mtDNA Single-Nucleotide Variants Associated with Type 2 Diabetes

Round 1

Reviewer 1 Report

In the present manuscript, Gaona and colleagues performed a secondary analysis of the complete mtDNA sequences of 1261 T2D patients and 1105 controls to determine whether certain mtDNA mutations are associated with T2D risk. Their results revealed correlations between m.1438A>G (controls: 32 [27.6%], T2D: 84 [72.4%]; OR: 2.46; 95%CI: 1.64-3.78; p<0.001), m.14766C>T (controls: 498 [36.9%], T2D: 853 [63.1%]; OR: 2.57, 95%CI: 2.18-3.04, p<0.001), and m.16519 T>C (controls: 363 [43.4%], T2D: 474 [56.6%]; OR: 1.24, 95%CI: 1.05-1.47, p=0.012) was significantly associated with an increased risk of diabetes. m.16189 T>C variable was not associated with T2D (controls: 148 [13.39] T2D: 171 [13.56%]; OR: 1.03; 95%CI: 0.815-1.31; p=0.83). These results provide further evidence for an association between certain mtDNA polymorphisms and the development of T2D, which is likely to be related to association rules and topological patterns and three-dimensional amplification associated with regions of change, rather than sequence point mutations.

Comments and suggestions:

-          I recommend that you also include the variant number "rs" in the abstract. This will make it easier to attract the readers' interest.

-          The Introduction section is very long (3 pages) and should be shortened to a maximum of 1 page. More descriptions here would fit better in the Discussion section. I suggest rewriting and shortening it.

-          The Materials and Methods section is incomplete.

o   It lacks a precise description of the sample population.

o   What are the definitions of the control and case (T2D) selection criteria?

o   A full description of the statistical methods used is missing. What statistics were used to calculate the OR and for which factors was the OR corrected?

o   Since several polymorphisms were analysed at the same time, was there a test correction (corrected p-value)?

-          Descriptive characterisation of sample populations is missing. Such as age, gender, BMI, medication habits, and other parameters.

-          The authors refer to Tables 1 and 2, but these are not included in the supplementary data or the manuscript.

-          Several variants associated with T2D were identified by the authors. I propose the calculation of a genetic risk score as a method to summarize individual effects. This method would provide an opportunity to develop a risk SNP panel.

-          In Table 1S, I propose a statistical comparison of haplotype frequencies and indication of p-values.

-          How are haplotypes associated with the risk of T2D?

-          The Discussion section lacks a presentation of limitations and strengths.

I recommend a thorough revision of the manuscript and, if possible, the inclusion of more complex statistical analyses.

Author Response

Current Issues in Molecular Biology Editor-in-Chief

Dear peer reviewer

          I am pleased send you the modifications and changes to the manuscript of our original research article entitled “mtDNA single nucleotide variants associated with type 2 diabetes" by García Gaona E., García Gregorio A., García Jiménez C., López-Olaiz MA., Mendoza-Ramírez P., Fernandez-Guzman D:, Pillado Sánchez RA, Soto Pacheco AD, Zuñiga LY., Sánchez-Parada  MG., González Santiago AE., Román Pintos LM., Castañeda Arellano R., Hernández-Ortega LD., Mercado-Sesma AR.,  Orozco Luna FJ, Villa Angulo C, Villa Angulo R  and your humble servant, Baptista Rosas RC. for consider publication. It is important to mention that recently this paper was invited by Ms. Norah Tang, Managing Editor.

            The comments and suggestions that were taken into consideration are the following:

Reviewer 1

-          I recommend that you also include the variant number "rs" in the abstract. This will make it easier to attract the readers' interest.

The dbSNP database IDs ("rs") were added to each of the variants in the abstract according to the peer-reviewer's recommendation.

-          The Introduction section is very long (3 pages) and should be shortened to a maximum of 1 page. More descriptions here would fit better in the Discussion section. I suggest rewriting and shortening it.

      The introduction section was reviewed, and multiple corrections were made to enhance the flow of the text. Paragraphs within the introduction were reorganized, some paragraphs were relocated to the discussion section, redundant information was removed, and certain paragraphs were summarized. The section has been condensed to a total of 2 pages. The changes can be seen in the manuscript; they were highlighted and added using the track changes tool.

      -          The Materials and Methods section is incomplete.

      According to the peer eviewer's recommendations, we added  a paragraph and the original text was corrected.

o   It lacks a precise description of the sample population.

The sequences were obtained from the NCBI Nucleotide database where cases with type 2 diabetes and controls were identified based on information provided in the metadata of each sequence. As they come from different sources, the database does not provide specific information about each patient from which the biological sample for mtDNA extraction was obtained, such as age, gender or specific criteria. To obtain details of the diagnostic criteria used and population data, it is recommended to consult references for each sequence that are provided in Table supplementary_3 as additional material.

o   What are the definitions of the control and case (T2D) selection criteria?

They were defined from sequences metadata provided in the NCBI Nucleotide database: REFERENCE (including AUTHORS, TITLE, JOURNAL, and COMMENT) and FEATURES (including organism, organelle, mol_type, and isolation_source info).

o   A full description of the statistical methods used is missing. What statistics were used to calculate the OR and for which factors was the OR corrected?

The odds ratio was calculated from the fourfold (2x2) contingency tables (variant presence-absence vs. T2D-control) and odds ratio corrected were estimated by logistic regression with a 95% confidence interval without considering missings in the calculation.

o   Since several polymorphisms were analysed at the same time, was there a test correction (corrected p-value)?

Yes, que use corrected p-value with Yates method for continuity in Chi square with goodness of fit.

-          Descriptive characterisation of sample populations is missing. Such as age, gender, BMI, medication habits, and other parameters.

The sequences were obtained from the NCBI Nucleotide database where cases with type 2 diabetes and controls were identified based on information provided in the metadata of each sequence. As they come from different sources, the database does not provide specific information about each patient from which the biological sample for mtDNA extraction was obtained, such as age, gender or specific criteria. To obtain details of the diagnostic criteria used and population data, it is recommended to consult references for each sequence that are provided in Table supplementary_3 as additional material.

-          The authors refer to Tables 1 and 2, but these are not included in the supplementary data or the manuscript.

We apologize for the oversight. The tables were included in our original submission. We are resending them for your reference. Thank you for bringing this to our attention.

-          Several variants associated with T2D were identified by the authors. I propose the calculation of a genetic risk score as a method to summarize individual effects. This method would provide an opportunity to develop a risk SNP panel.

According to the reviewer's recommendations, we added a generalized linear analysis and estimated the area under the ROC curve to estimate a risk score. This new process is incorporated into the manuscript in materials and methods and in documents, in addition to adding Figure 3.

-          In Table 1S, I propose a statistical comparison of haplotype frequencies and indication of p-values.   How are haplotypes associated with the risk of T2D?

In general, the polymorphisms m.73A>G, m.1189T>C, m.1193T>C, m.1420T>C, m.1438A>G, m.2667T>C, m.3027T>C, m.10398A> G and m.16519T>C are associated with a higher risk of developing type 2 diabetes; while the variants m.1811A>G and m.14766C>T and m.16126T>C are associated with a protective effect of lower risks of developing the disease (they are more frequent in the control population).

-          The Discussion section lacks a presentation of limitations and strengths.

I recommend a thorough revision of the manuscript and, if possible, the inclusion of more complex statistical analyses.

To deepen the statistical analysis of the findings provided by the chi-square test with goodness of fit, four additional statistical tests were carried out:

1. A proportion test was added to compare the proportion of successes in independent groups. A new table was attached to the draft in Table supplementary S2.
2. Generalized Logistic regression model were performed to examine the relationship between categorical variables controlling for other variants in other positions. This will allow you to evaluate the effect of a categorical variable on the probability of a binary or categorical outcome, taking into account other predictor variables such as the association between various polymorphisms. A multinomial logistic regression analysis was also constructed to analyze the association between them and understand how the categories of the dependent variable diabetes are related to the predictor variables related to the presence of various mitochondrial polymorphisms. A new table was attached to the draft in Figure supplementary S3.
3. From the regression model, a Receiver Operating Characteristic (ROC) curve was constructed and the area under the curve (AUC) was estimated by calculating the sensitivity and specificity of the variants as a whole in a patient with type 2 diabetes. A new chart was attached to draft as Figure supplementary S3.
4. Two new post-hoc tests were included, designed based on significant differences between the categorical variables presence/absence of polymorphism in a given position in the chi-square test. The standardized residual test and Bonferroni adjustment were performed to control type I error and counteract the problem of multiple comparisons. Standardized residuals indicate how much the observed frequencies deviate from the expected frequencies under the null hypothesis where large values in absolute value indicate significant discrepancies. Two new tables were attached to the draft in Tables supplementaries S4 and S5.

We appreciate your consideration.

Author Response File: Author Response.pdf

Reviewer 2 Report

Abstract Section:

The abstract provides a comprehensive overview of the study and its findings. However, there are a few minor improvements and clarifications the authors can consider:

• "...were significantly associated with diabetes probability." It could be rephrased as "...were significantly associated with the likelihood of developing diabetes."
• The sentence "The variant m.16189T>C Previously reported in multiple studies in different populations, it was not found to be associated with T2D in our analysis (controls: 148 [13.39] T2D: 171..." can be improved by removing "it."
• The final sentence can be rephrased for clarity: "These results provide evidence suggesting a link between specific mtDNA polymorphisms and T2D, possibly related to association rules, topological patterns, and three-dimensional conformations associated with regions where the changes occur, rather than specific point mutations in the sequence."

These changes enhance the clarity and readability of the abstract.

Introduction Section

The introduction is quite detailed, but its length is acceptable for the research paper. It provides a comprehensive overview of the topic and sets the stage for the study. However, there are a few minor grammar and readability improvements.

For example,

The authors should consider rephrasing the final sentence: The primary objective of this study was to determine the frequency of mtDNA polymorphisms in patients with and without T2D and their potential association.

 Is the reference citation style in accordance with the journal's guidelines?

Material and Methods Section

Overall, the section appears to be well-documented, but breaking down some of the technical details and providing context can enhance clarity.

Results Section

The Results section provides a detailed account of the study's findings. It appears clear and well-structured. However, here are a few suggestions to improve clarity and readability:

-       In the first paragraph, consider rephrasing the sentence: "After the graphical analysis and the statistical test to explore normality, it is shown that the distribution of the frequency of variants has a non-parametric distribution." This sentence could be clarified by stating that the frequency distribution of variants does not follow a normal distribution.

Discussion Section

Overall, the discussion is informative and well-structured but could benefit from a bit more detail in certain areas and some structural adjustments to enhance clarity and readability.

-       Are there no citations in the first paragraph of this section?

-       The authors should be cautious of repeating the same information or phrases unnecessarily. For example, paragraphs 13 and 20 appear to contain similar or redundant information.  

paragraph 13 (”These discrepancies could be attributed to differences in the populations studied ............”)  

paragraph 20 (”These discrepancies could be attributed to differences in the populations studied,..........”)

-       While the authors touch on the potential clinical implications of their findings, they could expand on this aspect. They should discuss how the discovered mitochondrial variants might impact clinical practice, diagnosis, or treatment of T2D.

-       Are there any limitations of the study?

Conclusions Section

This section is clear and effectively summarizes the current state of research in this area. However, to enhance it, emphasize your study's contribution, reiterate the complexity of the subject, and provide more specific insights into the implications of your findings for future research and clinical practice.

Author Response

Dear peer reviewer

            I am pleased send you the modifications and changes to the manuscript of our original research article entitled “mtDNA single nucleotide variants associated with type 2 diabetes" by García Gaona E., García Gregorio A., García Jiménez C., López-Olaiz MA., Mendoza-Ramírez P., Fernandez-Guzman D:, Pillado Sánchez RA, Soto Pacheco AD, Zuñiga LY., Sánchez-Parada  MG., González Santiago AE., Román Pintos LM., Castañeda Arellano R., Hernández-Ortega LD., Mercado-Sesma AR.,  Orozco Luna FJ, Villa Angulo C, Villa Angulo R  and your humble servant, Baptista Rosas RC. for consider publication. It is important to mention that recently this paper was invited by Ms. Norah Tang, Managing Editor.

             The comments and suggestions that were taken into consideration are the following:

Reviewer 2

The abstract provides a comprehensive overview of the study and its findings. However, there are a few minor improvements and clarifications the authors can consider:

• "...were significantly associated with diabetes probability." It could be rephrased as "...were significantly associated with the likelihood of developing diabetes."

            The paragraph was corrected according to the recommendations of the peer review

• The sentence "The variant m.16189T>C Previously reported in multiple studies in different populations, it was not found to be associated with T2D in our analysis (controls: 148 [13.39] T2D: 171..." can be improved by removing "it."

• The final sentence can be rephrased for clarity: "These results provide evidence suggesting a link between specific mtDNA polymorphisms and T2D, possibly related to association rules, topological patterns, and three-dimensional conformations associated with regions where the changes occur, rather than specific point mutations in the sequence."

             .

These changes enhance the clarity and readability of the abstract.

All changes based on the peer review recommendations were clearly noted through track changes and notes added to the footer of the draft.

Introduction Section

The introduction is quite detailed, but its length is acceptable for the research paper. It provides a comprehensive overview of the topic and sets the stage for the study. However, there are a few minor grammar and readability improvements.

For example,

The authors should consider rephrasing the final sentence: The primary objective of this study was to determine the frequency of mtDNA polymorphisms in patients with and without T2D and their potential association.

The introduction section was reviewed, and multiple corrections were made to enhance the flow of the text. Paragraphs within the introduction were reorganized, some paragraphs were relocated to the discussion section, redundant information was removed, and certain paragraphs were summarized. The section has been condensed to a total of 2 pages.

 Is the reference citation style in accordance with the journal's guidelines?

The style of our references citations has been changed according to the reference list and citations style guide for MDPI Journals version 5

Material and Methods Section

Overall, the section appears to be well-documented, but breaking down some of the technical details and providing context can enhance clarity.

The materials and methods section was reviewed according to the recommendations of the peer review and details were added to make it more explicit and several of its parts were rewritten to make the description of the procedures clearer. All statistics and graphs were made using R version 4.0.3.

Results Section

The Results section provides a detailed account of the study's findings. It appears clear and well-structured. However, here are a few suggestions to improve clarity and readability:

-       In the first paragraph, consider rephrasing the sentence: "After the graphical analysis and the statistical test to explore normality, it is shown that the distribution of the frequency of variants has a non-parametric distribution." This sentence could be clarified by stating that the frequency distribution of variants does not follow a normal distribution.

            The sentence was revised to address the reviewers' comments.

Discussion Section

Overall, the discussion is informative and well-structured but could benefit from a bit more detail in certain areas and some structural adjustments to enhance clarity and readability.

-       Are there no citations in the first paragraph of this section?

The first paragraph was reviewed, changes were made to enhance the text, and the appropriate citations were added.

The following text:

“Although there is a well-established relationship between mitochondrial physiology and T2D, mitochondrial dysfunction can hurt glucose metabolism and contribute to the development of the disease; changes in the sequence of the mitochondrial genome could have some role in the related pathophysiology, have been found recurrently and associated with specific populations. Some of the ways that mitochondrial dysfunction can be linked to T2D are related to decreased energy production, oxidative stress, altered metabolism of fatty acids, inflammation, and cell signaling. Mitochondria are responsible for the production of adenosine triphosphate (ATP), the primary source of energy for cells, and dysfunction can reduce ATP production, which can affect cells' ability to metabolize glucose and regulate blood sugar levels properly. In the same way, this mitochondrial dysfunction can lead to increased production of reactive oxygen species, resulting in oxidative stress.Oxidative stress can damage cells and tissues, including the insulin-producing beta cells of the pancreas, leading to decreased insulin secretion and insulin resistance, critical features of T2D” was replaced by:

“Although a well-established relationship exists between mitochondrial physiology and Type 2 Diabetes (T2D), mitochondrial dysfunction can impair glucose metabolism and contribute to the disease's development (Szendroedi et al., 2007).Mitochondrial dysfunction is intricately linked to T2D through several pathways, which include diminished energy production, oxidative stress, the altered metabolism of fatty acids, inflammation, and aberrations in cell signaling. Mitochondria play a pivotal role in synthesizing adenosine triphosphate (ATP), which is the cellular mainstay of energy. Consequently, any dysfunction may diminish ATP production, impairing the cells' capacity to metabolize glucose and regulate blood sugar levels effectively. Concurrently, mitochondrial dysfunction can also augment the production of reactive oxygen species, engendering oxidative stress. This stress has the potential to inflict damage on cells and tissues, notably the insulin-producing beta cells in the pancreas. Such damage could precipitate a decline in insulin secretion and foster insulin resistance, both of which are crucial characteristics of T2D (Kelley et al., 2002; Sangwung et al., 2020; Schrauwen & Hesselink, 2004).”

Paragraphs that were repeated were removed, changes were made to some sentences to improve the wording, some words were replaced, a section was moved from the introduction to the discussion, and two new paragraphs were added delving into the current evidence surrounding the identified polymorphisms.

-       The authors should be cautious of repeating the same information or phrases unnecessarily. For example, paragraphs 13 and 20 appear to contain similar or redundant information.  

paragraph 13 (”These discrepancies could be attributed to differences in the populations studied ............”)  

paragraph 20 (”These discrepancies could be attributed to differences in the populations studied,..........”)

Paragraphs with repetitive or redundant information were corrected or removed.

-       While the authors touch on the potential clinical implications of their findings, they could expand on this aspect. They should discuss how the discovered mitochondrial variants might impact clinical practice, diagnosis, or treatment of T2D.

It is discussed in the conclusion section the importance of the research of the mitochondrial SNPs such as the improvement of this area such as the personalized medicine.

-       Are there any limitations of the study?

A brief section was added summarizing some of the limitations found in our research, as well as areas for improvement for future investigations.

Conclusions Section

This section is clear and effectively summarizes the current state of research in this area. However, to enhance it, emphasize your study's contribution, reiterate the complexity of the subject, and provide more specific insights into the implications of your findings for future research and clinical practice.

            We appreciate your consideration. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I accept the authors' answers to my questions/comments.