**4. Genetic aspect of migraine**

Migraine is an episodic and disabling neurological disorder affecting roughly 14% of the pop‐ ulation. The two most prevalent forms are migraine without aura (MO) and migraine with aura (MA). Migraine tends to run in families and has a strong genetic basis, with heritability estimates of 40–57%. In the rare monogenic subtype of migraine, familial hemiplegic migraine (FHM), three causative genes have been identified. There is, however, no significant asso‐ ciation between these genes and MO and/or MA. Many linkage studies and candidate gene studies have suggested causative genes in MO and MA, but few have been replicated. Recent attempts using genome‐wide association studies (GWAS) have yielded four single nucleo‐ tide polymorphisms (SNPs) that are significantly associated with migraine and recently, three additional SNPs have shown convincing association as well [90–93].

Recently, several studies have been used to identify genetic variants either causing migraine or conferring vulnerability to the disease. The array‐based technologies and second‐generation DNA sequencing has provided novel analysis to genetic database. In general, rare variants are sought by DNA sequencing in multigenerational families with many affected individuals. These studies were previously performed using a linkage approach, followed by refinement of the linkage region and targeted Sanger sequencing of candidate genes. On the other hand, genome‐wide association studies (GWAS) allowed the determination of the case‐control or family‐based association studies in large samples [93].

Many studies are conducted for understanding of molecular genetic basis of MA, MO and FHM well. Especially GWAS has given very important results for these diseases. The present work does not represent a systematic review but rather aims to provide thorough coverage of this area of investigation. Migraine can be part of known genetic disorders, displaying mul‐ tiple manifestations and often involving various organs.

Migraine is associated with some of genetic syndromes. These diseases are CADASIL cere‐ bral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, COL4A1 retinal arteriolar tortuosity and leukoencephalopathy, CRV cerebroretinal vasculop‐ athy, CSD cortical spreading depression, FASPS familial anticipated sleep phase syndrome, HERNS hereditary endotheliopathy with retinopathy, nephropathy, stroke, HVR hereditary vascular retinopathy, MELAS mitochondrial encephalomyopathy, lactic acidosis, stroke‐like episodes, RVCL retinal vasculopathy and cerebral leukodystrophy These disorders, though rare, may lead to a better comprehension of the mechanisms underlying more common forms of idiopathic migraine. Other forms of typical FHM due to mutations in novel FHM candi‐ date genes have been recently described. Mutations resulting in an FHM phenotype have been identified in the PRRT2 (proline‐rich transmembrane protein 2) gene, located on human chromosome 16p11 and encoding for an axonal protein associated with the exocytosis protein complex [94–99].

Studies shown that FHM is related to CACNA1A gene which encoded by 19p13, which pro‐ duces voltage‐dependent (P/Q) Cav 2.1 channel, α1A subunit Over 70 missense mutations with "gain of function" effect. ATP1A2 encoded by 1q21‐23 that produce to Na<sup>+</sup> ‐K<sup>+</sup> ATPase α2 subunit responsible for FHM 2 disease. FHM3 is related to SCN1A gene. In conclusion, famil‐ ial forms of MA and in particular FHM, are due to rare inherited or sporadic genetic variants endowed with high penetrance. These mutations are affecting the transmembrane electro‐ chemical gradient by enhancing extracellular glutamate concentrations which are related with neuronal excitability [100, 101]. We can explain migraine‐associated genes in four groups and other effective mechanisms (**Figure 2**).

#### **4.1. Candidate genes**

acetaldehyde, is responsible for most of the alcohol breakdown in the liver. And Rainero and colleagues investigated the association of genetic variants within the ADH4 gene with CH susceptibility and phenotype. They suggested that CH was associated with the ADH4 gene or a linked locus. For rs1126671 polymorphism, the carriage of the AA genotype, in comparison with remaining genotypes, was associated with a significantly increased disease risk of 2.33 times. [84] But the results were not confirmed in Swedish population. The data from this study did not support an association of the ADH4 SNPs rs1126671 and rs1800759 with CH [85].

Only about 70% of migraine and CH patients report significant treatment responses to triptans, which are agonists at 5‐HT1B/D receptors belonging to the family of G protein‐coupled recep‐ tors. A C825T polymorphism identified in the gene for the G protein β3 (Gβ3) subunit (GNB3) has been associated with an enhanced signal transduction via GPCR [86]. It was investigated whether a common polymorphism in the gene for the G protein β3 subunit (GNB3 C825T) modu‐ lates responder rates to triptans among a large cohort of Caucasian CH patients. It was suggested that pain relief by triptans is significantly modulated by a common genetic GNB3 variant [88].

Also mutations of the P/Q type calcium channel alpha 1 subunit (CACNA1A) gene on chro‐ mosome 19p13 have been shown to cause several neurological disorders with a wide clinical spectrum, mainly episodic diseases. Missense mutations of the gene cause familial hemiplegic migraine (FHM) and it is also likely to be involved in the more common forms of migraine. It was investigated whether the CACNA1A gene is also a candidate gene for CH. In this study an association analysis of an intragenic polymorphic (CA)n‐repeat with marker D19S1150 and a (CAG)n‐repeat in the 3'UTR region was performed, in 75 patients with CH in Swedish population. But it was found that genotypes and allele frequencies were similarly distributed in patients and controls. Also linkage disequilibrium between the two markers was similar in patients and controls. And it was suggested that any significance of the CACNA1A gene in CH is unlikely [88]. Similarly Haan J et al. suggested that there is no involvement of the

Migraine is an episodic and disabling neurological disorder affecting roughly 14% of the pop‐ ulation. The two most prevalent forms are migraine without aura (MO) and migraine with aura (MA). Migraine tends to run in families and has a strong genetic basis, with heritability estimates of 40–57%. In the rare monogenic subtype of migraine, familial hemiplegic migraine (FHM), three causative genes have been identified. There is, however, no significant asso‐ ciation between these genes and MO and/or MA. Many linkage studies and candidate gene studies have suggested causative genes in MO and MA, but few have been replicated. Recent attempts using genome‐wide association studies (GWAS) have yielded four single nucleo‐ tide polymorphisms (SNPs) that are significantly associated with migraine and recently, three

Recently, several studies have been used to identify genetic variants either causing migraine or conferring vulnerability to the disease. The array‐based technologies and second‐generation DNA sequencing has provided novel analysis to genetic database. In general, rare variants

calcium channel gene (CACNA1A) mutations in a Dutch family with CH [89].

additional SNPs have shown convincing association as well [90–93].

**4. Genetic aspect of migraine**

12 Current Perspectives on Less-known Aspects of Headache

**(1) Neurological genes:** This group candidate genes encode (a)ion channels (calcium chan‐ nel, voltage‐dependent, P/Q type, alpha 1A subunit [CACNA1A], voltage‐potassium inter‐ mediate/small conductance calcium‐activated channel and subfamily N, member 3 [KCCN3]) (b) Na+/K+‐ATPase subunits, (c) molecules involved in the synthesis, release and binding of neuropeptides (calcitonin gene‐related peptide) or neurotransmitters (glutamate, GABA, dopamine, serotonin) relevant to neuronal excitation and/or to nociception. Some case‐control association studies have yielded positive results, as known 5‐HT‐related genes, MAOA, dopa‐ mine‐related genes, although most studies have been negative especially for the former two

**Figure 2.** Genetic database of migraine disease.

gene families. Nonetheless, a thorough screening of 150 brain‐expressed genes involved in ion homeostasis (channels, transporters, exchangers and accessory subunits) identified three genes encoding potassium channels associated with migraine, namely KCNK18, KCNG4 and KCNAB3 [100, 101].

**(2) Vascular genes:** These associated genes (ACE, MTHFR, NOTCH3, EDNRA) are involved in blood pressure regulation, endothelial cell function, vasoconstriction and vasodilation. Many vascular genes associated with migraine also confer risk for stroke and heart disease. These functional variants in some of vascular genes may cause migraine. Angiotensin con‐ verting enzyme (ACE) plays a key role in the maintenance of blood pressure and vessel wall tension. The D‐D ("deletion‐deletion") common variant located in the ACE gene (human chr. 17q23) increases ACE enzymatic activity, as well as the frequency and duration of MA attacks. MTHFR is a key component of the remethylation of homocysteine to methionine, as it catalyzes the conversion of 5,10‐methylenetetrahydrofolate to 5‐methyltetrahydrofolate. Rare loss‐of‐function mutations in the MTHFR gene, located in human chromosome 1p36.22, can lead to hyperhomocysteinemia due to decreased enzyme activity [102–109].

NOTCH3 encodes for a transmembrane receptor regulating vascular development and dif‐ ferentiation during embryogenesis, as well as contributing to vascular integrity in adults. In addition to rare NOTCH3 mutations producing MA within the context of CADASIL, also common variants are significantly associated with migraine. Hence, NOTCH3 may play a broader role also in the pathogenesis of common migraine, well beyond rare forms associ‐ ated with CADASIL. Endothelial genes assessed for association with migraine encode for endothelin‐1 (EDN1), endothelin receptor type A and B (EDNRA and EDNRB), inducible NO synthase (NOS2), endothelial NO synthase (NOS3) and vascular endothelial growth factor (VEGF) [110–114].

**(3) Hormonal genes:** These group genes are related with estrogen and progesterone metabo‐ lism especially relating to menstrual migraine. However, results from studies of genetic asso‐ ciation between these genes and migraine were published in the later study, three estrogen receptor 1 (ESR1) haplotypes were significantly associated with the disorder (*P* < 0.05 or 0.01). In addition to ESR1, six other hormonal genes have been investigated, estrogen receptor 2 (ESR2), progesterone receptor (PGR) androgen receptor (AR), follicle stimulating hormone receptor (FSHR), nuclear receptor interacting protein 1 (NRIP1) and cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) [115–119].

**(4) Inflammatory genes:** Recent studies shown that neurogenic inflammation, with activation of mast cells and macrophages accompanied by the release of proinflammatory cytokines may play an important role in the pathogenesis of migraine. Especially tumor necrosis factor alfa (TNF‐α) gene variants is positive associated with migraine [120, 121]

#### **4.2. Family linkage studies and GWAS**

Many GWAS and classical linkage studies have been performed for migraine either using a genome‐wide approach or targeting specific regions using microsatellite markers. Also, mito‐ chondrial dysfunction in migraine that increased influx of calcium increases oxidative stress, that muscle biopsy of patients with migraine may show mitochondrial abnormalities, that mtDNA polymorphisms may be increased in migraine patients and that riboflavin, coen‐ zyme‐Q, niacin and carnitine, all agents used in the treatment of MIDs, exhibit a beneficial effect for migraine [122].

Recent GWAS studies have shown four SNPs, located on chromosome 8q22.1, 2q37.1, 12q13.3 and 1p36.32, which are associated with MA and/or MO. Although, some meta‐analysis con‐ firmed that the same results in independent populations. In another recent GWAS, three addi‐ tional SNPs located at 1q22 and 3p24. However, all of these associated studies shown that the moderate of change in risk for migraine. On the other hand, the pedigree‐based GWAS in an isolated population of Norfolk Island with a high prevalence of migraine and several novel variants in migraine susceptibility were identified [117–120].

#### **4.3. Epigenetic**

gene families. Nonetheless, a thorough screening of 150 brain‐expressed genes involved in ion homeostasis (channels, transporters, exchangers and accessory subunits) identified three genes encoding potassium channels associated with migraine, namely KCNK18, KCNG4 and

**(2) Vascular genes:** These associated genes (ACE, MTHFR, NOTCH3, EDNRA) are involved in blood pressure regulation, endothelial cell function, vasoconstriction and vasodilation. Many vascular genes associated with migraine also confer risk for stroke and heart disease. These functional variants in some of vascular genes may cause migraine. Angiotensin con‐ verting enzyme (ACE) plays a key role in the maintenance of blood pressure and vessel wall tension. The D‐D ("deletion‐deletion") common variant located in the ACE gene (human chr. 17q23) increases ACE enzymatic activity, as well as the frequency and duration of MA attacks. MTHFR is a key component of the remethylation of homocysteine to methionine, as it catalyzes the conversion of 5,10‐methylenetetrahydrofolate to 5‐methyltetrahydrofolate. Rare loss‐of‐function mutations in the MTHFR gene, located in human chromosome 1p36.22, can

NOTCH3 encodes for a transmembrane receptor regulating vascular development and dif‐ ferentiation during embryogenesis, as well as contributing to vascular integrity in adults. In addition to rare NOTCH3 mutations producing MA within the context of CADASIL, also common variants are significantly associated with migraine. Hence, NOTCH3 may play a broader role also in the pathogenesis of common migraine, well beyond rare forms associ‐ ated with CADASIL. Endothelial genes assessed for association with migraine encode for

lead to hyperhomocysteinemia due to decreased enzyme activity [102–109].

KCNAB3 [100, 101].

**Figure 2.** Genetic database of migraine disease.

14 Current Perspectives on Less-known Aspects of Headache

Epigenetics role of many complex diseases including migraine has aroused curiosity.

The effect of methylated DNA, methylated cytosines in the human D‐loop of mitochondrial DNA (mtDNA), acetylation have shown differences between healthy controls and neurode‐ generative and age‐related diseases. Given comorbidities with migraine and the suggestive link between mitochondrial dysfunction and the lowered threshold for triggering a migraine attack, mitochondrial methylation may be a new avenue to pursue. New epigenetic approach of to solve the complex background of neurological diseases are very important [121–124].

The success of migraine genetic investigations will largely rely upon their capacity on one hand to apply the methodological approaches most apt to respond to each specific experimen‐ tal question on the other hand, on their capacity to integrate multiple levels of phenotypic, functional and genetic information, in accordance with the complexity of the disorder itself. Environmental factors, such as early and recent life events, hormones and inflammation, can indeed act upon a genetically vulnerable background to trigger the onset and determine the progression of the disease.
