Matrix Gla protein polymorphism rs1800801 associates with recurrence of ischemic stroke

Philipp Hendrix; Nelson Sofoluke; Matthew Adams; Saran Kunaprayoon; Ramin Zand; Amy N. Kolinovsky; Thomas N. Person; Mudit Gupta; Oded Goren; H. Lester Kirchner; Clemens M. Schirmer; Natalia S. Rost; James E. Faber; Christoph J. Griessenauer

doi:10.1371/journal.pone.0235122

Peer Review History

Original SubmissionMarch 16, 2020
29 May 2020 Decision Letter - Aurel Popa-Wagner, Editor PONE-D-20-07538 Matrix Gla protein polymorphism rs1800801 associates with recurrence of ischemic stroke PLOS ONE Dear Dr. Hendrix, 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. Please submit your revised manuscript by Jul 13 2020 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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Please ensure that you refer to Table 3 in your text; if accepted, production will need this reference to link the reader to the Table. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions 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 Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. 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 Reviewer #2: Yes ****** 4. 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 Reviewer #2: Yes ****** 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: In the Genotyping Recurrence Risk of Stroke (GRECOS) project, however, Fernándes-Cadenas et al. found an association of the single nucleotide polymorphism (SNP) rs1800801 in the matrix carboxyglutamatic acid Gla protein (MGP) gene with first-year recurrent ischemic stroke in Spanish Caucasians. MGP is an extracellular matrix protein involved in inhibition of bone and vascular calcification. Since a recent a meta-analysis highlighted the SNP rs1800801 (12-15038788C-T, G>A) in Caucasians to increase the risk for vascular calcification and atherosclerotic disease, the authors investigated the impact of rs18008001 on the risk for first-year recurrent stroke in a North American Caucasian population. This is a good study conducted on a acceptable no of patients. However, since age is the only non-modifiable risk factor for stroke I would like to know the age-dependence (if any) of the SNP rs1800801 among the enrolled patients. Further, the authors shall discuss the age-dependency of extracellular matrix protein involved in inhibition of bone and vascular calcification. Reviewer #2: -what is the normal distribution of the AA genotype of MGP SNP rs1800801 in general healthy Caucasian population of North America? Is it comparable with the population of Spain? -in Table 1 – Patients characteristics - in the stroke recurrent group: 44.3% had stroke in the past (vs. 7.6%), which may be in relation with genetic association (family history), associated with metabolic syndrome (80% with BMI>25, in both groups), and procoagulant disorders. Also it seem that the recurrence appear in nonsmokers group(43% vs. 25.3%). How would you comment on the lack of correlation between smoking and vascular complications? Studies did show an increase of MGP in smokers (Silaghi CN et al, Clin Chim Acta 2019) and the correlation with cardiovascular complications. - how would you comment on the fact that mortality was less probable in the recurrent group (even with prior strokes) than in the other group (1.26% vs. 2.43%). - why should be taken in consideration the AA genotype frequency of SNP rs1800801, representing 13% of genotypes (vs.39.5% and 47.4%), from 7.4% of patients that will have recurrence in the first year, and these events are less harmful than in the other group? How powerful is the message for introducing AA genotype of SNP rs1800801 as a risk tool, in comparison with other contributing factors to vascular complications? If other risk factors are not described, they should be at least mentioned – SNP for adiponectin, etc. -rows 70-71, 190 – „MGP is an extracellular matrix protein involved in inhibition of bone and vascular calcification” - is misleading information. MGP is not produced by bone cells, but by chondrocytes and fibroblasts, smooth muscle cells (Price PA, 1988, 1992). MGP does not have a role in bone remodeling cycle. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: 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.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0235122.r001
Revision 1
7 Jun 2020 Author Response Reviewer #1: In the Genotyping Recurrence Risk of Stroke (GRECOS) project, however, Fernándes-Cadenas et al. found an association of the single nucleotide polymorphism (SNP) rs1800801 in the matrix carboxyglutamatic acid Gla protein (MGP) gene with first-year recurrent ischemic stroke in Spanish Caucasians. MGP is an extracellular matrix protein involved in inhibition of bone and vascular calcification. Since a recent a meta-analysis highlighted the SNP rs1800801 (12-15038788C-T, G>A) in Caucasians to increase the risk for vascular calcification and atherosclerotic disease, the authors investigated the impact of rs18008001 on the risk for first-year recurrent stroke in a North American Caucasian population. This is a good study conducted on an acceptable no of patients. We appreciate the reviewer´s positive comment on our study. However, since age is the only non-modifiable risk factor for stroke I would like to know the age-dependence (if any) of the SNP rs1800801 among the enrolled patients. We appreciate the reviewer´s comment. We assessed age-dependent distribution of SNP rs1800801 genotypes in our cohort. We did not find age differences among AA, GA, GG genotypes in our cohort (mean 69 – 68 – 67; median 70 – 70 – 68; min/max/IQR 24/93/17 – 22/96/20 – 28/99/19, respectively). We also assessed age groups and dichotomized for cut-off values (age ≥ 60 as in ABCD2/California scores or age ≥ 69 as present study median) and did not find any differences. In our logistic regression model, impact of age among enrolled patients was assessed both as a quantitative (continuous) variable and a qualitative (age groups) variable. We agree that age is an important risk factor for stroke recurrence, which is also displayed by ABCD2, ESRS, and California risk score to predict recurrent events. However, the herein presented study is a retrospective database analysis, which should not compete with prospective clinical risk stratification. Further, the authors shall discuss the age-dependency of extracellular matrix protein involved in inhibition of bone and vascular calcification. The reviewer addresses an interesting issue. To date, the mechanisms of vascular calcification and the interplay of MGP have not been elucidated. We added a statement on the potential players in the pathological mechanisms. Elaborating on unsupported hypotheses, however, would side track the readers´ attention from the genetic association study. Thus, we limited the additional information on this aspect. Reviewer #2: -what is the normal distribution of the AA genotype of MGP SNP rs1800801 in general healthy Caucasian population of North America? Is it comparable with the population of Spain? The references (ref) and alternative (alt) allele frequencies can be obtained from reference [14] in the manuscript: https://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=1800801. The minor allele frequencies (MAFs) appear to be similar for most ethnicities and range from 0.27 – 0.37, whereas a significant decline has been observed in some Asian subgroups with MAF < 0.07. Therefore, we expect the MAF from our North American cohort (only Caucasians) and the GRECOS cohort to be in equal ranges. -in Table 1 – Patients characteristics - in the stroke recurrent group: 44.3% had stroke in the past (vs. 7.6%), which may be in relation with genetic association (family history), associated with metabolic syndrome (80% with BMI>25, in both groups), and procoagulant disorders. Also it seem that the recurrence appear in nonsmokers group (43% vs. 25.3%). How would you comment on the lack of correlation between smoking and vascular complications? Smoking status between the two groups (1st-year recurrent vs no recurrent) was equally distributed as displayed in Table 1. In the 1st-year recurrent group 25.3% were current smokers, whereas 43.0% had never smoked. In the no recurrent group 21.2% were current smokers and 38.5% had never smoked. We cannot replicate that non-smoking should be the predominant driver for stroke recurrence. Additionally, we do not report a lack of correlation between smoking and vascular complications; as such analysis is not part of the herein presented manuscript. In our study, smoking status was not associated with 1st-year stroke recurrence. However, in our cohort we do find that never-smokers have significant decreased odds of suffering from carotid stenosis, coronary heart disease, and peripheral vascular disease (all OR < 1, p < 0.05). That smoking status was not associated with 1st-year stroke recurrence could be attributed to multiple factor: i) data was obtained from a retrospective database, thus smoking status could not be analyzed in a quantitative but only a qualitative measure, ii) effects of former-smoking could counterbalance both current and never smoking depending on how long smoking has been stopped, or iii) smoking might not be a predominant factor for early stroke recurrence. Additional studies need to investigate the role of smoking for early stroke recurrence. Studies did show an increase of MGP in smokers (Silaghi CN et al, Clin Chim Acta 2019) and the correlation with cardiovascular complications. - how would you comment on the fact that mortality was less probable in the recurrent group (even with prior strokes) than in the other group (1.26% vs. 2.43%). The 1.3% vs 2.4% mortality rates are those at patient discharge. Discharge outcomes rates are prone to bias. Duration of hospital stay, complications during hospital stay, and discharge location have to be taken into consideration when interpreting discharge outcomes. Therefore, standardized follow-ups such as 90-day follow-ups are mandatory to evaluate outcome. Here, at 90-day follow-up, mortality rates are 7.8% vs 5.9% which reflects the burden of a recurrent stroke event. - why should be taken in consideration the AA genotype frequency of SNP rs1800801, representing 13% of genotypes (vs.39.5% and 47.4%), from 7.4% of patients that will have recurrence in the first year, and these events are less harmful than in the other group? As mentioned above, recurrent strokes are not less harmful event that no recurrent strokes. The AA genotype was significantly associated with occurrence of a first-year stroke recurrence. How powerful is the message for introducing AA genotype of SNP rs1800801 as a risk tool, in comparison with other contributing factors to vascular complications? If other risk factors are not described, they should be at least mentioned – SNP for adiponectin, etc. In the results section we demonstrate that we could not improve predictability of stroke recurrence with the integration of SNP rs1800801, i.e. the AUC are almost identical. In the discussion section we mention that we did not find this genetic polymorphism to be of additional predictive value and thus far clinical variables appear to be most accurate. -rows 70-71, 190 – „MGP is an extracellular matrix protein involved in inhibition of bone and vascular calcification” - is misleading information. MGP is not produced by bone cells, but by chondrocytes and fibroblasts, smooth muscle cells (Price PA, 1988, 1992). MGP does not have a role in bone remodeling cycle. We agree with the reviewer that the abstract from our reference reports vascular smooth muscle cell and chondrocyte calcification. We do not mention that MGP is synthesized by osteoblasts. However, a detailed look into the reports also mentioned by the reviewer (Fraser and Price 1988, J Biochem) reveals: “It should be noted in this context that MGP is in fact made by cartilage as well as bone.“ Additionally, Cancela and Price 2001: Matrix Gla protein in Xenopus laevis: molecular cloning, tissue distribution, and evolutionary considerations. J. Bone Miner. Res. extract MGP from bones and the role of MGP in bone remodeling is under investigation. However, this is beyond the scope of the manuscript. Our cited reference refers to enchondral ossification in mice and thus the term “bone calcification” is likely not precise. We corrected this according https://doi.org/10.1371/journal.pone.0235122.r002
10 Jun 2020 Decision Letter - Aurel Popa-Wagner, Editor Matrix Gla protein polymorphism rs1800801 associates with recurrence of ischemic stroke PONE-D-20-07538R1 Dear Dr. Griessenauer, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Aurel Popa-Wagner Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0235122.r003
Formally Accepted
16 Jun 2020 Acceptance Letter - Aurel Popa-Wagner, Editor PONE-D-20-07538R1 Matrix Gla protein polymorphism rs1800801 associates with recurrence of ischemic stroke Dear Dr. Griessenauer: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Aurel Popa-Wagner Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0235122.r004

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