Cardiac Autonomic Neuropathy Is Not Associated with Apolipoprotein E Gene Isoforms in the Kazakh Population: A Case–Control Study
, Alisher Aitkaliyev 1,†, Tamara Vochshenkova 1,‡, Balzhan Kassiyeva 1,‡ and Valeriy Benberin 1,2,‡
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript "The FTO and APOE Genes as Risk Factors for the Development of Cardiac Autonomic Neuropathy in Individuals of Kazakh Nationality" investigates the association of FTO and APOE gene polymorphisms in patients with cardiac autonomic neuropathy (CAN) thereby aiming to identify early markers for predicting the presence of cardiac autonomic neuropathy in Kazakh population. The study identified the association of a rs429358 polymorphism in APOE gene and increased risk of cardiac neuropathy development regardless of diabetes as well as the association of TC genotype of the rs429358 of APOE gene and the AA genotype of the rs12149832 polymorphism of the FTO gene in patients without diabetes. The results of the study show an overview of genetic involvement in development of cardiac autonomic neuropathy in Kazakh population and will be helpful in developing therepeutic intervention in patients with CAN.
After going through the manuscript, I have following comments for the authors.
1. The study spanned over three years (2017 to 2020). Were the clinical parameters of the patients taken only once or multiple times over the duration of the study. Were similar procedures followed during taking and documenting the clinical parameters of the patients and controls?
2. Please discuss whether the identified polymorphisms associated with development of cardiac autonomic neuropathy in Kazakh population were also identified in other populations?
3. Is the data on frequency of cardiac autonomic neuropathy in Kazakh population available?
4. Please briefly discuss the therepeutic relevance of the study.
Comments on the Quality of English LanguageMinor grammatical corrections and syntax adjustments suggested.
Author Response
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Comment Number |
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The study spanned over three years (2017 to 2020). Were the clinical parameters of the patients taken only once or multiple times over the duration of the study? Were similar procedures followed during taking and documenting the clinical parameters of the patients and controls? |
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Author’s Response |
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Patients were selected upon admission, and clinical parameters of the patients were taken once during selection. And the procedures were the same for patients and controls. |
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Please discuss whether the identified polymorphisms associated with development of cardiac autonomic neuropathy in Kazakh population were also identified in other populations? |
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Author’s Response |
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The identified polymorphisms associated with the development of cardiac autonomic neuropathy in Kazakh population were not identified in other populations in part due to the lack of study of APOE gene in association with cardiac autonomic neuropathy. However, there were reports of APOE gene associations with other cardio-metabolic syndromes such as type 2 diabetes mellitus, atherosclerosis, and coronary heart disease (lines 78-81). |
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Is the data on frequency of cardiac autonomic neuropathy in Kazakh population available? |
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Author’s Response |
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To our knowledge, there have unfortunately been no reports or research on the frequency of cardiac autonomic neuropathy in the Kazakh population. |
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Please briefly discuss the therapeutic relevance of the study. |
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Author’s Response |
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The lines 297-298 in the Conclusion section offer the possibility of conducting preventive measures based on the results. |
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsMulti Digital Publishing Institute (MDPI): Diagnostics - 3104904
Title: Article
The FTO and APOE Genes as Risk Factors for the development of Cardiac Autonomic Neuropathy in Individuals of Kazakh Nationality
The authors decided to investigate associations between well-known SNPs in the FTO gene and isoforms of APOE genes and the development of Cardiac autonomic neuropathy (CAN) with or without diabetes mellitus diagnosis in individuals in Kazakhstan by a case-control study with a cohort of patients in the therapeutic department of the medical center Hospital of the Presenten’s Affairs Administration of the Republic of Kazakhstan during the period of September 2017 to August 2020.
The authors with their data stated that they found that patients without CAN had a statistically higher association with AA genotype of the rs12149832 of FTO, and TC genotype in rs429358 of APOE in patients without DM although they found a statistically stronger association between FTO SNPs and CAN in patients with DM previously.
Overall comments:
The title is slightly misleading since the authors investigated SNPs in those genes, and not the coding regions of those genes. However, it seems as though it is commonly done, but it is important that the authors understand the differences.
Cardiac autonomic neuropathy (CAN) is a condition that occurs when the autonomic nerve fibers that control the heart and blood vessels are damaged.
This damage can lead to abnormalities in heart rate and blood pressure and can increase the risk of cardiovascular events. CAN is a microvascular complication that's often associated with long-term type 2 diabetes, but it can also develop before a diagnosis.
CAN can result in a number of clinical manifestations, including:
· Resting tachycardia
· Orthostasis
· Exercise intolerance
· Intraoperative cardiovascular liability
· Silent myocardial infarction (MI)
· Increased risk of mortality
Risk factors for the development of CAN:
· Diabetes mellitus.
· Amyloidosis, porphyria, hypothyroidism, and cancer.
· Other factors include hypertension, dyslipidemia, obesity, age, sex, body mass index (BMI), and cigarette smoking.
· Alcoholism can also impair cardiac autonomic nervous activity.
A little more biological mechanistic explanation of microvascular disease in diabetes mellitus (DM) should be considered since hyperglycemia and dyslipidemia are just phenotypes.
Mechanisms for microvascular disease in diabetes include the pathologic effects of AGE accumulation, overproduction of endothelial growth factors, and abnormal stimulation of the PKC and polyol pathways and the RAS.
Cade WT. Diabetes-related microvascular and macrovascular diseases in the physical therapy setting. Phys Ther. 2008 Nov;88(11):1322-35. doi: 10.2522/ptj.20080008. Epub 2008 Sep 18. PMID: 18801863; PMCID: PMC2579903.
Furthermore, involvement of parasympathetic nervous system:
Damage to the Vagus nerve, the longest parasympathetic nerve that control about 75% of parasympathetic nerve activities, can cause resting tachycardia and a decrease in parasympathetic tone. Early autonomic dysfunction may not have symptoms and can only be detected by abnormal heart rate variability (HRV) indices.
CAN leading to dysregulation of the cardiovascular system, resulting in symptoms such as dizziness, palpitations, lightheadedness etc.
At an advanced stage, both parasympathetic and sympathetic nervous systems can be affected, leading to denervation of the nervous system in the heart. This can manifest as unresponsiveness to exercise, stress, or sleeping – which can be dangerous.
Lines 48-49: with minor changes in blood glucose levels …. Obesity.
This does not explain the underlying mechanism. Please consider editing because there are so many cellular processes that occur between those phenotypes and the resulting phenotype which is CAN.
Although this is a single nucleotide variant (SNP)-association studies, not functionally related studies, it is always important to know about the genes involved in the study, and understand about the genes in question, FTO and apolipoprotein E (APOE).
It is important to differentiate “association” vs “causality”.
Association does not necessarily mean causality. Therefore, whenever association studies are conducted, it is important that one is not looking at a causal relationship to the disease in questions (CAN).
Especially, if SNPs in introns are the ones used in a study, not all have any functional significance to the biological system.
There may be another genetic site which may be in linkage disequilibrium which actually has a role in the disease in question.
Please keep these issues in mind when performing association studies.
Furthermore, both genes have some specific disorders associated with coding variants.
· FTO (italicized): alpha-ketoglutarate dependent dioxygenase
https://www.ncbi.nlm.nih.gov/gene/79068
Summary:
This gene is a nuclear protein of the AlkB related non-heme iron and 2-oxoglutarate-dependent oxygenase superfamily, but the exact physiological function of this gene is not known.
Other non-heme iron enzymes function to reverse alkylated DNA and RNA damage by oxidative demethylation.
Studies in mice and humans indicate a role in nervous and cardiovascular systems and a strong association with body mass index, obesity risk, and type 2 diabetes.
FTO protein expressed most in the brain, adrenal, thyroid, lungs, endometrium etc. (heart is not one of top organs with a high expression from the studies performed in the past).
Monogenic disorder caused by FTO
HGMD: over 20 variants have been reported (https://www.hgmd.cf.ac.uk/ac/all.php)
Most common phenotypes listed are growth retardation, developmental delay, and facial dysmorphism as in the publication below.
Growth retardation, developmental delay, and facial dysmorphism:
Biallelic mutations in FTO lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems.
Daoud H, Zhang D, McMurray F, Yu A, Luco SM, Vanstone J, Jarinova O, Carson N, Wickens J, Shishodia S, Choi H, McDonough MA, Schofield CJ, Harper ME, Dyment DA, Armour CM. Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay. J Med Genet. 2016 Mar;53(3):200-7. doi: 10.1136/jmedgenet-2015-103399. Epub 2015 Sep 16. PMID: 26378117.
From the article citation below.
Carnevali L, Graiani G, Rossi S, Al Banchaabouchi M, Macchi E, Quaini F, et al. (2014) Signs of Cardiac Autonomic Imbalance and Proarrhythmic Remodeling in FTO Deficient Mice. PLoS ONE 9(4): e95499. https://doi.org/10.1371/journal.pone.0095499.
Our data indicate that FTO knockout mice were characterized by (i) higher heart rate values during resting and stress conditions, (ii) heart rate variability changes (increased LF to HF ratio), (iii) larger vulnerability to stress-induced tachyarrhythmias, (iv) altered ventricular repolarization, and (v) cardiac hypertrophy compared to wild-type counterparts. We conclude that FTO deficiency in mice leads to an imbalance of the autonomic neural modulation of cardiac function in the sympathetic direction and to a potentially proarrhythmic remodeling of electrical and structural properties of the heart.
Although this is a study in mice, it seems to indicate that FTO gene may be important in cardiac autonomic stability.
The authors should mention such study to provide a stronger reason for studying CAN associated with FTO.
It is less credible what the authors have mentioned in the manuscript to connect FTO to CAN.
· APOE: apolipoprotein E
Apolipoprotein E (apoE) is a protein that's a protein component of many lipoproteins, including very low-density lipoproteins (VLDL), intermediate-density lipoproteins, chylomicrons, and high-density lipoproteins (HDLs).
Apolipoprotein E is an important ligand for three cell surface receptors: the low-density lipoprotein (LDL) receptor, the low-density lipoprotein-related protein (LRP), and the very-low-density lipoprotein (VLDL) receptor.
Please be aware that here, APOE polymorphisms are well known ones, and also they are in coding regions, different from the SNPs in FTO.
Dysbetalipoproteinemia
Javvaji A, Can AS, Sharma S. Dysbetalipoproteinemia. [Updated 2024 Feb 2]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK567738/
APOE (e2/e2) biallelic isoform e2 is found in patients with dysbetalipoproteinemia, which used to be known as the WHO hyperlipoproteinemia type III, or Frederickson Classification type III. However, there is typically a secondary factor present in the manifestation of dysbetalipoprotenemia such as DM, hypothyroidism, obesity etc. Currently, the underlying mechanisms of the development of the condition are still not deciphered clearly though it was originally found that IDL (intermediate-density lipoprotein) is elevated, and the lipid phenotype shows similar elevations in TC and TG.
The mechanisms of vascular pathology in dysbetatliporprotenemia may be due to the elevation of IDL/remnant particles or high apoB-100 with the vascular endothelium.
Gain-of-function variant in APOE is also found in individuals with dysbetalipoproteinemia.
It is important to consider the connection between the apoE metabolism and CAN.
It may be that vascular disease ultimately affects the nervous system, but it is important to consider the biology behind the association.
There is also an article on an association between apoM and CAN (FYI)
Safi M, Borup A, Stevns Hansen C, Rossing P, Thorsten Jensen M, Christoffersen C. Association between plasma apolipoprotein M and cardiac autonomic neuropathy in type 1 diabetes. Diabetes Res Clin Pract. 2022 Jul;189:109943. doi: 10.1016/j.diabres.2022.109943. Epub 2022 Jun 8. PMID: 35690270.
Please keep in mind the information above before performing any association studies.
Furthermore, information about SNPs.
SNPs are the most common type of genetic variation in humans, also known as genetic markers, occurring in at least 1% of the population and roughly once in every 1,000 nucleotides. This means that a person's genome typically contains around 4 to 5 million SNPs.
While genome wide association studies have identified important gene associations with certain diseases, please remember about what are being studied.
FTO intron variants:
rs9939609
rs8050136
rs1121980
rs1421085
rs17817449
rs3751812
Recommend italicizing gene names.
To perform an association study, the number of participant pool is too small to be the valid study. Association studies are typically performed in a large population or large group of people with a condition compared to a large group of individuals as control.
Having only a total of 300 people with the use of SNPs in an intron which are not translated into protein would be too small to have any credible finding.
Furthermore, SNPs in FTO are non-translated, but isoforms in APOE are protein-coding, and have been functionally studied, and some disorders have been associated with credibility (dysbetalipoproteinemia and Alzheimer’s disease).
It would be important to present all genotypes found in patients even in an appendix.
Please see the below for additional comments.
Specific comments:
1. Introduction
Please refer to the notes on FTO and APOE genes above.
Lines 64-65: Please clarify what the authors mean by, “The rs17817449 polymorphism is also located in the first intron of FTO (italicize), which is the region closely associated with obesity in humans.”
What do the authors mean by “the region closely associated”?
Do they mean to say that “the SNP which has been found associated with obesity by multiple researchers? This is a study of association study using various SNPs, not the region of the FTO gene. There is a big difference, and since FTO is not a causal gene for obesity, it does not make sense the way it is stated so recommend rewording this sentence.
Lines 84-85: Not clear what the authors mean by, “…associated with fat metabolism; …” referencing #24.
Please clarify. The authors on #24 were studying osteoarthritis and BMI. Not really clear for this reference to be used to describe fat metabolism. Do they mean BMI being related to fat metabolism? BMI is calculated weight (kg) / height (m2), not fat% assessment by DEXA.
Line 86 and Lines 94-95: It is incorrect to state that “The Apolipoprotein E (APOE) gene is also involved in lipid metabolism disorder and insulin resistance processes…” Yes, APOE gene product is involved in lipid metabolism disorder, or more specifically, a lipoprotein disorder and also being a ligand for several receptors, as above. NOT, involved in insulin resistance processes, specifically as the authors state (it may be indirectly somehow leads to insulin resistance, but apoE is not involved in insulin resistance processes. If this has been reported, please provide a reference with an appropriate study to document this. Yes, cardiovascular disease risk is high in dysbetalipoproteinemia; however, as its disease development, a secondary factor(s) including the environmental factor is important. Not everyone with APOE (e2/e2) develops dysbatalipoproteinemia. TG metabolism is very much modulated by dietary intake for the exogeneous pathway of TG metabolism.
Lines 96-97: Please see above for the details on apoE.
Lines 100-102: APOE (e3) is the most common type in the United States. APOE (e4) is the one which has been implicated in conditions listed. However, once dysbetalipoproteinemia develops, patients can develop premature cardiovascular disease. However, typically APOE (e2) has a lower risk of subclinical atherosclerosis. Therefore, some secondary factor probably triggers changes as stated above.
2. Research Design and Methods
2.1 Study design and patient selection
Lines 137-140: Please provide more details of acronyms used for Holter monitor evaluation.
Heart rate variability (HRV) is a respected measure used in the assessment of cardiac autonomic neuropathy (CAN)
Lines 140-141: It was not clear why 3 or more being the cut off of Holter monitor results.
Methods of CAN assessment in clinical practice include assessment of symptoms and signs, cardiovascular reflex tests based on HR and BP, short-term electrocardiography (ECG), QT interval prolongation, HR variability (24 h, classic 24 h Holter ECG), ambulatory BP monitoring, HR turbulence, baroreflex sensitivity, muscle sympathetic nerve activity, catecholamine assessment and cardiovascular sympathetic tests, heart sympathetic imaging.
Serhiyenko VA, Serhiyenko AA. Cardiac autonomic neuropathy: Risk factors, diagnosis and treatment. World J Diabetes. 2018 Jan 15;9(1):1-24. doi: 10.4239/wjd.v9.i1.1. PMID: 29359025; PMCID: PMC5763036.
Lines 142-144: It would be great to describe the criteria that was used to diagnosis DM.
One reading of fasting glucose level may or may not be a good indication of how DM control is or diagnosing DM. HgbA1C would be a better parameter to gauze on whether DM is under control or if the patient is taking a medication to manage DM, then one glucose reading may not be helpful. Not sure this was clearly stated in the manuscript.
Especially with the complications associated with DM, the duration of DM becomes important. Typically, it may take 5 – 10 years from the original diagnosis of DM for complications to develop although depends on if the individual was diagnosed earlier from the onset.
DM is a very heterogenous condition so that the years from diagnosis become important.
Although there may be variability in DM diagnosis and treatment between countries so that it is difficult to assess how it is in Kazakhstan.
Furthermore, it is not clear if any patient was treated for dyslipidemia.
Lines 154-155: “rhythmic disturbances of cardiac electrical activity” means arrhythmia, correct?
2.2. Genotyping
2.3. Statistical analysis
2.4. Ethics
3. Results:
Lines 218-219: Not clear about the age of patients with CAN clearly older than the control because only 1 year without any SD so difficult to assess how the distributions are in those groups.
Lines 228-229: Please clarify that the authors have quantified low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) rather than LDL and HDL (lipoproteins).
Please present FTO genotypes of patients without CAN if not presented in a table. Since “general cohort” seems to be the total patients (CAN and without CAN).
Then, please clarify labeling in Table 2.
This table only contains patients with CAN, so cases, correct? Then, maybe use another term for “general cohort” to include “case” since it indicates different from what it meant in Table 1. A bit confusing how they patients with CAN vs patients without CAN. Or are you including everyone?
To the readers who are not experts in statistics, it seems a bit confusing.
Table 3: Are the patients without CAN included in this cohorts? If so, the genotypes should be separated between CAN and control cases.
e.g., How many people with CAN had rs12149832 (G/G), (G/A) or (A/A) vs people without CAN, not just OR and CI?
For APOE, please specify the e2, e3, and e4 alleles found in patients.
It seems as though the analysis was performed, not according to the three types of allele. If each type of allele found in each patient is presented, it may be easier to understand the authors presentation.
Table 4: Please explain about models of inheritance.
rs429358 should be presented as alleles not separately from the other SNP. As the authors stated there are three types of alleles in APOE. APOE e2 (rs7412-T, rs429358-T) APOE e3 (rs7412-C, rs429358-T) or APOE e4 (rs7412-C, rs429358-C).
Each allele should be handled together, not separately although exceptions may exist.
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Allele |
e2 |
e3 |
e4 |
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Haplotype: rs429358 |
T |
T |
C |
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Amino acid: 112 |
Cystine |
Cystine |
Arginine |
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Haplotype: rs7412 |
T |
C |
C |
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Amino acid: 158 |
Cystine |
Arginine |
Arginine |
In order to have rs429358 to be T/C the allele has to include APOE (e2/e4) or APOE (e3/e4). Please present all genotypes, isoform combinations identified in patients in the study.
Tables 1 and Table 2:
Low-density lipoprotein cholesterol (LDL-C)
High-density lipoprotein cholesterol (HDL-C)
Please clarify that Table 2 only represent patients with CAN. From the table itself, “general cohort” labeling was confusing. The use of case or total vs case or control.
Please consider another subgroup name for “general cohort”, can just use “total patients with CAN”, instead.
4. Discussion
It is not surprising that conflicting results are found in the previous and current studies because the cohort was too small.
Although the authors stated that “The sample size was determined using the statistical software Epi Info.”
My understanding is that you would want to at least have 1:4 (case : control ration) in most cases. Please reassess and determine that your cohort was enough in size to determine any association, using the SNPs in FTO.
Hong EP, Park JW. Sample size and statistical power calculation in genetic association studies. Genomics Inform. 2012 Jun;10(2):117-22. doi: 10.5808/GI.2012.10.2.117. Epub 2012 Jun 30. PMID: 23105939; PMCID: PMC3480678.
Furthermore, as stated above, non-coding SNPs were used in FTO, and FTO is a causal gene for a condition, “Growth retardation, developmental delay, and facial dysmorphism”.
In addition, isoforms in APOE are coding, and there are some diseases associated with APOE (e2/e2) and APOE (e4/e4).
Please clarify how DM cases were identified. It would be helpful to state the diagnostic criteria set forth by the Ministry of Health of the Republic of Kazakhstan.
Finally, SNPs are thought of as genetic markers, and many often do not have any biological function, especially being in intron. However, some intron variants can have important functions in various regulations of genes (often in vicinity). Therefore, careful planning and planning are required to present results of an association study to be credible.
Thank you very much for allowing me to review this manuscript.
Sincerely,
Comments for author File: Comments.pdf
Author Response
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The title is slightly misleading since the authors investigated SNPs in those genes, and not the coding regions of those genes. However, it seems as though it is commonly done, but it is important that the authors understand the differences. |
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Agreed. We have changed the title of the article as per your recommendation. |
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Cardiac autonomic neuropathy (CAN) is a condition that occurs when the autonomic nerve fibers that control the heart and blood vessels are damaged. This damage can lead to abnormalities in heart rate and blood pressure and can increase the risk of cardiovascular events. CAN is a microvascular complication that's often associated with long-term type 2 diabetes, but it can also develop before a diagnosis. CAN can result in a number of clinical manifestations, including: · Resting tachycardia · Orthostasis · Exercise intolerance · Intraoperative cardiovascular liability · Silent myocardial infarction (MI) · Increased risk of mortality Risk factors for the development of CAN: · Diabetes mellitus. · Amyloidosis, porphyria, hypothyroidism, and cancer. · Other factors include hypertension, dyslipidemia, obesity, age, sex, body mass index (BMI), and cigarette smoking. · Alcoholism can also impair cardiac autonomic nervous activity. |
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Your recommendations have been incorporated into the text in the "Introduction" section: Lines – 33-39. |
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A little more biological mechanistic explanation of microvascular disease in diabetes mellitus (DM) should be considered since hyperglycemia and dyslipidemia are just phenotypes. Mechanisms for microvascular disease in diabetes include the pathologic effects of AGE accumulation, overproduction of endothelial growth factors, and abnormal stimulation of the PKC and polyol pathways and the RAS. Cade WT. Diabetes-related microvascular and macrovascular diseases in the physical therapy setting. Phys Ther. 2008 Nov;88(11):1322-35. doi: 10.2522/ptj.20080008. Epub 2008 Sep 18. PMID: 18801863; PMCID: PMC2579903. |
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Thank you for the recommendation and the reference! The facts have been included in the text and the reference list: Lines – 49-53. |
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Furthermore, involvement of parasympathetic nervous system: Damage to the Vagus nerve, the longest parasympathetic nerve that control about 75% of parasympathetic nerve activities, can cause resting tachycardia and a decrease in parasympathetic tone. Early autonomic dysfunction may not have symptoms and can only be detected by abnormal heart rate variability (HRV) indices. CAN leading to dysregulation of the cardiovascular system, resulting in symptoms such as dizziness, palpitations, lightheadedness etc. At an advanced stage, both parasympathetic and sympathetic nervous systems can be affected, leading to denervation of the nervous system in the heart. This can manifest as unresponsiveness to exercise, stress, or sleeping – which can be dangerous. Lines 48-49: with minor changes in blood glucose levels …. Obesity. This does not explain the underlying mechanism. Please consider editing because there are so many cellular processes that occur between those phenotypes and the resulting phenotype which is CAN. |
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We absolutely agree with you and have decided to include the data in those lines: Lines – 56-64. |
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Although this is a single nucleotide variant (SNP)-association studies, not functionally related studies, it is always important to know about the genes involved in the study, and understand about the genes in question, FTO and apolipoprotein E (APOE). It is important to differentiate “association” vs “causality”. Association does not necessarily mean causality. Therefore, whenever association studies are conducted, it is important that one is not looking at a causal relationship to the disease in questions (CAN). Especially, if SNPs in introns are the ones used in a study, not all have any functional significance to the biological system. There may be another genetic site which may be in linkage disequilibrium which actually has a role in the disease in question. Please keep these issues in mind when performing association studies. Furthermore, both genes have some specific disorders associated with coding variants. · FTO (italicized): alpha-ketoglutarate dependent dioxygenase https://www.ncbi.nlm.nih.gov/gene/79068 |
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Following your recommendations, we have decided not to include data on FTO gene polymorphisms for the lack of functional significance and cannot be interpreted as risk factors, as this could potentially mislead readers.
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Summary: This gene is a nuclear protein of the AlkB related non-heme iron and 2-oxoglutarate-dependent oxygenase superfamily, but the exact physiological function of this gene is not known. Other non-heme iron enzymes function to reverse alkylated DNA and RNA damage by oxidative demethylation. Studies in mice and humans indicate a role in nervous and cardiovascular systems and a strong association with body mass index, obesity risk, and type 2 diabetes. FTO protein expressed most in the brain, adrenal, thyroid, lungs, endometrium etc. (heart is not one of top organs with a high expression from the studies performed in the past). Monogenic disorder caused by FTO HGMD: over 20 variants have been reported (https://www.hgmd.cf.ac.uk/ac/all.php) Most common phenotypes listed are growth retardation, developmental delay, and facial dysmorphism as in the publication below. Growth retardation, developmental delay, and facial dysmorphism: Biallelic mutations in FTO lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems. Daoud H, Zhang D, McMurray F, Yu A, Luco SM, Vanstone J, Jarinova O, Carson N, Wickens J, Shishodia S, Choi H, McDonough MA, Schofield CJ, Harper ME, Dyment DA, Armour CM. Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay. J Med Genet. 2016 Mar;53(3):200-7. doi: 10.1136/jmedgenet-2015-103399. Epub 2015 Sep 16. PMID: 26378117. From the article citation below. Carnevali L, Graiani G, Rossi S, Al Banchaabouchi M, Macchi E, Quaini F, et al. (2014) Signs of Cardiac Autonomic Imbalance and Proarrhythmic Remodeling in FTO Deficient Mice. PLoS ONE 9(4): e95499. https://doi.org/10.1371/journal.pone.0095499. Our data indicate that FTO knockout mice were characterized by (i) higher heart rate values during resting and stress conditions, (ii) heart rate variability changes (increased LF to HF ratio), (iii) larger vulnerability to stress-induced tachyarrhythmias, (iv) altered ventricular repolarization, and (v) cardiac hypertrophy compared to wild-type counterparts. We conclude that FTO deficiency in mice leads to an imbalance of the autonomic neural modulation of cardiac function in the sympathetic direction and to a potentially proarrhythmic remodeling of electrical and structural properties of the heart. Although this is a study in mice, it seems to indicate that FTO gene may be important in cardiac autonomic stability. The authors should mention such study to provide a stronger reason for studying CAN associated with FTO. It is less credible what the authors have mentioned in the manuscript to connect FTO to CAN. |
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Author’s Response |
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First and foremost, we express our immense gratitude for the detailed description of the functions and mechanisms of the FTO gene, including the references! However, based on your comments and recommendations, we have decided not to include data on FTO gene polymorphisms for the lack of functional significance and cannot be interpreted as risk factors, as our conclusions might mislead readers. |
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Apolipoprotein E (apoE) is a protein that's a protein component of many lipoproteins, including very low-density lipoproteins (VLDL), intermediate-density lipoproteins, chylomicrons, and high-density lipoproteins (HDLs). Apolipoprotein E is an important ligand for three cell surface receptors: the low-density lipoprotein (LDL) receptor, the low-density lipoprotein-related protein (LRP), and the very-low-density lipoprotein (VLDL) receptor. Please be aware that here, APOE polymorphisms are well known ones, and also they are in coding regions, different from the SNPs in FTO. Dysbetalipoproteinemia Javvaji A, Can AS, Sharma S. Dysbetalipoproteinemia. [Updated 2024 Feb 2]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK567738/ APOE (e2/e2) biallelic isoform e2 is found in patients with dysbetalipoproteinemia, which used to be known as the WHO hyperlipoproteinemia type III, or Frederickson Classification type III. However, there is typically a secondary factor present in the manifestation of dysbetalipoprotenemia such as DM, hypothyroidism, obesity etc. Currently, the underlying mechanisms of the development of the condition are still not deciphered clearly though it was originally found that IDL (intermediate-density lipoprotein) is elevated, and the lipid phenotype shows similar elevations in TC and TG. The mechanisms of vascular pathology in dysbetatliporprotenemia may be due to the elevation of IDL/remnant particles or high apoB-100 with the vascular endothelium. Gain-of-function variant in APOE is also found in individuals with dysbetalipoproteinemia. It is important to consider the connection between the apoE metabolism and CAN. It may be that vascular disease ultimately affects the nervous system, but it is important to consider the biology behind the association. There is also an article on an association between apoM and CAN (FYI) Safi M, Borup A, Stevns Hansen C, Rossing P, Thorsten Jensen M, Christoffersen C. Association between plasma apolipoprotein M and cardiac autonomic neuropathy in type 1 diabetes. Diabetes Res Clin Pract. 2022 Jul;189:109943. doi: 10.1016/j.diabres.2022.109943. Epub 2022 Jun 8. PMID: 35690270. |
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Author’s Response |
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We also extend our sincere thanks for the detailed description of the functions and mechanisms of the APOE gene, along with the references! Your recommendations have been incorporated into the text: Lines – 68-87. |
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Reviewers Comment |
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Please keep in mind the information above before performing any association studies. Furthermore, information about SNPs. SNPs are the most common type of genetic variation in humans, also known as genetic markers, occurring in at least 1% of the population and roughly once in every 1,000 nucleotides. This means that a person's genome typically contains around 4 to 5 million SNPs. While genome wide association studies have identified important gene associations with certain diseases, please remember about what are being studied. FTO intron variants: rs9939609 rs8050136 rs1121980 rs1421085 rs17817449 rs3751812 Recommend italicizing gene names.
To perform an association study, the number of participant pool is too small to be the valid study. Association studies are typically performed in a large population or large group of people with a condition compared to a large group of individuals as control. Having only a total of 300 people with the use of SNPs in an intron which are not translated into protein would be too small to have any credible finding. Furthermore, SNPs in FTO are non-translated, but isoforms in APOE are protein-coding, and have been functionally studied, and some disorders have been associated with credibility (dysbetalipoproteinemia and Alzheimer’s disease). It would be important to present all genotypes found in patients even in an appendix. |
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Author’s Response |
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Based on your comments and recommendations, we have decided not to include data on FTO gene polymorphisms that lack functional significance and cannot be interpreted as risk factors, as our conclusions might mislead readers. The genotypes were included as an Appendix 1. |
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Reviewers Comment |
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1. Introduction Please refer to the notes on FTO and APOE genes above. Lines 64-65: Please clarify what the authors mean by, “The rs17817449 polymorphism is also located in the first intron of FTO (italicize), which is the region closely associated with obesity in humans.” What do the authors mean by “the region closely associated”? Do they mean to say that “the SNP which has been found associated with obesity by multiple researchers? This is a study of association study using various SNPs, not the region of the FTO gene. There is a big difference, and since FTO is not a causal gene for obesity, it does not make sense the way it is stated so recommend rewording this sentence. Lines 84-85: Not clear what the authors mean by, “…associated with fat metabolism; …” referencing #24. Please clarify. The authors on #24 were studying osteoarthritis and BMI. Not really clear for this reference to be used to describe fat metabolism. Do they mean BMI being related to fat metabolism? BMI is calculated weight (kg) / height (m2), not fat% assessment by DEXA. |
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Author’s Response |
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As we have decided not to include data on FTO gene polymorphisms that lack functional significance and cannot be interpreted as risk factors, all the aforementioned lines have been removed from the text. |
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Reviewers Comment |
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Line 86 and Lines 94-95: It is incorrect to state that “The Apolipoprotein E (APOE) gene is also involved in lipid metabolism disorder and insulin resistance processes…” Yes, APOE gene product is involved in lipid metabolism disorder, or more specifically, a lipoprotein disorder and also being a ligand for several receptors, as above. NOT, involved in insulin resistance processes, specifically as the authors state (it may be indirectly somehow leads to insulin resistance, but apoE is not involved in insulin resistance processes. If this has been reported, please provide a reference with an appropriate study to document this. Yes, cardiovascular disease risk is high in dysbetalipoproteinemia; however, as its disease development, a secondary factor(s) including the environmental factor is important. Not everyone with APOE (e2/e2) develops dysbatalipoproteinemia. TG metabolism is very much modulated by dietary intake for the exogeneous pathway of TG metabolism. |
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Author’s Response |
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Yes, we agree that the statements we made are still controversial. Therefore, we have completely removed the sentence “The Apolipoprotein E (APOE) gene is also involved in lipid metabolism disorder and insulin resistance processes…” along with the related paragraph. Instead, we have added more detailed explanations regarding dysbetalipoproteinemia: Lines – 82-87. |
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Reviewers Comment |
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Lines 96-97: Please see above for the details on Apoe. Lines 100-102: APOE (e3) is the most common type in the United States. APOE (e4) is the one which has been implicated in conditions listed. However, once dysbetalipoproteinemia develops, patients can develop premature cardiovascular disease. However, typically APOE (e2) has a lower risk of subclinical atherosclerosis. Therefore, some secondary factor probably triggers changes as stated above. |
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Author’s Response |
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Following your recommendation, we have also added more information about each isoform in the lines 82-87. |
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Reviewers Comment |
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2. Research Design and Methods 2.1 Study design and patient selection Lines 137-140: Please provide more details of acronyms used for Holter monitor evaluation. Heart rate variability (HRV) is a respected measure used in the assessment of cardiac autonomic neuropathy (CAN) Lines 140-141: It was not clear why 3 or more being the cut off of Holter monitor results. Methods of CAN assessment in clinical practice include assessment of symptoms and signs, cardiovascular reflex tests based on HR and BP, short-term electrocardiography (ECG), QT interval prolongation, HR variability (24 h, classic 24 h Holter ECG), ambulatory BP monitoring, HR turbulence, baroreflex sensitivity, muscle sympathetic nerve activity, catecholamine assessment and cardiovascular sympathetic tests, heart sympathetic imaging. Serhiyenko VA, Serhiyenko AA. Cardiac autonomic neuropathy: Risk factors, diagnosis and treatment. World J Diabetes. 2018 Jan 15;9(1):1-24. doi: 10.4239/wjd.v9.i1.1. PMID: 29359025; PMCID: PMC5763036. |
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Author’s Response |
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The information has been added to the lines – 114-137. |
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Reviewers Comment |
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Lines 142-144: It would be great to describe the criteria that was used to diagnosis DM. One reading of fasting glucose level may or may not be a good indication of how DM control is or diagnosing DM. HgbA1C would be a better parameter to gauze on whether DM is under control or if the patient is taking a medication to manage DM, then one glucose reading may not be helpful. Not sure this was clearly stated in the manuscript. Especially with the complications associated with DM, the duration of DM becomes important. Typically, it may take 5 – 10 years from the original diagnosis of DM for complications to develop although depends on if the individual was diagnosed earlier from the onset. DM is a very heterogenous condition so that the years from diagnosis become important. Although there may be variability in DM diagnosis and treatment between countries so that it is difficult to assess how it is in Kazakhstan. Furthermore, it is not clear if any patient was treated for dyslipidemia. |
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Author’s Response |
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The criteria for diagnosing diabetes have been added to the lines 143-146. |
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Reviewers Comment |
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Lines 154-155: “rhythmic disturbances of cardiac electrical activity” means arrhythmia, correct? |
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Author’s Response |
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Yes, correct. |
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Reviewers Comment |
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Lines 218-219: Not clear about the age of patients with CAN clearly older than the control because only 1 year without any SD so difficult to assess how the distributions are in those groups. |
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Author’s Response |
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Data on the average age and standard deviation (SD) have been added to the results section on the lines 214-217. |
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Reviewers Comment |
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Lines 228-229: Please clarify that the authors have quantified low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) rather than LDL and HDL (lipoproteins). |
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Author’s Response |
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Yes, we determined the concentrations of LDL-C and HDL-C. Corrections have been made in the test. Thank you for the clarification! |
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Reviewers Comment |
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Please present FTO genotypes of patients without CAN if not presented in a table. Since “general cohort” seems to be the total patients (CAN and without CAN). |
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Author’s Response |
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Since the results on CAN have been excluded from the article, the response to this comment will no longer be relevant. |
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Reviewers Comment |
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Then, please clarify labeling in Table 2. |
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Author’s Response |
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All the tables have been labelled. |
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Reviewers Comment |
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This table only contains patients with CAN, so cases, correct? Then, maybe use another term for “general cohort” to include “case” since it indicates different from what it meant in Table 1. A bit confusing how they patients with CAN vs patients without CAN. Or are you including everyone? To the readers who are not experts in statistics, it seems a bit confusing. |
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Author’s Response |
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Following the recommendation of another reviewer, we have decided against dividing the groups into subgroups. Now, there are only patients with CAN in the case group and a control group without CAN. |
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Reviewers Comment |
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Table 3: Are the patients without CAN included in this cohorts? If so, the genotypes should be separated between CAN and control cases. e.g., How many people with CAN had rs12149832 (G/G), (G/A) or (A/A) vs people without CAN, not just OR and CI? For APOE, please specify the e2, e3, and e4 alleles found in patients. It seems as though the analysis was performed, not according to the three types of alleles. If each type of allele found in each patient is presented, it may be easier to understand the authors presentation. |
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Author’s Response |
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The tables have been completely revised due to the following reasons: 1. Data related to the FTO gene have been removed from the analysis and deleted. 2. Subgroup analysis has been excluded from the results. 3. Following your recommendation, the statistical analysis for APOE has been recalculated and analyzed according to the isoforms e2, e3, and e4. |
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Reviewers Comment |
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Table 4: Please explain about models of inheritance. |
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Author’s Response |
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Table 4 has now become Table 3. Specifying the model, only the co-dominant model was applied. |
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22.
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Reviewers Comment |
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rs429358 should be presented as alleles not separately from the other SNP. As the authors stated there are three types of alleles in APOE. APOE e2 (rs7412-T, rs429358-T) APOE e3 (rs7412-C, rs429358-T) or APOE e4 (rs7412-C, rs429358-C). Each allele should be handled together, not separately although exceptions may exist.
In order to have rs429358 to be T/C the allele has to include APOE (e2/e4) or APOE (e3/e4). Please present all genotypes, isoform combinations identified in patients in the study. |
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Author’s Response |
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Following your recommendation, the statistical analysis for APOE has been recalculated and analyzed based on the isoforms e2, e3, and e4. |
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23.
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Reviewers Comment |
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Tables 1 and Table 2: Low-density lipoprotein cholesterol (LDL-C) High-density lipoprotein cholesterol (HDL-C) |
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Author’s Response |
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Corrections have been made in the text regarding the notation for LDL-C and HDL-C, as per your comments. |
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24.
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Reviewers Comment |
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Please clarify that Table 2 only represent patients with CAN. From the table itself, “general cohort” labeling was confusing. The use of case or total vs case or control. Please consider another subgroup name for “general cohort”, can just use “total patients with CAN”, instead. |
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Author’s Response |
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Following the recommendation of another reviewer, we have decided not to divide the groups into subgroups. Now, there are only patients with CAN in the case group and a control group without CAN. |
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25.
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Reviewers Comment |
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4. Discussion It is not surprising that conflicting results are found in the previous and current studies because the cohort was too small. Although the authors stated that “The sample size was determined using the statistical software Epi Info.” My understanding is that you would want to at least have 1:4 (case : control ration) in most cases. Please reassess and determine that your cohort was enough in size to determine any association, using the SNPs in FTO. Hong EP, Park JW. Sample size and statistical power calculation in genetic association studies. Genomics Inform. 2012 Jun;10(2):117-22. doi: 10.5808/GI.2012.10.2.117. Epub 2012 Jun 30. PMID: 23105939; PMCID: PMC3480678. Furthermore, as stated above, non-coding SNPs were used in FTO, and FTO is a causal gene for a condition, “Growth retardation, developmental delay, and facial dysmorphism”. |
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Author’s Response |
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Since the results on FTO gene polymorphisms have been excluded from the analysis, responses to these comments are likely no longer relevant. |
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26.
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Reviewers Comment |
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In addition, isoforms in APOE are coding, and there are some diseases associated with APOE (e2/e2) and APOE (e4/e4). |
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Author’s Response |
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As a result of reprocessing the statistical analysis based on APOE gene isoforms, our results showed an association between the e4 isoform and CAN. The conclusions have been revised, and additional information about the potential pathogenetic role of APOE e4 has been included in the discussion. |
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27.
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Reviewers Comment |
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Please clarify how DM cases were identified. It would be helpful to state the diagnostic criteria set forth by the Ministry of Health of the Republic of Kazakhstan. |
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Author’s Response |
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The criteria for diagnosing diabetes have been added to the lines 143-146, which is according to the protocol set by the Ministry of Health of the Republic of Kazakhstan. |
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28.
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Reviewers Comment |
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Finally, SNPs are thought of as genetic markers, and many often do not have any biological function, especially being in intron. However, some intron variants can have important functions in various regulations of genes (often in vicinity). Therefore, careful planning and planning are required to present results of an association study to be credible. |
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Author’s Response |
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We greatly appreciate your feedback and have made efforts to correct similar errors in our work. However, only you can assess how well these corrections have been implemented. In future research, we will consider all recommendations and plan our studies more carefully. |
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsQuestions on study design and methods. The authors state that "The sample size was determined using the statistical software Epi Info. The parameters included an estimated control group frequency of 30%, an odds ratio of 2.0, a 95% confidence level, and an 80% statistical power." It is not clear for which indicators and what they calculated in this way. For all nucleotide sequence variants analysed? Judging by the result (for example, for rs429358 the group size is formally insufficient, and for others too). Especially if we take into account the opinion of Donahue MP, Allen AS. Genetic association studies in cardiology. Am Heart J. 2005 Jun;149(6):964-70. doi: 10.1016/j.ahj.2005.03.011. PMID: 15976776. For all other indicators? Or for some specific ones? Which ones? I recommend removing this sentence ("The sample size was determined..."). Another sentence raises questions: "The allele and genotype distributions were in Hardy-Weinberg equilibrium." Hardy-Weinberg equilibrium estimates genotype frequencies.
Genetic polymorphism is a phenomenon, not a sequence change. According to modern international terminology, it is supposed to speak not of polymorphisms but of sequence variants. See http://varnomen.hgvs.org/
A significant drawback of the study is the initially small groups, which are then divided according to the presence/absence of diabetes. And on these groups the authors still try to perform multivariate analyses. This leads to the fact that the carriers of a combination of a number of factors turn out to be single and completely random. Consequently, in my opinion, the likelihood of reproducing the results of this analysis in larger studies is very low.
Author Response
Dear Reviewer 3,
Thank you very much for your comments and recommendations! Thanks to your recommendations, our manuscript has drastically improved. Here are our point-by-point responses to your comments. Moreover, grammatical corrections and syntax adjustments were made to address the quality of the English Language.
The given lines in the revised manuscript are indicated in a “simple mark-up” form of tracked changes.
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Comment Number |
Reviewers Comment |
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1. |
Questions on study design and methods. The authors state that "The sample size was determined using the statistical software Epi Info. The parameters included an estimated control group frequency of 30%, an odds ratio of 2.0, a 95% confidence level, and an 80% statistical power." It is not clear for which indicators and what they calculated in this way. For all nucleotide sequence variants analysed? Judging by the result (for example, for rs429358 the group size is formally insufficient, and for others too). Especially if we take into account the opinion of Donahue MP, Allen AS. Genetic association studies in cardiology. Am Heart J. 2005 Jun;149(6):964-70. doi: 10.1016/j.ahj.2005.03.011. PMID: 15976776. For all other indicators? Or for some specific ones? Which ones? I recommend removing this sentence ("The sample size was determined..."). Another sentence raises questions: "The allele and genotype distributions were in Hardy-Weinberg equilibrium." Hardy-Weinberg equilibrium estimates genotype frequencies. |
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Author’s Response |
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Based on your review and those of other reviewers, we decided not to include the results related to the polymorphisms of the FTO gene in our article, as they do not have any functional significance individually. Additionally, the sample size is insufficient to assess the impact of polymorphisms in the non-coding region.
Moreover, we decided not to divide an already small group into subgroups. Now, there are only patients with CAN in the case group and a control group without CAN.
Following your recommendation, we also removed the sentence 'The sample size was determined...' At the suggestion of another reviewer, we recalculated the results for the APOEgene by combining the two polymorphisms based on isoforms. Since only allele variants were calculated, the assessment of distribution according to Hardy-Weinberg equilibrium was not performed. |
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2. |
Reviewers Comment |
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Genetic polymorphism is a phenomenon, not a sequence change. According to modern international terminology, it is supposed to speak not of polymorphisms but of sequence variants. See http://varnomen.hgvs.org/ |
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Author’s Response |
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Due to major changes in the revised manuscript, the study now focuses more on E4 isoforms rather than on single polymorphisms or sequence variants. |
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Reviewers Comment |
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A significant drawback of the study is the initially small groups, which are then divided according to the presence/absence of diabetes. And on these groups the authors still try to perform multivariate analyses. This leads to the fact that the carriers of a combination of a number of factors turn out to be single and completely random. Consequently, in my opinion, the likelihood of reproducing the results of this analysis in larger studies is very low. |
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Author’s Response |
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Thanks to your raised concerns, we decided not to divide an already small group into subgroups, - lines 99-100 in Materials and Methods. Now, there are only patients with CAN in the case group and a control group without CAN. |
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsMulti Digital Publishing Institute (MDPI): Diagnostics – 3104904_R1
Title: Article
APOЕ (e4/?) as a risk factor for developing cardiac autonomic neuropathy in individuals of Kazakh Nationality
Overall comments:
The manuscript has been revised completely to present a study, just using APOE isoforms only.
The study is a case-control study to investigate the association between the APOE isoforms and their association with cardiac autonomic neuropathy comparing 147 patients and 153 control individuals.
Cardiac autonomic neuropathy (CAN) is a condition that occurs when the autonomic nerve fibers that control the heart and blood vessels are damaged.
This damage can lead to abnormalities in heart rate and blood pressure and can increase the risk of cardiovascular events. CAN is a microvascular complication that's often associated with long-term type 2 diabetes, but it can also develop before a diagnosis.
This version is much clearer, and since APOE isoforms are associated with certain diseases as the authors mentioned, it may be an interesting choice to study.
However, please use APOE (e/e) as a unit, not by a single allele because each individual has two parental alleles or two genes of APOE.
Please see the below for additional comments.
Specific comments:
1. Background
Lines 45:” impacts” here, do the authors mean to say “CAN impacts in the development of heart diseases…”
Line 47: “please clarify “This” here”. CAN often results from tissue damages so maybe the authors are referring to this type of developed damage?
Line 47-48: maybe the authors were trying to say, “CAN is a result of microvascular complication linked to long-term type 2 diabetes, “
Line 52-53: the authors were trying to say, “Risk factors for the development of CAN include …”
Line 62: may be a word may be better to say, “the factors associated with the development of CAN include…” since the factors listed do not seem to be connected directed via certain mechanisms.
Lines 73-74: please revise the sentence to clarify the authors’ point in this sentence.
An example:
“…there is some evidence that parasympathetic dysfunction can occur with minimal fluctuations in blood glucose levels and in the absence of insulin resistance or obesity.”
Line 76: maybe reversing the order of, “reduced parasympathetic tone and resting tachycardia”, maybe a better order?
Line 88: maybe the authors mean, “an irreversible stage or point?”
Lines 88-89: A little confusing sentence here because genetic make-up in individuals does not change much in lifetime.
“Therefore, the use of genetic variants associated with the development of CAN may be a way to identify individuals who have a high risk to develop CAN?” OR something similar maybe more what the authors were intended to write?
It is important for the authors to be aware that the presence of APOE (e2/e2) does not automatically result in dysbetalipoproteinemia. Another factor or other factors such as diabetes, hypothyroidism, obesity etc. are often present when individuals are diagnosed with this condition. Once patients develop type III or dysbetalipoproteinemia, they are at risk of developing atherosclerotic cardiovascular disease.
143-144: Please revise the sentence. ApoE does not really play those roles (formation and secretion of lipoproteins). It is a component of a number of lipoproteins as the authors mentioned earlier. More role in lipoprotein binding to several receptors as the authors mentioned as a ligand.
Line 145: recommend using “isoforms” than “alleles” since they are better known as isoforms…although the use of “alleles” is not incorrect.
Lines 148-153: the authors seem to be repeating earlier statement so no need for this?
Line 151: APOE (e4/e4) not just one allele.
Lines 158-159: Recommend revising the sentence, “It has been previously reported…” should be elaborated with the other statement commenting about the use of genetics. The authors should use this to make their point about the use of APOE (e4/e4) isoform.
Lines 202-203: Recommend revising the sentence.
“…, 147 individuals who had features consistent with CAN, based on … ?” although this is a bit overlapping with Lines 225-226.
Line 228: Please spell out “HT”, hypertension?
Line 233: The diagnosis of diabetes mellitus (DM) in the United States as below (FYI).
· Blood sugar levels
A blood sugar level of 200 mg/dL (11.1 mmol/L) or higher, regardless of when you last ate, suggests diabetes. This is especially true if you also have symptoms of diabetes, such as frequent urination, extreme thirst, weight loss, blurred vision, or fatigue.
· Fasting blood sugar test
A fasting level of 126 mg/dL (7 mmol/L) or higher on two separate tests is diagnosed as diabetes. A level between 100 to 125 mg/dL (5.6 to 6.9 mmol/L) is diagnosed as prediabetes, and less than 100 mg/dL (5.6 mmol/L) is considered healthy.
· Oral glucose tolerance test (OGTT)
This test involves drinking 75g of anhydrous glucose and then measuring your blood sugar levels two hours later. A level of 200 mg/dL (11.1 mmol/L) or higher is considered diagnostic.
· Hemoglobin A1c
A level of 6.5% or higher is considered diagnostic.
If multiple samples are not used, HgbA1C value of > 6.5% probably more desirable in my opinion though this is just a side comment.
Line 248: please provide a unit for Hb.
2.2 Genotyping:
Line 289 and Line 294: please add “APOE” as it should be specified as APOE isoforms.
Was the genotype analyzed as biallelic?
· APOE (e2/e2), APOE (e2/e3), APOE (e2/e4), APOE (e3/e3), APOE (e3/e4), and APOE (e4/e4)?
Line 329: … compared to the “non-CAN” be better than “non-neuropathy’ or DM neuropathy was also separated?
Line 335-337: Biallelic analyses should be performed since each individual carries two alleles, not a separate allele.
Thus, each one should be paired for analysis and cannot be separated.
Line 381: the same comment as above.
4. Discussion:
Line 424: As above, analysis should have been performed as a pair (biallelic isoform combination), not a single allele because an individual has two alleles, never one allele of APOE.
Lines 423-429: Recommend streamlining the finding.
Line 446: Recommend replacing “mutation” with “isoform pair” or the genotype APOE (e/e).
Lines 451-452: Please clarify the sentence. Which are the allelic variants?
Lines 445-447: Please review the role of APOE in LDL clearance. The main ligand for LDL is apolipoprotein B attachment to LDL receptors.
Although lipoprotein differentiation is never exact, but typically LDL do not carry apoE.
Yang LG, March ZM, Stephenson RA, Narayan PS. Apolipoprotein E in lipid metabolism and neurodegenerative disease. Trends Endocrinol Metab. 2023 Aug;34(8):430-445. doi: 10.1016/j.tem.2023.05.002. Epub 2023 Jun 24. PMID: 37357100; PMCID: PMC10365028.
APOE isoforms differentially affect LCAT-dependent cholesterol esterification as below (FYI).
Vitali C, Pavanello C, Turri M, Lund-Katz S, Phillips MC, Catapano AL, Baragetti A, Norata GD, Veglia F, Calabresi L. Apolipoprotein E isoforms differentially affect LCAT-dependent cholesterol esterification. Atherosclerosis. 2023 Oct;382:117266. doi: 10.1016/j.atherosclerosis.2023.117266. Epub 2023 Aug 30. PMID: 37725860.
Although other investigators might not have investigated APOE as a biallelic form, each individual has two alleles so when studying humans, it is important to remember that two alleles per gene is present, and APOE (e/e) should be examined together. Otherwise, the relationship between other parameters such as lipid levels should be analyzed against a pair of alleles present in an individual, not separately.
Line 482: recommend replacing “linkage” with “association” or some word similar since linkage in genetic has another meaning.
Line 486: recommend replacing “mutations” with “isoforms”
Line 487: recommend replacing “pathogenic significance” since isoforms are not really pathogenic variants though some disease associations have been identified, but they are not pathogenic variants for a monogenic disorder. (associations or risk).
Line 491: recommend replacing “genetic markers” to another word such as “underlying isoform in APOE associated with CAN”.
This is an association type of study, and the authors have not elucidated the underlying biological mechanisms in the development of CAN in the paper.
Line 500: The conclusion should be in APOE (e/e) biallelic form, not one allele as stated above.
Thank you very much for allowing me to review this manuscript.
Sincerely,
Comments for author File: Comments.pdf
Author Response
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Dear Esteemed Reviewer,
Once again, we sincerely thank you for your valuable comments and recommendations! Thanks to your review, our manuscript has significantly improved. We cannot overstate how much your suggestions have helped us gain greater clarity in understanding the mechanisms and pathogenesis of this disease. This experience has been immensely valuable in expanding our knowledge and skills.
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3. Point-by-point response to Comments and Suggestions for Authors |
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Comment (C): APOЕ (e4/?) as a risk factor for developing cardiac autonomic neuropathy in individuals of Kazakh Nationality |
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Response (R): Since statistical significance was found only for the APOE ε4 isoform, we have titled the article "APOE ε4 as a Risk Factor for Developing Cardiac Autonomic Neuropathy in Individuals of Kazakh Nationality." However, if you strongly recommend focusing the analysis solely on the APOE isoforms (ε2/ε2), (ε2/ε3), (ε2/ε4), (ε3/ε3), (ε3/ε4), and (ε4/ε4), we are prepared to revise the title accordingly.
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Overall comments:
The manuscript has been revised completely to present a study, just using APOE isoforms only.
The study is a case-control study to investigate the association between the APOE isoforms and their association with cardiac autonomic neuropathy comparing 147 patients and 153 control individuals.
Cardiac autonomic neuropathy (CAN) is a condition that occurs when the autonomic nerve fibers that control the heart and blood vessels are damaged.
This damage can lead to abnormalities in heart rate and blood pressure and can increase the risk of cardiovascular events. CAN is a microvascular complication that's often associated with long-term type 2 diabetes, but it can also develop before a diagnosis.
This version is much clearer, and since APOE isoforms are associated with certain diseases as the authors mentioned, it may be an interesting choice to study.
However, please use APOE (e/e) as a unit, not by a single allele because each individual has two parental alleles or two genes of APOE.
Please see the below for additional comments.
C: 1. Background
Lines 45:” impacts” here, do the authors mean to say “CAN impact in the development of heart diseases…”
R: Yes
C: Line 47: “please clarify “This” here”. CAN often results from tissue damages so maybe the authors are referring to this type of developed damage?
R: We have paraphrased that line: Line 33.
C: Line 47-48: maybe the authors were trying to say, “CAN is a result of microvascular complication linked to long-term type 2 diabetes.
R: Yes, that’s correct. Based on your recommendation, we have rephrased the sentence: lines 34-35.
C: Line 62: may be a word may be better to say, “the factors associated with the development of CAN include…” since the factors listed do not seem to be connected directed via certain mechanisms.
R: Yes, that’s correct. Based on your recommendation, we have rephrased the sentence: lines 38-39.
C: Lines 73-74: please revise the sentence to clarify the authors’ point in this sentence.
An example:
“…there is some evidence that parasympathetic dysfunction can occur with minimal fluctuations in blood glucose levels and in the absence of insulin resistance or obesity.”
R: Based on your recommendation, we have rephrased the sentence: lines 53-54.
C: Line 76: maybe reversing the order of, “reduced parasympathetic tone and resting tachycardia”, maybe a better order?
R: Based on your recommendation, we have swapped the phrases “reduced parasympathetic tone” and “resting tachycardia.”
C: Line 88: maybe the authors mean, “an irreversible stage or point?”
R: Yes, we meant to say "an irreversible stage." The sentence has been revised accordingly.
C: Lines 88-89: A little confusing sentence here because genetic make-up in individuals does not change much in lifetime.
“Therefore, the use of genetic variants associated with the development of CAN may be a way to identify individuals who have a high risk to develop CAN?” OR something similar maybe more what the authors were intended to write?
R: Based on your recommendation, we have rephrased the sentence: lines 65-66.
C: It is important for the authors to be aware that the presence of APOE (e2/e2) does not automatically result in dysbetalipoproteinemia. Another factor or other factors such as diabetes, hypothyroidism, obesity etc. are often present when individuals are diagnosed with this condition. Once patients develop type III or dysbetalipoproteinemia, they are at risk of developing atherosclerotic cardiovascular disease.
R: Thank you for the feedback! Based on your recommendation, we have reviewed the paragraph and revised the text on lines 80-84.
C: 143-144: Please revise the sentence. ApoE does not really play those roles (formation and secretion of lipoproteins). It is a component of a number of lipoproteins as the authors mentioned earlier. More role in lipoprotein binding to several receptors as the authors mentioned as a ligand.
-Спасибо за замечание! Абсолютно с Вами согласны. Решили убрать из предложения данное утверждение (formation and secretion of lipoproteins).
Line 145: recommend using “isoforms” than “alleles” since they are better known as isoforms…although the use of “alleles” is not incorrect.
R: We replaced "alleles" with "isoforms."
C: Lines 148-153: the authors seem to be repeating earlier statement so no need for this?
R: The text has been paraphrased.
C: Line 151: APOE (e4/e4) not just one allele.
R: We corrected е4 to е4/е4.
C: Lines 158-159: Recommend revising the sentence, “It has been previously reported…” should be elaborated with the other statement commenting about the use of genetics. The authors should use this to make their point about the use of APOE (e4/e4) isoform.
Lines 202-203: Recommend revising the sentence.
“…, 147 individuals who had features consistent with CAN, based on … ?” although this is a bit overlapping with Lines 225-226.
R: The corresponding changes have been made in the specified lines – lines 106-108, and 143.
C: Line 228: Please spell out “HT”, hypertension?
R: The word fully spelled out – line 169.
C: Line 233: The diagnosis of diabetes mellitus (DM) in the United States as below (FYI).
- Blood sugar levels
A blood sugar level of 200 mg/dL (11.1 mmol/L) or higher, regardless of when you last ate, suggests diabetes. This is especially true if you also have symptoms of diabetes, such as frequent urination, extreme thirst, weight loss, blurred vision, or fatigue.
- Fasting blood sugar test
A fasting level of 126 mg/dL (7 mmol/L) or higher on two separate tests is diagnosed as diabetes. A level between 100 to 125 mg/dL (5.6 to 6.9 mmol/L) is diagnosed as prediabetes, and less than 100 mg/dL (5.6 mmol/L) is considered healthy.
- Oral glucose tolerance test (OGTT)
This test involves drinking 75g of anhydrous glucose and then measuring your blood sugar levels two hours later. A level of 200 mg/dL (11.1 mmol/L) or higher is considered diagnostic.
- Hemoglobin A1c
A level of 6.5% or higher is considered diagnostic.
If multiple samples are not used, HgbA1C value of > 6.5% probably more desirable in my opinion though this is just a side comment.
R: Thank you for the information! In our country, according to the protocol, the diagnosis of diabetes mellitus is made when the fasting glucose level is ≥ 7 mmol/L and the hemoglobin A1c (HbA1c) level is ≥ 6.1% (48 mmol/mol): lines 176-178.
C: Line 248: please provide a unit for Hb.
R: The unit of measurement is either in % or mmol/mol.
C: 2.2 Genotyping:
Line 289 and Line 294: please add “APOE” as it should be specified as APOE isoforms.
R: We added “APOE”.
C: Was the genotype analyzed as biallelic?
R: Genotyping was performed for two polymorphisms, rs7412 and rs429358; however, the analysis was conducted as a biallelic assessment.
C: APOE (e2/e2), APOE (e2/e3), APOE (e2/e4), APOE (e3/e3), APOE (e3/e4), and APOE (e4/e4)?
R: Initially, the analysis was conducted for one allele of each polymorphism, such as the C allele of rs7412 and the C allele of rs429358. Subsequently, the analysis was carried out based on the aforementioned isoforms (genotypes). For example, results showing a CC genotype for rs7412 and a TT genotype for rs429358 were interpreted as the ε3/ε3 isoform.
C: Line 329: … compared to the “non-CAN” be better than “non-neuropathy’ or DM neuropathy was also separated?
R: Based on your recommendation, we have rephrased it as “non-CAN”.
C: Line 335-337: Biallelic analyses should be performed since each individual carries two alleles, not a separate allele.
Thus, each one should be paired for analysis and cannot be separated.
Line 381: the same comment as above.
R: A biallelic analysis was conducted, taking both alleles into account. However, there are studies where the alleles were also analyzed individually.
C: 4. Discussion:
Line 424: As above, analysis should have been performed as a pair (biallelic isoform combination), not a single allele because an individual has two alleles, never one allele of APOE.
R: A biallelic analysis was performed, considering both alleles. However, some studies have also analyzed the alleles individually.
C: Lines 423-429: Recommend streamlining the finding.
R: Thank you for the feedback! The conclusion has been revised in the specified lines: lines 295-313.
C: Line 446: Recommend replacing “mutation” with “isoform pair” or the genotype APOE (e/e).
R: Replaced “mutation” to “isoform pair”.
C: Lines 451-452: Please clarify the sentence. Which are the allelic variants?
R: Added the information: line 319.
C: Lines 445-447: Please review the role of APOE in LDL clearance. The main ligand for LDL is apolipoprotein B attachment to LDL receptors.
Although lipoprotein differentiation is never exact, but typically LDL do not carry apoE.
Yang LG, March ZM, Stephenson RA, Narayan PS. Apolipoprotein E in lipid metabolism and neurodegenerative disease. Trends Endocrinol Metab. 2023 Aug;34(8):430-445. doi: 10.1016/j.tem.2023.05.002. Epub 2023 Jun 24. PMID: 37357100; PMCID: PMC10365028.
R: Thank you for the feedback! We have made the corresponding changes to these lines regarding the role of APOE in LDL clearance and have included the referenced source: lines 323-326.
C: APOE isoforms differentially affect LCAT-dependent cholesterol esterification as below (FYI).
Vitali C, Pavanello C, Turri M, Lund-Katz S, Phillips MC, Catapano AL, Baragetti A, Norata GD, Veglia F, Calabresi L. Apolipoprotein E isoforms differentially affect LCAT-dependent cholesterol esterification. Atherosclerosis. 2023 Oct;382:117266. doi: 10.1016/j.atherosclerosis.2023.117266. Epub 2023 Aug 30. PMID: 37725860.
R: The additions have been made on lines 354-357.
C: Although other investigators might not have investigated APOE as a biallelic form, each individual has two alleles so when studying humans, it is important to remember that two alleles per gene is present, and APOE (e/e) should be examined together. Otherwise, the relationship between other parameters such as lipid levels should be analyzed against a pair of alleles present in an individual, not separately.
R: I would like to clarify: do you recommend completely excluding the analysis of alleles ε2, ε3, and ε4, and focusing solely on the analysis of genotypes?
In this version of the manuscript, we have included the analysis of ε4 and drawn conclusions based on it. However, if you strongly recommend focusing solely on the analysis of APOE (ε2/ε2), APOE (ε2/ε3), APOE (ε2/ε4), APOE (ε3/ε3), APOE (ε3/ε4), and APOE (ε4/ε4), we can revise the results and conclusions of the study accordingly.
C: Line 482: recommend replacing “linkage” with “association” or some word similar since linkage in genetics has another meaning.
R: Thank you for the recommendation! We have replaced "linkage" with "association".
C: Line 486: recommend replacing “mutations” with “isoforms”
R: Thank you for the recommendation! We have replaced "mutations" with "isoforms".
C: Line 487: recommend replacing “pathogenic significance” since isoforms are not really pathogenic variants though some disease associations have been identified, but they are not pathogenic variants for a monogenic disorder. (associations or risk).
R: Based on your recommendation, we have replaced "pathogenic significance" with "association" in the specified lines.
C: Line 491: recommend replacing “genetic markers” to another word such as “underlying isoform in APOE associated with CAN”.
R: Based on your recommendation, we have replaced "genetic markers" with "underlying isoform in APOE associated with CAN”.
C: This is an association type of study, and the authors have not elucidated the underlying biological mechanisms in the development of CAN in the paper.
R: We have attempted to address the biological mechanisms in lines 303-313 as much as possible.
C: Line 500: The conclusion should be in APOE (e/e) biallelic form, not one allele as stated above.
R: We have added information on all isoforms in the conclusion: lines 386-387.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors made necessary corrections and additions to the manuscript.
Author Response
Thank you for your hard work!
We cannot overstate how much your suggestions have helped us gain greater clarity in understanding the mechanisms and pathogenesis of this disease. This experience has been immensely valuable in expanding our knowledge and skills.
Round 3
Reviewer 2 Report
Comments and Suggestions for AuthorsMulti Digital Publishing Institute (MDPI): Diagnostics – 3104904-R2
Title: Article
APOЕ (e4/?) as a risk factor for developing cardiac autonomic neuropathy in individuals of Kazakh Nationality
Overall comments:
The manuscript has been revised completely to present a study, just using APOE isoforms only.
The study is to investigate the association between the APOE isoforms and their association with cardiac autonomic neuropathy comparing 147 patients and 153 control individuals in a case-control study.
This version is much clearer, and since APOE isoforms are associated with certain diseases as the authors mentioned, it may be an interesting choice.
However, as above, please use APOE (e/e) as a unit, not by a single allele.
The interpretation of results should be in the biallelic form, not by one allele, which does not exist in humans, except in people who have the whole APOE gene deleted on one allele.
The results of this study cannot be presented as a monoallelic form since each individual has two genes, one inherited from the father and another inherited from the mother, except in very rare cases. Thus, the results seem as though negative with isoforms of APOE.
Lines 78-81: this section needs to be after the APOE gene is introduced.
Line 98: As the authors mention about the study by Chang Gao et al., the genotype has to be presented as those authors did APOE (e2/e3), not separately.
Lines 348 – 351: The conclusion does not make sense. The analyses of APOE should be done biallelically which the authors also added, but the conclusion should be in the bi-allelic form. However, it does not seem as though any combinations which included APOE e4 was not found to be in association with CAN. (APOE (e2/e4), APOE (e3/e4) or APOE (e4/e4)) so it should be presented as no association between APOE alleles in individuals with CAN. It is possible that the cohort was too small to identify any difference. Regardless, one cannot present just one of the two alleles has a significant association.
This is totally different from SNPs which include just one site, and two or more alternative nucleotide changes, not like APOE case in which you need to see two sites.
There may be other SNPs which are more in association with CAN in the genome, but it is always difficult to perform any type of association studies in a small cohort.
GWAS data:
https://www.ebi.ac.uk/gwas/efotraits/EFO_1000783
No data on cardiac autonomic neuropathy exists, but diabetic neuropathy has some entries.
Please look at this website and see if new SNPs should be considered.
https://medlineplus.gov/genetics/gene/retreg1/
Furthermore, it may also be helpful to learn about monogenic autonomic neuropathy.
https://medlineplus.gov/genetics/condition/hereditary-sensory-and-autonomic-neuropathy-type-ii/#causes
Thank you very much for allowing me to review this manuscript.
Sincerely,
Comments for author File: 
Comments.docx
Author Response
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Dear Esteemed Reviewer,
Once again, we sincerely thank you for your valuable comments and recommendations! Thanks to your review, our manuscript has significantly improved.
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C: The manuscript has been revised completely to present a study, just using APOE isoforms only.
The study is to investigate the association between the APOE isoforms and their association with cardiac autonomic neuropathy comparing 147 patients and 153 control individuals in a case-control study.
This version is much clearer, and since APOE isoforms are associated with certain diseases as the authors mentioned, it may be an interesting choice.
However, as above, please use APOE (e/e) as a unit, not by a single allele.
Response (R): Following your recommendation, we have made changes and now use APOE (ε/ε) throughout.
C: The interpretation of results should be in the biallelic form, not by one allele, which does not exist in humans, except in people who have the whole APOE gene deleted on one allele.
R: Following your recommendation, we have revised the interpretation of the results and retained only the biallelic form.
C: The results of this study cannot be presented as a monoallelic form since each individual has two genes, one inherited from the father and another inherited from the mother, except in very rare cases. Thus, the results seem as though negative with isoforms of APOE.
R: Thank you for the recommendation! Thus, our results found no association with the APOE gene isoforms.
C: Lines 78-81: this section needs to be after the APOE gene is introduced.
R: Based on your recommendation, we have rearranged paragraphs – lines 67-25.
C: Line 98: As the authors mention about the study by Chang Gao et al., the genotype has to be presented as those authors did APOE (e2/e3), not separately.
R: Recommendations have been noted.
C: Lines 348 – 351: The conclusion does not make sense. The analyses of APOE should be done biallelically which the authors also added, but the conclusion should be in the bi-allelic form. However, it does not seem as though any combinations which included APOE e4 was not found to be in association with CAN. (APOE (e2/e4), APOE (e3/e4) or APOE (e4/e4)) so it should be presented as no association between APOE alleles in individuals with CAN. It is possible that the cohort was too small to identify any difference. Regardless, one cannot present just one of the two alleles has a significant association.
R: In the conclusion, the analysis was presented as the absence of an association between APOE alleles and CAN – lines 305-308.
C: This is totally different from SNPs which include just one site, and two or more alternative nucleotide changes, not like APOE case in which you need to see two sites.
R: Thank you for the clarifications!
C: association studies in a small cohort.
GWAS data:
https://www.ebi.ac.uk/gwas/efotraits/EFO_1000783
No data on cardiac autonomic neuropathy exists, but diabetic neuropathy has some entries.
Please look at this website and see if new SNPs should be considered.
https://medlineplus.gov/genetics/gene/retreg1/
Furthermore, it may also be helpful to learn about monogenic autonomic neuropathy.
https://medlineplus.gov/genetics/condition/hereditary-sensory-and-autonomic-neuropathy-type-ii/#causes
R: We completely agree with you and are grateful for the information provided! We will certainly take all these points into account in future research.
Author Response File: Author Response.pdf
