CLUSTER HEADACHE GENOME-WIDE ASSOCIATION STUDY AND META-ANALYSIS IDENTIFIES EIGHT LOCI AND IMPLICATES SMOKING AS CAUSAL RISK FACTOR 141 7 for each gene and tissue. The significance threshold was determined at p <1 x 10-9. Details on data sources and methods are described previously.17, 18 Transcriptome-wide association study analysis (TWAS-FUSION) To identify genes whose expression is significantly associated with CH, the CH meta-analysis results were integrated with gene expression data from single tissues (Table S5) using TWAS-FUSION.19 TWAS expression weights were computed using five linear models (Table S5), followed by crossvalidation to determine the best performing model for a given gene. The imputed gene expression was then used to test for association with CH, taking into account the LD structure and Bonferroni correcting for the number of genes tested for the given tissue. A joint/conditional analysis was performed to test for the significance of GWAS signals after removing TWAS-significant signals (expression weight from TWAS). Each variant association from the CH GWAS meta-analysis was conditioned on the joint model and a p value for conditional analysis results was obtained by permutation testing. Fine-mapping of causal gene sets (FOCUS) FOCUS20 took as input the CH meta-analysis results, the previously calculated TWAS expression prediction weights and LD-information for all SNPs in the risk regions, and estimated the probability for any given set of genes to explain the respective TWAS signal. FOCUS was run for chromosomes 1,2,6,7 and 17, in which TWAS-Fusion showed suggestive association of genes with tissues. Genetically driven DNA methylation scan (MetaMeth) Association between CH and genetically driven DNA methylation (DNAm) was assessed using the MetaMeth function in EstiMeth (v1.1).21 EstiMeth includes 86,710 models reflecting a robust genetically driven signal at methylation of 5'-C-phosphate- G-3' (CpG) sites in whole blood.21 The approach was applied to the CH meta-analysis results, and significance was set at p value < 0.05 after false discovery rate (FDR) correction. Each CpG was paired with its annotated gene(s) and represented in a Miami plot using the R-project (https://www.R-project.org/) ggplot package.22 Protein-altering variants (VEP-Ensembl) At deCODE Genetics (Iceland), for each of the lead CH variants it was determined if it was in high LD (r2 > 0.80, based on the Icelandic genotype data) with protein-altering (coding or splice) variants with moderate or high impact, as annotated using release 100 of the Ensembl Variant Effect Predictor (VEP-Ensembl) tool.23 Gene set and tissue enrichment analyses Genes prioritized by at least one of the five methods were used as input to the GENE2FUNC tool implemented in FUMA24 to examine enrichment in differentially expressed gene (DEG)
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