6 CHAPTER 6 120 Replication Analysis Association analysis was performed using a mixed logistic regression model implemented in SAIGE (v0.35.8.3), where CH was modeled as the dependent variable, and the genetic variants as the independent variable. Sex and the first eight principal components were included as covariates. From each independent significant locus (p < 5 x 10-8) in the discovery sample, the lead SNP, or a proxy SNP, was selected for replication. To correct for multiple testing, Bonferroni correction was applied for the number of loci tested (n = 4). Sex stratified analysis Analyses stratified for men and women were performed in SNPTEST to examine possible sexspecific genetic effects. Both models were adjusted for the first four principal components. Previously reported cluster headache loci The 9 different SNPs which have previously been reported for a significant association with CH were tested for association in our discovery analysis, see details in Table 4.8-10, 13, 27-31 P-values were adjusted for multiple-testing using Bonferroni correction. Univariate LD-score regression Linkage Disequilibrium Score Regression (LDSC v1.0.1) was used to estimate the proportion of a true polygenic signal versus confounding factors such as population stratification, and to calculate SNP-based heritability.32 Variants present in the HapMap 3 reference set were used, after excluding variants (1) with large-effect, explaining > 1% of phenotype variation, or variants in LD with such; (2) with MAF ≤ 0.01 or imputation INFO score ≤ 0.9; and (3) in the HLA region. Heritability estimates were converted to the liability scale assuming a population prevalence of CH of 0.1%.6 Colocalization analysis To test whether the association signals for CH and migraine, on chromosome 6 near UFL1/ FHL5, are consistent with a shared causal variant, we used a Bayesian colocalization procedure using the R package ‘coloc’ with default settings.33 This test generates posterior probabilities for each locus weighting the evidence for five competing hypotheses regarding the sharing of causal variants, namely H0 (no causal variant for either trait); H1 or H2 (a causal variant only for trait one or two); H3 (distinct causal variants, for each trait); and H4 (a single causal variant common to both traits). The analysis assumes a single causal SNP for each trait. For CH we used the summary statistics from the discovery cohort and for migraine we used the summary statistics from Gormley et al.34 without 23andMe (30,465 migraine cases and 143,147 controls); both populations are of European ancestry. Colocalization was tested for the region between the two nearest recombination hotspots.
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