in introduction and association with diseases. Clearly give in tables allele and all genotypes. Methods applied should be described clearly, with references. Results, emphasize important revelations clearly, and give concrete not descriptive results. 2. The authors should avoid using confusing structure, and explain methods and protocols used clearly and simply. genotyping should be given more clearly, regardless the process in which they are involved in. one term should be used in the whole manuscript polymorphism or variant Association test should be written more clearly 3. Results should not be descriptive, than clearly emphasis the difference with statistical parameters Table 1, family history, positive (%) 4. genome control-give explanation of the term and reference 5. Give results of HW disequilibrium in CME group, what is observed and what expected freq 6. multi loci comparison, how it was calculated by cumulative effect, gene-gene interaction, should be explained It is not clear 7. >0.05 should be changed to ns-non significant, keep consistency in presenting results through manuscript 8. instead loci, authors could use a term variants in the whole manuscript, they use different terms for the same thing and make confusion with that 9. Titles of the tables should be clear, concise and without sufficient information 10. Sentence Epidemiological studies, cell cultures, and animal models have positively related the low numbers of tandem repeats on the promoter region of ALOX5 to an increased risk of CMA, reinforcing our findings (24-26, 30) is very confusing Smaller and larger alleles than the LPA*8 have been associated with the pro thrombotic activity, reinforcing the our findings of the contribution of LPA to atherothrombosis development (40). It is not clear what is smaller or larger alleles, 11. instead of reinforcing authors should say which is in accordance with our results 12. MMP9 is mainly regulated at transcriptional level; polymorphisms located on the promoter region have been associated with MI, ischemic stroke, and coronary artery disease (38, 42), lung disorders and carcinoma should be added: References, e.g. Gene-environment interaction between the MMP9 C-1562T promoter variant and cigarette smoke in the pathogenesis of chronic obstructive pulmonary disease. 13. collective gene participation, could be multifactorial nature of disease 14. results of genotypes should be given also for IL-6, vWA, eNOS, HMOX1, and NOX-4 15. reinforce, cornerston-too many repeats in the manuscript 16. Weaknesses were the modest sample size, sample size is quite satisfying and should not be mentioned as weekness Authors could negelect limitations of the study mentioned. 17. title table 1. characteristics of the groups table 2 first columns give data of the groups than p-values of comparisons, title should be short and concise table 3 give OR (95% CI) format per column 18. Authors should Proof read the manuscript with native English editor Minor comments 1. gene-variants, at-risk, multi-ethnic Forty-two, at-susceptibility, complex-diseases, etc. no need for dashes 2. Biological pathways, biochemical pathways 3. Page4 morbid-, population-, ancestral-? 4. evaluated starting with the questions do you smoke or drink alcohol? evaluated by questionnaire 5. (units of alcohol consumed) * (standard drink unit) * (frequency of drink habits by week) this is confusing 6.The genetic structure for the nine polymorphism, this is confusing 7. Titles Genotyping assessment, should be Genotyping or Determination of genotypes Differences between the groups according to cardiovascular diseases could be Characteristics of (analzyed) groups Allele and genotype disparities among the studied groups, could be distribution of alleles and genotypes among groups Statistical population genetic parameters, very confusing (define group or population in paper) Multi-loci comparisons among the evaluated groups, could be

Comments to the Author(s)
The authros indicate in section 3.1 first paragraph "Classical risk factors such as diabetes type 2, hypercholesterolemia, body mass index, age, and family history of CVDs were obtained from the clinical records of these 408 individuals" My comment: healthy controls were selected among healthy blood donors and they should not have a clinical record at the moment of sampling. Please, modify as necessary. I have no further coments or questions Review form: Reviewer 2

Comments to the Author(s)
The authors corresponded well to the reviewers' suggestions. Just some more minor comments: 1. page 10/4 MMP9 italic 2. page 12/39-13/11 No need for mentioning questions from questioner, just say that in smokers/ex-smokers cumulative cigarette consumption is expressed by pack-years (standard well-known unit) till the occurrence of disease. The same is for alcohol consumption. 3. Table 2, as previously suggested first give columns with subjects than p values for all comparisons, footnote is not corresponding to the symbols in the table, give the no. and % of reference category, for LPA, usually denoted as 1, or eliminate non-carriers, genotypes are usually presented as X/X, or X/(X)….what is X in ALOX5? 4. indicate genes shown in tables s7-15 5. s16-23 column 1 is only allele, genes are indicated in subsequent columns 6. hoja worksheet is empty 7. Table 3. Clinical manifestation of AT is only above yes and no Footnotes are not corresponding to the symbols in the table, OR and 95% CI can be given in one column using OR (95% CI) formant, keep one or two decimals in the tables consistently give the no of subjects and %-age in yes/no column, and for LPA eliminate non-carriers as they are not needed. 8.Suggestions: Title Table 1 Characteristics of study participants  Title Table 2. and 3 Association of genetic risks with AT. Genetic risks is not good term as it predisposes the risk of the diseases, as already established, it could be changed to genetic factors. In table 2 only p values are shown while in table 3 only ORs, both values should be shown in both tables. Keep consistency and simplicity within the whole manuscript.

12-Nov-2019
Dear Dr Gomez, On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-190775.R1 entitled "ALOX5, LPA, MMP9 and TPO genetic variants increase atherothrombosis susceptibility in middle-aged Mexicans." has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
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• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-190775.R1 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Your manuscript has returned from peer review, and the referees' comments can be found below. Specifically, please address the comment made by Referee #1 on the sampling, and the minor comments made by Referee #2 when submitting your revision.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) The authors indicate in section 3.1 first paragraph "Classical risk factors such as diabetes type 2, hypercholesterolemia, body mass index, age, and family history of CVDs were obtained from the clinical records of these 408 individuals" My comment: healthy controls were selected among healthy blood donors and they should not have a clinical record at the moment of sampling. Please, modify as necessary. I have no further comments or questions Reviewer: 2 Comments to the Author(s) The authors corresponded well to the reviewers' suggestions. Just some more minor comments: 1. page 10/4 MMP9 italic 2. page 12/39-13/11 No need for mentioning questions from questioner, just say that in smokers/ex-smokers cumulative cigarette consumption is expressed by pack-years (standard well-known unit) till the occurrence of disease. The same is for alcohol consumption. 3. Table 2, as previously suggested first give columns with subjects than p values for all comparisons, footnote is not corresponding to the symbols in the table, give the no. and % of reference category, for LPA, usually denoted as 1, or eliminate non-carriers, genotypes are usually presented as X/X, or X/(X)….what is X in ALOX5? 4. indicate genes shown in tables s7-15 5. s16-23 column 1 is only allele, genes are indicated in subsequent columns 6. hoja worksheet is empty 7. Table 3. Clinical manifestation of AT is only above yes and no Footnotes are not corresponding to the symbols in the table, OR and 95% CI can be given in one column using OR (95% CI) formant, keep one or two decimals in the tables consistently give the no of subjects and %-age in yes/no column, and for LPA eliminate non-carriers as they are not needed. 8.Suggestions: Title Table 1 Characteristics of study participants  Title Table 2. and 3 Association of genetic risks with AT. Genetic risks is not good term as it predisposes the risk of the diseases, as already established, it could be changed to genetic factors. In table 2 only p values are shown while in table 3  Decision letter (RSOS-190775.R2)

28-Nov-2019
Dear Dr GOMEZ, It is a pleasure to accept your manuscript entitled "ALOX5, LPA, MMP9 and TPO genetic variants increase atherothrombosis susceptibility in middle-aged Mexicans." in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
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Comment 4.
Lines 49-51, p.5, "one gene was involved in coagulation mechanism" vWA is a STR of intron 40 of the von Willebrand factor gene (Table S2) and not "the gene involved in coagulation mechanism" This tetranucleotide repeat is an excellent marker for genetic studies. 7. >0.05 should be changed to ns-non significant, keep consistency in presenting results through manuscript 8. instead loci, authors could use a term variants in the whole manuscript, they use different terms for the same thing and make confusion with that 9. Titles of the tables should be clear, concise and without sufficient information Thank you for these suggestions; all of them have been clarified in the main text.
10. Sentence Epidemiological studies, cell cultures, and animal models have positively related the low numbers of tandem repeats on the promoter region of ALOX5 to an increased risk of CMA, reinforcing our findings (24-26, 30) is very confusing Smaller and larger alleles than the LPA*8 have been associated with the pro thrombotic activity, reinforcing the our findings of the contribution of LPA to atherothrombosis development (40). It is not clear what is smaller or larger alleles, 11. instead of reinforcing authors should say which is in accordance with our results.
Thank you for these suggestions; all of them have been clarified in the main text.
MMP9 is mainly regulated at transcriptional level; polymorphisms located on the promoter region have been associated with MI, ischemic stroke, and coronary artery disease (38, 42), lung disorders and carcinoma should be added… We appreciate this suggestion; we have incorporated the references suggested.