CHAPTER 1 16 1 The HCRTR2 gene encodes the G-protein coupled receptor hypocretin type 2 receptor that binds neuropeptides hypocretin-1 and -2 in the central nervous system. Such causal role of hypocretins makes sense as they have been implicated in sleep and arousal as well as pain modulation,102 and levels were reported to be lower in CSF of patients with cluster headache.103 However, initially positive genetic findings for HCRTR2 associations104-106 were not replicated in betterpowered studies.101, 107 Genes involved in circadian rhythmicity have also been investigated, but no association could be found.108 Genome-wide association studies As a result of the improvement in DNA technology and the advancement of cost-effective genotyping platforms GWAS has become the method of choice to identify gene variants in complex traits in an untargeted approach in the last decade. Typically, in GWAS, several millions of single nucleotide polymorphisms (SNPs) are tested for association with a disorder by assessing differences in allele frequencies between large numbers of patients and controls. Of note, only common variants with a low to high minor allele frequency (≥0.01) are interrogated. Since 2010, the International Headache Genetics Consortium (IHGC; www.headachegenetics. org/) has conducted several migraine GWAS, and with the increasing sample sizes, the number of associated gene variants steadily expanded. For cluster headache the first GWAS was performed in a very small, Italian study investigating patients with cluster headache.109 They found a suggestive association with genetic variants in ADCYAP1R1 and MME,109 but the findings were not be replicated in a larger Swedish sample.110 The hope is that larger GWAS will yield variants robustly associated with cluster headache. Next-generation sequencing A large part of the genetic variance and heritability in common diseases cannot be explained (usually referred to as “missing heritability”) with a GWAS approach alone. One reason is that rarer variants (MAF<0.01), potentially with higher effect sizes, are not well interrogated by genotyping arrays typically utilised in the GWAS approach. Such mediate-effect-size variants can be identified using a next-generation sequencing (NGS) approach, i.e. by the simultaneous large-scale sequencing of the coding exons (whole-exome sequencing; WES) or the entire genome (whole-genome sequencing; WGS). In addition, the simultaneous sequencing of RNA transcripts (“transcriptome”; RNA-seq), either of bulk tissue or of its single nuclei can shed light on molecular mechanisms. Only a few NGS studies have been performed in migraine thus far. Until now, WES was typically applied to cohorts of patients with HM, testing several hundred cases in an attempt to either find causal mutations in known HM genes or novel HM genes in patients that are negative for mutations in CACNA1A, ATP1A2, and SCN1A. Until now results have not led to additional
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