PONE-D-21-27176The Research on Association of the ANRIL Gene with Ischemic Stroke: the Evidence From a Comprehensive Meta-AnalysisPLOS ONE

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Reviewer #1: In the manuscript entitled “The Research on Association of the ANRIL Gene with Ischemic Stroke: the Evidence From a Comprehensive Meta-Analysis”, the authors explored whether f the All the data used in the published GWASA papers should be included, if cannot, please state the reasons.

The following papers can be cited and followed for the meta-analytic procedures to improve the quality. (if the data is not enough available, at least DISCUSSION should be added as the LIMITATION of this study with enough citation to support the viewpoints):

Ref 1: Wu Y, et al. Multi-trait analysis for genome-wide association study of five psychiatric disorders. Transl Psychiatry. 2020 Jun 30;10(1):209.

Ref 2: Jiang L, et al. Sex-Specific Association of Circulating Ferritin Level and Risk of Type 2 Diabetes: A Dose-Response Meta-Analysis of Prospective Studies..J Clin Endocrinol Metab. 2019 Oct 1;104(10):4539-4551.

Ref 3: Xu M, Lin Z. Genetic influences of dopamine transport gene on alcohol dependence: a pooled analysis of 13 studies with 2483 cases and 1753 controls.Prog Neuropsychopharmacol Biol Psychiatry. 2011 Jul 1;35(5):1255-60.

Ref 4: Xu M, Sham P, Ye Z, Lindpaintner K, He L. A1166C genetic variation of the angiotensin II type I receptor gene and susceptibility to coronary heart disease: collaborative of 53 studies with 20,435 cases and 23,674 controls.Atherosclerosis. 2010 Nov;213(1):191-9.

Ref 5: Xu M, et al. Quantitative assessment of the effect of angiotensinogen gene polymorphisms on the risk of coronary heart disease. Circulation. 2007 Sep 18;116(12):1356-66

Trans-ethnitic meta-analysis can be referred to Ref 1.

Subgroup analyses based on sex, age, race, gene dosage can be referred to References 2. and Quality assessment score can also to referred to Refences 3-5.

I am wondering if the authors may integrate genotype data with eQTL from GTEX or pQTLs is to explore the causality of the genetic variant in the development of stroke. But I strongly suggest to do causal inference analysis to see if the associated Genetic Polymorphisms in this gene are causally triggering the development of stroke through mediating the expression of this gene in specific tissues. If cannot, please discuss the limitations in the Discussion in detail with additional citations to support the viewpoints. For these reasons, the following papers regarding causal inference between genetic varients,inter-mediator phenotype and disease outcome can be cited and followed.

Reference 1: Fuquan Zhang, Ancha Baranova, Chao Zhou, et al. Causal influences of neuroticism on mental health and cardiovascular disease. Human Genetics. 2021 May 1

Reference 2:Fuquan Zhang, et al. Genetic evidence suggests posttraumatic stress disorder as a subtype of major depressive disorder. Journal of Clinical Investigation. 2021 May 30

Reference 3:Wang, X. et al. Genetic support of a causal relationship between iron status and type 2 diabetes: a Mendelian randomization study. The Journal of clinical endocrinology and metabolism, doi:10.1210/clinem/dgab454 (2021).

I am not sure if the genetic polymorphism can be used for predict stroke based on a machine learning model. In the PRECISION MEDICINE era, deep learning or machine learning is a hot topic in classification and prediction of diseases based on biomarkers. The authors may discuss the possibility to use the genetic variants related to stroke for the prediction or early diagnosis of stroke. The authors may cite the following papers for discussion or follow the analytic procedures to construct machine learning prediction models.

Reference 1:Yu H, et al. LEPR hypomethylation is significantly associated with gastric cancer in males.Exp Mol Pathol. 2020 Oct;116:104493.

Reference 2:Liu M, et al. A multi-model deep convolutional neural network for automatic hippocampus segmentation and classification in Alzheimer's disease.Neuroimage. 2020 Mar;208:11645

I suggest one paragraph in the DISCUSSION section to elucidate the potential biological regulation mechanisms regarding how the genetic variant to affect the stroke outcome. The following papers clearly disclosed some genes indications whose abnormal expressions are mediated through mRNA modifications. The recent progress in N4-Acetylcytidine on RNA expression is also playing key role on the human diseases. I suggest the authors discussing this mRNA modifications/ N4-Acetylcytidine with their findings in the DISCUSSION section because the knowledge needs to be updated.

Reference1: Jin G, et al. The Processing, Gene Regulation, Biological Functions, and Clinical Relevance of N4-Acetylcytidine on RNA: A Systematic Review. Mol Ther Nucleic Acids. 2020. PMID: 32171170

Reviewer #2: 1. I am not sure if the 15 SNPs in ANRIL Gene are tagSNPs or not. Only tagSNPs that are unrelated with each other are informative, and it is better that the tagSNPs may cover this whole gene region.

2. Multiple test correction should be conducted .

3. Statiscial power should be calculaed with proper method to ensure less type 2 errors.

3. subgroup analysis based on sex, age should be conducted.

4. I would suggest collecting published GWAS data to redo the meta-analysis if it is possible.

5. For the genetic varients with significant association with the phenotype, deep discussion about the potential biological mechanisms involved in the phenotype is need. Especially, you had better see if this kind of SNP is causal or not.. Especially the potential causal effects for the SNPs with strong association signals should be explored.

All the figures are not clear and need to be well tailored for publishing.

The language should be polished further.

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Reviewer #1: No

Reviewer #2: No

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Genetic association of ANRIL with susceptibility to Ischemic Stroke: a comprehensive meta-analysis

PONE-D-21-27176R1

Dear Dr. Liu,

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Kind regards,

Mingqing Xu

Academic Editor

PLOS ONE

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