Round 1

Reviewer 1 Report

1, The sample size is too small，it is difficult to prove significantly difference and not representative of the whole ACS population. 

2, In the method part, authors should describe in detail how the control group was selected，usually the control group should be matched at least with case group by sex, age, etc. But the age did not matched. 

Author Response

Dear Prof. Dr. Rafael Franco,

Please, find attached our revised manuscript cimb-1941282 entitled, “Role of single nucleotide polymorphism L55M in the Paraoxonase 1 gene as a risk and prognostic factor in acute coronary syndrome” by Krastina Doneva-Basheva et al., submitted for publication as an original publication in the  Journal “Current Issues in Molecular Biology ”

In the revised manuscript we have added all corrections according to the comments and criticisms of the reviewers. We also removed from the original text the figures 4A, 4B and 4C and added as supplementary data.

We added additionally a new table, according to a recommendation of a reviewer and several references (5 more).

In the letter below we added our answers addressed to each of the points in the reviews.

Thank you for your time and assistance.

Sincerely,

Corresponding Author

Prof. Tatyana Vlaykova

Medical Faculty

Trakia University, Stara Zagora, Bulgaria

[email protected]

Reviewer 1

1, The sample size is too small, it is difficult to prove significantly difference and not representative of the whole ACS population.

Dear reviewers, we agree with the criticism about the small study group (77) , which cannot give a representation of the whole population of individuals with ACS. Generally, the clinical study with 9 year follow-up  included a bigger patents’  group of 172, but suitable biological material for DNA analyses and successful genotyping was possible for those 77 patients. We would like to note that this is the first time that the prevalence of PON1 L55M has been studied in the Bulgarian population. Your remark is pertinent and will give us reason to proceed with future studies of the enzyme,and its polymorphisms in a larger sample of the population.

2, In the method part, authors should describe in detail how the control group was selected, usually the control group should be matched at least with case group by sex, age, etc. But the age did not matched – it is done!

“The control group has been selected according to the case-control method during preventive examinations among employees of the Faculty of Medicine, after signed informed consent by the participants. All control subjects have been surveyed for comorbidities, and those with a history of ischemic disease have been excluded.”

We agree that the best for case-control studies is matching the patients and controls by age and sex, but obtaining controls with higher age without any CVD is too difficult. To avoid the bias of differences in age and sex, when the Logistic regression analyses were performed, we added the sex and age as covariates (confounding factors) in multiple Logistic regression analyses (Tables 3 ad 4)

Author Response File: Author Response.docx

Reviewer 2 Report

This is a clinical study, which aimed to explore the possible role of PON1L55M, (rs 854560, 163T > A) SNP as a predisposing factor for acute coronary syndrome and to assess its potency as a prognostic biomarker for short (1 year) survival and for median (5 years) and 9-year long patients’ outcome. The authors indicated that the variant M allele and the M allele genotypes (LM+MM) of the PON1 L55M polymorphism are risk factors for acute coronary syndrome, especially for patients with STEMI. 

This paper was not organized and it had too many typos. This reviewer has some criticisms as described below. 

Major comments:

1.     Material and methods. How did the authors select control subjects. The number of ACS patients and controls were not large. How did the authors exclude the bias, especially in the aspect of PON1? 

2.     The authors should include left ventricular ejection fraction data, as well as cardiac biomarkers, such as troponin, and analyze. 

3.     Table 4. The authors enrolled small number of patients and controls. How did the authors evaluate the prevalence of genotype distribution without bias? It is very difficult to set appropriate control subject. 

4.     From Figure 2, the authors suddenly analyze ACS patients with and without genotypes. This issue should be mentioned in the Methods section. 

5.     If the authors would like to compare ACS patients with and without genotypes, they should show their clinical background divided into groups. 

Minor comments:

6.     There were no page number. 

7.     There were too many typos. For example, In the Results section of Abstract, “hadabout” might be “had about”. In the Introduction section, “foundto” might be “found to”. “PON1is” should be “PON1 is”. Just before Material and methods section, “itspotency” supposed to be “its potency”. Table 1, current smokers, control group; “926.1” may be “(26.1). 

Author Response

To: Prof. Dr. Rafael Franco
Editor-in-Chief of

Current Issues in Molecular Biology 

Date: 01.11.2022

Dear Prof. Dr. Rafael Franco,

Please, find attached our revised manuscript cimb-1941282 entitled, “Role of single nucleotide polymorphism L55M in the Paraoxonase 1 gene as a risk and prognostic factor in acute coronary syndrome” by Krastina Doneva-Basheva et al., submitted for publication as an original publication in the  Journal “Current Issues in Molecular Biology ”

In the revised manuscript we have added all corrections according to the comments and criticisms of the reviewers. We also removed from the original text the figures 4A, 4B and 4C and added as supplementary data.

We added additionally a new table, according to a recommendation of a reviewer and several references (5 more).

In the letter below we added our answers addressed to each of the points in the reviews.

Thank you for your time and assistance.

Sincerely,

Corresponding Author

Prof. Tatyana Vlaykova

Medical Faculty

Trakia University, Stara Zagora, Bulgaria

[email protected]

Reviewer 2

Major comments:

1. Material and methods. How did the authors select control subjects. The number of ACS patients and controls were not large. How did the authors exclude the bias, especially in the aspect of PON1?

Dear reviewers, we agree with the criticism about the small study group (77) , which cannot give a representation of the whole population of individuals with ACS. Generally, the clinical study with 9 year follow-up  included a bigger patents’  group of 172, but suitable biological material for DNA analyses and successful genotyping was possible for those 77 patients. We would like to note that this is the first time that the prevalence of PON1 L55M has been studied in the Bulgarian population. Your remark is pertinent and will give us reason to proceed with future studies of the enzyme, and its polymorphisms in a larger sample of the population.

The control group has been selected according to the case-control method during preventive examinations among employees of the Faculty of Medicine, after signed informed consent by the participants. All control subjects have been surveyed for comorbidities, and those with a history of ischemic disease have been excluded.

We agree that the best for case-control studies is matching the patients and controls by age and sex, but obtaining controls with higher age without any CVD is too difficult. To avoid the bias of differences in age and sex, when the Logistic regression analyses were performed, we added the sex and age as covariates (confounding factors) in multiple Logistic regression analyses (Tables 3 ad 4)

2. The authors should include left ventricular ejection fraction data, as well as cardiac biomarkers, such as troponin, and analyze.

We added in Table 1 the data for cardiac biomarkers (troponin, CPK, CPK-MB, LVEF).

We also analyzed the relation the PON1genotypes with those markers.

3. Table 4. The authors enrolled small number of patients and controls. How did the authors evaluate the prevalence of genotype distribution without bias? It is very difficult to set appropriate control subject.

The control group has been selected according to the case-control method during preventive examinations among employees of the Faculty of Medicine, after signed informed consent by the participants. All control subjects have been surveyed for comorbidities, and those with a history of ischemic disease have been excluded.

We agree that the best for case-control studies is matching the patients and controls by age and sex, but obtaining controls with higher age without any CVD is too difficult, that is why our control group is with lower age.  To avoid the bias of differences in age and sex, when the Logistic regression analyses were performed, we added the sex and age as covariates (confounding factors) in multiple Logistic regression analyses (Tables 3 ad 4)

4. From Figure 2, the authors suddenly analyze ACS patients with and without genotypes. This issue should be mentioned in the Methods section. If the authors would like to compare ACS patients with and without genotypes, they should show their clinical background divided into groups.

Dear reviewer, I think there is some misunderstanding. We have included in all analyses ONLY patients and controls which were genotyped  – 77 patients with ACS, among them - 53 with ST-elevation myocardial infarction, STEMI, 14 with non-ST-elevation myocardial infarction, NSTEMI and 10 with unstable angina, UA.

Minor comments:

5. There were no page number. –DONE
6. 6. There were too many typos. For example, In the Results section of Abstract, “hadabout” might be “had about”. In the Introduction section, “foundto” might be “found to”. “PON1is” should be “PON1 is”. Just before Material and methods section, “itspotency” supposed to be “its potency”. Table 1, current smokers, control group; “926.1” may be “(26.1).

Thank you for helping us to improve the outlook of the manuscript. We tried to correct the typing errors.

Author Response File: Author Response.pdf

Reviewer 3 Report

The study contains a very small number of patients (77) and only one genetic variant is typed, the novelty of the study is unclear. In addition, the age of the controls and patients differed significantly.

Why PON1 L55M variant was selected not Q192R to study as prognostic factor if the association of PON1 192R variant with cardiovascular disease is well established from meta-analyses?

Authors need to decode all abbreviations (STEMI, NSTEMI, UA etc) when they are first mentioned, and try not to use it in abstract.

Material and method section do not include information how control individuals were included. There is need to explain the age of the controls and patients differed significantly. What examination to make sure they have not  cardiovascular pathology was done? Were they followed-up for 9 years too and was it checked if somebody from the control group developed ACS?

“criteria fulfilling the diagnostic algorithm for STEMI, NSTEMI, UA according to European guidelines” needs reference.

Material and method section must include how GRACE score was calculated (with reference). And why this score? Author need to expand information about patient group: angiography, how many vessels were affected, was the group  heterogenous in it, what surgery was done.

Table 2 shifted and cannot be analyzed carefully.

Results section.

Genotypes % need to be incorporated in table 3.

What is the purpose of listing some differences without statistical significance?

Table 5 needs “N” – number of patients in all subgroups including for each genotype. Are the data normally distributed?

Data on number of survived patients is better to represent as a table.

“shows that although not significantly, the patients with LL genotype have unfavorable prognosis” – what is the purpose of representing figures 3 without significance in the main text, not in sup/materials?

Why after fig 3A fig 3C is next to be mentioned?

What is significance for analysis represented on figures 4? P value  is not mentioned now. What is the purpose of figures?

Discussion section.

What is the possible reason of genotype specific differences of TC and TAG levels appear only in patients with NSTEACS, please discuss not only list it again.

“All these markers are previously described in a vast number of publications as 102 prognostic factors for survival of patients with heart diseases, which proves their 103 usefulness in the clinical practice” needs a references.

Author Response

To: Prof. Dr. Rafael Franco
Editor-in-Chief of

Current Issues in Molecular Biology 

Date: 01.11.2022

Dear Prof. Dr. Rafael Franco,

Please, find attached our revised manuscript cimb-1941282 entitled, “Role of single nucleotide polymorphism L55M in the Paraoxonase 1 gene as a risk and prognostic factor in acute coronary syndrome” by Krastina Doneva-Basheva et al., submitted for publication as an original publication in the  Journal “Current Issues in Molecular Biology ”

In the revised manuscript we have added all corrections according to the comments and criticisms of the reviewers. We also removed from the original text the figures 4A, 4B and 4C and added as supplementary data.

We added additionally a new table, according to a recommendation of a reviewer and several references (5 more).

In the letter below we added our answers addressed to each of the points in the reviews.

Thank you for your time and assistance.

Sincerely,

Corresponding Author

Prof. Tatyana Vlaykova

Medical Faculty

Trakia University, Stara Zagora, Bulgaria

[email protected]

Reviewer 3

1. The study contains a very small number of patients (77) and only one genetic variant is typed, the novelty of the study is unclear.

We got the same criticism about the small number of patients also from the other reviewers.

We agree with that criticism about the small study group (77) , which cannot give a representation of the whole population of individuals with ACS. Generally, the clinical study with 9 year follow-up  included a bigger patents’  group of 172, but suitable biological material for DNA analyses and successful genotyping was possible for those 77 patients. We would like to note that this is the first time that the prevalence of PON1 L55M has been studied in the Bulgarian population. Your remark is pertinent and will give us reason to proceed with future studies of the enzyme, and its polymorphisms in a larger sample of the population.

About choosing only one of the SNPs, PON1 L55M , but not the other one also, we may admit that the PON1 Q192R rs662 has been studied much extensively in many other papers, while  the data for PON1 L55M in ACS is more limited. On the other hand the SNP, PON1 L55M  is found to influence more the expression of the gene for PON1, while according to the functional studies PON1 Q192R influence  mainly the activity (arhylesterase activity mainly). So, having in mind all mentioned above, as well the limited budget of the project which supported this study, we designed our case-control study only with the less explored SNP, PON1 L55M.

2. In addition, the age of the controls and patients differed significantly.

We agree that the best for case-control studies is matching the patients and controls by age and sex, but obtaining controls with higher age without any CVD is too difficult, that is why our control group is with lower age.  To avoid the bias of differences in age and sex, when the Logistic regression analyses were performed, we added the sex and age as covariates (confounding factors) in multiple Logistic regression analyses (Tables 3 ad 4)

3. Why PON1 L55M variant was selected not Q192R to study as prognostic factor if the association of PON1 192R variant with cardiovascular disease is well established from meta-analyses?

About choosing only one of the SNPs, PON1 L55M , but not the other one also, we may admit that the PON1 Q192R rs662 has been studied much extensively in many other papers, while  the data for PON1 L55M in ACS is more limited. On the other hand the SNP, PON1 L55M  is found to influence more the expression of the gene for PON1, while according to the functional studies PON1 Q192R influence  mainly the activity (arhylesterase activity mainly). So, having in mind all mentioned above, as well the limited budget of the project which supported this study, we designed our case-control study only with the less explored SNP, PON1 L55M.

4. Authors need to decode all abbreviations (STEMI, NSTEMI, UA etc) when they are first mentioned, and try not to use it in abstract. – DONE
5. Material and method section do not include information how control individuals were included. There is need to explain the age of the controls and patients differed significantly. What examination to make sure they have not cardiovascular pathology was done?

DONE – page 3, line 35 “All control subjects have been surveyed for comorbidities, and those with a history of ischemic disease have been excluded.”

6. Were they followed-up for 9 years too and was it checked if somebody from the control group developed ACS?

The individuals of the control group have not been followed for 9 years. This was not the aim of our study. It is important to follow-up the patients. But information on concomitant cardiovascular diseases of controls was collected before collection of the biological material. In this sense, there are no persons in the control group who have experienced ACS at the time of taking the biological material or before.

7. “criteria fulfilling the diagnostic algorithm for STEMI, NSTEMI, UA according to European guidelines” needs reference. We added a reference.
8. McDonagh TA, Metra M, Adamo M, Gardner RS, Baumbach A, Böhm M, Burri H, Butler J, Čelutkienė J, Chioncel O, Cleland JGF, Coats AJS, Crespo-Leiro MG, Farmakis D, Gilard M, Heymans S, Hoes AW, Jaarsma T, Jankowska EA, Lainscak M, Lam CSP, Lyon AR, McMurray JJV, Mebazaa A, Mindham R, Muneretto C, Francesco Piepoli M, Price S, Rosano GMC, Ruschitzka F, Kathrine Skibelund A; ESC Scientific Document Group. 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur Heart J. 2021 Sep 21;42(36):3599-3726. doi: 10.1093/eurheartj/ehab368. Erratum in: Eur Heart J. 2021 Oct 14;: PMID: 34447992.
9. Material and method section must include how GRACE score was calculated (with reference). And why this score?

We added in the text a short information for the Global Registry of Acute Coronary Events (GRACE) score, which is important for predicting in-hospital and 6 months mortality of patients with ACS. We added a reference as well as an internet link with calculator for GRACE score assessment.

24. https://www.outcomes-umassmed.org/grace/acs_risk2/index.html
25. Kumar D, Ashok A, Saghir T, Khan N, Solangi BA, Ahmed T, Karim M, Abid K, Bai R, Kumari R, Kumar H. Prognostic value of GRACE score for in-hospital and 6 months outcomes after non-ST elevation acute coronary syndrome. Egypt Heart J. 2021 Mar 6;73(1):22. doi: 10.1186/s43044-021-00146-9. PMID: 33677742; PMCID: PMC7937004.

9. Author need to expand information about patient group: angiography, how many vessels were affected, was the group heterogenous in it.

We added in Table 1 the number of the patients with the number of vessels affected and explanation in material and methods about clinical assessment methods.

10. Table 2 shifted and cannot be analyzed carefully. -Corrected

Results section.

11. Genotypes % need to be incorporated in table 3.

According to the recommendations, we corrected the Table 3 and 4 and instead the frequency we replaced % of genotype distribution.

12. What is the purpose of listing some differences without statistical significance?

The main reason for this is to show that the analyses have been performed and from those analyses the results are negative – no statistically significant. We think that each results is important, although not significant, because this shows that there is no relationship. So this could avoid some misinterpretations with factors that are not included in the results (if we omit the negative results).  

13. Table 5 needs “N” – number of patients in all subgroups including for each genotype.

Corrected

14. Are the data normally distributed?

Most of the data were with non-normal distribution, that is why the p-values from Mann-Whitney U or Kruskal-Walis tests were presented and given in the tables. However for easy reading  by most of the readers of papers, we decided to present the central values of the  continuous variables as mean±SEM (standard error of mean) instead of median and range or  interquartile range

15. Data on number of survived patients is better to represent as a table.

Thank you for the suggestion. We added an additional table 6

16. “shows that although not significantly, the patients with LL genotype have unfavorable prognosis” – what is the purpose of representing figures 3 without significance in the main text, not in sup/materials?

Thank you for this comments!  We agree that generally that figures with non-significant results is better to be added as supplementary data. But still we think that giving figure 3 we can visualize the difference in the Kaplan-Maier survival curves, because although not significant (possibly, because of the low number of patients) it is visible the tendency for better survival of patients with LM+MM genotypes.

According to your comments we can add the Kaplan-Maier survival curves in figure 4 as supplementary data.

17. Why after fig 3A fig 3C is next to be mentioned?

We corrected: “The difference in patients with STEMI is less distinguishing (Figure 3B), but more pronounce for patients with NSTEACS (p=0.115) (Figure 3C). “

18. What is significance for analysis represented on figures 4? P value is not mentioned now. What is the purpose of figures?

We wanted to show that with the time the possible effect of the genotype becomes less important. But you are right, the information in the text is enough and according to your comments we can add the Kaplan-Maier survival curves in figure 4 as supplementary data.

Discussion section.

19. What is the possible reason of genotype specific differences of TC and TAG levels appear only in patients with NSTEACS, please discuss not only list it again.

The observed difference in the levels of TC and TAG between the carriers of different genotypes could be explain with the functional effect of the variant PON1 M55 allele, being associated with lower enzyme concentration than 55L allele which further might lead to lower defense role against hypercholesterolemia and trigliceridemia.

We may admit that the observed statistically significant difference only in patients with NSTEACS, but in STEMI, might be due to the bias of the small group of NSTEACS, only 24 patients.

20. “All these markers are previously described in a vast number of publications as 102 prognostic factors for survival of patients with heart diseases, which proves their 103 usefulness in the clinical practice” needs a references.

We added additional references:

36. Bachar A, Benmessaoud FA, Diatta A, Fadoum H, Haroun AE, Oukerraj L, Cherti M. Predictive factors of heart failure in acute coronary syndrome: Institutional cross-sectional study. Ann Med Surg (Lond). 2022 Aug 10;81:104332. doi: 10.1016/j.amsu.2022.104332. PMID: 36147130; PMCID: PMC9486600.
37. Pramudyo M, Bijaksana TL, Yahya AF, Putra ICS. Novel scoring system based on clinical examination for prediction of in-hospital mortality in acute coronary syndrome patients: a retrospective cohort study. Open Heart. 2022 Oct;9(2):e002095. doi: 10.1136/openhrt-2022-002095. PMID: 36229139; PMCID: PMC9562746.
38. Pramudyo M, Yahya AF, Martanto E, Tiksnadi BB, Karwiky G, Rafidhinar R, Putri GNI. Predictors of In-Hospital Mortality in Patients with Acute Coronary Syndrome in Hasan Sadikin Hospital, Bandung, Indonesia: A Retrospective Cohort Study. Acta Med Indones. 2022 Jul;54(3):379-388. PMID: 36156467.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors answered my considers properly with reasonable explanation. 

Author Response

Thank you very much for your recommendations: we made some changes in English language and the style according to the corrections a native English speaker has done.

Author Response File: Author Response.docx

Reviewer 2 Report

This reviewer has no further comment. 

Author Response

Thank you very much for your recommendations: we made some changes in English language and the style according to the corrections a native English speaker has done.

Author Response File: Author Response.docx

Reviewer 3 Report

Abstract again includes conclusions based on not significant data:

How than you can explain these conflicting facts: M is a risk allele, but bad prognosis is characteristic of LL genotype? Doesn't such facts encourage you to reconsider the results and perhaps stop relying and drawing conclusions on the basis of nonsignificant data? Again nonsignificant data we see on figures 3. I don't see the meaning of figures 3.

For example PON1 Q192R might be modifying the results? Authors need to conduct additional experiments, to genotype PON1 Q192R and maybe to measure PON1 activity.

These is from the abstract:

“M allele had about 2.5-fold higher risk for developing ACS than those which are homozygous of the wild-type L allele (LL genotype)”

“Although not significantly, the ACS patients with LL genotype have unfavorable prognosis in comparison with patients with M allele genotypes, which is more pronounced for patients with NSTEACS.”

Additional to Q1: Was PON1 Q192R studied in the Bulgarian population?

“On the other hand the SNP, PON1 L55M is found to influence more the expression of the gene for PON1, while according to the functional studies PON1 Q192R influence mainly the activity (arhylesterase activity mainly).” – Do you mean, PON1 gene expression is more important for cardiovascular disease progression than its real activity?

Additional to Q2: What is the age of the first clinical symptoms in the patient group?

Additional to Q4: What is CPK-MB, AMI, LVEF?

Author Response

Reviewer 2

Thank you very much for your recommendations, comments and criticisms. We appreciate them because they help to improve our manuscript.

According the language -  we made some changes in English language and the style according to the corrections a native English speaker has done.

Further ate our answers and corrections we made according to your review.

Abstract again includes conclusions based on not significant data:

1) How than you can explain these conflicting facts: M is a risk allele, but bad prognosis is characteristic of LL genotype? Doesn't such facts encourage you to reconsider the results and perhaps stop relying and drawing conclusions on the basis of nonsignificant data? Again nonsignificant data we see on figures 3. I don't see the meaning of figures 3.

These is from the abstract:

“M allele had about 2.5-fold higher risk for developing ACS than those which are homozygous of the wild-type L allele (LL genotype)”

“Although not significantly, the ACS patients with LL genotype have unfavorable prognosis in comparison with patients with M allele genotypes, which is more pronounced for patients with NSTEACS.”

Thank you once again for your criticism. We agree that we should not make strong conclusions on the bases of insignificant results. In this respect we made the following changes:

1. a) We changed the abstract omitting those insignificant results
2. b) Figures 3A, 3B, 3C were added to the supplementary data
3. c) We changed the text in the result part:

“The Kaplan-Maier survival curve after one year of following of the patients shows that there was no significant difference in the survival of the patients with LL genotype (mean survival period of 17.81 months) and those patients with M allele genotypes (LM+MM, mean survival period of 20.31 months, p=0.553, Log rank test) (Figure 3A- supplementary). A lack of significant difference between carriers of different genotypes was obtained both in the subgroup of patients with STEMI (Figure 3B- supplementary), and with NSTEACS (p=0.115) (Figure 3C- supplementary).

”

How than you can explain these conflicting facts: M is a risk allele, but bad prognosis is characteristic of LL genotype? –

It is a very difficult point for finding explanation. ACS, as all CVD, is a multifactorial disease with variety of risk factors, and one of them could be the different level of expression of PON1 due to the L55M SNP. But the expression is influenced not only by the gene variants, but also by different signaling pathways triggered by cytokines and/or other signaling molecules. We can hypothesize that in early stages of developing of CVD, the effect of the PON1 gene variant can have stronger effect on the gene expression and the variant M  allele can lead to stronger influence on the level of PON1 enzyme molecules (to lower enzyme), and thus to stronger oxidative modification of LDL complexes and higher risk for CVD. In further periods after the acute coronary event, other factors might influence the progression of the disease.

2) For example PON1 Q192R might be modifying the results? Authors need to conduct additional experiments, to genotype PON1 Q192R and maybe to measure PON1 activity.

Yes, we agree that the other SNP, PON1 Q192R might be modifying factor for the results, but we were not able to explore this SNP in the panel of patients we have enrolled.

3) Additional to Q1: Was PON1 Q192R studied in the Bulgarian population?

As we know, so far there is no other study on the association of PON1 SNP (neither  for Q192R or for L55M) in Bulgarian population.

4) “On the other hand the SNP, PON1 L55M is found to influence more the expression of the gene for PON1, while according to the functional studies PON1 Q192R influence mainly the activity (arhylesterase activity mainly).” – Do you mean, PON1 gene expression is more important for cardiovascular disease progression than its real activity?

The enzyme activity (enzyme concentration) in the biological fluids/tissue depends both of the enzyme protein level (determined by the gene expression in cells and secretion if the enzyme is in blood) and on the activity of the enzyme (affinity to the substrate or stability of enzyme-substrate complex). In this respect both factors (enzyme activity and enzyme level) determine the real activity i.e. enzyme concentration (U/l) which quite commonly is replaced with the term enzyme activity (real activity).

So, I response to your question, I must say that the real activity (enzyme concentration, U/l)  is the most important for the cardiovascular disease progression, which depends both on the enzyme level (expression influenced by L55M  SNP) and on the enzyme activity (enzyme substrate affinity influenced by Q192R).

5) Additional to Q2: What is the age of the first clinical symptoms in the patient group?

Unfortunately, we have only the age of the patients when they were admitted to the hospital because of the ACS. The patients were asked about the time of the first symptoms, but they are in periods of hours.

The patients have not been questioned about the time of earlier symptoms of any other CVD, as hypertonia. 

6) Additional to Q4: What is CPK-MB, AMI, LVEF?

We added these abbreviations in the text under the tables:

CPK-MB – MB fraction of CPK (heart fraction of CPK)

LVEF – Left ventricular ejection fraction

BMI – body mass index

Author Response File: Author Response.docx