With a lifetime prevalence of 16%, migraine affects 75 million Europeans. It can be very disabling for the individual and is a large economic burden to society [
1]. Unraveling the genetics of migraine is therefore highly relevant. Migraine is a complex disorder caused by several genes and environmental factors [
2,
3]. A higher concordance of migraine in monozygotic than in dizygotic twins, and the 1.9-3.8 fold higher risk of migraine among first degree relatives of affected individuals, indicates an important genetic component [
2‐
5] Twin studies show a heritability of 34%–65% [
5,
6]. Heritability of migraine with typical aura (MA) that affects approximately one third of migraineurs is higher than for migraine without aura (MO), and evidence supports that MA and MO have different, though somewhat overlapping, etiology [
7,
8]. The diagnosis of migraine is based solely on patients’ history in the absence of validated biomarkers.
Causal mutations in three genes have been identified for familial hemiplegic migraine (FHM), a rare and severe, autosomal dominantly inherited subtype of migraine with aura [
9‐
11]. However, these genes are apparently not involved in the prevalent types of migraine [
12,
13]. Several linkage studies and association studies using a candidate-gene approach have failed to identify any robust association between genetic variants and the prevalent types of migraine (see Table
1 for explanation of terms and methods mentioned) [
14]. Recently, a large meta-analysis including almost 60,000 affected subjects demonstrated 44 independent common single nucleotide polymorphisms (SNPs) associated with migraine [
15]. Odds ratios (ORs) ranged between 0.85 and 1.11, and the mechanisms of action in migraine are unknown. It is now clear that no common variants are associated with a medium or high risk of migraine. Thus, common variants cannot explain much of the observed heritability of migraine. In general, only 1.5%–50% of heritability of common complex diseases and traits can be explained by common variants [
16]. In other words, variants with medium or high risk must be rare and therefore not possible to capture by genome-wide association studies (GWAS) in unrelated case–control samples [
17]. Migraine sometimes clusters in families with an inheritance pattern that often looks dominant. MA in particular seems to aggregate in large families [
2,
3]. It is reasonable to think that rare genetic variants or mutations with medium to high effect size play an important role in these large families [
18]. The same could be the case for families with multiple individuals affected by MO. A search for rare variants conferring a medium to high risk of migraine should therefore focus on families with many affected with migraine. Thus, it is hypothesized that the prevalent types of migraine can be oligogenic or even monogenic inherited, as is also hypothesized for many other common complex diseases [
18]. If oligogenetic inherited, susceptibility is due to a specific combination of rare variants and unrelated cases represent a wide range of different combinations. These would be impossible to capture by GWAS alone. In a family, the combination of variants is probably specific to that one family, and because of this clustering of variants it will increase the chance to find it. Linkage analysis is the method of choice when studying monogenic disorders, but linkage studies are difficult in case of oligogenic inheritance. Previous linkage analyses in migraine families have shown association between several loci and MA and MO with LOD > 3 [
19‐
25]. None of these associations have been consistently replicated and the causal genetic variants remain to be identified, but it shows that the family approach is promising for migraine. Based on an a-priori hypothesis about the involvement of the TRESK potassium channel (encoded by the KCNK18 gene) in MA, Lafreniére et al. 2010 sequenced the entire gene region (i.e. a candidate-gene approach) and identified five variants in a case–control sample, of which one rare variant was subsequently shown to segregate perfectly with MA in a large multigenerational family (eight affected and 8 unaffected members) [
26]. However, this association has later become questioned [
26‐
28]. The technology of next-generation sequencing (NGS) now provides an affordable tool to investigate genetic variation in the entire exome or genome [
29]. In theory, a family based study design and genome sequencing can embrace the search for rare variants with both high and medium effect size, whether monogenic or oligogenic. The family approach using NGS has not yet been used in migraine genetic research on a larger scale, but we and probably several other groups now have ongoing studies. We therefore judge it timely to investigate how the family approach and NGS has been used successfully in other common complex disorders, and to deduce from that the possibilities for its application in migraine.
Table 1
Explanation of genetic methods and terms
Single nucleotide polymorphism (SNP) | A SNP is a substitution of a single base pair in the genome that occur in >1% of a population, a so called common variant [ 84, 85]. SNPs that occur in <1% of a population are considered rare. On average, there is one SNP for every 0.75-1.91 kb throughout the genome [ 86, 87]. Many of these reside outside protein-coding areas. A proportion of these will reside in other functional elements [ 88]. <1% of SNPs lead to changes in protein function [ 89]. After completion of especially the HapMap project and The 1000 Genomes Project, the vast majority of SNPs and structural variants are now mapped throughout the genome [ 86, 87, 90‐ 92]. More than 38 million SNPs are identified and these are estimated to constitute more than 95% of all common SNPs [ 91]. The SNPs known to date are gathered in public databases like dbSNP [ 33] . |
LOD-score | LOD = logarithm of the odds. A measure of the probability of two genetic loci to be located close to each other on a chromosome and thereby the likelihood for them to be inherited together (be linked). A LOD-score on > 3 means that the likelihood for two loci to be located close (and be linked) is 1,000 times the likelihood of no linkage [ 93]. |
Genome wide association study (GWAS) | The rationale is to find variants that happen to occur more often than by chance in the genomes of individuals with a specific phenotype. It is carried out by an association analysis on genotyped cases and controls. SNPs are most widely used as genetic marker. Genomes are genotyped at specific points in the DNA where the chosen markers are localized if present. Every SNP represents a block of genes, a haplotype. These are inherited together more often than by chance. They are said to be in linkage disequilibrium [ 85]. Tag-SNPs present in the sample are tested for association with a phenotype of interest, e.g. migraine, by comparing the frequencies of the SNPs in cases vs. controls. |
Nest generation sequencing (NGS) | Sequencing of the nucleotides in the entire exome or genome by whole exome or whole genome sequencing (WES or WGS, see below) |
Whole exome sequencing (WES) | WES is sequencing of every nucleotide in all exomes in a genome. Exomes are the protein coding part of DNA. This means that the remaining part of DNA in between the exomes is not sequenced. |
Whole genome sequencing (WGS) | WGS is complete sequencing of the entire genome consisting almost 3 billion base pairs [ 89]. Thus, also non-coding parts of the DNA are sequenced. Non protein coding DNA contains many functional elements with influence on gene expression and regulation e.g. RNA coding sequences, transcription factor binding sides, regions of modification or with influence on chromatin (the DNA, RNA and proteins that chromosomes are made of) structure and other interacting regions [ 88]. |
Linkage-analysis | Attempts to find chromosome segments that are shared between affected family members. Thus, no prior hypothesis of involved loci is needed. To screen for shared DNA blocks, markers are needed. Often, sets of microsatellite-markers are used [ 94]. Microsatellites contain a short sequence of base pairs that are repeated a variable number of times. Every microsatellite represents a block of DNA, a haplotype. Thus, having a specific microsatellite means having a specific haplotype. The aim is then to find linkage between a phenotype e.g. a disease and a haplotype. If a haplotype segregates with a disease in a family, they are probably linked. |
Haplotype | Each gene has a specific position on a chromosome, a so called locus. A haplotype is a combination of gene alleles at a chromosome that are inherited together more often than by chance. On average haplotypes span 25,000 nucleotides [ 84, 85]. Haplotypes are longer for newer and inbred or isolated populations and shorter for old or very outbred populations [ 91]. |
Sanger sequencing | A classic method to sequence every nucleotide in a DNA fragment of interest. The method includes the use of modified nucleotides labeled radioactively or by fluorescence and gel electrophoresis [ 95]. More precise sequencing with fewer read errors that WES/WGS. It is used to confirm findings in WES/WGS. |
Phasing and imputation | Imputation is performed with different kinds of software and is a way to predict not genotyped variants, located between genotyped variants in haplotyped blocks, by using a reference sample where a greater number of variants are genotyped [ 96]. Phasing means to sort out which genotypes are placed on the paternal respectively the maternal chromosome [ 97]. |
Identity by descent (IBD) | Genomic regions that are identically inherited from parents to more than one child. This means that the siblings will share the DNA combination in that region [ 63]. IBD can prevail over many generations and reveal the familial relationship (a common ancestor) between very distantly related individuals. |