Predicting the causal relationship between polyunsaturated fatty acids and cerebral aneurysm risk from a Mendelian randomization study

Reviewer #1 (Public Review):

Summary:

The authors performed two-sample MR combined with sensitivity analyses and colocalization to test the effect of PUFA on cerebral aneurysms. They found that genetically predicted omega-3 and DHA decreased the risk for intracranial aneurysm (IA) and subarachnoid haemorrhage (SAH) but not for unruptured IA (uIA).

Strengths:

PUFA on the risk of cerebral aneurysms is of clinical importance; the authors performed multiple sensitivity analyses to ensure MR fulfils its assumptions.

Reviewer #2 (Public Review):

Summary:

In the manuscript, Yu et al reported a two-sample Mendelian randomization study to evaluate the causation between polyunsaturated fatty acids (PUFA) and cerebral aneurysm, based on summary statistics from published genome-wide association studies. The authors identified that omega-3 fatty acids and Docosahexaenoic acid decreased the risk for intracranial aneurysm (IA) and aneurysmal subarachnoid haemorrhage (aSAH). COLOC analysis suggested that the acids and IA, aSAH likely share causal variants in gene fatty acid desaturase 2.

Strengths:

The methodology is sound, with appropriate sensitivity analysis.

Weaknesses:

The results did not provide significant novel findings.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

(1) In my opinion, the major weakness is the selection of IVs, the same IVs should be used for each exposure, especially when the outcomes (IA, SAH, and uIA) are closely related. The removal of IVs was inconsistent, for example, why was LPA rs10455872 removed for SAH but not for uIA? (significantly more IVs were used for uIA). The authors should provide more details for the justification of the removal of IVs other than only indicating "confounder" in supplementary tables. The authors should also perform additional analyses including all IVs and IVs from other PUFA GWAS.

We apologized for our negligence. We reconducted a two-sample MR analysis following the removal of rs10455872 from the uIA, which yielded unaltered ORs and 95% confidence intervals. The P-value was once again found to be statistically insignificant. These results demonstrate the robustness of our MR analyses and indicate that this SNP does not exert an influence on the overall results. (see Figure 4)

For SNP selection, we adhered rigorously to the established Mendelian randomization analysis process for the screening of instrumental variables. "Confounder" is mean that a current explicit influencer that is explicitly associated with the outcome variable. Following the removal of such confounding SNPs, the analysis of heterogeneity and pleiotropy is repeated on several occasions in MR analysis using radical MR, MRPRESSO, IVW-radical and Egger-radical, with each iteration involving the removal of the corresponding anomalous SNPs until all instances of pleiotropy and heterogeneity have been eliminated, it can be observed that the final single-nucleotide polymorphism (SNP) for each group is not identical. Therefore, It can be observed that the final SNPs for each group is not identical.

(2) In addition, it seems that the SNPs in the FADS locus were driving the MR association, while FADS is a very pleiotropic locus associated with many lipid traits, removing FADS could attenuate the MR effect. The authors should perform a sensitivity analysis to remove this locus.

Thanks for the reviewer’s suggestion. In our revised manuscript, We reconducted MR analysis of the positive results after the removal of the FADS2 and its SNPs within 500 kb of the FADS2 locus. This analysis demonstrated that there was no significant causal pathogenic association between PUFA and IA, aSAH. This result validated that SNP: rs174564 was a significant factor driving the causal association between PUFAs and CA. (See page 6, line155-157 and Figure 8)

(3) Instead of removing multiple "confounder" IVs which I think may bias the MR results due to very closely related lipid traits, the authors should perform multivariable MR to identify independent effects of PUFAs to IA, conditioning on other PUFAs and/or other lipids.

Thanks for the reviewer’s suggestion. In our revised manuscript, we employed MVMR through adjust for HDL cholesterol, LDL cholesterol, total cholesterol and triglycerides, to remove bias from closely related lipid traits. The application of MVMR analysis serves to reinforce the robustness of our conclusions. (See page 6, line151-153 and Figure5-7)

(4) Colocalization was not well described, the authors should include the colocalization results for each locus in a supplementary table. They also mentioned "a large PP for H4 (PP.H4 above 0.75) strongly supports shared causal variants affecting both gene expression and phenotype". The authors should make sure that the colocalization was performed using the expression data of each gene or using the GWAS summary of each PUFA locus.

I apologize for our negligence. We have added the detailed results of the COLOC for each locus in the supplementary table. (See supplementary table 6)

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) I suggest the authors consult Borges et al., 2022 (doi: 10.1186/s12916-022-02399-w) for PUFA IV selection, and perform sensitivity analysis based on Borges et al., 2022 IVs and another PUFA GWAS (such as J Kettunen et al., 2016, doi: 10.1038/ncomms11122).

Thanks for the reviewer’s suggestion. In order to provide further evidence of the robustness of the results of our analyses, we conducted MVMR and a sensitivity analysis after excluding SNPs within 500 kb of the FADS2 locus, as recommended by Borges et al. (2022). (See page 6, line151-157 and Figure 5-8)

In regard to the article by J. Kettunen et al. (2016), we found that the validation dataset from which the article was sourced was insufficient in terms of sample size and lacked the requisite statistical efficacy to be used for validation purposes.

(2) The authors justified that colocalization is to determine if "PUFAs are mediators in the hereditary causative route of cerebral aneurysm", which I don't think is the case.

Colocalization is to determine whether an MR estimate is not confounded by LD.

I apologize for our incorrect description. We have made careful modification in our revised manuscript, as follows: “There is consistent evidence that PUFAs have a beneficial causal effect on cerebral aneurysm. In order to determine an MR estimate is not confounded by LD, we used COLOC to identify shared causal SNP between PUFAs and cerebral aneurysms”. (See page 7-8, line 215-217)

(3) Supplementary tables 2-4 were a bit confusing to me, I suggest the authors provide one supplementary table for each exposure.

Thanks for the reviewer’s suggestion. Supplementary tables 2_1-2_5 shows the exposure data for the five PUFAs associated with IA, supplementary tables 3_1-3_5 shows the exposure data for the five PUFAs associated with aSAH and supplementary tables 4_1-4_5 shows the exposure data for the five PUFAs associated with UIA. Each exposure is represented by a distinct table.

(4) Figure 1 legend: I can't find multivariable MR in the figure/method.

I apologize for our negligence. In our revised manuscript, we have added the MVMR methodology. We also have modified Figure 1 and Figure 1 legend. (See Figure 1, Figure 1 legend and page 6, line 151-153)

(5) LOO analysis was mentioned in methods and results but I could not find the results for LOO.

I apologize for our negligence. In our revised manuscript, we have described the results of the LOO, as follows: “The leave-one-out plot demonstrates that there is a potentially influential SNP (rs174564) driving the causal link between PUFA and cerebral aneurysm.” (See page 7, line 209-210)

(6) Finally, the authors should proofread their manuscript as many sentences are difficult to read, such as:

Line 183: "...MR methods revealed consistency", "However, there was no any causal relationship..."

Line 200: "For achieve that..."

I apologize for our incorrect description. We have modified these descriptions in our revised manuscript, as follows: “The results demonstrated consistency in the outcomes and directionality of the various MR methods employed” and “In order to determine an MR estimate is not confounded by LD, we used COLOC to identify shared causal SNP between PUFAs and cerebral aneurysms”. (See page 7, line 187-188 and line 215-217).

Reviewer #2 (Recommendations For The Authors):

(1) Are there any previous epidemiological studies on the association between PUFA and cerebral aneurysm? It will be helpful to introduce this background.

Thanks for the reviewer’s suggestion. The epidemiology of PUFA with aneurysm in other sites, such as the abdominal aorta, are described in the Introduction section. Although there is a paucity of large-scale multicenter clinical epidemiological studies examining the relationship between PUFAs and cerebral aneurysms, we are endeavoring to infer a prior association between PUFAs and cerebral aneurysms with the aid of Mendelian randomization analysis.

(2) The authors performed a leave-one-out analysis but did not explain much about the results. The leave-one-out analysis seems to provide some evidence that some SNP is driving the results, like rs174564 in Supplementary Figure 5-1.

I apologize for our negligence. In our revised manuscript, we have described the results of the leave-one-out analysis, as follows: “The leave-one-out plot demonstrates that there is a potentially influential SNP (rs174564) driving the causal link between PUFA and cerebral aneurysm”. (See page 7, line209-214)”.

(3) In the discussion (line 211), the authors mentioned omega-6 fatty acids increased the risk of IA and aSAH, omega-3 fatty acids decreased the risk for IA and aSAH, but omega-6 by omega-3 decreased the risk of IA and aSAH. This seems to be different from the figures.

I apologize for our incorrect description. We have modified this description in our revised manuscript, as follows: “We demonstrated that the omega-3 fatty acids, DHA and, omega-3-pct causally decreased the risk for IA and aSAH. And omega-6 by omega-3 causally increased the risk of IA and aSAH”. (See page 8, line228-230)

Minor:

(4) Some grammar errors need to be checked, such as:

In line 200, "For achieve that, we tested for shared causative SNPs between PUFAs and cerebral aneurysm using COLOC".

In line 123, "Fourth, to eliminate unclear, palindromic and associated with known confounding factors (body mass index (McDowell et 125 al., 2018), blood pressure (Sun et al., 2022), type 2 diabetes (Tian et al., 2022), high-density lipoprotein (Huang et al., 2018)) SNPs."

I apologize for our incorrect description. We have modified these descriptions in our revised manuscript, as follows: “Fourth, remove SNPs that are obscure, palindromic, and linked to recognized confounding variables (body mass index (McDowell et al., 2018), blood pressure (Sun et al., 2022), type 2 diabetes (Tian et al., 2022), high-density lipoprotein (Huang et al., 2018))” and “In order to determine an MR estimate is not confounded by LD, we used COLOC to identify shared causal SNP between PUFAs and cerebral aneurysms”. (See page 5, line 124-127 and page 7 line215-217)