GENETIC SUSCEPTIBILITY LOCI IN GENOME-WIDE ASSOCIATION STUDY OF CLUSTER HEADACHE 119 6 additive allelic effects. The model was adjusted for sex. In addition, the model was adjusted for the first four principal components to minimize effects of confounding and population stratification. A Manhattan and a quantile-quantile (QQ) plot for test statistics were generated using R v3.6.1 (R Foundation for Statistical Computing, Vienna, Austria). We determined lead SNPs that were independent from each other at r2 < 0.1 and further apart than 500 kb, with association p < 5 × 10−8. Positional gene mapping and fine mapping of significant loci was performed using Functional Mapping and Annotation v1.3.6 (FUMA), Probabilistic Identification of Causal SNPs (PICS), and Locuszoom.21-23 The proportion of variance explained by a given SNP was calculated using Nagelkerke pseudo R2. Patient recruitment and data generation in the replication stage Cases were recruited at the Norwegian Advisory Unit on Headaches,St Olav’s Hospital,Trondheim (Norway) between 2005 and 2016, with the inclusion criterion being the definite diagnosis of CH according to ICHD-2 or ICHD-3,24 made by a neurologist with special competence in headache disorders to minimize misclassification. As controls we used a random subset of 1,800 adult participants from the Nord-Trøndelag Health Study (HUNT) who did not have CH defined by the ICD-10 diagnosis G44.0 (Cluster headache syndrome) or the ICD-9 diagnosis 346.2 (Migraine variants, including cluster headache). 25 A sample of 159 CH cases were genotyped with the Illumina Infinium CoreExome-24 v1.1. Calling was performed with Genome Studio 2.0, using the cluster file from the largest batch of 58,996 HUNT All-in controls (see below).The analysis followed the Genome Studio quality protocol,17 and the CHARGE best practice calling of the HumanExome Bead chip.18 The HUNT control samples were genotyped on three different Illumina HumanCoreExome arrays (HumanCoreExome12 v1.0, HumanCoreExome12 v1.1 and UM HUNT Biobank v1.0), and called as described elsewhere.26 Markers with high missingness rates (≥ 2%), monomorphic variants and those failing the HardyWeinberg equilibrium were excluded. Individuals with high missingness rates (≥ 2%) or whose inferred sex contradicted with reported sex were excluded. A second round of quality control was performed after merging cases and all HUNT controls, excluding variants that were monomorphic, deviated from Hardy-Weinberg equilibrium or had different genotype rate between cases and controls. Individuals were excluded if they had missingness ≥ 2%, outlying heterozygosity rate or were duplicates. Population outliers and non-European samples were excluded. No overt population substructure between cases and controls was observed (data not shown). A total of 69,440 individuals passed quality control, including 144 cases. A dataset including the 144 cases and 1,800 randomly selected controls was imputed using Minimac3 (v2.0.1) and the Hapmap r22 CEU panel. Variants with minor allele count < 3 or with imputation quality r2 < 0.3 were excluded, resulting in 2,363,678 well-imputed variants for 144 cases (38 women and 106 men) and 1,800 controls (952 women and 848 men). The study was approved by the local ethics committees. Written informed consent was obtained from all participants.
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