Causal links of α-thalassemia indices and cardiometabolic traits and diabetes: MR study

Causal MR study links α-thalassemia-related erythrocyte traits to cardiometabolic risks and diabetes susceptibility in Taiwan.

spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): This manuscript studied 16p13.3region, covering a known deletion for alpha-thalassemia (monogenic disorder), for its genetic association with blood-related traits (RBC count, MCV, MCH, HB) and cardiometabolic traits in Taiwanese population.They found three lead SNPs (single nucleotide polymorphism) associated with blood-related traits and those SNPs are highly correlated with -SEA deletion, suggesting being used as a surrogate marker.They further conducted a stepwise linear regression with 3 SNPs on MCV and MCH.Additionally, they performed the association of genetic variants on 16p13.3 with cardiometabolic traits using the imputed genetic data, followed by a Mendelian randomization.I found several concerns.I have three major comments.1. Regarding the stepwise linear regression, given that -SEA is the causal genetic factor and 3 SNPs and -SEA are all in high LD, the stepwise linear regression to identify independent variants might be meaningness.2. Line 275.Why only PGAP6 was used/included in GWAS imputed data analysis?Is it only available in the GWAS imputed data?Even though, two other variants do not completely agree/match with WGS data, if their imputation quality is good, we want to see the results of them.Also, I don't think you applied the same exclusion criteria on other variants (other than those 3 variants) in imputed data.Also, it looks like PGAP6 has the lowest priority from the previous sections.3. I might have missed but it is unclear why MCV and MCH were selected to be examined more with cardiometabolic traits.It is not clear what's the goal of MR analysis in this manuscript.Is it for the causal relationship between RBC parameters and cardiometabolic traits?If so, you need to use all genetic variants associated with each trait and bidirectional MR can be performed.If you are interested in examining alpha-thalassemia triggered cascade of RBC parameters and cardiometabolic traits, then, a mediation analysis (rs375498877 -> RBC -> cardiometabolic trait; rs375498877 -> cardiometabolic trait -> RBC) looks more appropriate.
In Abstract, it will be good to mention -SEA mutation (in the first sentence), which can easily justify to perform regional association at 16p13.3.Table 3.Why only conducted only for MCV and MCH?As the homozygous form minor-alleles did not present in this data, remove those in the label (e.g., TT vs TC vs CC -> TT vs TC).Pg 8. Line 134 -what do you mean by specific to Asians?It looks like it means that the alternative allele is very rare in other Asian.Please clarify in the sentence and show those allele frequencies and -SEA mutation rate in this study and in other race.How did you define 3 SNPs as lead snps?Pg 16.Line 261: is this sensitivity for -SEA deletion detection by NPRL3 rs191086839 risk allele carrier?Please make it clear.
In Table 5, G_A -T_B and IV_A -T_B have the same p-value when using the same covariates, as expected.This probably indicates that MR is not a good choice for this.
In Abstract, it will be good to mention -SEA mutation (in the first sentence), which can easily justify to perform regional association at 16p13.3.
Response: We have mentioned it in the revised Abstract section (in the first sentence).Alpha-thalassemia, a microcytic anemia caused by reduced α-globin synthesis, is common in Southeast Asian and Chinese populations due to the (--SEA ) mutation.Response: We have added the rationale in the revised Result section: MCV and MCH were thus selected for in-depth examination with cardiometabolic traits based on their most significant association with 16p13.3 and their impact on erythrocyte count and Hb level.
Table 3.Why only conducted only for MCV and MCH?As the homozygous form minor-alleles did not present in this data, remove those in the label (e.g., TT vs TC vs CC -> TT vs TC).
Response: MCV and MCH were selected for in-depth examination with cardiometabolic traits based on their most significant association with 16p13.3 and their impact on erythrocyte count and Hb level.Additionally, we have taken away the labels for the homozygous minor-alleles genotype.Response: As already shown in supplemental Table 1, the minor allele frequencies of these 3 SNVs among European, American, and African populations are all <0.0001.Lead SNVs are defined and selected as the SNVs with the lowest p-value for the targeted region exhibiting genome-wide significant associations.We have clarified these two issues in the revised version as follows: Our findings revealed that three lead SNVs (with the smallest, i.e., most significant, p-values in this region), namely NPRL3 rs191086839, LUC7L rs372755452, and PGAP6 rs375498857, were specific to Asians (with minor allele frequencies ranging from 0.0177 to 0.0218 among TWB and east Asian populations vs. all <0.0001 among other ethnic populations).
Pg 16.Line 261: is this sensitivity for -SEA deletion detection by NPRL3 rs191086839 risk allele carrier?Please make it clear.
In Table 5, G_A -T_B and IV_A -T_B have the same p-value when using the same covariates, as expected.This probably indicates that MR is not a good choice for this.
Response: Thank you for this important comment.The association between the genetic variant of the exposure variable (G_A) and the outcome variable (T_B) reflects the observational or epidemiological association between the exposure and the outcome.In MR, this association is used as evidence to inform causal inference about the effect of the exposure on the outcome.On the other hand, the association between the instrumental variable (IV_A) and the outcome variable (T_B) reflects the total causal effect of the exposure on the outcome.If the association between the genetic variant of the exposure variable (G_A) and the outcome variable (T_B) has the same p-value as the association between the instrumental variable (IV_A) and the outcome (T_B), it implies that the genetic variant is a valid instrument for the exposure variable in MR analysis.Specifically, it suggests that the genetic variant is strongly associated with the exposure of interest and, in turn, influences the outcome through the exposure.Thank you for submitting your revised manuscript entitled "Causal links of α-thalassemia indices and cardiometabolic traits and diabetes: MR study".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
Along with points mentioned below, please tend to the following: -please address the Reviewer's remaining comments -please exclude figures from the manuscript text and upload them separately -please upload all figure files as individual ones, including the supplementary figure files; all figure legends should only appear in the main manuscript file -please add the Twitter handle of your host institute/organization as well as your own or/and one of the authors in our system -please add your main, supplementary figure, and table legends to the main manuscript text after the references section If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
LSA now encourages authors to provide a 30-60 second video where the study is briefly explained.We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608).Corresponding or first-authors are welcome to submit the video.Please submit only one video per manuscript.The video can be emailed to contact@life-science-alliance.orgTo upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plexYou will be guided to complete the submission of your revised manuscript and to fill in all necessary information.Please get in touch in case you do not know or remember your login name.
To avoid unnecessary delays in the acceptance and publication of your paper, please read the following information carefully.

A. FINAL FILES:
These items are required for acceptance.
--An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max.200 characters including spaces).This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title.It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person.Author names should not be mentioned.

B. MANUSCRIPT ORGANIZATION AND FORMATTING:
Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.
**Submission of a paper that does not conform to Life Science Alliance guidelines will delay the acceptance of your manuscript.****It is Life Science Alliance policy that if requested, original data images must be made available to the editors.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original data images prior to final submission.****The license to publish form must be signed before your manuscript can be sent to production.A link to the electronic license to publish form will be sent to the corresponding author only.Please take a moment to check your funder requirements.****Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript.If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.**Thank you for your attention to these final processing requirements.Please revise and format the manuscript and upload materials within 7 days.
Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.
Sincerely, Novella Guidi, PhD Scientific Editor Life Science Alliance ------------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): The manuscript has been improved, but clarify of manuscript can be still improved as the manuscript covers multiple topics using different types of data (surrogate marker, lead snps with RBC traits and cardiometabolic traits, MR).Especially, the abstract and introduction of study objective (at the end of introduction) can be polished as it was not easy to follow the overall flow of the study and which data was utilized for each step.Some minor editing comments: Line 118.Rather than calling them lead SNPs, it is more accurate to call them significant SNPs (p Line 123."genome-wide significant association" : is this "genome-wide" or on "16p13.3"? Figure 2 legend.(line 134) "P-value adjusted for xxx" -> p-value was obtained from a linear regression of each RBC parameter with a genetic variant, adjusted for age, ... .In the figure, r2 coloring does not match with r2 in figure 3. Also, in line 128-129, is this r2 from the data of this study or from other reference database?Line 211.This sentence reads like that there are 1474 participants with -SEA deletion.
Line 407-408.Isn't the surrogate marker -SEA be based on sensitivity, rather than the association with MCV/MCH? 1.The manuscript has been improved, but clarify of manuscript can be still improved as the manuscript covers multiple topics using different types of data (surrogate marker, lead snps with RBC traits and cardiometabolic traits, MR).Especially, the abstract and introduction of study objective (at the end of introduction) can be polished as it was not easy to follow the overall flow of the study and which data was utilized for each step.
Response: We would like to express our sincere gratitude for the incredibly detailed review of our manuscript.Your thoughtful and meticulous examination of our work has provided invaluable insights and suggestions for improvement, which we truly appreciate.
The abstract has been rewritten to enhance the clarity of our study's extensive content and to make it easier for readers to follow the overall flow of the study and understand which data were utilized at each step, as follows: Our study aimed to investigate if genetic variants around 16p13.3's HBA1 locus, associated with erythrocyte indices and HbA1c levels, predict α-thalassemia-related erythrocyte indices, cardiometabolic traits, and diabetes risk in Taiwanese individuals.We analyzed Taiwan Biobank data, including whole-genome sequencing (WGS) from 1493 participants and genotyping arrays from 129,542 individuals.First, we performed regional association analysis using WGS data to identify genetic variants significantly associated with erythrocyte indices, confirming their linkage disequilibrium with the α 0 thalassemia --SEA deletion mutation, a common cause of α-thalassemia in Southeast Asian populations.Deletion mutation sequencing further validated these variants' association with α-thalassemia.Subsequently, we analyzed genotyping array data, revealing associations between specific genetic variants and cardiometabolic traits, including lipid profiles, HbA1c levels, bilirubin levels, and diabetes risk.
Utilizing Mendelian randomization, we established causal relationships between α-thalassemia-related erythrocyte indices and cardiometabolic traits, elucidating their role in diabetes susceptibility.Our findings highlight genetic variants around the α-globin genes as surrogate markers for common α-thalassemia mutations in Taiwan, emphasizing the causal links between α-thalassemia-related erythrocyte indices, cardiometabolic traits, and heightened diabetes risk.
The description of the introduction of study objective has also been adjusted as follow: Our study was primarily designed to uncover and elucidate the causal relationships between α-thalassemia-related erythrocyte indices, cardiometabolic it only allows the selection of a general Asian population without specifying Chinese ethnicity.This is why you observed discrepancies in the coloring.
6. Line 211.This sentence reads like that there are 1474 participants with -SEA deletion.
Response: Thanks, we have clarified this sentence as follow: we conducted tests to detect the presence of this deletion in 1474 participants who had WGS data and genomic DNA available for analysis.
7. Line 407-408.Isn't the surrogate marker -SEA be based on sensitivity, rather than the association with MCV/MCH?
Response: Thank you for this important comment.As per the reviewer's observation, the utilization of the SNP as a surrogate marker for --SEA is based on its co-inheritance with the disease gene (--SEA ).This co-inheritance is supported by strong evidence from both the LD analysis and sensitivity/specificity tests.We acknowledge that our initial description may have been unclear, leading to confusion.Our intention was to convey that among the three SNPs strongly linked with the --SEA mutation and potentially usable as its surrogate marker, NPRL3 rs191086839 emerged as the strongest and independent candidate genetic predictor of MCV and MCH following a multivariate stepwise regression analysis.Furthermore, our analysis finds support in previous functional annotation studies of this chromosomal region, which encompasses the α-globin locus, as mentioned in the Discussion.To address the reviewer's concern, we have adjusted our description as follow: "Therefore, considering the robust evidence of co-inheritance, as reflected in the linkage disequilibrium analysis and the context of surrogate markers traditionally being associated with sensitivity and specificity, we propose NPRL3 rs191086839 as a strong and independent candidate genetic surrogate marker for the α 0 -thalassemia deletion mutation --SEA in Taiwanese individuals."The final published version of your manuscript will be deposited by us to PubMed Central upon online publication.Your manuscript will now progress through copyediting and proofing.It is journal policy that authors provide original data upon request.
Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript.If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.***IMPORTANT: If you will be unreachable at any time, please provide us with the email address of an alternate author.Failure to respond to routine queries may lead to unavoidable delays in publication.***Scheduling details will be available from our production department.You will receive proofs shortly before the publication date.Only essential corrections can be made at the proof stage so if there are any minor final changes you wish to make to the manuscript, please let the journal office know now.

DISTRIBUTION OF MATERIALS:
Authors are required to distribute freely any materials used in experiments published in Life Science Alliance.Authors are encouraged to deposit materials used in their studies to the appropriate repositories for distribution to researchers.
You can contact the journal office with any questions, contact@life-science-alliance.orgAgain, congratulations on a very nice paper.I hope you found the review process to be constructive and are pleased with how the manuscript was handled editorially.We look forward to future exciting submissions from your lab.Sincerely, Novella Guidi, PhD Scientific Editor Life Science Alliance

Fig 3 .
Fig 3. What's the rationale or any biological mechanism to draw the directed diagram between alpha-thalassemia, MCV, MCH and other traits (total cholesterol, etc)?

Fig 3 .
Fig 3. What's the rationale or any biological mechanism to draw the directed diagram between alpha-thalassemia, MCV, MCH and other traits (total cholesterol, etc)?

Pg 8 .
Line 134 -what do you mean by specific to Asians?It looks like it means that the alternative allele is very rare in other Asian.Please clarify in the sentence and show those allele frequencies and -SEA mutation rate in this study and in other race.How did you define 3 SNPs as lead snps?
Science Alliance Manuscript #LSA-2023-02204-TRR Dr. Yu-Lin Ko Taipei Tzu Chi Hospital The Division of Cardiology, department of Internal Medicine, Cardiovascular Medical Center 289 Jianguo Road, Xindian City New Taipei, Taiwan Dear Dr. Ko, Thank you for submitting your Research Article entitled "Causal links of α-thalassemia indices and cardiometabolic traits and diabetes: MR study".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.

Table 5 .
The main findings were consistent with the results obtained from PGAP6 rs375498857.
Adjustment for age, sex, current smoking status, BMI, and other possible confounders, such as systolic BP, total cholesterol, HbA1c, UA, eGFR and AST.3.I might have missed but it is unclear why MCV and MCH were selected to be examined more with cardiometabolic traits.It is not clear what's the goal of MR analysis in this manuscript.Is it for the causal relationship between RBC parameters and cardiometabolic traits?If so, you need to use all genetic variants associated with each trait and bidirectional MR can be performed.