The Leucocyte Telomere Length, GSTM1 and GSTT1 Null Genotypes and the Risk of Chronic Obstructive Pulmonary Disease

Round 1

Reviewer 1 Report

In this original study, the authors explored the link between the Glutathione 26 S-transferase (GST) polymorphisms and the potential link with short telomere length and the risk of COPD. It is an interesting study, coherent and well structured with novel results. However, i am concerned by some major issues:

1. Did the authors made a multivariate analysis controlling for age and sex? I noticed that there was a difference in female predominance in the control group. As telomere lentgh is affected by sex and age, this difference may have affected the results.

2. The authors measured telomere length with qPCR and not using FISH method, which is the most accurate. Why did they not use FISH? Please justify it and include it in the limitation section in the last paragraph of the discussion.

3. The authors do no present the limitations of their study. The most important include the sex difference between the two groups and the use of qPCR instead of FISH method. Please include them.

Author Response

Reviewer 1

 

Question: Did the authors made a multivariate analysis controlling for age and sex? I noticed that there was a difference in female predominance in the control group. As telomere lentgh is affected by sex and age, this difference may have affected the results.

Answer: Thank you for the comment and suggestion for enriching the results. We did the logistic regression analysis with counfaunding factors such as sex and age and we added those results  in Table 3. Indeed telomere lenght is affected by the age and sex as we have described in our previous article: Tacheva T, Zienolddiny S, Dimov D, Vlaykova D, Vlaykova T. 2021. The leukocyte telomere length, single nucleotide polymorphisms near TERC gene and risk of COPD. PeerJ 9:e12190 https://doi.org/10.7717/peerj.12190. “A near-marginal significance for shorter telomeres in COPD patients above 60 years compared to controls in the same subgroup was found. In COPD patients aged above 60 years which also are current/ex-smokers, telomeres were shorter in man compared to women.”.

Here we are focusing on the relation of GSTM1 and GSTT1 polymorphisms with the concentration of tolal glutathione and the LTL shortening.

 

Question: The authors measured telomere length with qPCR and not using FISH method, which is the most accurate. Why did they not use FISH? Please justify it and include it in the limitation section in the last paragraph of the discussion.

Answer: In the laboratory where the measurement of LTL was performed, the qPCR method was available. This is why we did not use the FISH method. Efficiency was more than 95%. As qPCR method is a robust method, we chose to run the samples in duplicate.

 

Question: The authors do no present the limitations of their study. The most important include the sex difference between the two groups and the use of qPCR instead of FISH method. Please include them.

Answer: The limitations were added in section Discussion. It is quite difficult to find control individuals above 60 years without any chronic diseases. Most of the male individuals had different chronic diseases which might reflect the results. This is why we have a predominance of females in the control group. But as you have suggested and we discussed above, we considered the sex as counfaunding factor in the logistic analysis.

 

Dear Editor-in-Chief,

Please find enclosed our revised manuscript, “ The leucocyte telomere length, GSTM1 and GSTT1 null geno-types and the risk of COPD” by Tanya Tacheva et al.

In this version, we added changes that we did according to the recommendations of the reviewers.

Further, in the letter you will find all answers to the comments of both reviewers.

We hope that the manuscript will fulfill the high requirements and standard of the journal and will create interest in readers of the journal.

All of the authors declare that they have all participated in the design, execution, and analysis of the paper, and that they have approved the final version. Additionally, there are no conflicts of interest in connection with this paper, and the material described is not under publication or consideration for publication elsewhere.

 

Thank you for your time and assistance.

Sincerely,

 

Corresponding Author

Prof. Tatyana Vlaykova,

Dept. Chemistry and Biochemistry,

Medical Faculty, Trakia University,

Stara Zagora, Bulgaria;

Dept. Medical Biochemistry,

Medical University - Plovdiv,

Plovdiv, Bulgaria

e-mail:  [email protected]

Reviewer 2 Report

This manuscript (Manuscript ID: cimb-1837919) explores the link between GSTM1 and GSTT1 gene polymorphisms, LTL and COPD risk. The authors propose that individual carriers of at least one null genotype have higher risk of developing COPD. However, the potential link between of GSTM1 and GSTT1 polymorphism and LTL in the development and severity of COPD remains still unclear.

 

Major Comments:

1)      It would be helpful if data regarding leukocyte telomere length and GSH levels are presented in a table.

2)      In Table 3 the genotype “at least one non-null” is inconsistent with the text “at least one null”. Please explain or correct.

3)      The authors speculate that the loss of enzymatic activity in carriers of the null genotypes (GSTM1 and GSTT1) result in altered antioxidant defence and detoxification of ROS and their products. Have the authors determined the enzymatic activity of other GST isoenzymes in the carriers of this study? Please elaborate the contribution of the enzymatic activity of other isoenzymes in GSTM1 and GSTT1 null genotypes.

4)      The discussion section should be thoroughly revised. In its current form reads as an enriched introduction section. Please focus on discussing the major findings.

5)      Please provide the rationale for exploring the correlation between serum total glutathione and LTL in COPD patients.

 

Minor comments

1)      Lines 58-59 please add relevant citation

2)      Lines 72-74 please add relevant citation

3)      Lines 74-76 please refer to the original study

4)      Please correct legend in Figure 1 (null GSTM1 genotype – individual 1 and 4) and incorporate M1-0, T1-0, M+, T+ into the legend.

5)      Line 134: Please Indicate the positive and negative controls that are included in PCRs.

6)      Figure legends 2, 3 and 4 should be more informative.

Author Response

Reviewer 2

Question: It would be helpful if data regarding leukocyte telomere length and GSH levels are presented in a table.

Answer: The data are presented in an additional table (Table 4). However the original text with the total glutathione (ng/ml) and LTL (bp) remain the section for the results because we think it is more adequate and easier to compare the values directly instead of searching in the table and then again continue reading the main text. In addition in the text the p-values are also given.

Owing to your comment we found we have missed to add the comparrison of total glutathione in GSTM1 and GSTT1 combined genotypes which was added in Results section and in Table 4:

“Controls with non-null genotype of both GSTM1 and GSTT1 had higher total glutathione in the serum in contrast with those caring at least one null genotype (15.72 ± 2.44 ng/ml vs. 7.71 ±1.75 ng/ml, p=0.018, Figure 5). In COPD patient there was a lack of differ-ence in the serum levels of total glutathione between the genotypes (non-null: 9.62±2.59 ng/ml vs. at least one null: 13.15±2.15 ng/ml, p=0.365).”

Question: In Table 3 the genotype “at least one non-null” is inconsistent with the text “at least one null”. Please explain or correct.

Answer: This is a technical mistake. We corrected the text in the table 3: instead of “null” it is now “at least one null”. What is written in the text is the correct one.

 

Question: The authors speculate that the loss of enzymatic activity in carriers of the null genotypes (GSTM1 and GSTT1) result in altered antioxidant defence and detoxification of ROS and their products. Have the authors determined the enzymatic activity of other GST isoenzymes in the carriers of this study? Please elaborate the contribution of the enzymatic activity of other isoenzymes in GSTM1 and GSTT1 null genotypes.

Answer: We did not determine the activity of GST isoenzymes, becuse the measurement of the enzyme activity requires the fresh serum/plasma, as we have done and described in earler paper (>>>>>>>>>). However for the current study, which is cross-sectional not prospective stydy, we used a bank of total DNA extracted during the years. We added into the Discussion section some comments about this topic.

 

Question: The discussion section should be thoroughly revised. In its current form reads as an enriched introduction section. Please focus on discussing the major findings.

Answer: The dicsussion was revised. Some parts have been removed although part of the text is in connection to the explanation of the results.

Question: Please provide the rationale for exploring the correlation between serum total glutathione and LTL in COPD patients.

Answer: Thank you for focusing our attention in this direction. The fact that oxidative stress is a major factor in COPD pathogenesis and telomere shortening suggests that antioxidants may have plausible effect in the prevention of both. Indeed antioxidants have been shown to improve the symptoms in COPD and delay ageing. As being one of the most important antioxidants in the body, we hypothesize glutathione might reflect the LTL. Even more glutathione is a co-substrate  of GSTs in the conjugation reactions for detoxification of variaty of compounds, including derivatives of oxidation of lipids and protein. This was added in Introduction section.

 

Question: Lines 58-59 please add relevant citation

Answer: The comments are considered and changes were done.

 

Question: Lines 72-74 please add relevant citation

Answer: The comments are considered and changes were done.

 

Question: Lines 74-76 please refer to the original study

Answer: The comments are considered and changes were done.

 

Question: Please correct legend in Figure 1 (null GSTM1 genotype – individual 1 and 4) and incorporate M1-0, T1-0, M+, T+ into the legend.

Answer: The comments are considered and changes were done.

 

Question: Line 134: Please Indicate the positive and negative controls that are included in PCRs.

Answer: The comments are considered and changes were done.

 

Question: Figure legends 2, 3 and 4 should be more informative.

Answer: The comments are considered and changes were done.

 

Dear Editor-in-Chief,

Please find enclosed our revised manuscript, “ The leucocyte telomere length, GSTM1 and GSTT1 null geno-types and the risk of COPD” by Tanya Tacheva et al.

In this version, we added changes that we did according to the recommendations of the reviewers.

Further, in the letter you will find all answers to the comments of both reviewers.

We hope that the manuscript will fulfill the high requirements and standard of the journal and will create interest in readers of the journal.

All of the authors declare that they have all participated in the design, execution, and analysis of the paper, and that they have approved the final version. Additionally, there are no conflicts of interest in connection with this paper, and the material described is not under publication or consideration for publication elsewhere.

 

Thank you for your time and assistance.

Sincerely,

 

Corresponding Author

Prof. Tatyana Vlaykova,

Dept. Chemistry and Biochemistry,

Medical Faculty, Trakia University,

Stara Zagora, Bulgaria;

Dept. Medical Biochemistry,

Medical University - Plovdiv,

Plovdiv, Bulgaria

e-mail:  [email protected]

Round 2

Reviewer 1 Report

The authors have improved their manuscript. However there are still some issues that concern me.

First of all, I would suggest to remove the limitations paragraph from the conclusion to the last paragraph in discussion.

Secondly, in Table 4, the level of significance is not presented when comparing COPD patients and controls. I would propose the authors modify the orientation of the Table in order to be more clear the comparison between patients and controls.

Author Response

Answers to the Reviewer 1.

Thank you very much for your assessment and for the new comments which have the goal to improve our manuscript. According to them we have made some new changes:

1) First of all, I would suggest to remove the limitations paragraph from the conclusion to the last paragraph in discussion.

We changed the position of the limitations to the end of Discussion part.

2) Secondly, in Table 4, the level of significance is not presented when comparing COPD patients and controls. I would propose the authors modify the orientation of the Table in order to be more clear the comparison between patients and controls.

We prepared a new version of the Table 4, when we added all p-values of the analyses for LTL and Total glutathione  between individuals with different genotypes in both separate groups of patients and controls), which is the main aim of the analyses. We also added the p-values of the differences between controls and patients with the same genotypes.

Reviewer 2 Report

After reviewing the revised manuscript with the title: The leucocyte telomere length, GSTM1 and GSTT1 null genotypes and the risk of Chronic Obstructive Pulmonary Disease, Manuscript ID cimb-1837919, it is apparent that the authors have incorporated the required comments / changes in the revised manuscript. I suggest a minor comment below.

Minor comment:

Please indicate significant terms in table 4.

Author Response

Answers to the Reviewer 2.

Thank you very much for your assessment and for the new comments which have the goal to improve our manuscript. According to them we have prepared a new version of the Table 4, when we added all p-values of the analyses for LTL and Total glutathione  between individuals with different genotypes in both separate groups of patients and controls), which is the main aim of the analyses. We also added the p-values of the differences between controls and patients with the same genotypes