Correlation of Eight (8) Polymorphisms and Their Genotypes with the Risk Factors of Cardiovascular Disease in a Black Elderly Population

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review this interesting paper.

 

Comment 1:

The results presented from lines 172-185 are overly complex and difficult to understand. Please simplify this section and avoid making judgments about whether values have increased or decreased, particularly for results that are not statistically significant.

 

Comment 2:

The linear regression analysis conducted lacks clarity regarding the covariates used. Additionally, please present the statistical results in terms of beta coefficients and standard errors. While statistical values are provided for rs28362286, there is a lack of statistical metrics for the more significant rs67608943, which makes interpretation challenging.

 

Comment 3:

Table 1 is quite difficult to read. Please revise the table to enhance clarity. Furthermore, rs67608943 appears to be a monomorphic marker; if this is the case, it should be excluded from the results. I also suggest presenting the information in a chart format instead of a table for better visualization.

 

Comment 4:

The discussion section appears to be overly lengthy concerning non-significant SNPs. It would be beneficial to focus the discussion around the PCSK9 SNPs and either reduce or remove the content regarding the other SNPs.

 

Comment 5:

The study limitations are noted, but the discussion is too brief. Issues such as sample size and the focus on an older demographic should be elaborated upon. This study primarily serves as a pilot involving an African population, making it difficult to derive broader significance beyond the initial findings.

Author Response

Comment 1: The results presented from lines 172-185 are overly complex and difficult to understand. Please simplify this section and avoid making judgments about whether values have increased or decreased, particularly for results that are not statistically significant.

Response 1: We thank reviewer 1 for drawing our attention to the clarity in this part of the paper. The reviewer’s comment has helped us to considerably improve this section, which now reads:

The genotype frequencies of the polymorphisms for the CVR factors are shown in Table 1. Subgroup analysis showed that some of the genotype carriers had higher levels of the CVR factors than others. Overall, the Lipo (a) levels were found to be elevated in this elderly population. The Lipo (a) levels were higher in the TA genotype carriers of the rs675 polymorphism than the T genotype carriers. According to the South African Hypertension Society (SAHS) guidelines, this population is hypertensive (>140/90 mm Hg) with higher BP readings found in the G genotype carriers of the rs699 polymorphism compared to the AG genotype carriers. The TC, TG, LDL-C and Lipo (a) levels were found to be elevated in the carriers of the T, GT, GT and G genotypes of the rs1968905 polymorphism. The TC, TG, LDL-C and Lipo (a) were also elevated in the carriers of the TC, T, C and C genotypes of the rs247616 polymorphism, respectively. The glucose and insulin levels were elevated especially in the C genotype carriers of the rs1801278 polymorphism. The Hyc levels were elevated in this population with the highest levels present in the carriers of the GA genotype than the A and G genotype of the rs1805087. The PCSK9 levels were low in this population with the highest levels present in the carriers of the CA genotype of rs28362286 than the C genotypes of both PCSK9 polymorphisms.

 

Comment 2: The linear regression analysis conducted lacks clarity regarding the covariates used. Additionally, please present the statistical results in terms of beta coefficients and standard errors. While statistical values are provided for rs28362286, there is a lack of statistical metrics for the more significant rs67608943, which makes interpretation challenging.

Response 2: We appreciate this comment from the reviewer. We have added further explanations on the linear regression analysis in the data analysis section as per below:

Multilinear regression analysis was conducted to assess the relationship between the polymorphism genotypes and the CVR markers. Eight polymorphisms were examined: rs675 (ApoA-IV), rs699 (Angiotensinogen (AGT)), rs247616 & rs1968905 (CETP), rs1801278 (IRS-1), rs1805087 (MTHFR) and rs28362286 & rs67608943 (PCSK9) due to their influence on the gene activity that affects the concentrations of corresponding CVR markers in blood. We assessed the normal distribution of the residuals, checked for multicollinearity and homoscedasticity, and evaluated the priori power to analyze the findings. Subsequently, we utilized a right-tailed F test to assess the statistical significance of the regression model.

The findings presented in this paper are from a broader multidisciplinary research project that further explored these relationships using other statistical tools, For example:

Chalwe, JM et al., (2023) Available from: https://doi.org/10.3390/nu15112470 

While these factors are not discussed in this paper, they have been referenced and discussed in other reports cited in this paper. The aim of this study was to simply correlate eight (8) polymorphisms and their genotypes with the risk factors of CVD in a black elderly population.

 

Comment 3: Table 1 is quite difficult to read. Please revise the table to enhance clarity. Furthermore, rs67608943 appears to be a monomorphic marker; if this is the case, it should be excluded from the results. I also suggest presenting the information in a chart format instead of a table for better visualization.

Response 3: We appreciate this comment from the reviewer. We also acknowledge that there may be some inconsistency on this point.

However, we would like to maintain the current format of the results in Table 1 in order to align with the structure of our discussion. It's also worth noting that the other two reviewers have not raised any comments or concerns about Table 1 nor requested us to change it into a chart.

 

Comment 4: The discussion section appears to be overly lengthy concerning non-significant SNPs. It would be beneficial to focus the discussion around the PCSK9 SNPs and either reduce or remove the content regarding the other SNPs.

Response 4: The reviewer's comment is much appreciated by us. While we recognize that certain SNPs seemed to lack significance, it's important to note that this research is a component of a long-term study, and we aim to uphold consistency in the findings presented.

 

Comment 5: The study limitations are noted, but the discussion is too brief. Issues such as sample size and the focus on an older demographic should be elaborated upon. This study primarily serves as a pilot involving an African population, making it difficult to derive broader significance beyond the initial findings.

Response 5: We would like to thank reviewer 1 for drawing our attention to the clarity in this part of the paper. The reviewer’s comment has helped us to considerably improve this section, which now reads:

Our research was limited to participants who visited the elderly daycare center in Sharpeville. We specifically focused on examining eight (8) polymorphisms: rs675 (ApoA-IV), rs699 (Angiotensinogen (AGT)), rs247616 & rs1968905 (CETP), rs1801278 (IRS-1), rs1805087 – (MTHFR) and rs28362286 & rs67608943 (PCSK9). Similarly, in assessing the cardiovascular risk (CVR) factors, we only analyzed Apo A-IV, ApoB, HDL-C, hcy, insulin, Lipo (a), LDL-C, TC and TG. Lastly, the use of a convenient sample method resulted in a small population size, impacting the generalizability of our findings. Consequently, the findings of this report are specific to this particular setting and cannot be applied to the broader population.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Chalwa and colleagues shows how polymorphisms and genotypes of specific genes are associated with risk factors for CVD. The manuscript is well-developed and includes a discussion in which the authors address each polymorphism analyzed in the context of its association with a risk factor for CVD. Although the manuscript is well written, some things must be corrected before publication.

 

Why were these polymorphisms selected, mostly one from each gene, except for CEPT and PCSK9, where two polymorphisms were selected?

 

Why are not all results regarding the association of all analyzed SNPs with all investigated risk factors for cardiovascular diseases shown in Table 1, but only selected ones? This should be explained in more detail.

 

In the Methods section, the authors state that genotyping was performed using a massARRAY genotyping system and MALDI-TOF. Subsequently, TaqMan probes and a BioRad CFX real-time PCR system were used. If TaqMan probes had been used, the entire analysis would probably have been performed with a BioRad instrument, not a MassARRAY instrument and MALDI-TOF. The authors should be more precise here, as it is unclear from what has been written so far how the genotyping was performed.

 

The authors often state the genotype as an allele. For example, line 22, the AG allele of the rs699 polymorphism is mentioned, but it is a genotype. On the other hand, allele G is referred to as a genotype (line 269). This inconsistency runs through the entire manuscript, and the authors should check and correct everything in detail (lines 178-179, 180, 183, 185, 243, 269, 270, 272, 314, 315, 317-319, 353, 354, 380, 403).

 

The abstract should directly mention which genes the analyzed SNPs mentioned are to be found in, as in the introduction (lines 109-110), because it was only written on page 4, lines 155-158.

 

The authors use different abbreviations for triglycerides. In line 168, for example, the abbreviation is TG, while in line 169, it is Trig. The entire manuscript should be harmonized, and a single abbreviation should be agreed upon. The abbreviation Trig is used in lines 169, 248, 409 and 414.

 

When abbreviations such as Lipo (a) (line 174) and Hyc (line 182) are mentioned for the first time in chapter 3.1, the authors should write their full names and not wait until the discussion or below table to clarify what they are precisely.

 

Table 1 should be referenced in the text of the manuscript itself.

 

In Table 1, for polymorphism rs1801278, there should be a zero before the dot when the p-value is shown.

 

Line 266: The authors mention the RAS system, but it is not entirely clear what it is or what it is for, which needs to be clarified.

 

Lines 269-270: 'The allele carriers of the G genotype had higher...' The sentence is incomprehensible and should be rephrased.

 

Line 288 - the LDLR gene does not code for LDL. As stated in the following sentence, LDLR is the gene that codes for the LDL receptor. Correct or rephrase the sentence.

 

Line 412 - if the p-value is minimal, write p<0.001, not p=0.000.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Comment 1: Why were these polymorphisms selected, mostly one from each gene, except for CEPT and PCSK9, where two polymorphisms were selected?

Response 1: We are grateful for the reviewer's comment. Below, we will explain the rationale behind the selection of these specific SNPs.

The co-authors of this paper initiated research on the elderly population more than ten years ago, focusing on investigating various cardiovascular risk factors such as homocysteine, lipids, and glucose. The genetic aspects of these factors were incorporated later as part of ongoing and longitudinal studies. The specific polymorphisms for this study were chosen because the gene products they affect had already been examined as independent markers previously. The methodologies for these factors had already been established and optimized. Two SNPs each for CEPT and PCSK9 were included due to funding from another project aimed at exploring these genes alongside other markers. The objective of this study was to establish correlations between the polymorphisms and their genotypes with the cardiovascular disease risk factors in the elderly population.

 

Comment 2: Why are not all results regarding the association of all analyzed SNPs with all investigated risk factors for cardiovascular diseases shown in Table 1, but only selected ones? This should be explained in more detail.

Response 2: We appreciate this comment from the reviewer.

The findings presented in this paper are from a multidisciplinary research project that examined various factors, including social demographics and blood biochemical markers.

While these factors are not discussed in this paper, they have been referenced and discussed in other reports cited in this paper. For example:

Oldewage-Theron WH et al., (2008). Available from: https://journals.co.za/doi/abs/10.10520/EJC35054

Grobler CJ (2015) Available from: https://openscholar.dut.ac.za/handle/10321/1421

Chalwe, JM et al., (2023) Available from: https://doi.org/10.3390/nu15112470

 

Comment 3: In the Methods section, the authors state that genotyping was performed using a massARRAY genotyping system and MALDI-TOF. Subsequently, TaqMan probes and a BioRad CFX real-time PCR system were used. If TaqMan probes had been used, the entire analysis would probably have been performed with a BioRad instrument, not a MassARRAY instrument and MALDI-TOF. The authors should be more precise here, as it is unclear from what has been written so far how the genotyping was performed.

Response 3: We appreciate this comment from the reviewer and have rephrased the method section to give more clarity as per below:

The genotyping was outsourced to Inqaba Biotechnical Industries (Pty) Ltd, located in Pretoria, South Africa, as previously referenced [14]. They utilized the Agena Bioscience MassARRAY genotyping system, which employs single base-extension or cleavage chemistry in conjunction with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. The following SNPs: rs675, rs699, rs247616, rs1968905, rs1801278, rs1805087, rs28362286, and rs67608943 were analyzed in each sample. The first step was Polymerase Chain Reaction (PCR), which was carried out using the Bio-Rad CFX Real-Time PCR System, located in Hercules, California, USA, following the manufacturer’s specifications. The primers and probes were obtained from Inqaba Biotech, located in Pretoria, South Africa, at concentrations of 10 μM. The amplification of the specific genomic DNA fragments was conducted using PCR. A Multiplex PCR cocktail was prepared according to the manufacturer’s instructions. A no-template control was included in every PCR reaction to detect false positive reactions. The products were analyzed using the MassARRAY Compact mass spectrometer and Agena real-time detection software, based in San Diego, United States. This was then followed by genotyping on the Agena MassARRAY platform, based in San Diego, United States. The genotyping was performed using the TaqMan® Pre-designed SNP Genotyping Assay Kit from Thermo Fisher Scientific, located in Foster City, CA, USA. The 20 µl reaction mix consisted of 1 µl of template DNA (15 ng/µl), 10 µl of TaqMan® Genotyping Master Mix (Cat. # 4371355), 1 µl of probe (TaqMan® Pre-designed SNP Genotyping Assay), and 8 µl of deionized water. Before the reaction, the probe was diluted in Tris EDTA buffer (10 mM Tris–HCl (pH 8.0), 0.1 mM EDTA) at a 1:1 ratio. The detection of rs675, rs699, rs247616, rs1968905, rs1801278, rs1805087, rs28362286, and rs67608943 was successful in the participants, and the genotypes of the SNPs were also determined.

 

Comment 4: The authors often state the genotype as an allele. For example, line 22, the AG allele of the rs699 polymorphism is mentioned, but it is a genotype. On the other hand, allele G is referred to as a genotype (line 269). This inconsistency runs through the entire manuscript, and the authors should check and correct everything in detail (lines 178-179, 180, 183, 185, 243, 269, 270, 272, 314, 315, 317-319, 353, 354, 380, 403).

Response 4: We appreciate this comment from the reviewer and completely agree. We have corrected the entire manuscript by replacing the term ‘allele’ with ‘genotype’.

 

Comment 5: The abstract should directly mention which genes the analyzed SNPs mentioned are to be found in, as in the introduction (lines 109-110), because it was only written on page 4, lines 155-158.

Response 5: We appreciate this comment from the reviewer and have addressed this by directly mentioning the genes where each of the analyzed are found in the abstract.

 

Comment 6: The authors use different abbreviations for triglycerides. In line 168, for example, the abbreviation is TG, while in line 169, it is Trig. The entire manuscript should be harmonized, and a single abbreviation should be agreed upon. The abbreviation Trig is used in lines 169, 248, 409 and 414.

Response 6: We appreciate this comment from the reviewer and have addressed it as per below.

We have had the entire manuscript harmonized by using the abbreviation for triglycerides as ‘TG’.

 

Comment 7: When abbreviations such as Lipo (a) (line 174) and Hyc (line 182) are mentioned for the first time in chapter 3.1, the authors should write their full names and not wait until the discussion or below table to clarify what they are precisely.

Response 7: We appreciate this comment from the reviewer and have addressed it as per below.

The abbreviations for Lipo(a) and Hyc are first mentioned in Chapter 2.4, line 126. We had initially spelled out their names in full.

 

Comment 8: Table 1 should be referenced in the text of the manuscript itself.

Response 8: We appreciate this comment from the reviewer and have addressed it as per below.

Table 1 has been referenced in the manuscript on Line 162 and Line 173.

 

Comment 9: In Table 1, for polymorphism rs1801278, there should be a zero before the dot when the p-value is shown.

Response 9: We appreciate this comment from the reviewer and have addressed it as per below.

In Table 1, for the rs1801278 polymorphism, leading zeros have been added before the decimal point when displaying the p-value.

 

Comment 10: Line 266: The authors mention the RAS system, but it is not entirely clear what it is or what it is for, which needs to be clarified.

Response 10: We appreciate this comment from the reviewer and have addressed it as per below.

The RAS is first mentioned in Line 259 and is described as follows:

“AGT is an α2-globulin and the precursor of angiotensin II and the substrate for renin. It is produced by hepatic parenchymal cells in the liver [37 & 38]. AGT is a part of the renin-angiotensin system (RAS), a hormone system that regulates blood pressure and fluid balance by salt retention and vasoconstriction [39 & 40].”

 

Comment 11: Lines 269-270: 'The allele carriers of the G genotype had higher...' The sentence is incomprehensible and should be rephrased.

Response 11: We appreciate this comment from the reviewer and have addressed it as per below.

The statement in Line 269-270 has been revised as per below:

The carriers of the G genotype of the rs699 SNP had elevated BP readings compared to the carriers of the AG genotype.

 

Comment 12: Line 288 - the LDLR gene does not code for LDL. As stated in the following sentence, LDLR is the gene that codes for the LDL receptor. Correct or rephrase the sentence.

Response 12: We entirely agree with the reviewer. To address this, we have removed the following statement:

“LDL is encoded by the LDLR gene.”

We hope that this provides clarity to the text.

 

Comment 13: Line 412 - if the p-value is minimal, write p<0.001, not p=0.000.

Response 13: We appreciate this comment from the reviewer and have addressed it as per below.

We have addressed this by correcting the p-value as p<0.001

 

Comment 14: Minor editing of English language required.

Response 14: We appreciate this comment from the reviewer and have addressed it as per below.

We resolved this issue by having the entire manuscript language edited and revised.

Reviewer 3 Report

Comments and Suggestions for Authors

Your research is the impetus for more extensive research that should confirm your findings. Research is particularly important for underdeveloped countries where the population needs to be directed to a healthy lifestyle in order to reduce the incidence of cardiovascular diseases. They used most of the biomarkers and thus opened a possible path towards the treatment of CVD in older populations (eg. arterial hypertension).

Through extensive discussion and adequate statistical methods, you have earned the right to publish this research.

Author Response

Comment 1: Your research is the impetus for more extensive research that should confirm your findings. Research is particularly important for underdeveloped countries where the population needs to be directed to a healthy lifestyle in order to reduce the incidence of cardiovascular diseases. They used most of the biomarkers and thus opened a possible path towards the treatment of CVD in older populations (eg., arterial hypertension).

Through extensive discussion and adequate statistical methods, you have earned the right to publish this research.

Response 1: We appreciate this feedback from the reviewer.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for your efforts in revising the manuscript. However, I have several concerns that I believe need to be addressed before the paper can be considered for publication.

Firstly, while the authors state that there are no issues with Table 1, I must point out that the rs67608943 SNP is monomorphic, with only the C allele present and a frequency of F < 0.001. Therefore, the reported p-value of < 0.001 is not logically tenable.

Furthermore, in Line 189, the authors assert that "The Lipo (a) levels were higher in the TA genotype carriers of the rs675 polymorphism than in the T genotype carriers." However, Table 1 shows no statistical significance, and the standard deviation for T-carriers is notably large. If this result were represented graphically, it would likely underscore the large SD, which may imply an unwillingness to present it clearly.

Similarly, in Line 191, the statement regarding the hypertensive population (BP > 140/90 mm Hg) and the comparison of BP readings between G genotype carriers and AG genotype carriers lacks statistical significance.

In Sub-section 3.1, the only claim that appears to have a significant association with CVR factors is regarding PCSK9 levels, specifically that "the PCSK9 levels were low in this population with the highest levels present in the carriers of the CA genotype of rs28362286 compared to the C genotypes of both PCSK9 polymorphisms." All other results presented seem to lack significance.

Moreover, in Sub-section 3.2, Lines 205-206 claim a significant association between the rs67608943 polymorphism (p < 0.001) and PCSK9 levels. This statement could lead to confusion, as it is illogical to derive statistical analysis from a monomorphic SNP where only one allele is detected.

I am concerned that the Discussion section is lengthy and relies heavily on results that lack statistical significance. I believe it is inappropriate to ascribe meaning to findings that are not statistically supported.

Overall, it seems that my initial review comments have not been adequately addressed. In my experience, the only result from this study that holds any significant weight is the finding related to rs28362286.

If the manuscript remains unchanged, I will have no option but to recommend rejection.

Thank you for your understanding.

Author Response

Comment 1: Firstly, while the authors state that there are no issues with Table 1, I must point out that the rs67608943 SNP is monomorphic, with only the C allele present and a frequency of F < 0.001. Therefore, the reported p-value of < 0.001 is not logically tenable.

Response 1: We express our gratitude to Reviewer 1 for highlighting the need for clarity in this section of the manuscript. The insightful feedback has significantly enhanced the results section. Consequently, we have undertaken a revision of the entire table. Furthermore, we have restructured some results as figures and included only the p-values pertinent to the associations described.

 

Comment 2: Furthermore, in Line 189, the authors assert that "The Lipo (a) levels were higher in the TA genotype carriers of the rs675 polymorphism than in the T genotype carriers." However, Table 1 shows no statistical significance, and the standard deviation for T-carriers is notably large. If this result were represented graphically, it would likely underscore the large SD, which may imply an unwillingness to present it clearly.

Response 2: We appreciate this comment from the reviewer. Accordingly, we have removed the aforementioned statement as Table 1 does not indicate statistical significance.

 

Furthermore, we have revised the entire manuscript to refrain from making determinations regarding increases or decreases in values, especially concerning results that lack statistical significance.

 

Comment 3: Similarly, in Line 191, the statement regarding the hypertensive population (BP > 140/90 mm Hg) and the comparison of BP readings between G genotype carriers and AG genotype carriers lacks statistical significance.

Response 3: We express our gratitude for the reviewer’s insightful comment. This section of the manuscript has been amended accordingly and now states:

‘Subgroup analysis indicated that certain genotype carriers exhibited variations in their concentrations of CVR factors compared to others; however, these differences did not reach statistical significance. For example, carriers of the G genotype of the rs699 polymorphism showed marginally different blood pressure readings compared to the AG genotype carriers.’

 

Comment 4: In Sub-section 3.1, the only claim that appears to have a significant association with CVR factors is regarding PCSK9 levels, specifically that "the PCSK9 levels were low in this population with the highest levels present in the carriers of the CA genotype of rs28362286 compared to the C genotypes of both PCSK9 polymorphisms." All other results presented seem to lack significance.

Response 4: We concur with the reviewer's observations. Consequently, we have eliminated the referenced statements and retained only the following:

"the PCSK9 levels were low in this population with the highest levels present in the carriers of the CA genotype of rs28362286 compared to the C genotypes of both PCSK9 polymorphisms."

 

Comment 5: Moreover, in Sub-section 3.2, Lines 205-206 claim a significant association between the rs67608943 polymorphism (p < 0.001) and PCSK9 levels. This statement could lead to confusion, as it is illogical to derive statistical analysis from a monomorphic SNP where only one allele is detected.

Response 5: We agree with the reviewer's observations. Section 3.2 has been revised accordingly and now states:

The results of the multiple linear regression analysis indicated a significant relationship between the rs28362286 polymorphism and the PCSK9 levels in this elderly population (F(1, 47) = 5.1, p = .0290, R² = 0.1, R² adjusted = 0.08). This suggests that the rs28362286 polymorphism had a strong direct association with PCSK9 levels. However, the analysis revealed that the other SNPs showed collectively non-significant relationships with cardiovascular risk factors.

 

Comment 6: I am concerned that the Discussion section is lengthy and relies heavily on results that lack statistical significance. I believe it is inappropriate to ascribe meaning to findings that are not statistically supported.

Response 6: We appreciate the reviewer's insightful comment. Consequently, we have condensed the discussion to focus solely on the statistically significant findings related to PCSK9.

 

Comment 7: Overall, it seems that my initial review comments have not been adequately addressed. In my experience, the only result from this study that holds any significant weight is the finding related to rs28362286.

Response 7: We have carefully considered the reviewers' comments and express our gratitude for their valuable insights. This feedback has significantly enhanced the quality of our manuscript.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed all the mentioned issues.

Author Response

Comment 1: The authors addressed all the mentioned issues.

Response 1: We would like to express our gratitude for the feedback provided by Reviewer 2.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Regrettably, despite previous feedback, Table 1 in this manuscript continues to present an association p-value of < 0.001 for the rs67608943 SNP, which is monomorphic. I find it difficult to understand why this issue remains unaddressed in the manuscript. Consequently, I must inform you that I will not be continuing my review of this paper.