Leukocyte Mitochondrial DNA A to G Polymorphism at 11084 is not a Risk Factor for Japanese Migraineurs

Abstract

Mitochondrial dysfunction has been reported in patients with migraine. We investigated leukocyte mitochondrial DNA 11084 A to G polymorphism in 166 Japanese migraineurs and 483 Japanese controls. The migraine group consisted of 43 patients suffering from migraine with aura (MWA) and 123 from migraine without aura (MOA). The frequency of the transition was 7.2% (12/166) in the migraine group and 7.3% (35/483) in the controls. The frequency of the transition was 4.7% in MWA and 8.1% in MOA. There was no significant difference among the groups (chi-square test). The mitochondrial DNA 11084 A to G transition was more common in Japanese subjects than reported in Caucasians; however, this polymorphism is not a genetic risk factor for migraine in Japanese patients.

Introduction

Mitochondrial dysfunction in migraine has been suggested (1), and mitochondrial genetic studies have been widely performed (1–5). A mitochondrial DNA11084 A to G transition has been reported in which a coding region of NADH dehydrogenase 4 (ND4), a subunit of mitochodrial complex I, causes amino acid replacement from Thr to Ala. This mutation was first reported as a cause of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome (6). Sakuta et al. (7) suggested that this mutation might be a polymorphism but did not investigate this mutation in migraine patients. In a small study of Japanese patients submitted as an abstract, 13 out of 53 (24.5%) migraineurs had mitochondrial DNA 11084 mutation (8). In contrast, the 11084 mutation has been absent in Caucasians with/without migraine headache (3, 5). The aim of this study was to elucidate whether the 11084 polymorphism is a risk factor for migraine headache in the Japanese population.

Subjects and method

One hundred and sixty-six migraineurs participated in this study after giving written consent. The diagnosis of migraine was established in accordance with the criteria of the International Headache Society (9). Forty-three subjects suffered from migraine with aura (MWA, mean age = 34.1 ± 11 (SD) years, male:female=13:30) and 123 suffered from migraine without aura (MOA, 37.1 ± 15.3 years; male:female=15:108). The control group consisted of 483 healthy subjects (45.5 ± 9.9 years; male:female=242:241). All subjects were Japanese. Genomic and mitochondrial DNA was extracted from venous blood leukocytes by the standard DNA extraction method. All samples were encoded and analysed blindly. Polymerase chain reaction, restriction fragment-length polymorphism (PCR-RFLP) analysis, was performed as follows. The sense oligonucleotide primer was 5′-CTC CTA CCC CTC ACA ATC ATG GCA AGC-3′ and the antisense primer was 5′-ATT AGT GCG ATG AGT AGG GGA AGG GAG C-3′, which was designed using Genetyx Ver. 8 Software (Software Development Co. Ltd, Tokyo, Japan). The PCR mixture (final volume 24 μl) consisted of 0.5 μM sense and antisense oligonucleotide, 250 μM dNPTs, 1 unit-Taq, and PCR buffer (TaKaRa-Taq, #R001, Takara, Japan) to which 1 μl (50 ng) of test DNA was added.

The PCR cycle parameters were: 5 min of initial denaturation at 94 °C, followed by 35 cycles of denaturation for 30 s at 94 °C, primer annealing for 30 s at 55 °C, and primer extension for 1 min at 72 °C. A final extension step was performed for 5 min at 72 °C. These primers generated a 281-bp PCR-product. The PCR products were digested by a restriction enzyme BsaM I (#R6991, Promega, Madison, Wisconsin, USA) at 65 °C for 4 h. The fragment derived from the A allele (wild) was not digested by BsaM I, whereas the fragment of the same length from the G allele (mutant) was digested by BsaM I into 169- and 112-bp fragments. The BsaM I-digested PCR fragments were electrophoresed in 1.5% agarose gels and stained with ethidium bromide. Sequencing of PCR-products from a part of the samples was carried out to confirm that this PCR method provided amplification of the target mitochondrial DNA region. The frequency of the mutation was evaluated using the gene counting method and the differences between the groups were analysed using the chi-square test.

Results

Thirty-five of 483 control subjects (7.3%) had mitochondrial DNA 11084 A to G transition. In the migraine group, 12 out of 166 (7.2%) had the mutation. There was no significant difference between the control and migraine groups (chi-square test). Two of 43 patients (4.7%) with MWA and 10 of 123 patients (8.1%) with MOA had the mutation. The frequency of the mutation in the migraine subgroups was also not significantly different from that of the controls (chi-square test, Yates' correction was applied as appropriate).

Discussion

Mitochondrial dysfunction in migraine has been hypothesized based on altered respiratory chain enzymatic activity, abnormal ³¹P-magnetic resonance spectroscopy, and elevated plasma lactate and pyruvate in migraine (1, 10, 11). Patients with mitochondrial disorders, especially in MELAS, often complain of migraine-like headache. Thus, to elucidate whether migraine is a monosymptomatic form of mitochondrial disorders, mitochondrial genetic studies have been performed. Some mutations of mitochondrial DNA were reported in patients with migraine (8), migrainous stroke (2) and cluster headache (12, 13). However, negative results of mitochondrial DNA study in common forms of migraine were reported (1, 3–5).

In this study, we investigated the mitochondrial DNA 11084 A to G transition, in a large group of Japanese migrainures and controls. The frequency of G allele (mutant) in migraine was 7.2%, which was same as in the controls. The 11084 mutation has been reported only in Japanese subjects, except for one case of MELAS and severe migraine (6).

We conclude that leukocyte mitochondrial DNA 11084 A to G transition is a common polymorphism in the Japanese population and is not a genetic risk factor for migraine in Japanese people.