Association of GSTT1, GSTM1 and GSTP1 (Ile105Val) mRNA Expression with Cardiometabolic Risk Parameters in Women with Breast Cancer and Comorbidities

Round 1

Reviewer 1 Report

The manuscript entitled «Association of GSTT1, GSTM1 and GSTP1 (Ile105Val) Polymorphisms with Cardiometabolic Risk Parameters in Women with Breast Cancer and Comorbidities» is devoted to the important problem of modern biomedical science – molecular genetic basis of cancer and other comorbidities. The manuscript is well-designed but there are some issues that must be solved before publication, mainly the problem with methodology that does not allow to fully assess the scientific significance of the obtained results.

Major issues:

1. It seems to be very confusing why the authors used qPCR (quantitative PCR) and RNA for the analysis of genetic polymorphism? Described protocol is used for gene expression analysis – it is impossible to use RNA for genetic polymorphism analysis. Moreover, the described ΔΔCt method and reference gene (GAPDH) are used also for gene expression analysis. In the current form, it seems impossible to obtain results corresponding to the subject of the manuscript. Authors must explain this issue.
2. Why the authors used TaqMan Fast Virus 1-Step Master Mix developed for viral nucleic acids have very low levels of target and optimized for high-sensitivity detection of viral targets. Authors used human nucleic acids not viral nucleic acids in the research.
3. Full characteristic of studied genes (gene name, reference SNP number, chromosomal position, nucleotide and amino change, primer sequence) must be presented in the separate table in the Material and methods section.
4. What methods or commercial kits were used for the assessment of biochemical profile?
5. It is very low number of patients (34 patients in the case group and 23 patients in the control group) for gene polymorphism associated research. Power calculations must be performed and the obtained results must be presented.
6. In the presented form Discussion section is very descriptive and has no critical discussion of obtained results. So, the possible pathophysiological pathways and mechanisms of obtained associations between genetic polymorphisms and other studied parameters must me discussed (e.g. how the Ile105Val polymorphism in the GSTP1 gene involved in BC susceptibility, LDL level in women and TC, VLDL and TG levels in the control group; how the GSTT1 gene can modulate the blood pressure; why GSTM1 gene associated with TC, VLDL-c and IA including BC, etc.).
7. The text of manuscript contains many dashes(-) in the middle of words (lines 44, 47 and further in the text). Authors must read the text carefully.
8. The text of manuscript must be proofread by native English speaker.

Minor issues:

1. Gene names through the text must be highlighted in italic.
2. Line 13: Glutathione S-transferase (GST) and line 17: GST – italic is not necessary.
3. In the Abstract, the full information about genes must be presented (gene name, nucleotide and amino change, reference SNP number).
4. In the Material and methods, subtitles should be added (e.g. Group description, Biochemical analysis, Molecular genetic testing, etc.).
5. Full exclusion criteria must be showed, not only voluntary What else?
6. Please add information about country and manufacturer of chemicals used in the research.
7. Results of the Kolmogorov-Smirnov test must be presented.
8. Did the distribution of genotypes and alleles correspond to the Hardy-Weinberg distribution and the population level?
9. Lines 195-196 «which suggests that 39.1% of women in this group might have a better response to chemotherapy treatment» – reference?
10. The conclusion «The phenotypes of these polymorphisms could be linked to 237 oxidative stress, low grade chronic inflammation» is not supported by the results of the study – the authors did not study markers of oxidative stress and inflammation.

Author Response

Major issues:

1. It seems to be very confusing why the authors used qPCR (quantitative PCR) and RNA for the analysis of genetic polymorphism? Described protocol is used for gene expression analysis – it is impossible to use RNA for genetic polymorphism analysis. Moreover, the described ΔΔCt method and reference gene (GAPDH) are used also for gene expression analysis. In the current form, it seems impossible to obtain results corresponding to the subject of the manuscript. Authors must explain this issue.

Response: Indeed, it is a study of gene expression because we used total RNA, so the ΔΔCt of each gene was obtained by RT-PCR. However, the aim of this study was not to quantify the expression but rather to determine the presence of these polymorphisms in this type of patients with comorbidities. The results were classified as present and absent, according to the expression and the results of the melting curve (modifications were made in lines 122-148).

2. Why the authors used TaqMan Fast Virus 1-Step Master Mix developed for viral nucleic acids have very low levels of target and optimized for high-sensitivity detection of viral targets. Authors used human nucleic acids not viral nucleic acids in the research.

Response: TaqMan Fast Virus 1-Step Master Mix is ​​designed for rapid and highly sensitive real-time RT-PCR, although its formulation provides improved detection of nucleic acids for viruses, its use was considered because it is ideal also for multiplex gene expression studies, due to its high sensitivity and specificity, target flexibility and consistent results. We clarify this information in lines 140-144.

3. Full characteristic of studied genes (gene name, reference SNP number, chromosomal position, nucleotide and amino change, primer sequence) must be presented in the separate table in the Material and methods section.

Response: We have completed this information partially. We already obtained the gene ID, chromosome position and nucleotide, we are working on the SNP number and aminoacid change. This information will be inserted in table 1.

4. What methods or commercial kits were used for the assessment of biochemical profile?

Response: Glucose and lipids profile tests were performed by Quest Diagnostics^Ò Clinical Analysis Laboratories in Toluca, Mexico. We are waiting for the specific information.

5. It is very low number of patients (34 patients in the case group and 23 patients in the control group) for gene polymorphism associated research. Power calculations must be performed and the obtained results must be presented.
6. Response: During the recruitment period (6 months), the population of women diagnosed with breast cancer at the in the Breast Cancer Detection Clinic (UNEME-DEDICAM; Toluca, State of Mexico) was 59, we performed a power analysis according to this number and we computed a n of 31 women with a confidence level of 90%. We increased the sample to 29 women with breast cancer which is closed to the computed n. We are adjusting the results in table 4 to this sample number.

7. In the presented form Discussion section is very descriptive and has no critical discussion of obtained results. So, the possible pathophysiological pathways and mechanisms of obtained associations between genetic polymorphisms and other studied parameters must be discussed (e.g. how the Ile105Val polymorphism in the GSTP1 gene involved in BC susceptibility, LDL level in women and TC, VLDL and TG levels in the control group; how the GSTT1 gene can modulate the blood pressure; w GSTM1 gene associated with TC, VLDL-c and IA including BC, etc.).

Response: we need to modify tables 3 and 4 in order to draw a better discussion for that.

8. The text of manuscript contains many dashes(-) in the middle of words (lines 44, 47 and further in the text). Authors must read the text carefully.

Response: In process

9. The text of manuscript must be proofread by native English speaker.

Response: In process

Minor issues:

1. Gene names through the text must be highlighted in italic.

Response: This has been revised.

2. Line 13: Glutathione S-transferase (GST) and line 17: GST – italic is not necessary.

Response: This has been revised.

3. In the Abstract, the full information about genes must be presented (gene name, nucleotide and amino change, reference SNP number). Response:

Response: We will add this information in table 1, we will include this information in the abstract if the word length allows it.

4. In the Material and methods, subtitles should be added (e.g. Group description, Biochemical analysis, Molecular genetic testing, etc.)

Response: This has been revised.

5. Full exclusion criteria must be showed, not only voluntary What else?

Response: in process

6. Please add information about country and manufacturer of chemicals used in the research.?

Response: in process

7. Results of the Kolmogorov-Smirnov test must be presented?

Response: in process

8. Did the distribution of genotypes and alleles correspond to the Hardy-Weinberg distribution and the population level? Response: in process.
9. Lines 195-196 «which suggests that 39.1% of women in this group might have a better response to chemotherapy treatment» – reference?

Response: It corresponds to reference number 19.

10. The conclusion «The phenotypes of these polymorphisms could be linked to 237 oxidative stress, low grade chronic inflammation» is not supported by the results of the study – the authors did not study markers of oxidative stress and inflammation.

Response: We agree with the reviewer we did not perform measurements of oxidative stress markers, the statement was hypothetical, we will modify that.

Author Response File: Author Response.docx

Reviewer 2 Report

Yizel Becerril-Alarcón and colleagues studied Association of GSTT1, GSTM1 and GSTP1 (Ile105Val) polymorphisms with cardiometabolic risk parameters in women with breast cancer and comorbidities and they concluded that there is an association between GST genotypes with metabolic parameters, specifically with serum lipids in Mexican women.

I have following major concern regarding the conclusion of study:

Concern 1.    
Authors included 34 patients with breast cancer and the control 23 patients in the study. From clinical perspective, the sample size of control is very small. To maintain statistical reliability, this is the just number of comparisons between the two groups. In other words, the number of patients intra group is quite small in table 4, and it cannot be denied that this result may be a coincidence. Authors should increase the sample size significantly to conclude their study as clinically relevant or need to delete the intra-group comparison.

Concern 2. 
To analyze including both groups in table 5 makes the focus of this study more unclear. The purpose of this study is to clear whether the frequency of the GST family polymorphisms is related to the presence of altered cardio-metabolic variables in women with BC. To investigate including both groups with and without breast cancer deviates from the purpose of this study.

Concern 3
The Mexican population is very unique, because the mixed rate of Latin and Indigena is very high. Probably lipids resistance and the incidence of cancer differ between Caucasian and Mongoloid. For example, in Japanese, the first leading cause of death is malignancy.
This is a genetic study.　Has the racial background of the candidates been investigated? I recommend you discuss about this issue.

Author Response

Major concerns

1. Authors included 34 patients with breast cancer and the control 23 patients in the study This is backwards. From clinical perspective, the sample size of control is very small. To maintain statistical reliability, this is the just number of comparisons between the two groups. In other words, the number of patients intra group is quite small in table 4, and it cannot be denied that this result may be a coincidence. Authors should increase the sample size significantly to conclude their study as clinically relevant or need to delete the intra-group comparison.

Response: During the recruitment period (6 months), the population of women diagnosed with breast cancer at the in the Breast Cancer Detection Clinic (UNEME-DEDICAM; Toluca, State of Mexico) was 59, we performed a power analysis according to this number and we computed a n of 31 women with a confidence level of 90%. We increased the sample to 29 women with breast cancer which is closed to the computed n. We are adjusting the results in table 4 to this sample number. 

2. To analyze including both groups in table 5 makes the focus of this study more unclear. The purpose of this study is to clear whether the frequency of the GST family polymorphisms is related to the presence of altered cardio-metabolic variables in women with BC. To investigate including both groups with and without breast cancer deviates from the purpose of this study.

Response: We deleted table 5

3. 3. The Mexican population is very unique, because the mixed rate of Latin and Indigena is very high. Probably lipids resistance and the incidence of cancer differ between Caucasian and Mongoloid. For example, in Japanese, the first leading cause of death is malignancy.
   This is a genetic study.　Has the racial background of the candidates been investigated? I recommend you discuss about this issue.

Response:  in process

Author Response File: Author Response.docx

Reviewer 3 Report

In this paper, authors  aimed to identify the frequency of Glutathione S-transferase genotypes and their association with clinical, body composition and biochemical parameters in women with breast cancer and metabolic comorbidities. The idea that breast cancer and cardiometabolic diseases shared some interactions and that the incidence and mortality of BC in women worldwide coulb be influenced by those parameters is interesting. Some concerns:

• in the clinical parameters of Table 1, the value of SBP that is higher in control group is a confoundent factor, as far as it is normal in both groups, and maybe it could be substitued with diagnosis of arterial hypertension (if applicable);
• no macroscopic statistical differences emerged from the Table 1 between the two groups, ant it deserves comments in the Discussion;
• in Table 4, what do the authors said for High/normal values? Could they explicate it in the Methods section, please? 
• authors commented about high blood pressure, hyper-glycemia, obesity, high body fat and dyslipidemia, however they did not give any data about uric acid serum, that it is known as a player in the pathogenesis of CV disease affecting vascular structure and function, as well expressed here "Antonini-Canterin F, Di Nora C, Pellegrinet M, Vriz O, La Carrubba S, Carerj S, Zito C, Matescu A, Ravasel A, Cosei I, Popescu BA. Effect of uric acid serum levels on carotid arterial stiffness and intima-media thickness: A high resolution Echo-Tracking Study. Monaldi Arch Chest Dis. 2019 Mar 27;89(1). doi: 10.4081/monaldi.2019.1007". I would suggest to add some comments on this.

Author Response

1. In the clinical parameters of Table 1, the value of SBP that is higher in control group is a confoundent factor, as far as it is normal in both groups, and maybe it could be substitued with diagnosis of arterial hypertension (if applicable).

Response: This is not applicable.

2. No macroscopic statistical differences emerged from the Table 1 between the two groups, ant it deserves comments in the Discussion

            Response:  in process

3. In Table 4, what do the authors said for High/normal values? Could they explicate it in the Methods section, please?

Response: This information was added in the table note.

4. Authors commented about high blood pressure, hyper-glycemia, obesity, high body fat and dyslipidemia, however they did not give any data about uric acid serum, that it is known as a player in the pathogenesis of CV disease affecting vascular structure and function, as well expressed here "Antonini-Canterin F, Di Nora C, Pellegrinet M, Vriz O, La Carrubba S, Carerj S, Zito C, Matescu A, Ravasel A, Cosei I, Popescu BA. Effect of uric acid serum levels on carotid arterial stiffness and intima-media thickness: A high resolution Echo-Tracking Study. Monaldi Arch Chest Dis. 2019 Mar 27;89(1). doi: 10.4081/monaldi.2019.1007". I would suggest to add some comments on this.

Response:  in process

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The major issue that calls into question the experimental design and the possibility to obtain reliable results, voiced earlier (the use of RNA to assess the genetic polymorphism of DNA), is still not resolved. It is still unclear how the evaluation of the gene expression can help to provide data on the frequency of certain genotypes in the studied genes (taking into account the possible post-translational changes). The authors need to correct study design, perform DNA extraction and evaluate polymorphic variants of the corresponding genes according to the generally accepted method.

Author Response

Response: We performed Real time PCR to qualitatively evaluate the mRNA expression of GST variants. We performed this type of PCR because it is more precise and robust to detect and quantitate mRNA expression levels, especially for this type of polymorphic variants.  We have modified the text throughout the manuscript to adjust this misunderstood information (title, abstract, introduction, results and discussion). In fact, we performed Real time RT-PCR, which is usually also known as qPCR and this method can be used for mRNA expression qualitative as well as quantitative evaluation. However, the quantification was not an intended objective for the investigation question we are trying to answer in this manuscript. We are reporting the data from the High Resolution Melting analysis for these variants, as described in the methods section. To prevent any confusion in the aims of this manuscript, we used the term “Real time PCR” because we are actually reporting cDNA data. This is the first study to report this kind of data and we explain this in the introduction and discussion sections.

Author Response File: Author Response.docx

Reviewer 2 Report

Author Yizel Becerril Alarcón is to be congratulated for a nicely presented review.

The authors addressed my concerns nearly in this version.

However, I feel that I have not received an accurate response from the author to my Comment 3, because I emphasized whether the racial background was investigated in this study.

I am waiting for the author's sincere response to this issue.

Author Response

Response: We did not evaluate the racial background to identify the Latin/Indigenous mix rate in this sample of women. But we performed the Hardy Weinberg test, showing an equilibrium in the sample to perform the analysis.  We clarified this information in lines 283-292.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have improved the paper after the revisions suggested.

Author Response

Response: We greatly appreciate your comments and revision for this manuscript. We uploaded the latest version of the manuscript.

Author Response File: Author Response.docx