6 CHAPTER 6 130 With respect to the other replicated loci, rs11579212 and rs10184573, which mapped to RP11815M8.1 and AC093590.1 respectively, they have not previously been related to disease, and their role in CH pathogenesis remains unclear. Although rs6541998 did not replicate in the small replication sample, two genes (POLR1B, TMEM87B) in the locus showed differential expression in CH compared to controls in RNA-seq analyses, whereas no such effect was seen in migraine data. POLR1B, encoding DNA-directed RNA polymerase I subunit RPA2, has been associated with Treacher Collins and TMEM87B, encoding transmembrane protein 87B may be involved in restrictive cardiomyopathy.43, 44 MERTK, the nearest gene, encodes a receptor tyrosine kinase of the TAM (Tyro3, Axl, MERTK) family, is among other tissues expressed in oligodendrocytes, astrocytes and microglia in the brain and has an effect on the immune response.45 Unfortunately, the number of associated genes with CH is not large enough to perform meaningful further downstream pathway analyses. Based on the regression coefficients, we found no evidence for a different effect for the lead SNPs between men and women. In conclusion, this GWAS of CH reveals four genetic risk loci for CH with unusually high effect sizes for a complex disorder, of which three replicated in an independent sample. One of the loci has previously been identified as a migraine risk locus. Our results suggest several genes to be involved in the pathogenesis of CH and offer a starting point for future research to elucidate the molecular mechanisms of this severe disease. Post-script paragraph Two parallel manuscripts (Harder et al. and O’Connor et al.), submitted to the journal, report the first replicated genomic loci associated with CH. Whereas Harder et al. investigated Dutch CH cases (n = 840) and controls (n = 1,457) and Norwegian CH cases (n = 144) and controls (n= 1,800), O’Connor et al. investigated UK cases (n = 852) and controls (n = 5,614) as well as Swedish cases (n = 591) and controls n = 1,134). The four loci reported by Harder et al. correspond to four loci reported by O’Connor et al., with the index variants reported in the two studies being in linkage disequilibrium with each other (D’ = 0.86 and r2 = 0.36 for rs11579212 and rs12121134; D’ = 0.98 and r2 = 0.95 for rs6541998 and rs4519530; D’ = 0.95 and r2 = 0.34 for rs10184573 and rs113658130; and D’ = 0.93 and r2 = 0.38 for rs2499799 and rs11153082, in the 1000 Genomes data for European populations). The independent discovery of the four loci in the two studies provides additional support that they represent genuine risk loci for cluster headache. Next, we combined the summary statistics from the four studies (Dutch, Norwegian, UK, Swedish) using inverse-variance weighted meta-analysis as implemented in METAL (with the ‘STDERR’ option), after harmonizing the datasets using EasyQC.46, 47 In total, 8,039,373 variants were analyzed. The association to CH remained significant for all eight index variants (in the four loci) reported in the two papers: rs11579212 (effect allele, EA: C), OR 1.31 (95% CI 1.21-1.41),
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