Skip to main content

Genetic variants in the NOTCH4 gene influence the clinical features of migraine



Recent studies suggested an important role for vascular factors in migraine etiopathogenesis. Notch4 belongs to a family of transmembrane receptors that play an important role in vascular development and maintenance. The aim of this study was to test the hypothesis that polymorphisms of the NOTCH4 gene would modify the occurrence and the clinical features of migraine.


Using a case–control strategy, we genotyped 239 migraine patients and 264 controls for three different non-synonymous polymorphisms (T320A, G835V, R1346P) of the NOTCH4 gene and for the (CTG) n-encoding polyleucine polymorphism in exon 1. Although the analyzed polymorphisms resulted not associated with migraine, the clinical characteristics of our patients were significantly influenced by the different NOTCH4 genotypes. Longer duration of disease and severity of neurovegetative symptoms during headache attacks were associated with the R1346P and G835V polymorphisms, respectively. In female patients, worsening of migraine symptoms at menarche was significantly correlated with T320A polymorphism.


Our study shows that genetic variations within the NOTCH4 gene significantly modify the clinical characteristics of migraine and may have a role in disease pathogenesis.


Migraine is a chronic and disabling neurovascular disorder that affects approximately 15% of the general population in Western Countries [1]. The diagnosis of migraine, as proposed by the International Headache Society (IHS), is mainly based on clinical features such as unilateral throbbing headache, hypersensitivity to sound and light, nausea and/or vomiting and focal neurological symptoms (aura). According to these criteria, migraine is classified into two main subtypes, migraine with aura (MA) and migraine without aura (MO) [2].

Migraine etiology is complex and multifactorial, involving both genetic and environmental factors that interact in a manner yet to be completely understood. Mutations in CACNA1A, ATP1A2 and SCN1A genes have been associated with familial hemiplegic migraine (FHM), a rare monogenic form of migraine [3]. Recently, mutations in the TRESK K2P potassium channel (KCNK18 gene) have been linked to familial migraine with aura [4]. Genetic association studies and genome-wide association studies (GWAS) suggested a role in migraine heritability for a large number of different genes, highlighting the genetic complexity of this disorder [5, 6]. However, up to now, no clear genetic association conferring major risk for developing migraine has been found.

Although the pathophysiology of migraine is not fully understood, cortical spreading depression (CSD), a depolarization wave that propagates across the brain cortex and activates the trigeminal nerve in animal models, is speculated to cause the neurological symptoms typical of migraine [7]. It has been suggested that CSD may be initiated by a vascular event, implying that a vascular endothelial dysfunction may increase migraine susceptibility [8]. Genetic studies provided evidence of an increased susceptibility to migraine in patients with polymorphisms in genes associated with endothelial function. The endothelin type A receptor (ETA) gene −231 A/A polymorphism and the Angiotensin I-converting enzyme (ACE) deletion/deletion (DD) polymorphism have been associated with migraine, both with and without aura [9, 10]. In addition, genes related to nitric oxide pathway and vascular permeability (like VEGF) resulted significantly associated with both migraine and aura [11].

NOTCH4 gene is located at 6p21.3, in the major histocompatibility complex (MHC), a chromosomal region intensively investigated in migraine [12, 13]. The gene encodes for a 2,003 amino acids long protein involved in a cell-cell signalling pathway strongly conserved during evolution. In situ hybridization revealed that NOTCH4 transcripts are primarily restricted to endothelial cells both in embryonic and adult life, suggesting a specific role for Notch4 during vascular development [14]. Mice mutants for the genes of the Notch system show important defect in vascular morphogenesis [15]. In addition, Notch4 plays a crucial role in the differentiation of glial cells, in the organization of neural processes [16] and in the regulation of neural stem cells quiescence, activation, and self-renewal [17].

Notch4 belongs to a family that in humans include four receptors (Notch1-4), binding with different ligands, such as Jagged1, Jagged2, Dll1 and Dll4 [15]. Mutations in the Notch pathway genes, like NOTCH2 and JAG1, are associated with Alagille syndrome, a multisystem disorder that includes cardiac and vascular abnormalities [18]. Mutations in NOTCH3 gene lead to a condition named cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) that is associated with a higher prevalence of migraine with aura [19].

The aim of the present work was to evaluate the association between migraine and the NOTCH4 gene. We studied several polymorphisms of the gene in a large cohort of Italian migraine patients recruited from a University-based Headache Center and in healthy controls.




A group of 239 consecutive unrelated migraine patients (165 females, 74 males; mean age ± SD = 40.6 ± 13.0 yrs), recruited from the Headache Center of the University of Torino (Italy), were selected for the study. The diagnosis of migraine was made, according to the ICHD-II criteria [2], by a neurologist specialized in headaches. One hundred and ninety (79.5%) patients fulfilled the diagnostic criteria for migraine without aura while forty nine patients (20.5%) for migraine with aura. Mean age at onset and duration of the disease were 18.1 ± 9.9 and 22.5 ± 13.5 yrs, respectively. The patients underwent an extensive physical and neurological examination. All the clinical characteristics of the patients were recorded using a standardized questionnaire. Psychological evaluation was performed by a psychologist with the use the Beck Depression Inventory (BDI) and the State and Trait Anxiety Inventory (STAI x-1 and STAI x-2) tests. Table 1 summarizes the demographic and clinical characteristics of migraine patients. A group of 264 age and sex matched healthy subjects (169 females, 95 males, mean age ± SD = 41.6 ± 12.8 yrs) was used as control. The controls were blood donors and were screened to exclude primary headaches. Both patients and controls were of Caucasian origin. Written informed consent was obtained from all participants and the study was approved by the local Ethics Committee. The same population was previously used to perform genetic association studies between DNA polymorphisms and migraine [20, 21].

Table 1 Demographic and clinical characteristics of migraine patients

DNA extraction and genotyping

We examined three non synonymous polymorphisms (T320A - rs422951, G835V - rs9267835, and R1346P - rs8192573) and the (CTG) n-encoding polyleucine polymorphism in exon 1 for NOTCH4 gene. All polymorphisms, selected from SNPs database of NCBI (, are located in regions of putative functional relevance.

Genomic DNA was extracted from 200 μl of peripheral blood using the QIAamp DNA mini Kit (QIAGEN). PCR reactions were performed with 90 ng of genomic DNA, 0.4 unit of Taq Gold DNA polymerase, 250 nM of each primer, 1.5 mM MgCl2 and 50 mM dNTPs. For non synonymous polymorphisms, PCR conditions used were: an initial denaturation at 95°C 10 min followed by 35 cycles 95°C for 1 min, annealing specific temperature for each couple of primers for 1 minute, then at 72°C for 1 min with a final extension at 72°C for 5 min. PCR products were electrophoresed on a 2% agarose TBE 1X gel and stained with ethidium bromide. All non synonymous polymorphisms were analyzed by enzymatic digestion. Genotyping for T320A, G835V, R1346P polymorphisms was performed by digesting PCR products with BsuRI, XmiI, SmaI restriction enzyme, respectively. Conversely, (CTG)n polymorphism was performed on ABI 3700 sequencer (Applied Biosystems, Inc); PCR product was obtained using the same mixture previously described and using the following primers: forward: 5- CCTGCCTGAAGAGGGACAG-3, reverse: 5-CCCCACTGATCATCCTCCTA-3. The forward primer was fluorescently labeled with FAM. Alleles were scored using GeneMarker v1.5 software package (SoftGenetics LLC –


Fisher’s exact test, χ 2 test and Monte Carlo Markov chain method (MCMC), were used to compare allelic (AF) and genotypic frequencies (GF) between cases and controls. Patients clinical characteristics were analyzed with ANOVA followed by Bonferroni correction for multiple comparisons. Statistical analysis was performed using Statistical Package for Social Sciences – version 20 (IBM SPSS Statistics 20.0 - August 2011 - SPSS Inc., Chicago Ill). The Hardy-Weinberg equilibrium was verified for observed genotype frequency in all tested populations in order to detect deviation from the expected genotype distribution and to detect genotyping errors. The level of statistical significance was taken at p<0.05.


Table 2 shows genotypic (GF) and allelic (AF) frequencies of the three non-synonymous coding sequence variants (T320A, G835V, R1346P) of the NOTCH4 gene in migraineurs and controls. GF and AF of the examined polymorphisms were not significantly different between cases and controls. Subsequently, we analyzed the recessive, dominant and additive models for different alleles without reaching any statistical difference.

Table 2 Genotype and Allele Frequencies of the NOTCH4 polymorphisms in migraine patients and controls

Table 3 shows allelic frequencies of the (CTG) n-encoding polyleucine polymorphism in cases and controls. No significant difference was found between the two groups.

Table 3 Allelic frequencies of the polymorphic CTG repeat in exon 1 of the NOTCH4 gene

When we divided the migraine patients into different subgroups (MA and MO, episodic and chronic migraine), according to IHCD-II criteria, no significant difference in the distribution of the genetic variants was found.

Finally, we compared the clinical characteristics (age of onset, duration of the disease, frequency of migraine as attacks for year, photophobia, phonophobia, nausea, vomiting, menstrual migraine, and variation at menarche) of our migraine patients according to different NOTCH4 genotypes. Interestingly, we found that the carriage of the allele T of the G835V polymorphism was associated with a higher frequency of vomiting (p=0,034), while carrying the allele C of R1346P polymorphism correlated with a longer duration of disease (p=0,020). When we analyzed our patients according to gender, we found that in females there was a significant association between the allele A of T320A and worsening of symptoms at menarche (p=0,016). When patients were divided into migraine with aura and without aura subgroups, no significant difference was found.


To the best of our knowledge, this is the first study that investigated the association between migraine and the NOTCH4 gene. We found that the analyzed polymorphisms are not significantly associated with the disease. Genotypic and allelic frequencies of the examined polymorphisms were similarly distributed between cases and controls. When patients were divided into migraine with aura and without aura subgroups, no significant difference was found. It is unlikely that genetic variations within the NOTCH4 gene contribute greatly to migraine susceptibility. In addition, we assessed the relation between different NOTCH4 genotypes and the clinical characteristics of migraine. Intriguingly, we found that several characteristics of the disease, like the presence of neurovegetative symptoms, the duration of the disease and the worsening of symptoms at menarche were influenced by NOTCH4 genotypes. Our data support a role for NOTCH4 as a disease-modifier gene in migraine.

There are some limitations in our study that deserve mention and suggest caution in the interpretation of the results. First of all, we performed our study in a sample of patients recruited from an Headache Center that may not be representative of the general migraine population [22]. In addition, the number of migraine with aura patients genotyped in our study may be too low for subgroups analysis. Finally, it is well known that results of genetic association studies must be viewed cautiously until they are confirmed by multiple replications studies [23, 24].

Our data are in accordance with several studies suggesting a significant involvement of vascular mechanisms in migraine etiopathogenesis. First of all, migraine symptoms are included in the phenotype of several genetic vasculopathies like cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL), retinal vasculopathy with cerebral leukodystrophy (RVCL) and hereditary infantile hemiparesis, retinal arteriolar tortuosity and leukoencephalopahty (HIHRATL) [25]. It is of interest to note that all the aforementioned disorders are mainly characterized by involvement of small vessels in the brain. Secondly, it is well known that several vascular genetic risk factors, like the methylenetetrahydrofolate reductase (MTHFR) gene and angiotensin I-converting enzyme (ACE) genes, are significantly associated with migraine [26, 27]. Finally, epidemiological studies have linked migraine with and without aura to a broader range of ischemic vascular disorders including angina, myocardial infarction, coronary revascularization, claudication and cardiovascular mortality [28].

How genetic and acquired vascular mechanisms might be involved in migraine pathogenesis is still matter of investigation. A dysfunction in endothelial function has been suggested as a key finding in migraine pathogenesis. The endothelium regulates numerous vascular functions. In response to specific stimuli, endothelial cells secrete local vasoactive mediators, including the vasodilator nitric oxide (NO) and vasoconstrictor endothelin-1 (ET-1) and tissue-type plasminogen activator (t-PA). Several observations suggest that endothelial function is abnormal in migraine patients, including an increased prevalence of anti-endothelial cell antibodies that may induce endothelial damage and raised plasma levels of ET-1 and von Willebrand factor [29]. Finally, endothelial progenitor cells were shown to be significantly reduced in migraine patients [30]. An increasing understanding of the role of endothelium dysfunction in migraine pathogenesis may suggest new strategy for disease treatment.

In conclusion, we found no evidence for a strong association between the NOTCH4 gene and migraine and our data does not support the hypothesis that the NOTCH4 gene is a major genetic risk factor for migraine. However, due to the relatively small number of subjects examined, replication studies in larger samples are required in order to confirm our data. Intriguingly, we found that genetic variants within NOTCH4, a gene involved in vascular maintenance and remodeling, significantly influence several clinical features of migraine. Our results further support a role for vascular genetic factor in the disease. Finally, we can hypothesize that the NOTCH4 gene may be particularly involved in some migraine subtypes. Additional studies in families segregating an atypical migraine phenotype are warranted to test this hypothesis.


  1. 1.

    Lipton RB, Scher AI, Kolodner K, Liberman J, Steiner TJ, Stewart WF: Migraine in the United States: epidemiology and patterns of health care use. Neurology 2002,58(6):885–894. 10.1212/WNL.58.6.885

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Headache Classification Subcommittee of the International Headache Society: The international classification of headache disorders: 2nd edn. Cephalalgia 2004,24(1):1–151. 10.1111/j.1468-2982.2004.00711.x

    Article  Google Scholar 

  3. 3.

    Schürks M: Genetics of migraine in the age of genome-wide association studies. J Headache Pain 2012,13(1):1–9. 10.1007/s10194-011-0399-0

    PubMed Central  PubMed  Article  Google Scholar 

  4. 4.

    Lafrenière RG, Cader MZ, Poulin JF, Andres-Enguix I, Simoneau M, Gupta N, Boisvert K, Lafrenière F, McLaughlan S, Dubé MP, Marcinkiewicz MM, Ramagopalan S, Ansorge O, Brais B, Sequeiros J, Pereira-Monteiro JM, Griffiths LR, Tucker SJ, Ebers G, Rouleau GA: A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura. Nat Med 2010,16(10):1157–1160. 10.1038/nm.2216

    PubMed  Article  Google Scholar 

  5. 5.

    Rudkjobing LA, Esserlind AL, Olesen J: Future possibilities in migraine genetics. J Headache Pain 2012,13(7):505–11. 10.1007/s10194-012-0481-2

    PubMed Central  PubMed  Article  Google Scholar 

  6. 6.

    Di Lorenzo C, Grieco GS, Santorelli FM: Migraine headache: a review of the molecular genetics of a common disorder. J Headache Pain 2012,13(7):571–80. 10.1007/s10194-012-0478-x

    PubMed Central  PubMed  Article  Google Scholar 

  7. 7.

    Bolay H, Reuter U, Dunn AK, Huang Z, Boas DA, Moskowitz M: Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model. Nat Med 2002, 8: 136–142. 10.1038/nm0202-136

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Parsons AA, Strijbos PJ: The neuronal versus vascular hypothesis of migraine and cortical spreading depression. Curr Opin Pharmacol 2003, 3: 73–77. 10.1016/S1471-4892(02)00016-4

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Tzourio C, El Amrani M, Poirier O, Nicaud V, Bousser MG, Alperovitch A: Association between migraine and endothelin type A receptor (ETA −231 A/G) gene polymorphism. Neurology 2001,56(2):1273–7.

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Kowa H, Fusayasu E, Ijiri T, Ishizaki K, Yasui K, Nakaso K, Kusumi M, Takeshima T, Nakashima K: Association of the insertion/deletion polymorphism of the angio- tensin I-converting enzyme gene in patients of migraine with aura. Neurosci Lett 2005,374(2):129–31. 10.1016/j.neulet.2004.10.041

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Gonçalves FM, Luizon MR, Speciali JG: Haplotypes in candidate genes related to nitric oxide pathway and vascular permeability associated with migraine and aura. J Headache Pain 2012,13(4):335–6. 10.1007/s10194-012-0438-5

    PubMed Central  PubMed  Article  Google Scholar 

  12. 12.

    Martelletti P, Lulli P, Morellini M, Mariani B, Pennesi G, Cappellacci S, Brioli G, Giacovazzo M, Trabace S: Chromosome 6p-encoded HLA-DR2 determination discriminates migraine without aura from migraine with aura. Hum Immunol 1999,60(1):69–74. 10.1016/S0198-8859(98)00087-1

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Rainero I, Grimaldi LM, Salani G, Valfre W, Rivoiro C, Savi L, Pinessi L: Association between the tumor necrosis factor-alpha −308 G/A gene polymorphism and migraine. Neurology 2004,62(1):141–143. 10.1212/01.WNL.0000101717.16799.8F

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Roca C, Adams RH: Regulation of vascular morphogenesis by Notch signaling. Genes Dev 2007,21(20):2511–2524. 10.1101/gad.1589207

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Krebs LT, Xue Y, Norton CR: Notch signaling is essential for vascular morphogenesis in mice. Genes Dev 2000,14(11):1343–1352.

    CAS  PubMed Central  PubMed  Google Scholar 

  16. 16.

    Harper JA, Yuan JS, Tan JB, Visan I, Guidos CJ: Notch signaling in development and disease. Clin Genet 2003,64(6):461–472. 10.1046/j.1399-0004.2003.00194.x

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Ables JL, Breunig JJ, Eisch AJ, Rakic P: Not(ch) just development: Notch signalling in the adult brain. Nat Rev Neurosci 2011,12(5):269–283. 10.1038/nrn3024

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  18. 18.

    Turnpenny PD, Ellard S: Alagille syndrome: pathogenesis, diagnosis and management. Eur J Hum Genet 2012,20(3):251–257. 10.1038/ejhg.2011.181

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  19. 19.

    Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, Alamowitch S, Domenga V, Cécillion M, Marechal E, Maciazek J, Vayssiere C, Cruaud C, Cabanis EA, Ruchoux MM, Weissenbach J, Bach JF, Bousser MG, Tournier-Lasserve E: Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature 1996,383(6602):707–710. 10.1038/383707a0

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Rainero I, Pinessi L, Salani G, Valfrè W, Rivoiro C, Savi L, Gentile S, Lo Giudice R, Grimaldi L: A polymorphism in the interleukin-1alpha gene influences the clinical features of migraine. Headache 2002, 42: 337–340. 10.1046/j.1526-4610.2002.02103.x

    PubMed  Article  Google Scholar 

  21. 21.

    Rainero I, Salani G, Valfré W, Savi L, Rivoiro C, Ferrero M, Pinessi L, Grimaldi LM: Absence of linkage between the interleukin-6 gene (−174 G/C) polymorphism and migraine. Neurosci Lett 2003,343(3):155–158. 10.1016/S0304-3940(03)00304-5

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Ziegler DK, Paolo AM: Self-reported disability due to headache: a comparison of clinic patients and controls. Headache 1996,36(8):476–480. 10.1046/j.1526-4610.1996.3608476.x

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Bird TD, Jarvik GP, Wood NW: Genetic association studies: genes in search of diseases. Neurology 2001,57(7):1153–1154. 10.1212/WNL.57.7.1153

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Marian AJ: Molecular genetic studies of complex phenotypes. Transl Res 2012,159(2):64–79. 10.1016/j.trsl.2011.08.001

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  25. 25.

    Stam AH, Haan J, van den Maagdenberg AM, Ferrari MD, Terwindt GM: Migraine and genetic and acquired vasculopathies. Cephalalgia 2009,29(9):1006–17. 10.1111/j.1468-2982.2009.01940.x

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    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(8):818–825. 10.1111/j.1468-2982.2007.01400.x

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Schürks M, Rist PM, Kurth T: MTHFR 677C>T and ACE D/I polymorphisms in migraine: a systematic review and meta-analysis. Headache 2010,50(4):588–599. 10.1111/j.1526-4610.2009.01570.x

    PubMed Central  PubMed  Article  Google Scholar 

  28. 28.

    Sacco S, Ricci S, Carolei A: Migraine and vascular diseases: a review of the evidence and potential implication for management. Cephalalgia 2012,32(10):785–795. 10.1177/0333102412451361

    PubMed  Article  Google Scholar 

  29. 29.

    Hamed SA, Hamed EA, Ezz Eldin AM, Mahmoud NM: Vascular risk factors, endothelial function, and carotid thickness in patients with migraine: relationship to migraine. J Stroke Cerebrovasc Dis 2010,19(2):92–103. 10.1016/j.jstrokecerebrovasdis.2009.04.007

    PubMed  Article  Google Scholar 

  30. 30.

    Rodríguez-Osorio X, Sobrino T, Brea D, Martínez F, Castillo J, Leira R: Endothelial progenitor cells: a new key for endothelial dysfunction in migraine. Neurology 2012,79(5):474–479. 10.1212/WNL.0b013e31826170ce

    PubMed  Article  Google Scholar 

Download references


The study was supported by grants from the “Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR)” and from Regione Piemonte (Italy).

Author information



Corresponding author

Correspondence to Elisa Rubino.

Additional information

Competing interest

The authors declare that they have no conflicting interest.

Authors’ contributions

ER and IR conceived and supervised the project and drafted the manuscript. PDM, MLG, SG, and SG were responsible for migraineurs patient collection. PF and SB carried out the molecular genetic studies. FG and AV performed the statistical analyses. LP edited the manuscript for intellectual content. All authors read and approved the final manuscript.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 2.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and Permissions

About this article

Cite this article

Rubino, E., Fenoglio, P., Gallone, S. et al. Genetic variants in the NOTCH4 gene influence the clinical features of migraine. J Headache Pain 14, 28 (2013).

Download citation


  • Migraine
  • Aura
  • NOTCH4 gene
  • Polymorphisms
  • Clinical features