Genetics of Migraine: Insights Into the Molecular Basis of Migraine Disorders

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Heidi G. Sutherland, PhD and Lyn R. Griffiths, PhD

Genomics Research Centre, Institute of Health and Biomedical Innovation, QUT, Musk Ave,

Kelvin Grove, QLD 4059, Australia

The authors declare no conflicts of interest.

Acknowledgements:

We wish to acknowledge the support of the Australian National Health and Medical Research

Council and the Migraine Research Foundation, NY, USA for supporting our migraine genetic and diagnostic research.

Key words:

Migraine; genetics; hemiplegic migraine; genome-wide association study

ABSTRACT

Migraine is a complex, debilitating neurovascular disorder, typically characterised by recurring, incapacitating attacks of severe headache often accompanied by nausea and neurological disturbances. It has a strong genetic basis demonstrated by rare migraine disorders caused by mutations in single genes (monogenic), as well as familial clustering of common migraine which is associated with polymorphisms in many genes (polygenic).

Hemiplegic migraine is a dominantly inherited, severe form of migraine with associated motor weakness. Family studies have found that mutations in three different ion channels genes, CACNA1A, ATP1A2 and SCN1A can be causal. Functional studies of these mutations has shown that they can result in defective regulation of glutamatergic neurotransmission and the excitatory/inhibitory balance in the brain, which lowers the threshold for cortical spreading depression, a wave of cortical depolarisation thought to be involved in headache initiation mechanisms. Other putative genes for monogenic migraine include KCKN18,

PRRT2, and CSNK1D, which can also be involved with other disorders. There are a number of primarily vascular disorders caused by mutations in single genes, which are often accompanied by migraine symptoms. Mutations in NOTCH3 causes cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) a hereditary cerebrovascular disease that leads to ischemic strokes and dementia, but in which migraine is often present, sometimes long before the onset of other symptoms. Mutations in the TREX1 and COL4A1 also cause vascular disorders, but often feature migraine. With respect to common polygenic migraine, genome-wide association studies have now identified single nucleotide polymorphisms at 38 loci significantly associated with migraine risk.

Functions assigned to the genes in proximity to these loci suggest that both neuronal and vascular pathways also contribute to the pathophysiology of common migraine. Further studies are required to fully understand these findings and translate them into treatment options for migraine patients.

INTRODUCTION

Migraine symptoms and diagnosis

Migraine is a complex, debilitating neurovascular disorder, typically characterized by recurring, incapacitating attacks of moderate or severe headache for 1-3 days and often accompanied by autonomic dysfunction, nausea and sometimes aura symptoms. The disorder affects at least 12% of the general population, including young children, with a marked preponderance of females (~3:1 ratio) [1, 2]. In addition to individual suffering, migraine has severe social and economic impacts. Data from the recent Global Burden of Disease study

(2013) ranks migraine as the most burdensome neurological disorder, and 6th highest cause of disability worldwide [3]. It ranks among the World Health Organisation top 12 of most disabling and undertreated disorders and is responsible for one of the highest socio-economic burdens of any brain ailment. It is often misdiagnosed because of lack of objective diagnostic tests and current treatments are not satisfactory or effective for everyone.

Migraine presents with variable clinical phenotypes which can be heterogeneous in the population. Characteristic clinical features of migraine include throbbing head pain (usually unilateral), nausea, vomiting, photophobia, phonophobia and often severe, neurological disturbances [4]. It is currently clinically diagnosed based on the International Classification of Headache Disorders 3rd Edition (ICHD-III) [4] which formally classifies migraine into two main subtypes; Migraine without Aura (MO) and Migraine with Aura (MA). These have substantial symptomatic overlap, however MA sufferers also experience distinguishing neurological phenomena that precede the headache phase of an attack. The aura can encompass various neurological symptoms, often visual, such as scintillating shapes, hallucinations or black spots, but can also affect sensory, speech, motor, brainstem and retinal functions. Overall, MO and MA sufferers account for ~70% and 20-30% of migraineurs, respectively. ICHD-III also describes other migraine sub-types: chronic migraine is diagnosed in patients who experience frequent or almost continuous migraine symptoms, and is often linked with medication overuse; more than 50% of females suffering from MO report a menstrual association which has led to the further classification of menstrual migraine [5,

6]; and hemiplegic migraine (HM) is a rare severe form of MA that also features motor weakness.

Pathophysiology of Migraine

Activation of the trigeminovascular system

The pathophysiology of migraine is only partially understood, but is believed to be caused by activation of the trigeminovascular system [7]. The headache phase of a migraine attack is thought to result from activation of nociceptors innervating the cranial blood vessels, which transmits a signal to the trigeminal bipolar neurons; this is further relayed to thalamic and cortical areas, producing the sensation of pain [8]. The signal from the perivascular neurons is transmitted by the vasoactive neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP) and nitric oxide, resulting in a downstream cascade of events that leads to the release of vasoactive inflammatory mediators, inflammation in the meninges and sensitization of pain relevant brainstem regions [9].

Cortical Spreading Depression

Trigeminal activation is a downstream event; how it is triggered is not well understood, but there is accumulating evidence that cortical spreading depression (CSD) is one route. CSD is a slowly propagating wave of neuronal and glial depolarisation accompanied by massive ion fluxes, that spreads across the brain cortex, and which is followed by a long-lasting suppression of neuronal activity [10, 11]. It coincides with, and is thought to underlie, the migraine aura [12, 13]. The onset of CSD has been demonstrated experimentally following noxious stimuli which lead to a build-up of glutamate in the neuronal environment, which then pathologically activates calcium and sodium channels, particularly the N-methyl-D- aspartate (NMDA) receptors [11]. Evidence from experimental animals suggests that CSD might not only cause migraine auras, but also play a pivotal role in headache initiation mechanisms [12-14]. CSD is associated with opening of Pannexin1 mega channels causing caspase-1 activation and HMGB1 release which initiates parenchymal inflammatory pathways and may provide the stimulus for sustained trigeminal activation [15]. Proof of a direct link between CSD and migraine in humans is still lacking, and the question of how

CSD itself is triggered, and details of the downstream effects needs further investigation.

Furthermore, the majority of migraine patients do not experience aura, so alternative triggers for trigeminovascular activation may also be important, such as cortical hyperexcitability or brain stem or hypothalamic dysfunction [16]. Clinical and neurophysiological studies have confirmed that individuals suffering from migraine display chronic hypersensitivity to sensory stimuli and or abnormal processing of sensory information [17-19]. Migraineurs exhibit cortical excitability [19, 20], which may make them more susceptible to CSD.

Dissection of the genetics of migraine, particularly the severe monogenic forms, has added support to this and animal models have aided in understanding some of the mechanisms.

Genetics of Migraine

Migraine is a complex disorder with many factors contributing to its presentation. Migraine can be triggered by environmental factors - stress, bright lights, sleep disturbances, physical exertion, food and drink products, and menses have all been reported as triggers [21, 22].

Fluctuating hormone levels may explain the increased prevalence of migraine in females and its variability across life span [23]. However, the disorder also has a large genetic component.

This is clear for some rare monogenic migraine subtypes which have been attributed to mutations in particular genes. Common migraine is also recognised as clustering in families with first-degree relatives of migraine sufferers having an increased risk of migraine (up to 4 times depending on sub-type) [24, 25]. Furthermore, twin studies have estimated a heritability of around 42% for migraine (ranging from 0.34 to 0.57) [26-28], reflecting a strong genetic influence. Common migraine is mostly polygenic with variants or polymorphisms in many genes contributing to susceptibility and recent genome-wide association studies (GWAS) have now identified numerous gene loci to be associated with migraine [29]. Understanding the genetic factors that contribute to a disorder deepens understanding of the mechanisms involved and importantly this information may be incorporated into prevention and treatment strategies for the disorder.

In Part I of this review we focus on the genes and mutations that have been identified as causing rare monogenetic forms of migraine, as well as disorders that have substantial migraine overlap, and summarise studies that have investigated how their function may relate to migraine susceptibility. In Part II we focus on the identification of loci implicated in common polygenic migraine, discussing the function of genes associated with these loci and how knowledge of the contribution of common variants on migraine may be further extended and used.

PART 1: MONOGENIC MIGRAINE DISORDERS

There are a number of rarer sub-types of migraine that are accompanied by distinctive neurological symptoms, as well other disorders in which migraine is often present in addition to the other characteristic symptoms. Mutations in particular genes have been found to be causal in a number of these disorders, and studies into the function of the genes and mutations identified have given valuable insights into migraine etiology.

Hemiplegic Migraine (HM)

Symptoms and diagnosis of HM

Hemiplegic migraine (HM) is a severe MA sub-type in which auras are characterised by motor symptoms such as temporary numbness or weakness, often affecting one side of the body (i.e. hemiparesis) [4, 30]. Individuals generally experience unusually severe migraine and extended aura episodes which may include confusion, fever, prolonged weakness, seizures, and coma. The phenotype is often severe and in rare cases can be fatal after minor head trauma [31]. Although most people with HM recover completely between episodes, neurological symptoms such as memory loss and problems with attention can last for weeks or months, and a small subgroup can develop mild but permanent difficulty coordinating movements (ataxia), which may worsen with time [32].

HM has an estimated prevalence of up to 0.01% in Europeans [32, 33]. Familial Hemiplegic

Migraine (FHM) is a sub-category of hemiplegic migraine (HM) and is diagnosed when there is at least one 1st or 2nd degree relative in the family who also suffers HM attacks [4].

Sporadic Hemiplegic Migraine (SHM) is diagnosed in individuals with no family history of the condition [34]. Estimates indicate that in the general population nearly two-thirds of HM cases are familial [33]. FHM follows an autosomal dominant pattern of inheritance, but shows variable expressivity and genetic heterogeneity with 70% to 90% penetrance. FHM is considered to be monogenic, but genetically heterogeneous, with three main causative genes

(CACNA1A, ATP1A2 and SCN1A) identified through family linkage studies [35]. FHM is therefore further classified into FHM1, 2 and 3 depending on which of these three genes mutations are detected in. The phenotypes of the three FHM sub-types are nearly identical clinically, although overlapping features may vary.

FHM1 due to mutations in the CACNA1A gene

The first FHM locus to be identified through positional cloning and mutation analysis in

FHM family pedigrees was the CACNA1A gene at chromosome (chr) 19p13 [36]. CACNA1A encodes the pore-forming 1 subunit of the neuronal voltage-gated Cav2.1 (P/Q-type) channels [37]. Cav2.1 channels are predominantly localised at the presynaptic terminals of cortical glutamatergic and GABAergic neurons in the cerebral cortex, trigeminal ganglia, brainstem nuclei and cerebellum where they play an important in controlling neurotransmitter release [38]. >25 causal mutations have been reported for FHM1; associated clinical severity and other symptoms differs with the various mutations [39] (also see Leiden Open Variation

Database http://chromium.lovd.nl/LOVD2). FHM1 mutations are usually missense and gain- of-function, leading to increased Ca2+ influx, which has the effect of enhancing glutamatergic neurotransmission [36, 40, 41].

Two transgenic FHM1 knock-in (KI) mouse models have been generated, expressing the

R192Q and S218L CACNA1A mutations. Mice with the milder R192Q mutation show no overt phenotype, while the more severe S218L mice exhibit cerebellar ataxia and spontaneous seizures in accordance with severity of the clinical symptoms observed in patients [42, 43]. Both FHM1 KI models show alterations in the cortical excitatory-inhibitory balance, synaptic plasticity, and CGRP-mediated trigeminal pain signalling, in addition to enhanced susceptibility to CSD [44, 45]. The decrease in threshold for induction and propagation of CSD is most likely due to enhanced synaptic release of glutamate as a result of the Cav2.1 channel gain-of-function mutation selectively affecting glutamatergic neurons, but not GABA-ergic inhibitory interneurons [46, 47]. Gene expression analysis of FHM1

R192Q mice revealed that CSD modulates inflammatory processes in both wild-type and mutant brains, but that an additional unique inflammatory signature becomes expressed after

CSD in the FHM1 mice [48]. This may lead to an increased activation of meningeal nociceptors and trigeminal ganglia and drive the activation of pain-related brain structures to cause migraine headache.

Interestingly FHM, like common migraine, shows a female preponderance [49]. Studies in

FHM1 transgenic mice have shown that increased susceptibility to CSD is reduced in males and further enhanced by female sex hormones, and this sex difference is abolished with ovariectomy and senescence [50]. Thus hormones are modifying factors which may explain some of the variable expressivity and penetrance of FHM mutations. A large-scale functional

RNAi screen in Caenorhabditis elegans for modifiers of unc-2, the worm orthologue of

CACNA1A which displays an uncoordinated phenotype, identified the TGF- and Notch signalling pathways as modifiers of CACNA1A [51]. This may have implications for understanding variability in phenotype and also possible therapeutic interventions, particularly as these pathways are relevant to other syndromes that overlap with FHM, e.g.

CADASIL (see below), as well as some of the common migraine susceptibility loci (Part II).

Episodic Ataxia 2 and Spinocerebellar Ataxia Type 6

Mutations in CACNA1A also cause episodic ataxia 2 (EA2) and spinocerebellar ataxia type 6

(SCA6) [36, 52], and there can be symptomatic overlap between the three allelic disorders.

~50% EA2 mutation carriers also suffered migraine [53], and episodic headaches and nausea are also common in SCA6 patients [54]. EA2 mutations include missense, truncating or aberrant splicing variants of CACNA1A [55], which have the opposite effect on the Cav2.1 channels to FHM, leading to decreased Ca2+ influx [45]. SCA6 is usually due to small expansions of a polyglutamine repeat in the COOH tail of CACNA1A [52]; pathogenesis of

SCA6 is thought to be an age-dependent process related to accumulation of mutant Cav2.1 channels which results in a toxic gain-of-function effect [56].

FHM2 due to mutations in the ATP1A2 gene

A second FHM gene was mapped to chr 1q23 and subsequent mutation screening identified mutations in the ATP1A2 gene to cause FHM [57]. ATP1A2 encodes the 2 subunit of the

Na+/K+ pump present in the membranes of cells of the central nervous system (CNS), as well as heart, skeletal and smooth muscle tissue [58]. In the CNS, it is mainly expressed on astrocytes at tripartite synapses, where it is co-distributed with the Excitatory Amino Acid

Transporter (EAAT) glutamate transporters [59], and is required for clearance of extracellular

K+ and production of the Na+ gradient used in the reuptake of glutamate [60]. There are >80 causal mutations linked to FHM2, with ~25 diagnosed in SHM, suggesting that de novo mutations are common at the ATP1A2 locus [61]. An overlap with epilepsy or seizures has been noted in about 15% of cases, as well as other pathologies including alternating hemiplegia of childhood, basilar migraine, sensorineural hearing loss, pulmonary arterial hypertension and reversible cerebral vasoconstriction [61] (and references therein). Most

FHM mutations are missense, mainly clustering in the catalytic P domain, the transmembrane domain, or in the central region between them. It is not clear whether ATP1A2 mutations in

FHM2 classify as gain- or loss-of-function as while many mutations abolish or largely reduce

Na+, K+ pumping, others cause more subtle effects including shifts in voltage dependence, kinetics, or apparent cation affinities [61].

Several Atp1A2 knockout (KO) mice models have been generated, as well as FHM2 KI mice carrying either the human W887R or G301R mutations [62]. Homozygous KO and KI mice die immediately after birth, while heterozygous KO mice display altered behaviour and neurological defects. Both Atp1A2 KI mice models display altered CSD, with W887R heterozygous mice more susceptible to CSD [63], while G301R heterozygotes showed a prolonged recovery phase following CSD [64]. Capuani et al. showed that W887R heterozygous mice have a reduced rate of glutamate and K+ clearance by cortical astrocytes during neuronal activity, as well as a reduced density of GLT-1a glutamate transporters in the astrocytic processes surrounding glutamatergic synapses, and that the consequent defective glutamate clearance facilitates CSD ignition [65]. Collectively these findings suggest that

ATP1A2 mutations in migraine primarily cause a disorder of glutamatergic neurotransmission with defective regulation of the excitatory/inhibitory balance in the brain which facilitates

CSD and downstream effects.

FHM3 due to mutations in the SCN1A gene

The third FHM locus to be identified was the SCN1A gene on chr 2q24 [66]. <5% of FHM patients (for which a molecular diagnosis has been made) have mutations in SCN1A. SCN1A encodes the 1 subunit of the neuronal voltage-gated sodium channel Nav1.1, which is critical for generation and propagation of action potentials [67]. SCN1A is well-known as an epilepsy gene with hundreds of truncating and missense mutations associated with epilepsy syndromes, including severe myoclonic epilepsy of infancy (SMEI, also known as Dravet syndrome) and generalised epilepsy with febrile seizures (GEFS+) [68, 69]. Functional studies of epileptic mutations has revealed mainly loss of function effects, which predict network hyperexcitability due to speciﬁcally reduced action potential ﬁring in inhibitory

GABAergic interneurons, in which NaV1.1 is predominantly expressed [70] (and references within). SCN1A KO mice suffer from ataxia and epileptic seizures [71, 72].

To date 10 SCN1A mutations have been reported to cause FHM3. Q1489K, L1649Q,

I1498M, F1661L, F1774S and L1642P were identified in pure FHM cases (L1642P showed early onset) [66, 70, 73-75], whereas others were linked with FHM in addition to other features such as epileptic seizures (L263V, T1174S and Q1489H) [76, 77], and elicited repetitive daily blindness (Q1489H and F1499L) [78]. In FHM3 mutations in SCN1A are missense, and usually lead to a gain-of-function effects such as an increased threshold-near persistent current, delayed entry into inactivation, and a faster recovery and higher channel availability during repetitive stimulation [70]. However, others exhibit loss-of-function effects in heterologous cell systems [79], and mutations such as T1174 can act in both a gain- and loss- of function manner, although with respect to FHM its gain-of-function in

GABAergic neurons may be important [77]. As NaV1.1 is the predominant channel in

GABAergic interneurons [71, 72], FHM3 SCN1A mutations predict increased firing of inhibitory GABAergic neurons, which could lead to higher extracellular potassium concentrations, enhanced glutamate release and triggering of CSD [70, 80]. FHM3 KI mouse models have yet to be reported, but will help to reveal how FHM3 mutations cause disease when expressed in a natural cellular context in a whole animal model.

Genetics of Sporadic Hemiplegic Migraine

The genetics of SHM (or sporadic cases) is not well understood. In some cases it results from de novo genetic mutations in the known FHM genes (which would become familial in future generations) [81-83]. The possibility of mosaicism in the transmitting parent, as has been observed in other neurogenetic disorders, including Dravet syndrome-causing SCN1A mutations, should be considered [84, 85]; high-depth next-generation sequencing (NGS) approaches would be required to identify this. However, SHM may also be due to alternative models of inheritance, other genes or mutations with lower penetrance, and/or gene and environment interactions, e.g. compound recessive mutations or a combination of multiple lower-risk genetic variants similar to common migraine [32].

Hemiplegic Migraine due to mutations in other genes

A number of HM individuals and families have not been able to be linked to the known FHM genes. Using an NGS approach that allows comprehensive sequencing of all the exons of each the CACNA1A, ATP1A2 and SCN1A genes (as well as TRESK and NOTCH3), our laboratory found mutations in the known FHM genes in only ~25% of FHM patients requesting a molecular diagnosis (unpublished data). It is possible that mutations may reside in non-coding regions of the known FHM genes, but it also suggests that there are likely to be additional genes that cause HM. Other genes have been implicated in HM: PPRT2, SLC2A1 and PNKD (see below); SLC1A3, which encodes the glial glutamate transporter EAAT1, and in which mutations are causal for episodic ataxia, type 6 [86]; and SLC4A4, encoding the sodium bicarbonate cotransporter NBCe1 [87]. However, the evidence for some of these as

FHM genes is still very limited.

PRRT2 and genes involved in paroxysmal disorders

Mutations in the Proline rich transmembrane domain protein 2 (PRRT2) gene have been implicated in a number of childhood-onset paroxysmal disorders (i.e. those that display a sudden attack or intensity of symptoms) including Paroxysmal kinesigenic dyskinesia (PKD), infantile convulsions with PKD (PKD/IC) and benign familial infantile epilepsy (BFIE).

More recently the spectrum of disorders that PRRT2 mutations are associated with has expanded and includes other paroxysmal conditions with movement, as well as migraine disorders, and has putatively been assigned as another HM locus [88, 89]. PRRT2 is expressed throughout the CNS and encodes a 430 amino acid protein with a proline-rich N- terminus and a C-terminal transmembrane domain. Its function is poorly understood although it is mainly sub-localised in axons where it associates with the GluA1 subunit (GRIA1) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors at the glutamatergic synapses [90]. It also interacts with SNAP25 [91], a t-SNARE protein that is involved in synaptic vesicle docking and exocytosis at presynaptic terminals and regulates voltage-gated calcium channels in glutamatergic neurons [92]. Thus PRRT2 appears to be involved in synaptic neurotransmitter release.

The majority of PRRT2 mutations are loss-of-function resulting in a truncated, or no protein leading to haploinsufficiency, and thus likely affect SNAP25/SNARE interaction and lead to increased vesicle release. The most common mutation is the c.649dupC (R217Pfs*8) frameshift mutation (>75% cases) [93], which causes nonsense-mediated decay of the transcript [94]. All the migraine or HM cases with PRRT2 mutations reported to date locate to the c.649/650 residue, and have either the common c.649dupC (R217Pfs*8), a c.649delC, or a c.650delG mutation [88]. However the phenotype can vary greatly between individuals and even between members of the same family with the same mutation [95], suggesting that other modifying genes or environmental factors play a role in disease expression.

Exercise induced dyskinesia (PED) and paroxysmal non-kinesigenic dyskinesia (PNKD) are other paroxysmal conditions which have been associated with mutations in the glucose transporter type 1 gene (SLC2A1, also known as EAAT2 and GLT-1) and PKND, respectively [93]. PED can be associated with migraine, hemiplegia, ataxia and epilepsy [96], and a novel PNKD deletion was identified in a pure FHM family [93]. While PRRT2,

SLC2A1 and PKND may genes may not necessarily be classified as true FHM genes [97], they should be considered in the molecular diagnosis of HM patients.

Migraine with Aura associated with mutation of KCNK18 encoding the TRESK channel

While the majority of the familial migraine disorders are associated with the severe HM phenotype, a monogenic form of typical MA was identified from a large multigenerational pedigree by Lafreniere et al. [98]. Sequencing of the TWIK-related spinal cord potassium channel (TRESK, encoded by the KCNK18) gene revealed a frameshift mutation

(F139Wfsx24) that results in loss of function that segregated perfectly with migraine in that family. TRESK is a member of the two pore domain potassium channel (K2P) family which are involved in the regulation of cellular electrical excitability. TRESK is expressed most abundantly in sensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia, and is also present in the CNS including the cortical region [99]. TRESK KO mouse models show that it functions to modify certain forms of nociceptive afferentation [100,

101]. Functional analysis of cells expressing the TRESK F139Wfsx24 mutation indicate that it has a dominant negative effect on plasma membrane localisation, and on whole-cell

TRESK currents resulting in hyperexcitability of trigeminal ganglion neurons [102]. Further sequencing of this gene in other migraine cases has suggested that the relationship between TRESK and migraine is complicated. While a loss of function TRESK mutation A34V was detected in a single migraine case, another loss of function mutation C110R was found in controls as well as migraine cases, suggesting that a single non-functional variant in TRESK alone is insufficient to cause migraine [103].

Vascular monogenic disorders with substantial migraine overlap

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL)

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy

(CADASIL) is the most common heritable cause of stroke and vascular dementia in adults

[104]. It is characterised by the middle-aged onset of cerebrovascular disease that often progresses to dementia. The presence of subcortical infarcts and leukoencephalopathy, best detected by MRI, are crucial for the diagnosis of CADASIL. Up to 40% of CADASIL patients have MA, and when present it is usually the first symptom experienced, and can present decades before the transient ischaemic attacks and ischaemic strokes that are the main manifestations of the disorder. CADASIL is autosomal dominant and has been found to be caused by mutations in NOTCH3 [105]. NOTCH3 is a single pass transmembrane domain receptor protein which features 34 epidermal growth factor repeats (EGFR) and three

Notch/Lin12 repeats in the extracellular domain, and seven ankyrin repeats in the intracellular portion of the protein. NOTCH3 is a homologue of the Drosophila notch protein, which via its interaction with cell-bound ligands establishes intracellular signalling pathways that are essential for cell fate decisions during embryogenesis and postnatal tissue homeostasis. NOTCH signalling is important in the development of most organs in vertebrates, and NOTCH3 is predominantly expressed in vascular smooth-muscle cells, particularly in small arteries [106]. It functions in cell-cell communication as a receptor for ligands Jagged1, Jagged2 and members of the Delta-like family to transduce extracellular signals into changes in gene expression via forming a transcriptional activator complex with

CSL or other co-activators to drive expression of target genes [107].

Each EGFR contains six cysteines and all CADASIL mutations reported to date (>150 in

>500 pedigrees) occur in exons 2-24 encoding the EGFR and lead to an odd number of cysteines within a given repeat [104]. CADASIL mutations appear to act via a gain of novel function (neomorphic) mechanism; rather than an effect on canonical NOTCH signalling, it is thought that changes in the number of cysteine residues may result in unpaired cysteines which participate in novel protein-protein interactions. In two different transgenic CADASIL mouse models, one a Notch3 KO and the other overexpressing the equivalent of the R90C mutation, susceptibility to CSD was enhanced [108]. Furthermore, both develop larger infarcts with an increased frequency of peri-infarct depolarisations upon experimental stroke

[109, 110]. Hyperexcitability resulting from genetically enhanced glutamatergic neurotransmission might be a common mechanism for both migraine pathogenesis and tissue sensitization to ischemia [44]. The details of how NOTCH3 mutations are linked to CSD and migraines (in particular MA) are yet to be fully elucidated, but these findings implicate vascular mechanisms.

Cerebral and hereditary angiopathy with vascular retinopathy and internal organ dysfunction (CHARIOT), also called Retinal Vasculopathy with Cerebral Leukodystrophy

(RVCL)

Cerebral and hereditary angiopathy with vascular retinopathy and internal organ dysfunction

(CHARIOT) is a progressive systemic small vessel disease characterised by progressive blindness due to vascular retinopathy, focal and global neurological symptoms due to cerebral mass and white matter lesions, and premature death. ~50% patients also have mild

Raynaud’s phenomenon and/or migraine as part of the clinical spectrum, which can proceed other symptoms by up to a decade [111]. CHARIOT has been found to be caused by mutations in TREX1 [112], a gene that is also mutated in autoimmunity diseases Aicardi-

Goutières syndrome, familial chilblain lupus, and systemic lupus erythematosus [113].

TREX1 is a nuclear protein with 3’ exonuclease activity that may play a role in immune regulation of self-tolerance via its DNA repair and proofreading, or degradation activities

[114, 115]. CHARIOT-related TREX1 variants have been exclusively reported to occur as frameshift mutations in the C-terminus, which is necessary for its normal endoplasmic reticulum localization [113]. They are therefore likely associated with a gain-of-function or toxic effect due to altered cellular distribution. The TREX1-V235fs mutation in patient lymphoblasts and KI mice causes dysregulation of the ER oligosaccharyltransferase complex which results in immune activation [116], although how this contributes to systemic vasculopathy or migraine susceptibility is unclear at present.

Hereditary infantile hemiparesis, retinal arteriolar tortuosity, and leukoencephalopathy

(HIHRATL)

Specific mutations in the COL4A1 gene cause a vascular disorder with hereditary infantile hemiparesis, retinal arteriolar tortuosity, and leukoencephalopathy (HIHRATL) [117]. In some individuals migraine (usually MA) is a prevalent symptom, particularly in those that carry the G652E mutation [118]. Collagen IV is a major component of the basement membrane, an extracellular matrix structure that provides structural support and influences cell behaviour and signalling. Col4a1 mutations in mice cause abnormal vascular development; intracellular accumulation of mutant collagen in vascular endothelial cells and pericytes triggers small vessel disease, recurrent hemorrhagic strokes and age-related macro- angiopathy [119].

Familial advanced sleep-phase syndrome (FASPS)

Familial advanced sleep-phase syndrome (FASPS) patients show severe disruption of the sleep-wake-cycle and other circadian rhythms. In two large pedigrees with the disorder mutations in the CSNK1D have been found to be causal [120]. Interestingly, the phenotype also co-segregated with MA. CSNK1D encodes CKIδ, a ubiquitous serine-threonine kinase that phosphorylates the circadian clock protein Per2 and other proteins involved in brain signalling [121]. Mice engineered to carry the CKIδ-T44A mutation were more sensitive to pain after treatment with the migraine trigger nitroglycerin, exhibited a reduced threshold for

CSD, and astrocytes from the mice displayed increased spontaneous and evoked calcium signaling [122]. The migraine-FASP connection advances the likely role of the hypothalamus in migraine [123]. Hypothalamic activation has been observed in the premonitory phase of migraine [124]. Furthermore, known migraine triggers present physiological stresses that the hypothalamus plays a role in regulating, suggesting hypothalamic dysfunction may be linked to migraine susceptibility [125].

Other family-based linkage studies

Linkage analyses have identified a number of genetic loci for overall migraine, MA, and MO suggesting that variants in genes with a strong effect may lie in these regions. E.g. overall migraine has been linked to chromosomes Xq24-28, Xp22, 6p12.2-21.1, 19p13, and 1q31;

MA to 4q24, 9q21-q22, 11q24, and 15q11-13; and MO to 14q21.1-22.3, 4q21, and 18q12

[126, 127] and references therein. In addition to these latent class analysis (LCA) and trait component analysis (TCA) to breakdown the MA/MO classification into more homogenous groups for analyses have identified other loci for specific features related to migraine [127].

However, the underlying genes from these chromosomal regions that may alter risk of migraine have yet to be identified.

Importance of molecular diagnosis for FHM and migraine-related disorders

FHM symptoms (e.g. paralysis on one side of the body) can be similar to stroke, although the onset of paralysis is usually slower in FHM as it corresponds to CSD and the related aura.

The characteristics of FHM can also make it difficult to distinguish from other inherited neurological disorders associated with migraine symptoms such as EA2, SCA6 and

CADASIL. It is important for clinicians to be able to differentiate these conditions to ensure appropriate clinical responses for patients; if a mutation in a particular gene is identified then treatments may be tailored accordingly. For example, mutations in CACNA1A (in FHM1 and

EA2) are typically responsive to acetazolamide [128, 129]. Other FHM sub-types can be more variable and calcium channel blockers are often used, with ATP1A2 mutations being successfully treated with flunarizine, and reports describing some efficacy of sodium valproate, lamotrigine, and verapamil in FHM sub-types [30]. There are some concerns using more standard migraine drugs like triptans and ergotamines in FHM due to stroke risk from the vaso-constriction, although as CSD in now thought to underlie aura this contraindication has been questioned and they have been used successfully [130]. For patients with the severe

FHM1 S218L missense mutation with which minor head trauma can lead to seizures, cerebral oedema, coma and sometimes death [31, 131], care and vigilance is required and patients can be advised to avoid contact sports. In the case of a NOTCH3 mutation, there would be more aggressive anti-stroke treatments and better surveillance for dementia than there would be for

FHM. Carbamazepine is used to control paroxysmal attacks in PKD, and also has been used with some success for migraine in individuals with PRRT2 mutations [132]. Detecting a causal mutation in an individual also allows for family screening and better clinical care for family members.

Summary and future directions

FHM is primarily considered to be a channelopathy disorder caused by dominant mutations in a range of ion channel genes. The overarching theme is that FHM mutations cause a disorder of glutamatergic neurotransmission with defective regulation of the excitatory/inhibitory balance in the brain, resulting in a higher susceptibility to CSD. TRESK is also an ion channel that also appears to affect hyperexcitability in neurons and while not an ion channel gene, PRRT2 also may be involved in neuronal excitability. There are also primarily vascular diseases which commonly feature migraine or HM as a symptom which supports a vascular component in the etiology of migraine, although the mechanisms by which these genetic vasculopathies give rise to migraine are still unclear. In genetic small vessel diseases such as CADASIL and CHARIOT, vascular changes, including endothelial dysfunction, may directly or via neuronal pathways increase migraine susceptibility [44]. The discovery of mutations in a gene that regulates circadian rhythms

(CSNK1D) and implicates the hypothalamus in migraine opens an interesting avenue of research into how migraine may result from stresses that induce disruption of homeostasis.

Mutations in monogenic migraine disorders described to date are usually coding variants and are often dominantly inherited. The fact that only ~25% of HM patients have a molecular diagnosis suggests that more mutations are to be found. These may be non-coding variants, i.e. regulatory mutations, but also it is likely that other monogenic migraine genes will be identified, e.g. there are >100 ion channel genes across the genome. NGS techniques will play an increasingly valuable role in identifying new migraine genes which could then be incorporated into diagnostics [133]. NGS provides a means for gene identification in FHM families that were too small for classical linkage analysis as well as facilitating screening of genomic regions for mutations that were previously found associated with migraine through linkage. Both whole exome and whole genome sequencing (WES and WGS) will play important roles, the latter to detect non-coding variants. However more efficient methods of identifying and testing pathogenicity of potentially damaging variants are needed as many variants of unknown significance are detected using these methods. To this end exciting developments in the use of induced pluripotent stem cell (iPSC) and CRISPR/Cas9 genome editing technologies will benefit functional studies. iPSCs will allow screening and functional analysis of mutations in cells that can be differentiated into various neuronal cell types [134,

135], or even brain organoids [136]. They can be isolated from patients and therefore mutations studied in the context of their genomic background. CRISPR-Cas9 genome editing will allow more refined and faster generation of mutants for functional analysis in both cell lines and animal models [137].

PARTII: COMMON MIGRAINE DUE TO POLYGENIC FACTORS

Common genetic variation and complex traits

Common forms of migraine are thought to be polygenic. Multiple predisposing genes and their variants contribute, each with small effect size, to the underlying risk of the disorder. On top of this there are the complex interactions of these genetic variations with each other and with lifestyle and environmental factors. Common genetic variation comprises single nucleotide polymorphisms (SNPs), small indels, short tandem repeats (STRs) and copy number variants (CNVs). Large efforts have been expended on investigating the role of SNPs in a myriad of traits, including migraine. The search for common variants that contribute to migraine is difficult because although each variant may contribute to an increase in susceptibility to the disorder, it is neither necessary, nor sufficient, to cause it. While monogenic migraine disorders have on the whole been approached from a familial background, discovering common variants that influence migraine risk relies on finding differences in allele frequencies between case and control cohorts composed of unrelated individuals. Significant differences in allele frequencies suggest either that the tested SNP is itself a susceptibility factor, or more often that the causal variant is in close proximity and does not segregate independently from the tested SNP, i.e. they are in linkage disequilibrium

(LD).

Studies of polymorphisms in candidate genes

Hypothesis-driven candidate gene association studies have been a popular approach to attempt to identify genes associated with migraine. The genes investigated by this method have mainly reflected the theories and pathways thought to be the basis for the disorder, thus the focus has been on genes involved in neurological, vascular, hormonal, and mitochondrial pathways [138, 139]. The candidate gene approach has also been used in conjunction with linkage studies to try and identify which gene within a genomic region linked to migraine in a pedigree may be driving the association signal. These studies have generally either attempted to cover the specific gene or loci using tagging SNPs or have selected known functional variants in genes thought to be related to migraine [127].

At least 185 polymorphisms in 98+ genes have been investigated in migraine with both positive and negative associations reported [139]. Polymorphisms in ion channel genes, including those causal for FHM have not been found to be particularly associated with common migraine [140]. The most studied polymorphism is the non-synonymous C667T variant (rs1801133) in the 5,10-methylenetetrahydrofolate reductase gene (MTHFR), which encodes a key enzyme in folate metabolism. Some meta-analyses found an association of the

T-allele with MA, but not MO, particularly in non-Caucasian populations [141-144].

However, this has not been supported by other meta-analyses or genome-wide association study (GWAS) data [145-147]. On the whole candidate genes studies have been conducted with small sample sizes and are consequently underpowered, are often not sufficiently corrected for multiple testing, and findings often not replicated in independent populations.

Notably a systematic re-evaluation of the most promising candidate gene SNPs, including those that have previously been reported to be positively associated with migraine, showed no clear evidence for involvement in migraine using International Headache Genetics

Consortium (IGHC) genome-wide marker data for 5175 clinic-based migraineurs and 13,972 controls [147].

Where are the polymorphisms that might influence susceptibility?

While there are some common functional variants in a gene that may affect the stability, structure, or activity of the encoded protein, the majority of SNPs that influence the association of a genomic loci to a particular trait are thought to do so via regulation of linked genes [148, 149]. Thus the limited success of candidate gene studies may also reflect on our poor knowledge of what regions of a gene are important for its regulation. Promoter SNPs and splice sites variants are relatively easy to identify, but as characterisation and annotation of the genome has improved, it has become clear that regulatory regions that control expression are often spread far and wide throughout gene loci – in introns, or large distances

5’ or 3’ to the gene, or may even reside in neighbouring genes. Genome-wide approaches are required to find these.

Rise of the genome-wide association study (GWAS)

GWAS have revolutionised how complex genetic traits are dissected. They offer a more unbiased approach that requires no prior knowledge of genes, pathways or biology and therefore can produce novel findings that can give fresh insights into the biology and pathways involved in a particular trait [150]. GWAS rely on genotyping hundreds of thousands of SNPs distributed across the genome; reduction in genotyping costs and high- throughput arrays with comprehensive SNP coverage has allowed GWAS to reach their potential. The tag SNPs on the arrays can serve as a proxy for any SNPs that are in strong LD and are tested for association with the trait in question. For a disorder such as migraine, genotypes obtained from a large cohort of cases are compared with those from a control cohort to identify alleles that are increased in frequency with the disease with stringent significance thresholds applied to p-values (below 5 x 10-8) to correct for multiple testing.

GWAS for many traits were initially disappointing. However, we now know that in general the effect sizes of SNPs that are significantly associated with most traits and complex disorders are small, and therefore large numbers of samples are required to detect associations of genome wide significance [150]. Collaborations and consortiums have played an important role in addressing this.

Migraine GWAS

Five major migraine GWAS have been reported to date. In 2010 the first migraine associated

SNP (rs1835740) was identified via a GWAS conducted in 2748 MA cases from three

European clinics with 10,747 controls, which was replicated in a further 3,202 cases and

40,062 controls from Europe [151]. rs1835740 is located between the metadherin MTHD/lyric and CPG gene plasma glutamate carboxypeptidase (PGCP) genes. Expression quantitative trait (eQTL) analysis revealed that the SNP correlates with increased MTHD expression, which has been shown to downregulate SLC1A2/EAAT2, the major glutamate transporter in the brain, suggesting a role that could plausibly be integrated with current concepts of migraine pathophysiology.

A second major GWAS for migraine was performed as part of the Women’s Genome Health

Study, involving 5122 migraine cases and 18,108 general population controls, and found three loci significantly associated with migraine at the genome-wide level which replicated in independent cohorts (TRPM8, LRP1 and PRDM16) [152]. TRPM8 is a cation channel, expressed on peripheral sensory neurons and sensory afferents innervating the meninges, which on activation by stimuli (TRPM8 is known as a sensor for cold sensation) allows influx of Ca2+ and Na+, resulting in membrane depolarisation and activation of second messenger cascades [153]. The transient receptor potential (TRP) family of channels participate in the sensory encoding of pain and therefore provides a link between pain-related pathways and migraine [154]. LRP1 interacts with and regulates the cellular distribution of

GluA1 receptors on neurons and therefore plays a role in modulating synaptic transmission through the NMDA glutamate receptors [155]. PRDM16 is mainly known for its role in the differentiation of brown fat [156], but may also play a role in neurogenesis in response to

Notch signalling [157, 158]. Thus these genes reflect the fact that neuronal functions, including ion channels and glutamatergic transmission, are also important in common migraine.

A third GWAS which focussed on clinic-based MO patients (2326 cases and 4580 controls) subsequently confirmed a role for LRP1 and TRPM8 and identified four additional novel migraine susceptibility loci (MEF2D, TGFBR2, PHACTR1 and ASTN2) [159]. While

MEF2D and ASTN2 may contribute to migraine susceptibility via their involvement in synapse and neuronal differentiation [160, 161], TGFBR2 may contribute via a vascular role as TGFβ receptor signalling is essential for normal brain vascular development [162].

PHACTR1 has both neuronal and vascular functions: it modulates PP1 activity to regulate activity of ion channels and signal transduction enzymes at synapses [163], has a role in endothelial cell function, and is a susceptibility locus for coronary artery disease (CAD)

[164], myocardial infarction (MI) [165] and other vascular disorders [166].

In 2013 Anttila and colleagues performed a meta-analysis of 29 clinic- and population-based

GWAS for the International Consortium of Headache Genetics (ICHG). This study, involving

23,285 cases and 95,425 controls, brought the number of loci significantly associated with migraine to thirteen, with the reporting of new loci near AJAP1, TSPAN2, and within FHL5,

C7orf10 and MMP16 [167]. Pathway analysis showed that the loci were particularly enriched near genes involved in synaptic or neuronal regulation, but many also have vascular functions. Several of the SNPs associated with migraine are located in DNase I hypersensitivity sites or known transcription factor binding motifs, supporting the idea that alterations in genetic regulation might be causative in migraine pathology.

The most recent effort by the ICHG involved both international academic collaborations and partnerships with the commercial entities 23andMe and deCODE, and included migraine cases assessed by questionnaire in addition to clinic-based cases, to increase sample size and boost statistical power. Analysis of 59,674 cases and 316,078 controls from 22 GWAS revealed 44 independent SNPs that map to 38 distinct genomic loci that are significantly associated with migraine risk [29], and included the majority of loci identified previously. MTDH, near AJAP1, and MMP16 were now only nominally significant, but these may be associated with specific features of migraine [168]. The loci currently associated with migraine susceptibility are summarised in Table 1, along with descriptions of what is currently understood of their function and other diseases the genes are implicated in. Effect sizes are small (OR 0.87-1.11) with some minor alleles increasing susceptibility to migraine

(OR >1) while others show a protective effect (OR <1) (Table 1). Analyses of genes linked to loci found that they were particularly enriched in those expressed in vascular tissues, as well as tissues with a smooth muscle component [29].

Insights into migraine from GWAS findings

What pathways are implicated in migraine?

An overview of the functions or pathways that migraine associated genes are involved in is shown in Figure 1. Many of the genes associated with migraine susceptibility, where function is known, have roles in neuronal development or glutamatergic neurotransmission and synaptic function. Furthermore, genomic loci that have been implicated in vascular function and diseases, as well as regulation of vascular tone also feature strongly among the migraine susceptibility loci identified from recent GWAS, underlining a central role for vascular aetiologies in common migraine [29, 169]. Other important pathways appear to be nitric oxide signalling and oxidative stress with the implication of loci near REST, GJA1, YAP1,

PRDM16, LRP1, and MRVI1. While FHM is primarily seen as a channelopathy, ion channel genes had been mostly missing from genetic associations in common migraine [140].

However, in addition to TPRM8, the ion channel KCKN5 was also identified as migraine susceptibility locus, as were other gene loci with roles in ion homeostasis (SLC24A3, ITPK1 and GJA1) [29] (Figure 1, Table 1). The presence of SNPs near JAG1, with the encoded protein JAGGED1 a ligand for NOTCH3, as well as NOTCH4 suggests further overlap with the monogenic disorders. In CADASIL NOTCH3 mutation KI mice some mutations disrupt the Jagged1-Notch3 interaction and downstream signalling via RBP/JK transcription factor pathway [170]. Also mutations in HTRA1 (near the migraine susceptibility GWAS SNP rs2223089) are causal for CARASIL which is a rare, recessive disorder with the same symptoms as CADASIL [171]. It may also be relevant that ITPK3 is located in a region that was previously mapped to a FHM locus in a large Spanish kindred, although no coding mutations in this or the other two genes in the region were detected by sequencing [172].

From GWAS loci to causal SNPs

Compared to candidate gene studies where understanding of the biology suggested which genes to test for association, GWAS yields genes for which there may be little known of their function, or it is difficult to relate their known function to the trait in question. Furthermore, associated SNPs are not usually causal and the causal variants have been notoriously difficult to find [150, 173]. Which gene is affected by the associated SNP/s is not always clear as they often reside outside gene boundaries. Even those within a gene may have an effect on alternative genes from a distance, as the body mass index associated SNPs in FTO have shown [174, 175]. Detailed analysis of the migraine associated variant rs12355831 located in the intron of ZDHHC6 [167] using RNA sequence data to find expression quantitative trait loci (eQTL), showed that it in fact tagged a functional variant in intron 20 of the neighbouring ACSL5 gene, which promotes skipping of exon 20 [176]. ACSL5 encodes a mitochondrial protein involved in the activation of long-chain fatty acids and this result suggests involvement of this pathway and the spliced ACSL-20 transcript in migraine. In an elegant study to dissect the role of an intronic SNP in PHACTR1, which is associated with

CAD and MI in addition to migraine, Beaudoin et al. fine-mapped the locus to prioritise a likely causal SNP at which alleles differential bound the transcription factor MEF2

[166].They then used CRISPR-Cas9 genome-editing methodology to show that deletion of the MEF2 binding site reduces PHACTR1 expression in endothelial cells, strengthening evidence for PHACTR1 in susceptibility of these traits and providing a mechanism for the

SNP effect. From the most recent GWAS, of the 38 loci, eQTL signals were only found at

HPSE2 and HEY2 [29]. This low number most likely reflects the fact that existing eQTL catalogues lack sufficient power, tissue specificity and development diversity to provide meaningful biological insight [177]. Expansion of gene expression resources, and further studies into understanding the function of the SNPs, as well as their target genes, will improve our general understanding of migraine pathophysiology.

Are migraine sub-types separate diseases?

Further increases in sample numbers will no doubt reveal more loci that contribute to migraine, as there are numerous sub-genome-wide significant peaks [29]. More importantly the increased power may allow dissection of migraine sub-types, or gender-specific differences. It has been questioned whether migraine subtypes are genetically separate or part of the same disease spectrum [25, 178]. Sub-type analysis by Gormley et al., revealed seven significantly associated genomic loci for MO (near TSPAN2, TRPM8, PHACTR1, FHL5,

ASTN2, near FGF6 and LRP1), but none for MA [29]. However, heterogeneity analysis implicated most of identified loci in both migraine sub-types and the lack of significant loci for MA was mainly due to smaller sample size. Other migraine sub-types such as menstrual migraine are yet to be considered as larger well-defined cohorts are required. Menstrual migraine remains in an Appendix in the ICHD-III [4], as it is unclear whether it should be classified separately to MO; better understanding of its genetic basis might resolve this.

Heterogeneity analysis of SNP effects across MA and MO subgroups of previously identified migraine susceptibility loci showed concordance in sub-types justifying meta-analysis of

GWAS data sets [179], however the reliance on large sample sizes means that disparate cohorts are put together and may hide true associations, making it more difficult to find genes specific for sub-types. There may also be selectivity in genetic associations for particular features of the migraine attack, e.g. pain character, duration, frequency, accompanying nausea, photophobia and triggers [168]. Thus the complexity of migraine pathology with features that may have different genetic causes does complicate the discovery of genuinely associated genes.

Co-morbidity of migraine with other diseases

As described in part I, some monogenic migraine disorders have a range of overlapping symptoms in addition to migraine, e.g. FHM is recognised to be co-morbid with vascular conditions [180], which suggests that common mechanisms exist between the disorders. In a similar vein common (polygenic) migraine is also co-morbid with a number of conditions including: vascular disorders such as stroke and heart disease [181-183]; neurological conditions such as epilepsy [184]; and psychiatric disorders such as anxiety, panic disorder, and depression [183, 185, 186]. Several theories have been proposed to explain the etiology of the association between migraine and co-morbid conditions, including unidirectional causal and bidirectional causal models, latent brain state models, and shared environmental or genetic risk factors [186]. With respect to the latter, a number of studies have shown that shared genetic factors are involved in co-morbid disorders, including for stroke [187], epilepsy, [188], and depression [189], with a number of GWAS migraine susceptibility loci overlapping between data sets, e.g. PHACTR1 is a susceptibility locus for both migraine,

CAD and MI. With respect to CAD and migraine, a shared genetic basis was found, but surprisingly in MO only, and the impact risk variants was in the opposite direction [190].

There is a bidirectional relationship between migraine and depression which can be explained, at least partly, by shared underlying genetic factors [189, 191]. However, using a genetic risk score analysis of GWAS SNP data Ligthart et al. [192] provided evidence that migraine with and without comorbid depression are genetically distinct disorders, with the implication being that for a subset of migraine patients with comorbid depression, migraine may be a symptom or consequence of the depression. Thus these patients’ migraine may require a different course of treatment to those with pure migraine and respond better to treatments that relate to the depression, e.g. the antidepressant drug amitriptyline is frequently used in migraine prevention [193]. Treatments that work in one comorbid condition may work in another, e.g. antiepileptic drugs are also effective in migraine and depression [194]. It is therefore important to delineate the comorbidities of migraine and recognise the genetic contribution because it can lead to better understanding of its pathophysiology and improve treatment strategies.

Ethnic-specific variation in migraine

The large migraine GWAS performed to date mainly included Caucasians. Some small replication studies have been carried out in Asian and Indian populations, with positive associations with some loci, e.g. LRP1 and PRDM16 [195-197]. However, there is the potential for ethnic-specific differences in migraine susceptibility, which may reflect SNPs present in a particular population. For instance the Norfolk Island population isolate which has a partly Polynesian genetic background has a high prevalence of migraine (25%) and a

GWAS for migraine revealed a number of novel loci of suggestive significance [198].

Variants with important functional consequences that are rare in European population may be much higher in other populations [199]; therefore, exploring genetic factors involved in migraine in alternative populations may yield different loci and provide further insights to disease mechanisms.

How much of the inheritance of migraine is now explained?

In common with the genetics underlying other complex human traits or disorders, such as obesity, hypertension and psychosis, the genomic loci identified so far only explain a part of the trait variance. Linkage disequilibrium analysis of the SNP data set from Gormley et al.

2 calculated the heritability (h g) captured for migraine to be 14.6%, with that for MO being

20.6% and for MA 10.6% [29]. Collecting phenotype and genotype data from such a large number of individuals was already a tour de force relying on extensive international collaborations and partnerships [169], but presumably as sample sizes increase the amount of heritability captured will also increase, particularly for MA, and other suggestive peaks which had not quite reached genome-wide significance will do so. However, apart from more SNPs other factors might also contribute to migraine heritability. There may be rare or family- specific variants with strong effects, and this might be particularly relevant to MA. The

TRESK and FASPS examples (Part I), as well as other family pedigrees with typical migraine mapped to a particular genomic region, but for which causal genes are not yet identified, support this notion. Structural variants may contribute to migraine susceptibility: this avenue has not been well explored in migraine and a study by Carreño et al. failed to find

CNVs of CACNA1A in FHM cases [82], but CNVs have been linked to neurodevelopmental disorders, e.g. deletions of SCN1A in epilepsy [200] (and references within). Epigenetic factors may also play a role: little has been reported in this regard to date, but epigenetic modifications, such as DNA methylation and histone modifications, may be a worthwhile avenue of exploration in migraine [201]. Environmental factors can trigger migraine and these interact with the genome via epigenetic mechanisms. There are monozygotic twins who are discordant for both common and familial hemiplegic migraine [202, 203], and DNA methylation variants have been associated with other complex neurological disorders such as schizophrenia, bipolar disorder and depression [204, 205], as well as pain sensitivity [206].

Accessibility of relevant tissues for human studies is difficult as epigenetic modifications are tissue specific, but there is some evidence that variants associated with phenotypes expressed in the brain, fat or vasculature can be also be detected in blood DNA [206-208].

Diagnostic potential for common variants in migraine

Much progress has been made in identifying common migraine risk variants from GWAS. As the effect sizes are too small to make definitive predictions of migraine outcome, it is yet to be seen whether this information will be useful diagnostically or to influence patient care.

However, there is the potential for it to be of used to identify sub-types of migraine that might benefit from particular treatment approaches as described above, or to stratify risk. For example, some studies have used susceptibility variants to calculate a genetic load, or additive polygenic risk score to show that a higher score correlates with a diagnosis of migraine [209, 210], or its severity [211], however sensitivity and specificity are low.

Where common variants may be useful is in understanding drug reactions and efficacy of therapies in individuals to allow treatments to be tailored more personally. For example, a single risk variant, rs2651899 in PRDM16, was significantly associated with efficacy of triptans with an odds ratio (OR) of treatment success of 1.3, and the OR was 2.6 in patients with a higher combined genetic score when 12 migraine susceptibility loci were considered

[212]. Also a clinical trial on the effect of folate and vitamin B supplementation on migraine symptoms found that common variants in enzymes of the folate pathway influenced treatment response [213]. Variants in the genes encoding drug targets, but also the proteins and enzymes that transport and metabolise drugs can affect their pharmacological utility [214-

216]. The effect of common variation on efficacies of migraine drugs and treatments has yet to be explored in a genome-wide manner, but may be particularly useful in understanding drug reactions in individuals and the goal of personalised medicine.

Summary and future directions

While there is limited overlap in the actual genes that cause either monogenic or polygenic migraine, there is commonality in the main pathways affected, i.e. neuronal and synapse development and function and glutamatergic neurotransmission, and vascular development and function (Figure 1). Therefore, the more tractable functional studies that monogenic disorders afford can inform on mechanisms and treatment strategies for common migraine. A greater appreciation for the vascular component in the risk of polygenic migraine that GWAS have brought may lead to increased focus on this aspect with regards to functional studies to understand mechanisms, as well as in prevention and treatment strategies. A key question that remains regards the role, if there is one, of CSD in MO patients that do not experience aura.

If it is not CSD, what are the mechanisms for generating migraine without it? Further dissection of the genetic factors involved in MO may contribute to answering this.

As mentioned in Part I, CRISPR-Cas9 and iPSC technologies will enable more high- throughput ways to investigate function of mutations that are linked to migraine, but they will also allow functional consequences of common variants to be studied more efficiently. Other emerging technologies such as longer read sequencing technologies (e.g. PacBio) will make it more feasible to study how large structural variation might contribute to migraine susceptibility, and high throughput epigenetic technologies (e.g. DNA methylation arrays and methods to assess DNA methylation or chromatin modifications combined with NGS approaches) will allow more comprehensive exploration of this aspect. In addition to the fascinating biological insights that studies into the genetic basis of migraine reveals, the end goal is improved outcomes for patients to reduce the personal and social burden of migraine.

References

1. Lipton RB, Bigal ME, Diamond M, Freitag F, Reed ML, Stewart WF: Migraine prevalence, disease burden, and the need for preventive therapy. Neurology. 2007;68:343-349. 2. Lipton RB, Stewart WF, Diamond S, Diamond ML, Reed M: Prevalence and burden of migraine in the United States: data from the American Migraine Study II. Headache. 2001;41:646-657. 3. Global Burden of Disease Study C: Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;386:743-800. 4. Headache Classification Committee of the International Headache S: The International Classification of Headache Disorders, 3rd edition (beta version). Cephalalgia. 2013;33:629-808. 5. Vetvik KG, Macgregor EA, Lundqvist C, Russell MB: Prevalence of menstrual migraine: a population-based study. Cephalalgia. 2014;34:280-288. 6. Pavlovic JM, Stewart WF, Bruce CA, Gorman JA, Sun H, Buse DC, Lipton RB: Burden of migraine related to menses: results from the AMPP study. J Headache Pain. 2015;16:24. 7. Olesen J, Goadsby, J.G., Ramadan, N.H., Tfelt-Hansen, P. and Welch, K.M.H. (Eds.): The Headaches third ed. 3rd edn: Lippincott Williams & Wilkins, Philadelphia; 2006. 8. Noseda R, Burstein R: Migraine pathophysiology: anatomy of the trigeminovascular pathway and associated neurological symptoms, cortical spreading depression, sensitization, and modulation of pain. Pain. 2013;154 Suppl 1:S44-53. 9. Levy D: Endogenous mechanisms underlying the activation and sensitization of meningeal nociceptors: the role of immuno-vascular interactions and cortical spreading depression. Curr Pain Headache Rep. 2012;16:270-277. 10. Leao AA: The slow voltage variation of cortical spreading depression of activity. Electroencephalogr Clin Neurophysiol. 1951;3:315-321. 11. Kramer DR, Fujii T, Ohiorhenuan I, Liu CY: Cortical spreading depolarization: Pathophysiology, implications, and future directions. J Clin Neurosci. 2016;24:22-27. 12. Bolay H, Reuter U, Dunn AK, Huang Z, Boas DA, Moskowitz MA: Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model. Nat Med. 2002;8:136-142. 13. Zhang X, Levy D, Noseda R, Kainz V, Jakubowski M, Burstein R: Activation of meningeal nociceptors by cortical spreading depression: implications for migraine with aura. J Neurosci. 2010;30:8807-8814. 14. Zhang X, Levy D, Kainz V, Noseda R, Jakubowski M, Burstein R: Activation of central trigeminovascular neurons by cortical spreading depression. Ann Neurol. 2011;69:855-865. 15. Karatas H, Erdener SE, Gursoy-Ozdemir Y, Lule S, Eren-Kocak E, Sen ZD, Dalkara T: Spreading depression triggers headache by activating neuronal Panx1 channels. Science. 2013;339:1092-1095. 16. Burstein R, Noseda R, Borsook D: Migraine: multiple processes, complex pathophysiology. J Neurosci. 2015;35:6619-6629. 17. Stankewitz A, Aderjan D, Eippert F, May A: Trigeminal nociceptive transmission in migraineurs predicts migraine attacks. J Neurosci. 2011;31:1937-1943. 18. Lang E, Kaltenhauser M, Neundorfer B, Seidler S: Hyperexcitability of the primary somatosensory cortex in migraine--a magnetoencephalographic study. Brain. 2004;127:2459-2469. 19. Aurora SK, Barrodale PM, Tipton RL, Khodavirdi A: Brainstem dysfunction in chronic migraine as evidenced by neurophysiological and positron emission tomography studies. Headache. 2007;47:996-1003; discussion 1004-1007. 20. Vecchia D, Pietrobon D: Migraine: a disorder of brain excitatory-inhibitory balance? Trends Neurosci. 2012;35:507-520. 21. Kelman L: The triggers or precipitants of the acute migraine attack. Cephalalgia. 2007;27:394-402. 22. Levy D, Strassman AM, Burstein R: A critical view on the role of migraine triggers in the genesis of migraine pain. Headache. 2009;49:953-957. 23. Vetvik KG, MacGregor EA: Sex differences in the epidemiology, clinical features, and pathophysiology of migraine. Lancet Neurol. 2016. 24. Russell MB, Iselius L, Olesen J: Migraine without aura and migraine with aura are inherited disorders. Cephalalgia. 1996;16:305-309. 25. Russell MB, Ulrich V, Gervil M, Olesen J: Migraine without aura and migraine with aura are distinct disorders. A population-based twin survey. Headache. 2002;42:332- 336. 26. Honkasalo ML, Kaprio J, Winter T, Heikkila K, Sillanpaa M, Koskenvuo M: Migraine and concomitant symptoms among 8167 adult twin pairs. Headache. 1995;35:70-78. 27. Mulder EJ, Van Baal C, Gaist D, Kallela M, Kaprio J, Svensson DA, Nyholt DR, Martin NG, MacGregor AJ, Cherkas LF, et al: Genetic and environmental influences on migraine: a twin study across six countries. Twin Res. 2003;6:422-431. 28. Polderman TJ, Benyamin B, de Leeuw CA, Sullivan PF, van Bochoven A, Visscher PM, Posthuma D: Meta-analysis of the heritability of human traits based on fifty years of twin studies. Nat Genet. 2015;47:702-709. 29. Gormley P, Anttila V, Winsvold BS, Palta P, Esko T, Pers TH, Farh KH, Cuenca- Leon E, Muona M, Furlotte NA, et al: Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine. Nat Genet. 2016;48:856-866. 30. Pelzer N, Stam AH, Haan J, Ferrari MD, Terwindt GM: Familial and sporadic hemiplegic migraine: diagnosis and treatment. Curr Treat Options Neurol. 2013;15:13-27. 31. Kors EE, Terwindt GM, Vermeulen FL, Fitzsimons RB, Jardine PE, Heywood P, Love S, van den Maagdenberg AM, Haan J, Frants RR, Ferrari MD: Delayed cerebral edema and fatal coma after minor head trauma: role of the CACNA1A calcium channel subunit gene and relationship with familial hemiplegic migraine. Ann Neurol. 2001;49:753-760. 32. Russell MB, Ducros A: Sporadic and familial hemiplegic migraine: pathophysiological mechanisms, clinical characteristics, diagnosis, and management. Lancet Neurol. 2011;10:457-470. 33. Lykke Thomsen L, Kirchmann Eriksen M, Faerch Romer S, Andersen I, Ostergaard E, Keiding N, Olesen J, Russell MB: An epidemiological survey of hemiplegic migraine. Cephalalgia. 2002;22:361-375. 34. Thomsen LL, Ostergaard E, Olesen J, Russell MB: Evidence for a separate type of migraine with aura: sporadic hemiplegic migraine. Neurology. 2003;60:595-601. 35. Silberstein SD, Dodick DW: Migraine genetics: Part II. Headache. 2013;53:1218- 1229. 36. Ophoff RA, Terwindt GM, Vergouwe MN, van Eijk R, Oefner PJ, Hoffman SM, Lamerdin JE, Mohrenweiser HW, Bulman DE, Ferrari M, et al: Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell. 1996;87:543-552. 37. Diriong S, Lory P, Williams ME, Ellis SB, Harpold MM, Taviaux S: Chromosomal localization of the human genes for alpha 1A, alpha 1B, and alpha 1E voltage- dependent Ca2+ channel subunits. Genomics. 1995;30:605-609. 38. Catterall WA: Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium. 1998;24:307-323. 39. de Vries B, Frants RR, Ferrari MD, van den Maagdenberg AM: Molecular genetics of migraine. Hum Genet. 2009;126:115-132. 40. Di Lorenzo C, Grieco GS, Santorelli FM: Migraine headache: a review of the molecular genetics of a common disorder. J Headache Pain. 2012;13:571-580. 41. Tottene A, Fellin T, Pagnutti S, Luvisetto S, Striessnig J, Fletcher C, Pietrobon D: Familial hemiplegic migraine mutations increase Ca(2+) influx through single human CaV2.1 channels and decrease maximal CaV2.1 current density in neurons. Proc Natl Acad Sci U S A. 2002;99:13284-13289. 42. van den Maagdenberg AM, Pietrobon D, Pizzorusso T, Kaja S, Broos LA, Cesetti T, van de Ven RC, Tottene A, van der Kaa J, Plomp JJ, et al: A Cacna1a knockin migraine mouse model with increased susceptibility to cortical spreading depression. Neuron. 2004;41:701-710. 43. van den Maagdenberg AM, Pizzorusso T, Kaja S, Terpolilli N, Shapovalova M, Hoebeek FE, Barrett CF, Gherardini L, van de Ven RC, Todorov B, et al: High cortical spreading depression susceptibility and migraine-associated symptoms in Ca(v)2.1 S218L mice. Ann Neurol. 2010;67:85-98. 44. Chen SP, Tolner EA, Eikermann-Haerter K: Animal models of monogenic migraine. Cephalalgia. 2016;36:704-721. 45. Ferrari MD, Klever RR, Terwindt GM, Ayata C, van den Maagdenberg AM: Migraine pathophysiology: lessons from mouse models and human genetics. Lancet Neurol. 2015;14:65-80. 46. Tottene A, Conti R, Fabbro A, Vecchia D, Shapovalova M, Santello M, van den Maagdenberg AM, Ferrari MD, Pietrobon D: Enhanced excitatory transmission at cortical synapses as the basis for facilitated spreading depression in Ca(v)2.1 knockin migraine mice. Neuron. 2009;61:762-773. 47. Pietrobon D, Moskowitz MA: Pathophysiology of migraine. Annu Rev Physiol. 2013;75:365-391. 48. Eising E, Shyti R, t Hoen PA, Vijfhuizen LS, Huisman SM, Broos LA, Mahfouz A, Reinders MJ, Ferrari MD, Tolner EA, et al: Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine. Mol Neurobiol. 2016. 49. Thomsen LL, Kirchmann M, Bjornsson A, Stefansson H, Jensen RM, Fasquel AC, Petursson H, Stefansson M, Frigge ML, Kong A, et al: The genetic spectrum of a population-based sample of familial hemiplegic migraine. Brain. 2007;130:346-356. 50. Eikermann-Haerter K, Dilekoz E, Kudo C, Savitz SI, Waeber C, Baum MJ, Ferrari MD, van den Maagdenberg AM, Moskowitz MA, Ayata C: Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1. J Clin Invest. 2009;119:99-109. 51. Pereira Mda C, Morais S, Sequeiros J, Alonso I: Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-beta and Notch Signaling Pathways as Modifiers of CACNA1A. ASN Neuro. 2016;8. 52. Zhuchenko O, Bailey J, Bonnen P, Ashizawa T, Stockton DW, Amos C, Dobyns WB, Subramony SH, Zoghbi HY, Lee CC: Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet. 1997;15:62-69. 53. Jen J, Kim GW, Baloh RW: Clinical spectrum of episodic ataxia type 2. Neurology. 2004;62:17-22. 54. Sinke RJ, Ippel EF, Diepstraten CM, Beemer FA, Wokke JH, van Hilten BJ, Knoers NV, van Amstel HK, Kremer HP: Clinical and molecular correlations in spinocerebellar ataxia type 6: a study of 24 Dutch families. Arch Neurol. 2001;58:1839-1844. 55. Mantuano E, Romano S, Veneziano L, Gellera C, Castellotti B, Caimi S, Testa D, Estienne M, Zorzi G, Bugiani M, et al: Identification of novel and recurrent CACNA1A gene mutations in fifteen patients with episodic ataxia type 2. J Neurol Sci. 2010;291:30-36. 56. Watase K, Barrett CF, Miyazaki T, Ishiguro T, Ishikawa K, Hu Y, Unno T, Sun Y, Kasai S, Watanabe M, et al: Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels. Proc Natl Acad Sci U S A. 2008;105:11987-11992. 57. De Fusco M, Marconi R, Silvestri L, Atorino L, Rampoldi L, Morgante L, Ballabio A, Aridon P, Casari G: Haploinsufficiency of ATP1A2 encoding the Na+/K+ pump alpha2 subunit associated with familial hemiplegic migraine type 2. Nat Genet. 2003;33:192-196. 58. Skou JC: The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim Biophys Acta. 1957;23:394-401. 59. Cholet N, Pellerin L, Magistretti PJ, Hamel E: Similar perisynaptic glial localization for the Na+,K+-ATPase alpha 2 subunit and the glutamate transporters GLAST and GLT-1 in the rat somatosensory cortex. Cereb Cortex. 2002;12:515-525. 60. Benarroch EE: Glutamate transporters: diversity, function, and involvement in neurologic disease. Neurology. 2010;74:259-264. 61. Friedrich T, Tavraz NN, Junghans C: ATP1A2 Mutations in Migraine: Seeing through the Facets of an Ion Pump onto the Neurobiology of Disease. Front Physiol. 2016;7:239. 62. Isaksen TJ, Lykke-Hartmann K: Insights into the Pathology of the alpha2- Na(+)/K(+)-ATPase in Neurological Disorders; Lessons from Animal Models. Front Physiol. 2016;7:161. 63. Leo L, Gherardini L, Barone V, De Fusco M, Pietrobon D, Pizzorusso T, Casari G: Increased susceptibility to cortical spreading depression in the mouse model of familial hemiplegic migraine type 2. PLoS Genet. 2011;7:e1002129. 64. Bottger P, Glerup S, Gesslein B, Illarionova NB, Isaksen TJ, Heuck A, Clausen BH, Fuchtbauer EM, Gramsbergen JB, Gunnarson E, et al: Glutamate-system defects behind psychiatric manifestations in a familial hemiplegic migraine type 2 disease- mutation mouse model. Sci Rep. 2016;6:22047. 65. Capuani C, Melone M, Tottene A, Bragina L, Crivellaro G, Santello M, Casari G, Conti F, Pietrobon D: Defective glutamate and K+ clearance by cortical astrocytes in familial hemiplegic migraine type 2. EMBO Mol Med. 2016;8:967-986. 66. Dichgans M, Freilinger T, Eckstein G, Babini E, Lorenz-Depiereux B, Biskup S, Ferrari MD, Herzog J, van den Maagdenberg AM, Pusch M, Strom TM: Mutation in the neuronal voltage-gated sodium channel SCN1A in familial hemiplegic migraine. Lancet. 2005;366:371-377. 67. Catterall WA: From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron. 2000;26:13-25. 68. Meng H, Xu HQ, Yu L, Lin GW, He N, Su T, Shi YW, Li B, Wang J, Liu XR, et al: The SCN1A mutation database: updating information and analysis of the relationships among genotype, functional alteration, and phenotype. Hum Mutat. 2015;36:573-580. 69. Zuberi SM, Brunklaus A, Birch R, Reavey E, Duncan J, Forbes GH: Genotype- phenotype associations in SCN1A-related epilepsies. Neurology. 2011;76:594-600. 70. Fan C, Wolking S, Lehmann-Horn F, Hedrich UB, Freilinger T, Lerche H, Borck G, Kubisch C, Jurkat-Rott K: Early-onset familial hemiplegic migraine due to a novel SCN1A mutation. Cephalalgia. 2016. 71. Ogiwara I, Miyamoto H, Morita N, Atapour N, Mazaki E, Inoue I, Takeuchi T, Itohara S, Yanagawa Y, Obata K, et al: Nav1.1 localizes to axons of parvalbumin- positive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation. J Neurosci. 2007;27:5903-5914. 72. Yu FH, Mantegazza M, Westenbroek RE, Robbins CA, Kalume F, Burton KA, Spain WJ, McKnight GS, Scheuer T, Catterall WA: Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci. 2006;9:1142-1149. 73. Chastan N, Lebas A, Legoff F, Parain D, Guyant-Marechal L: Clinical and electroencephalographic abnormalities during the full duration of a sporadic hemiplegic migraine attack. Neurophysiol Clin. 2016;46:307-311. 74. Weller CM, Pelzer N, de Vries B, Lopez MA, De Fabregues O, Pascual J, Arroyo MA, Koelewijn SC, Stam AH, Haan J, et al: Two novel SCN1A mutations identified in families with familial hemiplegic migraine. Cephalalgia. 2014;34:1062-1069. 75. Vanmolkot KR, Babini E, de Vries B, Stam AH, Freilinger T, Terwindt GM, Norris L, Haan J, Frants RR, Ramadan NM, et al: The novel p.L1649Q mutation in the SCN1A epilepsy gene is associated with familial hemiplegic migraine: genetic and functional studies. Mutation in brief #957. Online. Hum Mutat. 2007;28:522. 76. Castro MJ, Stam AH, Lemos C, de Vries B, Vanmolkot KR, Barros J, Terwindt GM, Frants RR, Sequeiros J, Ferrari MD, et al: First mutation in the voltage-gated Nav1.1 subunit gene SCN1A with co-occurring familial hemiplegic migraine and epilepsy. Cephalalgia. 2009;29:308-313. 77. Cestele S, Labate A, Rusconi R, Tarantino P, Mumoli L, Franceschetti S, Annesi G, Mantegazza M, Gambardella A: Divergent effects of the T1174S SCN1A mutation associated with seizures and hemiplegic migraine. Epilepsia. 2013;54:927-935. 78. Vahedi K, Depienne C, Le Fort D, Riant F, Chaine P, Trouillard O, Gaudric A, Morris MA, Leguern E, Tournier-Lasserve E, Bousser MG: Elicited repetitive daily blindness: a new phenotype associated with hemiplegic migraine and SCN1A mutations. Neurology. 2009;72:1178-1183. 79. Kahlig KM, Rhodes TH, Pusch M, Freilinger T, Pereira-Monteiro JM, Ferrari MD, van den Maagdenberg AM, Dichgans M, George AL, Jr.: Divergent sodium channel defects in familial hemiplegic migraine. Proc Natl Acad Sci U S A. 2008;105:9799- 9804. 80. Pellacani S, Sicca F, Di Lorenzo C, Grieco GS, Valvo G, Cereda C, Rubegni A, Santorelli FM: The Revolution in Migraine Genetics: From Aching Channels Disorders to a Next-Generation Medicine. Front Cell Neurosci. 2016;10:156. 81. de Vries B, Freilinger T, Vanmolkot KR, Koenderink JB, Stam AH, Terwindt GM, Babini E, van den Boogerd EH, van den Heuvel JJ, Frants RR, et al: Systematic analysis of three FHM genes in 39 sporadic patients with hemiplegic migraine. Neurology. 2007;69:2170-2176. 82. Carreno O, Corominas R, Serra SA, Sintas C, Fernandez-Castillo N, Vila-Pueyo M, Toma C, Gene GG, Pons R, Llaneza M, et al: Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies. Mol Genet Genomic Med. 2013;1:206-222. 83. Riant F, Ducros A, Ploton C, Barbance C, Depienne C, Tournier-Lasserve E: De novo mutations in ATP1A2 and CACNA1A are frequent in early-onset sporadic hemiplegic migraine. Neurology. 2010;75:967-972. 84. Simons C, Rash LD, Crawford J, Ma L, Cristofori-Armstrong B, Miller D, Ru K, Baillie GJ, Alanay Y, Jacquinet A, et al: Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy. Nat Genet. 2015;47:73-77. 85. Depienne C, Trouillard O, Gourfinkel-An I, Saint-Martin C, Bouteiller D, Graber D, Barthez-Carpentier MA, Gautier A, Villeneuve N, Dravet C, et al: Mechanisms for variable expressivity of inherited SCN1A mutations causing Dravet syndrome. J Med Genet. 2010;47:404-410. 86. Jen JC, Wan J, Palos TP, Howard BD, Baloh RW: Mutation in the glutamate transporter EAAT1 causes episodic ataxia, hemiplegia, and seizures. Neurology. 2005;65:529-534. 87. Suzuki M, Van Paesschen W, Stalmans I, Horita S, Yamada H, Bergmans BA, Legius E, Riant F, De Jonghe P, Li Y, et al: Defective membrane expression of the Na(+)- HCO(3)(-) cotransporter NBCe1 is associated with familial migraine. Proc Natl Acad Sci U S A. 2010;107:15963-15968. 88. Ebrahimi-Fakhari D, Saffari A, Westenberger A, Klein C: The evolving spectrum of PRRT2-associated paroxysmal diseases. Brain. 2015;138:3476-3495. 89. Riant F, Roze E, Barbance C, Meneret A, Guyant-Marechal L, Lucas C, Sabouraud P, Trebuchon A, Depienne C, Tournier-Lasserve E: PRRT2 mutations cause hemiplegic migraine. Neurology. 2012;79:2122-2124. 90. Schwenk J, Harmel N, Brechet A, Zolles G, Berkefeld H, Muller CS, Bildl W, Baehrens D, Huber B, Kulik A, et al: High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes. Neuron. 2012;74:621- 633. 91. Lee HY, Huang Y, Bruneau N, Roll P, Roberson ED, Hermann M, Quinn E, Maas J, Edwards R, Ashizawa T, et al: Mutations in the gene PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions. Cell Rep. 2012;1:2-12. 92. Condliffe SB, Corradini I, Pozzi D, Verderio C, Matteoli M: Endogenous SNAP-25 regulates native voltage-gated calcium channels in glutamatergic neurons. J Biol Chem. 2010;285:24968-24976. 93. Gardiner AR, Jaffer F, Dale RC, Labrum R, Erro R, Meyer E, Xiromerisiou G, Stamelou M, Walker M, Kullmann D, et al: The clinical and genetic heterogeneity of paroxysmal dyskinesias. Brain. 2015;138:3567-3580. 94. Wu L, Tang HD, Huang XJ, Zheng L, Liu XL, Wang T, Wang JY, Cao L, Chen SD: PRRT2 truncated mutations lead to nonsense-mediated mRNA decay in Paroxysmal Kinesigenic Dyskinesia. Parkinsonism Relat Disord. 2014;20:1399-1404. 95. Brueckner F, Kohl B, Puest B, Gassner S, Osseforth J, Lindenau M, Stodieck S, Biskup S, Lohmann E: Unusual variability of PRRT2 linked phenotypes within a family. Eur J Paediatr Neurol. 2014;18:540-542. 96. Bhatia KP: Paroxysmal dyskinesias. Mov Disord. 2011;26:1157-1165. 97. Pelzer N, de Vries B, Kamphorst JT, Vijfhuizen LS, Ferrari MD, Haan J, van den Maagdenberg AM, Terwindt GM: PRRT2 and hemiplegic migraine: a complex association. Neurology. 2014;83:288-290. 98. Lafreniere RG, Cader MZ, Poulin JF, Andres-Enguix I, Simoneau M, Gupta N, Boisvert K, Lafreniere F, McLaughlan S, Dube MP, et al: A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura. Nat Med. 2010;16:1157-1160. 99. Enyedi P, Czirjak G: Properties, regulation, pharmacology, and functions of the K(2)p channel, TRESK. Pflugers Arch. 2015;467:945-958. 100. Dobler T, Springauf A, Tovornik S, Weber M, Schmitt A, Sedlmeier R, Wischmeyer E, Doring F: TRESK two-pore-domain K+ channels constitute a significant component of background potassium currents in murine dorsal root ganglion neurones. J Physiol. 2007;585:867-879. 101. Chae YJ, Zhang J, Au P, Sabbadini M, Xie GX, Yost CS: Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK. Anesthesiology. 2010;113:1326-1337. 102. Liu P, Xiao Z, Ren F, Guo Z, Chen Z, Zhao H, Cao YQ: Functional analysis of a migraine-associated TRESK K+ channel mutation. J Neurosci. 2013;33:12810-12824. 103. Andres-Enguix I, Shang L, Stansfeld PJ, Morahan JM, Sansom MS, Lafreniere RG, Roy B, Griffiths LR, Rouleau GA, Ebers GC, et al: Functional analysis of missense variants in the TRESK (KCNK18) K channel. Sci Rep. 2012;2:237. 104. Chabriat H, Joutel A, Dichgans M, Tournier-Lasserve E, Bousser MG: Cadasil. Lancet Neurol. 2009;8:643-653. 105. Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, Alamowitch S, Domenga V, Cecillion M, Marechal E, et al: Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature. 1996;383:707- 710. 106. Prakash N, Hansson E, Betsholtz C, Mitsiadis T, Lendahl U: Mouse Notch 3 expression in the pre- and postnatal brain: relationship to the stroke and dementia syndrome CADASIL. Exp Cell Res. 2002;278:31-44. 107. Hori K, Sen A, Artavanis-Tsakonas S: Notch signaling at a glance. J Cell Sci. 2013;126:2135-2140. 108. Eikermann-Haerter K, Yuzawa I, Dilekoz E, Joutel A, Moskowitz MA, Ayata C: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy syndrome mutations increase susceptibility to spreading depression. Ann Neurol. 2011;69:413-418. 109. Arboleda-Velasquez JF, Zhou Z, Shin HK, Louvi A, Kim HH, Savitz SI, Liao JK, Salomone S, Ayata C, Moskowitz MA, Artavanis-Tsakonas S: Linking Notch signaling to ischemic stroke. Proc Natl Acad Sci U S A. 2008;105:4856-4861. 110. Arboleda-Velasquez JF, Manent J, Lee JH, Tikka S, Ospina C, Vanderburg CR, Frosch MP, Rodriguez-Falcon M, Villen J, Gygi S, et al: Hypomorphic Notch 3 alleles link Notch signaling to ischemic cerebral small-vessel disease. Proc Natl Acad Sci U S A. 2011;108:E128-135. 111. Stam AH, Kothari PH, Shaikh A, Gschwendter A, Jen JC, Hodgkinson S, Hardy TA, Hayes M, Kempster PA, Kotschet KE, et al: Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations. Brain. 2016. 112. Richards A, van den Maagdenberg AM, Jen JC, Kavanagh D, Bertram P, Spitzer D, Liszewski MK, Barilla-Labarca ML, Terwindt GM, Kasai Y, et al: C-terminal truncations in human 3'-5' DNA exonuclease TREX1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy. Nat Genet. 2007;39:1068-1070. 113. Rice GI, Rodero MP, Crow YJ: Human disease phenotypes associated with mutations in TREX1. J Clin Immunol. 2015;35:235-243. 114. Peschke K, Achleitner M, Frenzel K, Gerbaulet A, Ada SR, Zeller N, Lienenklaus S, Lesche M, Poulet C, Naumann R, et al: Loss of Trex1 in Dendritic Cells Is Sufficient To Trigger Systemic Autoimmunity. J Immunol. 2016;197:2157-2166. 115. Grieves JL, Fye JM, Harvey S, Grayson JM, Hollis T, Perrino FW: Exonuclease TREX1 degrades double-stranded DNA to prevent spontaneous lupus-like inflammatory disease. Proc Natl Acad Sci U S A. 2015;112:5117-5122. 116. Hasan M, Fermaintt CS, Gao N, Sakai T, Miyazaki T, Jiang S, Li QZ, Atkinson JP, Morse HC, 3rd, Lehrman MA, Yan N: Cytosolic Nuclease TREX1 Regulates Oligosaccharyltransferase Activity Independent of Nuclease Activity to Suppress Immune Activation. Immunity. 2015;43:463-474. 117. Breedveld G, de Coo IF, Lequin MH, Arts WF, Heutink P, Gould DB, John SW, Oostra B, Mancini GM: Novel mutations in three families confirm a major role of COL4A1 in hereditary porencephaly. J Med Genet. 2006;43:490-495. 118. Stam AH, Haan J, van den Maagdenberg AM, Ferrari MD, Terwindt GM: Migraine and genetic and acquired vasculopathies. Cephalalgia. 2009;29:1006-1017. 119. Jeanne M, Jorgensen J, Gould DB: Molecular and Genetic Analyses of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention. Circulation. 2015;131:1555-1565. 120. Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, Saigoh N, Saigoh K, Ptacek LJ, Fu YH: Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome. Nature. 2005;434:640-644. 121. Xu Y, Toh KL, Jones CR, Shin JY, Fu YH, Ptacek LJ: Modeling of a human circadian mutation yields insights into clock regulation by PER2. Cell. 2007;128:59- 70. 122. Brennan KC, Bates EA, Shapiro RE, Zyuzin J, Hallows WC, Huang Y, Lee HY, Jones CR, Fu YH, Charles AC, Ptacek LJ: Casein kinase idelta mutations in familial migraine and advanced sleep phase. Sci Transl Med. 2013;5:183ra156, 181-111. 123. Alstadhaug KB: Migraine and the hypothalamus. Cephalalgia. 2009;29:809-817. 124. Maniyar FH, Sprenger T, Monteith T, Schankin C, Goadsby PJ: Brain activations in the premonitory phase of nitroglycerin-triggered migraine attacks. Brain. 2014;137:232-241. 125. Ahn AH, Goadsby PJ: Migraine and sleep: new connections. Cerebrum. 2013;2013:15. 126. Schurks M: Genetics of migraine in the age of genome-wide association studies. J Headache Pain. 2012;13:1-9. 127. Maher BH, Griffiths LR: Identification of molecular genetic factors that influence migraine. Mol Genet Genomics. 2011;285:433-446. 128. Battistini S, Stenirri S, Piatti M, Gelfi C, Righetti PG, Rocchi R, Giannini F, Battistini N, Guazzi GC, Ferrari M, Carrera P: A new CACNA1A gene mutation in acetazolamide-responsive familial hemiplegic migraine and ataxia. Neurology. 1999;53:38-43. 129. Omata T, Takanashi J, Wada T, Arai H, Tanabe Y: Genetic diagnosis and acetazolamide treatment of familial hemiplegic migraine. Brain Dev. 2011;33:332- 334. 130. Artto V, Nissila M, Wessman M, Palotie A, Farkkila M, Kallela M: Treatment of hemiplegic migraine with triptans. Eur J Neurol. 2007;14:1053-1056. 131. Stam AH, Luijckx GJ, Poll-The BT, Ginjaar IB, Frants RR, Haan J, Ferrari MD, Terwindt GM, van den Maagdenberg AM: Early seizures and cerebral oedema after trivial head trauma associated with the CACNA1A S218L mutation. J Neurol Neurosurg Psychiatry. 2009;80:1125-1129. 132. Dale RC, Gardiner A, Branson JA, Houlden H: Benefit of carbamazepine in a patient with hemiplegic migraine associated with PRRT2 mutation. Dev Med Child Neurol. 2014;56:910. 133. Rudkjobing LA, Esserlind AL, Olesen J: Future possibilities in migraine genetics. J Headache Pain. 2012;13:505-511. 134. Nemeth AH, Kwasniewska AC, Lise S, Parolin Schnekenberg R, Becker EB, Bera KD, Shanks ME, Gregory L, Buck D, Zameel Cader M, et al: Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model. Brain. 2013;136:3106-3118. 135. Handel AE, Chintawar S, Lalic T, Whiteley E, Vowles J, Giustacchini A, Argoud K, Sopp P, Nakanishi M, Bowden R, et al: Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics. Hum Mol Genet. 2016;25:989-1000. 136. Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA: Cerebral organoids model human brain development and microcephaly. Nature. 2013;501:373-379. 137. Sander JD, Joung JK: CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32:347-355. 138. Gasparini CF, Sutherland HG, Griffiths LR: Studies on the pathophysiology and genetic basis of migraine. Curr Genomics. 2013;14:300-315. 139. Kondratieva N, Azimova J, Skorobogatykh K, Sergeev A, Naumova E, Kokaeva Z, Anuchina A, Rudko O, Tabeeva G, Klimov E: Biomarkers of migraine: Part 1 - Genetic markers. J Neurol Sci. 2016;369:63-76. 140. Nyholt DR, LaForge KS, Kallela M, Alakurtti K, Anttila V, Farkkila M, Hamalainen E, Kaprio J, Kaunisto MA, Heath AC, et al: A high-density association screen of 155 ion transport genes for involvement with common migraine. Hum Mol Genet. 2008;17:3318-3331. 141. Rubino E, Ferrero M, Rainero I, Binello E, Vaula G, Pinessi L: Association of the C677T polymorphism in the MTHFR gene with migraine: a meta-analysis. Cephalalgia. 2009;29:818-825. 142. Schurks M, Rist PM, Kurth T: MTHFR 677C>T and ACE D/I polymorphisms in migraine: a systematic review and meta-analysis. Headache. 2010;50:588-599. 143. Samaan Z, Gaysina D, Cohen-Woods S, Craddock N, Jones L, Korszun A, Owen M, Mente A, McGuffin P, Farmer A: Methylenetetrahydrofolate reductase gene variant (MTHFR C677T) and migraine: a case control study and meta-analysis. BMC Neurol. 2011;11:66. 144. Liu R, Geng P, Ma M, Yu S, Yang M, He M, Dong Z, Zhang W: MTHFR C677T polymorphism and migraine risk: a meta-analysis. J Neurol Sci. 2014;336:68-73. 145. Kaunisto MA, Kallela M, Hamalainen E, Kilpikari R, Havanka H, Harno H, Nissila M, Sako E, Ilmavirta M, Liukkonen J, et al: Testing of variants of the MTHFR and ESR1 genes in 1798 Finnish individuals fails to confirm the association with migraine with aura. Cephalalgia. 2006;26:1462-1472. 146. Todt U, Freudenberg J, Goebel I, Netzer C, Heinze A, Heinze-Kuhn K, Gobel H, Kubisch C: MTHFR C677T polymorphism and migraine with aura. Ann Neurol. 2006;60:621-622; author reply 622-623. 147. de Vries B, Anttila V, Freilinger T, Wessman M, Kaunisto MA, Kallela M, Artto V, Vijfhuizen LS, Gobel H, Dichgans M, et al: Systematic re-evaluation of genes from candidate gene association studies in migraine using a large genome-wide association data set. Cephalalgia. 2016;36:604-614. 148. Nicolae DL, Gamazon E, Zhang W, Duan S, Dolan ME, Cox NJ: Trait-associated SNPs are more likely to be eQTLs: annotation to enhance discovery from GWAS. PLoS Genet. 2010;6:e1000888. 149. Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, Reynolds AP, Sandstrom R, Qu H, Brody J, et al: Systematic localization of common disease- associated variation in regulatory DNA. Science. 2012;337:1190-1195. 150. Visscher PM, Brown MA, McCarthy MI, Yang J: Five years of GWAS discovery. Am J Hum Genet. 2012;90:7-24. 151. Anttila V, Stefansson H, Kallela M, Todt U, Terwindt GM, Calafato MS, Nyholt DR, Dimas AS, Freilinger T, Muller-Myhsok B, et al: Genome-wide association study of migraine implicates a common susceptibility variant on 8q22.1. Nat Genet. 2010;42:869-873. 152. Chasman DI, Schurks M, Anttila V, de Vries B, Schminke U, Launer LJ, Terwindt GM, van den Maagdenberg AM, Fendrich K, Volzke H, et al: Genome-wide association study reveals three susceptibility loci for common migraine in the general population. Nat Genet. 2011;43:695-698. 153. Dussor G, Cao YQ: TRPM8 and Migraine. Headache. 2016;56:1406-1417. 154. Julius D: TRP channels and pain. Annu Rev Cell Dev Biol. 2013;29:355-384. 155. Gan M, Jiang P, McLean P, Kanekiyo T, Bu G: Low-density lipoprotein receptor- related protein 1 (LRP1) regulates the stability and function of GluA1 alpha-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor in neurons. PLoS One. 2014;9:e113237. 156. Chi J, Cohen P: The Multifaceted Roles of PRDM16: Adipose Biology and Beyond. Trends Endocrinol Metab. 2016;27:11-23. 157. Kinameri E, Inoue T, Aruga J, Imayoshi I, Kageyama R, Shimogori T, Moore AW: Prdm proto-oncogene transcription factor family expression and interaction with the Notch-Hes pathway in mouse neurogenesis. PLoS One. 2008;3:e3859. 158. Endo K, Karim MR, Taniguchi H, Krejci A, Kinameri E, Siebert M, Ito K, Bray SJ, Moore AW: Chromatin modification of Notch targets in olfactory receptor neuron diversification. Nat Neurosci. 2011;15:224-233. 159. Freilinger T, Anttila V, de Vries B, Malik R, Kallela M, Terwindt GM, Pozo-Rosich P, Winsvold B, Nyholt DR, van Oosterhout WP, et al: Genome-wide association analysis identifies susceptibility loci for migraine without aura. Nat Genet. 2012;44:777-782. 160. Flavell SW, Kim TK, Gray JM, Harmin DA, Hemberg M, Hong EJ, Markenscoff- Papadimitriou E, Bear DM, Greenberg ME: Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection. Neuron. 2008;60:1022-1038. 161. Wilson PM, Fryer RH, Fang Y, Hatten ME: Astn2, a novel member of the astrotactin gene family, regulates the trafficking of ASTN1 during glial-guided neuronal migration. J Neurosci. 2010;30:8529-8540. 162. Nguyen HL, Lee YJ, Shin J, Lee E, Park SO, McCarty JH, Oh SP: TGF-beta signaling in endothelial cells, but not neuroepithelial cells, is essential for cerebral vascular development. Lab Invest. 2011;91:1554-1563. 163. Allen PB, Greenfield AT, Svenningsson P, Haspeslagh DC, Greengard P: Phactrs 1-4: A family of protein phosphatase 1 and actin regulatory proteins. Proc Natl Acad Sci U S A. 2004;101:7187-7192. 164. Consortium CAD, Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR, Ingelsson E, Saleheen D, Erdmann J, et al: Large-scale association analysis identifies new risk loci for coronary artery disease. Nat Genet. 2013;45:25- 33. 165. Myocardial Infarction Genetics C, Kathiresan S, Voight BF, Purcell S, Musunuru K, Ardissino D, Mannucci PM, Anand S, Engert JC, Samani NJ, et al: Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants. Nat Genet. 2009;41:334-341. 166. Beaudoin M, Gupta RM, Won HH, Lo KS, Do R, Henderson CA, Lavoie-St-Amour C, Langlois S, Rivas D, Lehoux S, et al: Myocardial Infarction-Associated SNP at 6p24 Interferes With MEF2 Binding and Associates With PHACTR1 Expression Levels in Human Coronary Arteries. Arterioscler Thromb Vasc Biol. 2015;35:1472- 1479. 167. Anttila V, Winsvold BS, Gormley P, Kurth T, Bettella F, McMahon G, Kallela M, Malik R, de Vries B, Terwindt G, et al: Genome-wide meta-analysis identifies new susceptibility loci for migraine. Nat Genet. 2013;45:912-917. 168. Chasman DI, Anttila V, Buring JE, Ridker PM, Schurks M, Kurth T, International Headache Genetics C: Selectivity in genetic association with sub-classified migraine in women. PLoS Genet. 2014;10:e1004366. 169. Gormley P, Winsvold BS, Nyholt DR, Kallela M, Chasman DI, Palotie A: Migraine genetics: from genome-wide association studies to translational insights. Genome Med. 2016;8:86. 170. Joutel A, Monet M, Domenga V, Riant F, Tournier-Lasserve E: Pathogenic mutations associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy differently affect Jagged1 binding and Notch3 activity via the RBP/JK signaling Pathway. Am J Hum Genet. 2004;74:338-347. 171. Hara K, Shiga A, Fukutake T, Nozaki H, Miyashita A, Yokoseki A, Kawata H, Koyama A, Arima K, Takahashi T, et al: Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease. N Engl J Med. 2009;360:1729-1739. 172. Cuenca-Leon E, Corominas R, Montfort M, Artigas J, Roig M, Bayes M, Cormand B, Macaya A: Familial hemiplegic migraine: linkage to chromosome 14q32 in a Spanish kindred. Neurogenetics. 2009;10:191-198. 173. Spain SL, Barrett JC: Strategies for fine-mapping complex traits. Hum Mol Genet. 2015;24:R111-119. 174. Smemo S, Tena JJ, Kim KH, Gamazon ER, Sakabe NJ, Gomez-Marin C, Aneas I, Credidio FL, Sobreira DR, Wasserman NF, et al: Obesity-associated variants within FTO form long-range functional connections with IRX3. Nature. 2014;507:371-375. 175. Claussnitzer M, Dankel SN, Kim KH, Quon G, Meuleman W, Haugen C, Glunk V, Sousa IS, Beaudry JL, Puviindran V, et al: FTO Obesity Variant Circuitry and Adipocyte Browning in Humans. N Engl J Med. 2015;373:895-907. 176. Matesanz F, Fedetz M, Barrionuevo C, Karaky M, Catala-Rabasa A, Potenciano V, Bello-Morales R, Lopez-Guerrero JA, Alcina A: A splice variant in the ACSL5 gene relates migraine with fatty acid activation in mitochondria. Eur J Hum Genet. 2016;24:1572-1577. 177. Schizophrenia Working Group of the Psychiatric Genomics C: Biological insights from 108 schizophrenia-associated genetic loci. Nature. 2014;511:421-427. 178. Nyholt DR, Gillespie NG, Heath AC, Merikangas KR, Duffy DL, Martin NG: Latent class and genetic analysis does not support migraine with aura and migraine without aura as separate entities. Genet Epidemiol. 2004;26:231-244. 179. Nyholt DR, International Headache Genetics C, Anttila V, Winsvold BS, Kurth T, Stefansson H, Kallela M, Malik R, Vries B, Terwindt GM, et al: Concordance of genetic risk across migraine subgroups: Impact on current and future genetic association studies. Cephalalgia. 2015;35:489-499. 180. Thomsen LL, Eriksen MK, Roemer SF, Andersen I, Olesen J, Russell MB: A population-based study of familial hemiplegic migraine suggests revised diagnostic criteria. Brain. 2002;125:1379-1391. 181. Spector JT, Kahn SR, Jones MR, Jayakumar M, Dalal D, Nazarian S: Migraine headache and ischemic stroke risk: an updated meta-analysis. Am J Med. 2010;123:612-624. 182. Schurks M, Rist PM, Shapiro RE, Kurth T: Migraine and mortality: a systematic review and meta-analysis. Cephalalgia. 2011;31:1301-1314. 183. Wang SJ, Chen PK, Fuh JL: Comorbidities of migraine. Front Neurol. 2010;1:16. 184. Sowell MK, Youssef PE: The Comorbidity of Migraine and Epilepsy in Children and Adolescents. Semin Pediatr Neurol. 2016;23:83-91. 185. Ligthart L, Penninx BW, Nyholt DR, Distel MA, de Geus EJ, Willemsen G, Smit JH, Boomsma DI: Migraine symptomatology and major depressive disorder. Cephalalgia. 2010;30:1073-1081. 186. Buse DC, Silberstein SD, Manack AN, Papapetropoulos S, Lipton RB: Psychiatric comorbidities of episodic and chronic migraine. J Neurol. 2013;260:1960-1969. 187. Malik R, Freilinger T, Winsvold BS, Anttila V, Vander Heiden J, Traylor M, de Vries B, Holliday EG, Terwindt GM, Sturm J, et al: Shared genetic basis for migraine and ischemic stroke: A genome-wide analysis of common variants. Neurology. 2015;84:2132-2145. 188. Winawer MR, Connors R, Investigators E: Evidence for a shared genetic susceptibility to migraine and epilepsy. Epilepsia. 2013;54:288-295. 189. Yang Y, Ligthart L, Terwindt GM, Boomsma DI, Rodriguez-Acevedo AJ, Nyholt DR: Genetic epidemiology of migraine and depression. Cephalalgia. 2016;36:679- 691. 190. Winsvold BS, Nelson CP, Malik R, Gormley P, Anttila V, Vander Heiden J, Elliott KS, Jacobsen LM, Palta P, Amin N, et al: Genetic analysis for a shared biological basis between migraine and coronary artery disease. Neurol Genet. 2015;1:e10. 191. Yang Y, Zhao H, Heath AC, Madden PA, Martin NG, Nyholt DR: Shared Genetic Factors Underlie Migraine and Depression. Twin Res Hum Genet. 2016;19:341-350. 192. Ligthart L, Hottenga JJ, Lewis CM, Farmer AE, Craig IW, Breen G, Willemsen G, Vink JM, Middeldorp CM, Byrne EM, et al: Genetic risk score analysis indicates migraine with and without comorbid depression are genetically different disorders. Hum Genet. 2014;133:173-186. 193. Couch JR, Amitriptyline Versus Placebo Study G: Amitriptyline in the prophylactic treatment of migraine and chronic daily headache. Headache. 2011;51:33-51. 194. Linde M, Mulleners WM, Chronicle EP, McCrory DC: Valproate (valproic acid or sodium valproate or a combination of the two) for the prophylaxis of episodic migraine in adults. Cochrane Database Syst Rev. 2013:CD010611. 195. An XK, Ma QL, Lin Q, Zhang XR, Lu CX, Qu HL: PRDM16 rs2651899 variant is a risk factor for Chinese common migraine patients. Headache. 2013;53:1595-1601. 196. Fan X, Wang J, Fan W, Chen L, Gui B, Tan G, Zhou J: Replication of migraine GWAS susceptibility loci in Chinese Han population. Headache. 2014;54:709-715. 197. Ghosh J, Pradhan S, Mittal B: Genome-wide-associated variants in migraine susceptibility: a replication study from North India. Headache. 2013;53:1583-1594. 198. Cox HC, Lea RA, Bellis C, Carless M, Dyer TD, Curran J, Charlesworth J, Macgregor S, Nyholt D, Chasman D, et al: A genome-wide analysis of 'Bounty' descendants implicates several novel variants in migraine susceptibility. Neurogenetics. 2012;13:261-266. 199. Minster RL, Hawley NL, Su CT, Sun G, Kershaw EE, Cheng H, Buhule OD, Lin J, Reupena MS, Viali S, et al: A thrifty variant in CREBRF strongly influences body mass index in Samoans. Nat Genet. 2016;48:1049-1054. 200. Fry AE, Rees E, Thompson R, Mantripragada K, Blake P, Jones G, Morgan S, Jose S, Mugalaasi H, Archer H, et al: Pathogenic copy number variants and SCN1A mutations in patients with intellectual disability and childhood-onset epilepsy. BMC Med Genet. 2016;17:34. 201. Eising E, N AD, van den Maagdenberg AM, Ferrari MD: Epigenetic mechanisms in migraine: a promising avenue? BMC Med. 2013;11:26. 202. Ulrich V, Olesen J, Gervil M, Russell MB: Possible risk factors and precipitants for migraine with aura in discordant twin-pairs: a population-based study. Cephalalgia. 2000;20:821-825. 203. Barros J, Barreto R, Brandao AF, Domingos J, Damasio J, Ramos C, Lemos C, Sequeiros J, Alonso I, Pereira-Monteiro J: Monozygotic twin sisters discordant for familial hemiplegic migraine. J Headache Pain. 2013;14:77. 204. Dempster EL, Pidsley R, Schalkwyk LC, Owens S, Georgiades A, Kane F, Kalidindi S, Picchioni M, Kravariti E, Toulopoulou T, et al: Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder. Hum Mol Genet. 2011;20:4786-4796. 205. Cordova-Palomera A, Fatjo-Vilas M, Gasto C, Navarro V, Krebs MO, Fananas L: Genome-wide methylation study on depression: differential methylation and variable methylation in monozygotic twins. Transl Psychiatry. 2015;5:e557. 206. Bell JT, Loomis AK, Butcher LM, Gao F, Zhang B, Hyde CL, Sun J, Wu H, Ward K, Harris J, et al: Differential methylation of the TRPA1 promoter in pain sensitivity. Nat Commun. 2014;5:2978. 207. Wang X, Zhu H, Snieder H, Su S, Munn D, Harshfield G, Maria BL, Dong Y, Treiber F, Gutin B, Shi H: Obesity related methylation changes in DNA of peripheral blood leukocytes. BMC Med. 2010;8:87. 208. Lund G, Andersson L, Lauria M, Lindholm M, Fraga MF, Villar-Garea A, Ballestar E, Esteller M, Zaina S: DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E. J Biol Chem. 2004;279:29147-29154. 209. Ran C, Graae L, Magnusson PK, Pedersen NL, Olson L, Belin AC: A replication study of GWAS findings in migraine identifies association in a Swedish case-control sample. BMC Med Genet. 2014;15:38. 210. Rodriguez-Acevedo AJ, Ferreira MA, Benton MC, Carless MA, Goring HH, Curran JE, Blangero J, Lea RA, Griffiths LR: Common polygenic variation contributes to risk of migraine in the Norfolk Island population. Hum Genet. 2015;134:1079-1087. 211. Esserlind AL, Christensen AF, Steinberg S, Grarup N, Pedersen O, Hansen T, Werge T, Hansen TF, Husemoen LL, Linneberg A, et al: The association between candidate migraine susceptibility loci and severe migraine phenotype in a clinical sample. Cephalalgia. 2016;36:615-623. 212. Christensen AF, Esserlind AL, Werge T, Stefansson H, Stefansson K, Olesen J: The influence of genetic constitution on migraine drug responses. Cephalalgia. 2016;36:624-639. 213. Menon S, Lea RA, Roy B, Hanna M, Wee S, Haupt LM, Oliver C, Griffiths LR: Genotypes of the MTHFR C677T and MTRR A66G genes act independently to reduce migraine disability in response to vitamin supplementation. Pharmacogenet Genomics. 2012;22:741-749. 214. Ferrari A, Sternieri E, Ferraris E, Bertolini A: Emerging problems in the pharmacology of migraine: interactions between triptans and drugs for prophylaxis. Pharmacol Res. 2003;48:1-9. 215. Schurks M, Kurth T, Stude P, Rimmbach C, de Jesus J, Jonjic M, Diener HC, Rosskopf D: G protein beta3 polymorphism and triptan response in cluster headache. Clin Pharmacol Ther. 2007;82:396-401. 216. Gentile G, Missori S, Borro M, Sebastianelli A, Simmaco M, Martelletti P: Frequencies of genetic polymorphisms related to triptans metabolism in chronic migraine. J Headache Pain. 2010;11:151-156.

Figure 1. Functions and pathways of genes associated with migraine. The majority of genes implicated in migraine have either a neuronal or vascular function. Genes that have been assigned to particular or putative functions are shown in the relevant box. Some genes have multiple functions and appear in overlapping sections (N.B. GJA1 appears twice to accommodate all its roles). Genes from GWAS loci are depicted in black, while genes involved in migraine-related monogenic disorders are depicted in red.