Transcriptome-wide association study for restless legs syndrome identifies new susceptibility genes

Restless legs syndrome (RLS) is a common neurological condition, with a prevalence of 5–15% in Central Europe and North America. Although genome-wide association studies (GWAS) have identified some common risk regions for RLS, the causal genes have yet to be fully elucidated. We conducted a transcriptome-wide association study involving 15,126 RLS cases and 95,725 controls, from the most recent meta-analysis of GWAS, and gene expression weights of GTEx v7 and the CMC dorsolateral prefrontal cortex tissue panels. We identified 13 associations (in 8 independent loci) at the transcriptome-wide significant level, of which 6 were not implicated in the previous GWAS: SKAP1, SLC36A1, CCDC57, FN3KRP, NCOA6/TRPC4AP. A fine-mapping approach prioritized CMTR1, RP1-153P14.5, PRPF6, and PPP3R1 – to our knowledge, the latter of which is the first RLS-associated gene directly implicated in dopaminergic pathways. Overall, our findings highlight the power of integrating gene expression data with GWAS to prioritize putative causal genes for functional follow-up studies.


5)
The authors should provide more detail on the GWAS summary statistics used in their study. More importantly, the authors should clarify whether these GWAS data the authors analyzed are all based on samples of European ancestry. 6) In Methods, the authors mention that they only used 17 tissue panels from GTEx. They authors should provide justifications on why they choose to analyze these 17 tissues, instead of the entire panel, involving 53 tissues. The authors seem to focus only on brain tissues. If that's the case, the authors should discuss reasons for making this choice. 7) Table 1: It would be useful to include chromosome and base pair position information for each gene. Also, it's not clear whether the TWAS Z-score refers to FUSION or S-PrediXcan results.
8) The authors should give more interpretations to Figure 2. 9) Figure 3 is very hard to parse -most of the red, blue, and green dots overlap, making this figure hard to understand. I recommend only coloring the gene models that pass the significance threshold, and leaving the rest of the dots in gray. Also, the author should consider using different color for odd and even chromosomes.
Minor comments: 1) Line 36: what does it mean to "assign genes"?
2) Line 37: the semicolon after "approaches" should be replaced with a comma.
3) There is a typo on line 116. Table 2, the authors should also report the total number of tests, 67156.

4) In Supplementary
We would like to thank the reviewers for their thoughtful review of our paper. Below we respond point by point to the comments made by the reviewers.

Reviewer #1 (Remarks to the Author):
Akçimen et al. report a TWAS for restless legs syndrome using published datasets and identify new genes. "This study is the first to impute the expression data and search for associations between gene expression and RLS from summary-level GWAS data. In addition to newly identified RLS associated genes, several pathways and associated phenotypes that were not reported in the previous post-GWAS analysis were identified using imputed expression data." 2-Line 23: The authors report "6 were not implicated in the previous GWAS". This is the main result of this study. Please indicate these genes. Are they new identified gene locus or reported locus with different gene prioritization?
Response: As recommended, the new genes have been listed in the revised abstract. We also indicated whether they are in newly identified loci or previously reported loci in results.

"We identified 13 associations at the transcriptome-wide significant level, of which 6
were not implicated in the previous GWAS: SKAP1, SLC36A1, CCDC57/FN3KRP, NCOA6/TRPC4AP." "Among the total 13 significant associations, 6 of them were not reported in the previous GWAS. These are SKAP1, that was identified in a previously known RLS locus of HOXB cluster, and the splicing events that were detected in the SLC36A1, CCDC57/FN3KRP, NCOA6/TRPC4AP genes."

Minor comments:
1-Line 19: A prevalence of 18% is very high, is it an extreme value? Please report the general prevalence.

Response: Epidemiological studies have shown that 5-15% of European and North
American populations suffer from RLS; in particular, an increased prevalence of up to 18% was reported in the Québec population. As suggested, we reported the general prevalence as follow: "Restless legs syndrome (RLS) is a common neurological condition, with a prevalence of 5-15% in Central Europe and North America." 2-Line 21: Please provide the transcriptional data in the abstract and make it clear previous published GWAS and expression datasets were used in this study.

Response:
We made it clear that we used the data from previous meta-analysis of GWAS and stated the gene expression datasets in the abstract. These were previously indicated in the methods, as well.
"We conducted a transcriptome-wide association study (TWAS) involving 15,126 RLS cases and 95,725 controls,which were obtained from the most recent meta-analysis of GWAS for RLS, and gene expression weights of GTEx 53 v7 and the CMC dorsolateral prefrontal cortex tissue panels." 3-Line 51-53: duplicated sentences.

Response:
We removed the second sentence.

Reviewer #2 (Remarks to the Author):
In this manuscript, the authors describe a transcriptome-wide association study (TWAS) of restless legs syndrome (RLS). They identified 13 gene-trait associations, performed TWAS finemapping to pinpoint the possible causal genes, and performed gene set enrichment analysis.
Overall, this manuscript could benefit from much more detailed description of their analysis and results.

Major comments:
1-In the abstract, the authors mention that they identified 13 associations at TWAS significant level. It's important to clarify whether these 13 associations are independent signals coming from different LD regions, or correlated signals coming from the same LD regions -in most cases, it's preferable to report the number of independent signals / regions.

Response:
We have clarified it in abstract. There are 13 associations in 7 different loci.
Additionally, the genes in the same loci were reported as CCDC57/FN3KRP, NCOA6/TRPC4AP. "We identified 13 associations (in 7 independent loci) at the transcriptome-wide significant level, of which 6 were not implicated in the previous GWAS: SKAP1, SLC36A1, CCDC57/FN3KRP, NCOA6/TRPC4AP." 2-Additionally, reporting the number of genes might be more informative than reporting the number of associations. How many of the 6 novel associations represent unique genes not discovered in previous GWAS?