Relative Leukocyte Telomere Length and Genetic Variants in Telomere-Related Genes and Serum Levels Role in Age-Related Macular Degeneration

Round 1

Reviewer 1 Report

The authors conducted a case-control study of Lithuanian subjects to evaluate associations 78 between relative leukocyte telomere length, genetic variants in telomere-related genes 79, and serum TERF-1 and TERF2 levels on the 81 risks of age-related macular degeneration development to assess the possibility of genetic variants of telomere length as one of the risk factors of AMD.

There are several publications showing the relevance between risk factors and the pathology of various types of diseases, including age-related diseases. However, it is still controversial the significance of genetic variants of telomere length in the pathology of AMD. The reviewer strongly recommends the authors address the comment below:

 

Major comment

As described in the general comment above, it is still controversial regarding genetic variants of telomere length as risk factor of AMD, as described in line 245. In addition, in line 261, the author described such chronic systemic inflammation has been reported in the pathogenesis of cardiovascular disease and AMD. It is reasonable that chronic systemic inflammation plays essential roles in AMD pathologies; however, further discussion is required to justify and support the rationale of data of this paper since it is thought that the cell cycle of retinal cells related to AMD pathologies is arrested. Please clarify the relevance of telomere length and AMD pathologies based on the pathologies of AMD including characteristics of cell types involved in AMD. 

Author Response

Dear Editor and Reviewers,

We kindly appreciate the revision of our manuscript. We have highlighted in yellow the changes we made in the manuscript. We hope that the revised manuscript will be acceptable for publication in your journal. Enclosed please also find attached our point-by-point response to the comments raised by the reviewers (editors).

1^st reviewer

Comments and Suggestions for Authors

The authors conducted a case-control study of Lithuanian subjects to evaluate associations 78 between relative leukocyte telomere length, genetic variants in telomere-related genes 79, and serum TERF-1 and TERF2 levels on the 81 risks of age-related macular degeneration development to assess the possibility of genetic variants of telomere length as one of the risk factors of AMD.

There are several publications showing the relevance between risk factors and the pathology of various types of diseases, including age-related diseases. However, it is still controversial the significance of genetic variants of telomere length in the pathology of AMD. The reviewer strongly recommends the authors address the comment below:

Major comment

As described in the general comment above, it is still controversial regarding genetic variants of telomere length as risk factor of AMD, as described in line 245. In addition, in line 261, the author described such chronic systemic inflammation has been reported in the pathogenesis of cardiovascular disease and AMD. It is reasonable that chronic systemic inflammation plays essential roles in AMD pathologies; however, further discussion is required to justify and support the rationale of data of this paper since it is thought that the cell cycle of retinal cells related to AMD pathologies is arrested. Please clarify the relevance of telomere length and AMD pathologies based on the pathologies of AMD including characteristics of cell types involved in AMD. 

 

Dear reviewer, thank you for your comment;

A paragraph added:

 

Drigeard Desgarnier et al. found telomere length differences in different human eye structures (56). Moreover, Bell et al. observed a unique telomere DNA expansion phenotype in the rod cells but not in other retinal cells (57).

Analysing the AMD pathogenesis, scientists suggested that the senescence of the RPE cells might play a role in AMDdevelopment (58) through several pathways, including oxidative stress response (59). Oxidative stress damages telomeres due to their guanine-rich DNA structure. While it can be more challenging to repair, in some cases, the telomerase may extend oxidative stress-shortened telomeres, preventing further RPE cell degeneration following AMD progression (60).

56. Drigeard Desgarnier, M.C.; Zinflou, C.; Mallet, J.D.; Gendron, S.P.; Méthot, S.J.; Rochette, P.J. Telomere Length Measurement in Different Ocular Structures: A Potential Implication in Corneal Endothelium Pathogenesis. Investig. Ophthalmol. Vis. Sci. 2016, 57, 5547–5555.
57. Bell W.R., Meeker A.K., Rizzo A., Rajpara S., Rosenthal I.M., Flores Bellver M., Aparicio Domingo S., Zhong X., Barber J.R., Joshu C.E., et al. A unique telomere DNA expansion phenotype in human retinal rod photoreceptors associated with ageing and disease. Brain Pathol. 2019;29:45–52. doi: 10.1111/bpa.12618.
58. Bhutto I., Lutty G. Understanding age-related macular degeneration (AMD): Relationships between the photoreceptor/retinal pigment epithelium/Bruch’s membrane/choriocapillaris complex. Mol. Asp. Med. 2012;33:295–317. doi: 10.1016/j.mam.2012.04.005.
59. Blasiak J. Senescence in the pathogenesis of age-related macular degeneration. Cell. Mol. Life Sci. 2020;77:789–805. doi: 10.1007/s00018-019-03420-x.
60. Blasiak J, Szczepanska J, Fila M, Pawlowska E, Kaarniranta K. Potential of Telomerase in Age-Related Macular Degeneration-Involvement of Senescence, DNA Damage Response and Autophagy and a Key Role of PGC-1α. Int J Mol Sci. 2021;22(13):7194. doi: 10.3390/ijms22137194.

Reviewer 2 Report

In my opinion, the manuscript is generally well written and the results are interesting. Nevertheless, I have the following comments:

1.    Material and methods

The division of the control group into two separate groups is incomprehensible, as the median age in both groups is similar. Please provide more detailed justification for this choice. It is also unclear whether control group II was extracted from control group I or whether they are completely different individuals.

There is no description of statistical analysis methods in the section.

It is necessary to check whether the genotypes tested were in concordance with the Hardy-Weinberg equilibrium.

2.    Results

Tables – lack of information on the test(s) used to determine the p value

TERF1 and TERF2 serum levels measuring - do the control groups also correspond to the previously designated groups I and II respectively?

In Figures 4-7 lack of information on the test(s) used to determine the p value.

 

Additionally, the study limitations should be clearly identified and discussed.

Author Response

Dear Editor and Reviewers, 

We kindly appreciate the revision of our manuscript. We have highlighted in yellow the changes we made in the manuscript. We hope that the revised manuscript will be acceptable for publication in your journal. Enclosed please also find attached our point-by-point response to the comments raised by the reviewers (editors). 

 

2nd reviewer 

In my opinion, the manuscript is generally well written and the results are interesting. Nevertheless, I have the following comments: 

1.    Material and methods 

The division of the control group into two separate groups is incomprehensible, as the median age in both groups is similar. Please provide more detailed justification for this choice. It is also unclear whether control group II was extracted from control group I or whether they are completely different individuals. 

 

 Two control groups were formed to ensure that the control and AMD group does not differ statisticaly signifcantly. 

Control group justification 

Control group I do not differ from Early AMD group (p>0.05), but is statistially significantly younger than Exudative AMD group (p<0.05), so the Control group II was formed, 

Control group II was formed using the same subjects from Control group I, only the younger subjects were excluded to ensure that there is no statistically significant difference between Exudative AMD and Control group II (p>0.05). 

  

There is no description of statistical analysis methods in the section. 

 

Statistical analysis 

Statistical analysis was performed using the SPSS/W 20.0 software (Statistical Package for the Social Sciences for Windows, Inc., Chicago, Illinois, USA). The data are presented as median with interquartile range (IQR) for continuous data. The normality of data distribution was checked using the Shapiro–Wilk test. For non-normally-distributed data, the Mann–Whitney test was used to compare the data between two groups. For normally-distributed data, the Pearson Chi-Square test was used to compare the data between two groups. Differences were considered statistically significant when p<0.05. 

Frequencies of genotypes and alleles, and gender are reported using absolute numbers with percentages in brackets. The distributions of the genotypes and alleles, male and female distributions between study groups were compared using the χ² test.  Binomial logistic regression analysis was performed to estimate the impact of genotypes on early, exudative AMD development and presented as odds ratios with it’s  95% confidence intervals. 

The best genetic model selection was based on the Akaike Information Criterion (AIC); therefore, the best genetic models were those with the lowest AIC values. 

It is necessary to check whether the genotypes tested were in concordance with the Hardy-Weinberg equilibrium. 

Hardy Weinberg equilibrium (HWE ) was evaluated for all SNPs in both control group I and control group II. Only one SNP TRF1 rs10107605 did not folow the HWE, which might be caused by low sample size in study groups. 

2.    Results 

Tables – lack of information on the test(s) used to determine the p value 

 Added: 

* χ² test. 

 TERF1 and TERF2 serum levels measuring - do the control groups also correspond to the previously designated groups I and II respectively? 

Yes, correspond to the previously designated groups I and II respectively. 

In Figures 4-7 lack of information on the test(s) used to determine the p value. 

Information added.  

Additionally, the study limitations should be clearly identified and discussed. 

 Study limitations 

While our study revealed significant results, it is still important to highlight that further studies with larger sample size are necessary to confirm such associations.  Moreover, other AMD risk factors should be included into future studies. 

 

Round 2

Reviewer 1 Report

The authors addressed the comment correctly. 

Author Response

Dear Reviewer,

thank you for your comment.

Best regards

Greta Gedvilaite