PONE-D-20-37687

LOXL1 gene polymorphisms are associated with exfoliation syndrome/exfoliation glaucoma risk: an updated meta-analysis

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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Reviewer #1: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors in this manuscript report a meta-analysis over the association of known risk alleles of the gene LOXL1 and XFS/XFG, including 36658 participants (cases and controls) of different ethnic groups. The research design is appropiate and the conclusions supported by the results. However, the authors should review the manuscript in depth. Mistakes and inaccuracies throughout the text must be addressed. References should be revised and some parts of the text should be rewritten or clarified. Moreover, extensive editing of English language is required.

Comments and Suggestions for Authors:

1. Human gene symbols must be italicized throughout the text. LOXL1 when you refer to the gene. LOXL1 for the protein.

2. Page 7, Abstract background, first sentence: “Lysyl oxidase-like 1 (LOXL1), a matrix cross-linking enzyme involved in elastic fiber formation, is a risk factor for exfoliation syndrome and exfoliation glaucoma” This sentence should be corrected.

Single nucleotide polymorphisms (SNPs) in the gene encoding the LOXL1 are the risk factors for exfoliation syndrome and exfoliation glaucoma”.

3. Page 8, Abstract results: “… was observed in the disease types-based subgroup” should be “were observed in the disease type-based subgroups”

4. Page 8, Abstract results: please clarify “…combined polymorphism…”

5. Page 8, Abstract results: “relative to” should be “related to”

6. Page 8, Introduction, paragraph 1: please clarify “occoecatio”. Is there another better known term with the same meaning?

7. Page 8, Introduction, paragraph 1: Reference 4 is before reference 3 in the text.

8. The article corresponding to reference number 3 in the list of references, has been retracted by the editor of International Journal of Ophthalmology. This reference should be removed from the article.

9. Page 9, paragraph 1: “intraocular pressure” should be “elevated intraocular pressure”

10. Page 9, paragraph 1: Add a comma “Relative to primary open-angle glaucoma, XFS-associated secondary open-angle glaucoma...”

Points 11-15 refer to the same paragraph of the introduction (Paragraph 3 of page 9 and 1 of page 10), which must be rewritten more neatly after an exhaustive revision. I think the authors try to show the discrepancies between some studies carried out in different populations of the same or different ethnic origin, but they fail to do it due to the mistakes and inaccuracies they commit.

11. Page 9, paragraph 3: reference 16 is wrong. The correct reference is: Thorleifsson G, Magnusson KP, Sulem P, Walters GB, Gudbjartsson DF, Stefansson H et al. Common sequence variants in the LOXL1 gene confer susceptibility to exfoliation glaucoma. Science. 2007;317(5843):1397-1400.

12. Page 9, paragraph 3: This statement is incorrect: “Dubey SK et al (17) reported that LOXL1 G (rs1048661) and T (rs3825942) alleles are XFS/XFG risk factors in Caucasians.” The work of Dubey et al was carried out in an Asian population no in Caucasians. Moreover, there is also some mistake regarding the risk alleles.

Dubey et al reported that the G allele of rs1048661, the G allele of rs3825942 and the T allele of rs2165241 were XFS/XFG risk factors in a South Indian population. Which is similar to that found in the original study conducted by Thorleifsson et al (2007) in Caucasians (Scandinavian population), as well as in most studies carried out in Caucasians, but different to that reported in most studies in Asians, where the alleles T and C of rs1048661 and rs2165241, respectively, are the risk alleles. References of these works should be added.

13. Pages 9-10, Paragraph 3 of page 9 and 1 of page 10: The sentence “De Juan-Marcos L et al (18) showed that LOXL1 rs2165241 polymorphism may be associated with XFS/XFG susceptibility” should be corrected.

De Juan-Marcos et al showed that the G allele of rs3825942 and the T allele of rs2165241 were XFS/XFG risk factors in a Spanish population. But no significant association between XFS/XFG and the SNP rs1048661 was observed. This last observation is the most relevant, since it shows a discrepancy with the majority of studies in Caucasian populations. References of these works should be added.

14. Page 10, paragraph 1: The statement “However, Tanito M et al (19) reported that LOXL1 G and T alleles are protective against XFS/XFG in Japanese populations. Similar observations were made by Chen L et al (20)” should be extended to more Asian studies, where G (rs1048661) and T (rs2165241) alleles are protective against XFS/XFG, and not only to the work of Chen L et al (20) in a Chinese population. References of these works should be added. See point 12.

15. Page 10, paragraph 1: Some reference should be made to the results found in the literature for the black race and discrepancies with other ethnicities.

16. Page 10, paragraph 2: The statement “Past genetic predisposition studies have been inconclusive” should be qualified.

Despite the existence of discrepancies between some studies related with the risk alleles of LOXL1 SNPs, it is widely accepted that LOXL1 gene is the most important genetic risk factor known so far for XFS/XFG.

17. Page 10, Material and methods: Please clarify the point “3) if they consisted of a complete number of genotypes among cases and controls”

18. Page 11, Statistical analyses: Please, replace “gene models” by “genetic models”

19. Page 11, Statistical analyses: Add software/s used to carry out the statistical analyses.

20. Page 12, Publication bias: References 22 and 23 are not the most suitable. I suggest the following references:

Peters JL, Sutton AJ, Jones DR, et al. Contour-enhanced meta-analysis funnel plots help distinguish publication bias from other causes of asymmetry. J Clin Epidemiol 2008;61:991–6.

Sterne J A, Gavaghan D, Egger M. Publication and related bias in meta-analysis: power of statistical tests and prevalence in the literature. J Clin Epidemiol 2000;53:1119–29.

21. Page 12, 3.1 Study characteristics: Please, correct Figure 1. According to the text, titles and abstracts where searched in step 1 and not in step 2 as showed in the Figure 1.

22. Page 12, 3.1 Study characteristics, line 6, “a previous meta-analysis” should be “in a previous meta-analysis”

23. Table 2: Please, specify what is N

24. I suppose that N is the number of studies, but in table 2, for rs1048661, N is 39 instead of 38 as showed in the text (page 13, line 1).

25. In Table 2 N is 17 for rs1048661, XFS, however, in Figure 2 only 16 studies are listed.

26. In Table 2 N is 21 for rs1048661 in Asians, however, in Figure 3 only 19 different studies are listed.

27. In Table 2 N is 18 for rs1048661 in Caucasians, however, in Figure 3 only 17 different studies are listed.

28. Please, clarify y/o correct the following discrepancies between the data of the tables 1 and 2:

- In table 2 the total number of cases (XFS/XFG) is 4900 for rs1048661, however, if the number of cases from each study listed in Table 1 are added, the total number of cases is 4828.

- In table 2 the total number of controls (XFS/XFG) is 10111 for rs1048661, however, if the number of controls from each study listed in Table 1 are added, the total number of cases is 10036.

- In table 2 the total number of cases (XFS/XFG) is 4305 for rs3825942, however, if the number of cases from each study listed in Table 1 are added, the total number of cases is 4233.

- In table 2 the total number of controls (XFS/XFG) is 9092 for rs3825942, however, if the number of controls from each study listed in Table 1 are added, the total number of cases is 9017.

29. Page 13, first paragraph: The authors claim that no associations were found between the SNP rs1048661 and XFS/XFG (total group). That is according to table 1 and Fig3 data but however, according to the data of figure 2, there is association. OR=1.73 �1.41, 2.12�.

Please, explain more in detail the two analysis realized for each SNP (by disease and by ethinicity) and why analysis considering ethnicity is chosen.

It should be clarify in the text that the data “G vs. T, OR:1.13,95%CI: 0.85-1.52, P:0.40” are shown in Fig3.

30. Page 13, second paragraph, “rs3825942” should be “rs2165241”.

31. Page 13, second paragraph, it should be clarify in the text that the data “T vs. C, OR: 1.61, 95%CI: 1.18-2.19, p:0.002” are shown in Fig5.

32. Page 13, second paragraph, “Fig4” should be “Fig4, Table 2”.

33. Page 14, first paragraph, “Fig5” should be “Fig5, Table 2”.

34. Page 14, second paragraph, it should be clarify in the text that the data “G vs. A, OR: 5.33, 95%CI: 3.49-8.16, p<0.001” are shown in Fig7.

35. Page 14, second paragraph, “Fig6” should be “Fig6, Table 2” and “Fig7” should be “Fig7, Table 2”

36. Page 14, second paragraph, the dot must be replaced by a comma. “…increased risks were identified among Caucasians (G vs. A, OR: 6.48, 95%CI: 3.67-11.44, P<0.001) and Asians (G vs. A, OR: 5.89, 95%CI: 3.79-9.16, p <0.001) (Fig 7), suggesting that…”

37. Figures 2-7 legends: The reader should be provided with more details about the figures. For instance, what the squares (or the diamonds) and their size indicate.

38. Figures 2-7 legends: Please, replace “LOXL1 gene polymorphism in rs…” by “LOXL1 gene polymorphism rs…”

39. Figure 8: Please, correct the leyend: Begg’s funnel plot for evaluation of publication bias in the selection of studies on the association between between exfoliation syndrome /exfoliation glaucoma risks and LOXL1 gene polymorphism, rs1048661(A), rs2165241(B) and rs3825942(C).

40. Page 15, Discussion: The statement “Various population studies have provided inconclusive evidence that LOXL1 single nucleotide polymorphisms are strongly

associated with XFS/XFG” should be qualified.

As mentioned in point 18, despite the existence of discrepancies between some studies related with the risk alleles of LOXL1 SNPs, it is widely accepted that LOXL1 gene is the most important genetic risk factor known so far for XFS/XFG.

Moreover, references 6, 70, 71 are not suitable.

41. Page 15, Discussion: Please, remove the reference 3 which has been retracted by the editor of International Journal of Ophthalmology.

42. Page 16, line 4: The number of participants was 36658 (cases + controls)

43. Page 16: Risk alleles should be specified in the sentence “XFS/XFG analysis by ethnicity revealed significantly high association between the 3 LOXL1 polymorphisms and XFS/XFG risk in Caucasians”.

“XFS/XFG analysis by ethnicity revealed significantly high association between the allele G of rs1048661, the allele T of rs2165241 and the allele G of rs3825942 and XFS/XFG risk in Caucasians”

44. The same should be done in the rest of the paragraph with all SNPs, since alleles that confer risk vary between ethnic groups. The risk alleles should be specified. For instance: “…the variant G of rs1048661 polymorphism may have potentially negative effects on…”

Moreover, some mistakes should be corrected: For instance: “In Asians, significantly increased XFS/XFG risk was associated with the two LOXL1 polymorphisms (rs3825942)” should be “In Asians, significantly increased XFS/XFG risk was associated with the allele G of rs3825942”.

Please, revise, correct and rewrite the paragraph in a more orderly way.

45. The lack of studies in Africans, specially about the SNP rs2165241 should be mentioned in the text.

46. In my opinion, mention should be made of the high frequencies of risk alleles in the controls and discuss their implications.

47. Page 16, Discussion: Differences and similarities with the results of other studies or with previous meta-analysis must be described and discussed in the manuscript. Does this work make any new contribution to the field or confirm the results of previous studies?

48. Page 16: Clarify the sentence: “…the differences in genetic susceptibility might be affected by ethnic factors and periods of glaucoma”.

Ethnic factors can affect genetic susceptibility, the genetic background is different between ethnicities. However, the stage of glaucoma can not affect genetic susceptibility. The genotype of an individual do not change, is the same throughout his life regardless of the stage of a disease

49. Page 17: Please, provide details of the studies that could support the hypothesis: “We hypothesized that the LOXL1 gene polymorphisms may upregulate LOXL1 production and that the high serum concentration of LOXL1 may contribute to XFS/XFG susceptibility”

50. Page 17: Please, correct the sentence“… hence the observed opposite rs1048661 risks in Asian vs Caucasians subjects”.

The effect of rs2165241 is also opposite in Asian and Caucasian

51. Page 17: Please, rewrite the sentence: “Heterogeneity involved in studies is generally in meta-analysis for the quality of the included research, population characteristics, experimental methods and other reasons”.

52. Page 17: Please, correct or clarify the sentence: “Thus, we speculated that there are several other factors leading to heterogeneity: Blood sample sampling and storage methods are different; the measurement method and experimental conditions are different; varying levels of illness severity”.

In my opinion the factors leading to heterogeneity listed by the authors are irrelevant to a genetic study. The genotype of an individual is not affected by factors such as blood sample sampling or storage method. The mentioned factors could be relevant for other types of studies, for instance when quantification of a protein in blood is required.

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Reviewer #1: No

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PONE-D-20-37687R1

LOXL1 gene polymorphisms are associated with exfoliation syndrome/exfoliation glaucoma risk: an updated meta-analysis

PLOS ONE

Dear Dr. Jie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The revised manuscript is improved, but the reviewer has identified remaining significant errors that need to be corrected. If these errors can be addressed we would review a revised manuscript submitted according to journal instructions.  

==============================

Please submit your revised manuscript by Apr 25 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Philip C. Trackman, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

The reviewer has noted an improvement in the manuscript, but several significant errors remain that need to be corrected. If you can adress these issues we will reconsider a revised manuscript with changes made tot he manuscript explained according to journal requirements.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The Authors made several important changes that increased the clarity of the manuscript. However, the authors should address following issues.

Comments and Suggestions for Authors:

1. Pag 1, Abstract Background, line 1: LOXL1 (do not use italics when referring to protein)

2. Pag 1, Abstract Results: The number of controls in the sentence “In total, 5022 cases and 10400 controls were included in this meta-analysis” should be corrected.

Table 1 show the number of XFS and XFG cases and controls in each study. Some studies report the results for XFS and XFG separately, in these studies the control group is the same for both diseases, but it was counted in duplicate. For example, in the case of the study of Panday et al., there is only a group control with 61 individuals.

For the same reason, the total number of participants must be corrected in Discussion, pag 19, first line.

3. Pag 2, Abstract results: Delete the word “only” in the sentence: “In addition, only rs1048661 and rs3825942 correlated with XFS/XFG susceptibility in Africans.

The SNP rs2165241 was not analized in Africans. Authors do not know if this SNP is associated with XFS/XFG.

4. Pag 3, Introduction: in the sentence “…as well as in most studies carried out in Caucasians [16,17]” delete reference 17, and add references of other studies in Caucasians

5. Pag 3-4, Introduction: Delete the word “where” in the sentence: “However, in most studies in Asians, where the alleles T and C of rs1048661 and rs2165241, respectively, are the risk alleles”.

6. Pag 4, Introduction: Please, change the sentence: “However, no significant association between XFS/XFG and SNP rs1048661 was observed” to “However, in contrast to what was observed in most Caucasian populations, no significant association between XFS/XFG and SNP rs1048661 was observed”.

7. Page 6, Study characteristics: Figure 1 has not been corrected according to the text.

Original figure 1 has been included in the revised manuscript

“Our initial literature search returned 197 articles. Upon browsing the titles and abstracts, 111 articles were excluded, leaving 86 articles that underwent full-text review”.

Titles and abstracts were browsed in the first step, not in the second as is shown in the figure 1.

8. Please, review the number of records excluded because they involved other LOXL1 gene polymorphisms. 32 in the text and 31 in the figure 1.

Review also the numbers in the text: “Although five articles [10,30-33] had been analyzed in a previous meta-analysis [34], we excluded them because three articles [10,30,31] did not achieve HWE in the control group, and two articles [32-33] reported the relationship between LOXL1 gene polymorphisms and primary open-angle glaucoma”. The numbers in the text must match those in the figure 1.

9. Figure 5 appears in the text before figure 4.

10. Figure 7 appears in the text before figure 6.

11. Discussion, pag 20: “Based on previous studies and the findings from the present meta-analysis, we hypothesized that the LOXL1 gene polymorphisms may upregulate LOXL1 production and that the high serum concentration of LOXL1 may contribute to XFS/XFG susceptibility [75,76]”.

Neither the present study nor the references provided by the authors support this hypothesis.

The works of Greene et al. (75) and Want et al. (76) have not studied the relationship between LOXL1 variants and dysregulation of LOXL1 production. According to Greene et al., (75) LOXL1 expression is downregulated in XFG due to DNA methylation and LOXL1 promoter methylation, while Want et al. (76) associate XFS with dysfunction in autophagy. In these studies there is no mention of LOXL1 variants

On the other hand, the present study has not analyzed the production of LOXL1 and its relationship with LOXL1 variants.

No indication has been provided to support that LOXL1 gene variants may upregulate LOXL1 production.

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Reviewer #1: No

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PONE-D-20-37687R2

LOXL1 gene polymorphisms are associated with exfoliation syndrome/exfoliation glaucoma risk: an updated meta-analysis

PLOS ONE

Dear Dr. Jie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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The reviewer has identified a few minor writing errors that need to be corrected, and the journal does not perform copy editing. When these have been corrected, your manuscript will be accepted. Thank you for submitting your work to PLoS One. 

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Philip C. Trackman, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

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Reviewer #1: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

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Reviewer #1: (No Response)

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Reviewer #1: This manuscript is acceptable for publication. However, some misprints should be corrected

Abstract, Background: “… in the gene encoding the LOXL1…” should be “… in the gene encoding LOXL1…”

Discussion, Pag 18: “… the allele T of rs2165241 had a potential protective effect on XFS/XFGS in Asians.” XFG not XFGS

Discussion, Pag 19: “However, we also found that LOXL1 the G allele of rs1048661..." should be “However, we also found that the G allele of rs1048661 ..."

Discussion, Pag 19: “Additionally, there was a significant association between LOXL1 gene polymorphisms and susceptibility to various disease types. These results affirmed the association between LOXL1 gene polymorphisms and XFS and XFG.” LOXL1 in italics when referring to the gene

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Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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LOXL1 gene polymorphisms are associated with exfoliation syndrome/exfoliation glaucoma risk: an updated meta-analysis

PONE-D-20-37687R3

Dear Dr. Jie,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Philip C. Trackman, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: