Migraine is a prevalent neurological disturbance with the incidence of 16% of general population and impact roughly 20% of adults, ¹,²,³,⁴ that involves women more than men by the percent of up to 12% of men and 24% of women in the general population. ⁵ Migraine without aura [MO] and migraine with aura [MA] in which headache is started with supplemen tary neurological symptoms, are two principal pattern of migraine disease. ⁶,⁷

Studies on twin and family have substantiated that migraine comprises a main genetic component that MA has more than MO. The disease is genetically elaborate because many genes and environmental factors play a part in this disorder ⁵,⁷ and half of the patients have an affected first degree relative that it manifests the strong familial aggregation. ⁶

Although some clinical heterogeneity are shown in the MA and MO subtypes, Mochi et al. ⁸ with segregation analysis proposed that there may be a common genetic etiology for MA and MO. ⁹

Familiar hemiplegic migraine [FHM] is one of the most powerful congenital migraine sort of headache which is a rare autosomal dominantly inherited subtype of migraine with aura ⁵ that many studies revealed association this kind of migraine with mutations in the CACNA1A gene in different ethnic groups. Ophoff et al.¹⁰ characterized the CACNA1A gene in preparation for a mutation search in neurologic disorders that map to 19p13. They found that the gene covers 300 kb with 47 exons. ¹¹

On the other hand, several reports ⁵,¹²,¹³ suggest the possible involvement of CACNA1A in non-hemiplegic migraine, but the other studies show contradictory results. ⁵,⁸,¹⁴,¹⁵

Another studies showed the role of CACNA1A gene in the causation of common migraine, ¹²,¹³ however, some others in East Asia ¹⁶ did not show any correlation role. There is no study from Middle East and west of Asia to show the prevalence and correlation of this gene.

On the other hand migraine may genetically heterogeneous, ⁴ and more studies in different families and populations are required for a definite conclusion.

Therefore, the aim of this study was to surveyed leukocyte genomic DNA mutation of CACNA1A in Iranian migraine patients with and without aura who have family history of migraine.

Patient selection

MA and MO subtype of migraine were detected by expert clinical neurologist, according to the International Headache Society criteria [Headache Classification Committee of the International Headache Society [IHS] [MA = criteria 1.2.1 and MO = criteria 1.1]. ¹⁷

Mutation analysis

Then turning to genetic studies [found mutated in FHM ¹⁰ ] were studied in 30 patients who had family history of migraine with or without aura in first degree relatives. Genomic DNA was isolated from peripheral EDTA-treated blood cells by Qiagene DNA Mini kit [cat No: 51304]. DNA of each patient was subjected to PCR amplification of the Exon 5, 16, 32 and 36 of CACNA1A gene. Our study focused on 4 of the 47 exons of the CACNA1A gene. Exons 5, 16, 32 and 36 were studied because missense mutations [S218L, T666M, R1667W, L1682P, W1683R and I1811L, 2] have been found in FHM families. ¹⁰,¹⁸,¹⁹,²⁰

Twenty five-mL PCR reactions were carried out using 50 μL containing 100-200 ng total DNA from the patient, 10 pmol of each primers Table 1, 2.5 mM MgCl2, 200 mM each of dNTP and 1 U Taq DNA polymerase [Roche Diagnostics, Mannheim, Germany]. The reaction mixture was amplified under following condition: 94°C for 30s, 60°C for 30s and 72°C for 45s for 35 cycles followed by one cycle of 72°C for 7 minutes, after initial denaturation 95°C for 6 minutes. The amplification products were detected on 2% agarose gel, run in 0.5X TBE at 110 V for 50 minutes and visualized under UV upon staining with 0.002 mg/mL ethidium bromide. Good quality PCR products were sequenced using the Big Dye Terminator sequencing kit [Applied Biosystems] and an ABI 3130 Genetic Analyzer [Applied Biosystems]. Sequence results were compared with the published sequence [GenBank no.X99897] by using Chromas and DNAMAN software. The primers sequences have been reported in Table 1.{Table 1}

Review

Additionally, we performed a narrative review of all studies that evaluated CACNA1A gene, non-hemiplegic migraine [MA and MO] and FHM. Key words were classic migraine, migraine with aura, migraine without aura, common migraine and familial hemiplegic migraine and CACNA1A gene, gene mapping, gene screening and polymorphism. We searched MEDLINE-PUBMED, ISI, Scopus and Cochrane databases up to December 2012. We searched references lists from articles identified by search as well as a key review article to identify additional articles. We indentified case control, case series.

Mutation analysis of the 4 exons of the CACNA1A gene in these patients revealed one polymorphism, but no mutations were identified in this gene. Direct sequencing revealed a polymorphism previously reported polymorphism ¹⁰,²¹ G to A transition in the exon 16 [nt2369, G®A] in 9 patients. In review, the correlation of FHM loci [CACNA1A gene] with MA and MO has been showed in different population and only small population from Caucasians presented this correlation.

In this study we found no CACNA1A gene mutations in Iranian patients. In accordance with our study, many researches had been reported such result. Table 2 showed all familial hemiplegic migraine [FHM] loci previously reported as candidate loci in migraine with typical aura [MA] and migraine without aura [MO] in different population. All of studies with linkage analysis [microsatellite marker in 19p13], mutations analysis on CACNA1A gene and related polymorphism were included in this table. Similar to our study, most of studies in western countries ⁹,¹⁴,²²,²³,²⁴,²⁵,²⁶,²⁷,²⁸,²⁹,³⁰ and east of Asia ¹⁶ showed no correlation with FHM loci and MA and MO. Only a few studies in Netherlands and Finland ⁵,¹² and a small subset of patients and families ³¹,³² explained this correlation. It appears the correlation of FHM loci with MA and MO is not depended to specific ethnic and only small population from Caucasians showed this correlation.{Table 2}

Hence, CACNA1A is not probable responsible gene for types of migraine. Other genes or loci that are involved are more suitable alternative genes for MA and MO migraine, ⁷ for example in a study it was found that a responsible gene for migraine was the insulin receptor gene INSR, which lies in a Chr19p13 region near CACNA1A. Five single-nucleotide polymorphisms within the insulin receptor gene were significantly associated with migraine. Five single-nucleotide polymorphisms within the insulin receptor gene were considerably related with migraine, but there are no practical outcomes of this polymorphism. ³³

However, it seems, there is possible limitation for unobtaining any mutation in the CACNA1A gene. At least 18 CACNA1A gene mutations in 47 exons have been identified in people with FHM1. ¹⁹,²⁰ In our studies and some others ²² only limited mutations of FHM loci in limited exons were analyzed. Therefore, more studies with larger sample size and covering all 47 exons of the CACNA1A gene are necessary to confirm this hypothesis. We found a known polymorphism (nt 2369 G A) in exon 16 in 9 patients. Many studies identified other polymorphisms in the coding region of FHM loci in the patients with migraine, a number of them with significant difference ¹⁴,³⁰,³⁴ and some others without significant differences compare with control. ³⁵,³⁶

As a result, CACNA1A is most likely not a major susceptibility gene for types of migraine in Iranian migraines. It is essential to study more on larger series of MA patients and covering all 47 exons of the CACNA1A gene to surely include or exclude whether migraine and FHM share common genetic defects.

This work was founded by Grant No. 290028 from the deputy for Research, University of Medical Sciences and Isfahan, Iran. We would like to thank.