Abstract
Several studies have suggested that iron metabolism may be involved in the pathogenesis of migraine. Using a case-control design, we performed an association study in a cohort of Italian migraine patients to evaluate whether a particular allele or genotype of the haemochromatosis gene (HFE) would modify the occurrence and clinical features of the disease. We genotyped 256 migraine patients and 237 healthy age-, sex- and ethnicity-matched controls for the C282Y and H63D polymorphisms of the HFE gene. Phenotype and allele frequencies of both polymorphisms were similarly distributed in migraine patients and controls. The patients carrying the DD genotype of the H63D polymorphism showed a later age at onset of the disease and an increased number of migraine attacks. Our data suggest that the HFE gene is not a major disease gene for migraine. However, the H63D polymorphism of the HFE gene may be considered a modifying genetic factor in migraine.
Introduction
Migraine is a common headache disorder characterized by various combinations of neurological, gastrointestinal and autonomic symptoms (1). In Western countries, migraine affects up to 18% of women and 6% of men and significantly impairs the patient's quality of life (2, 3). The aetiology of migraine is still not completely clarified. Several studies have provided evidence that genetic factors play a major role in migraine but, at present, the number and types of genes responsible for the disease are still not clearly understood (4).
Recent studies have suggested that iron metabolism may be involved in the pathophysiology of migraine. Welch and colleagues reported elevated iron concentrations in the periaqueductal grey matter of patients with migraine and chronic daily headaches and demonstrated a significant correlation between duration of the illness and iron deposition (5). A significant positive association between plasma haemoglobin concentrations and migraine prevalence was found (6). Finally, a large Norwegian population-based study showed a high migraine prevalence in women with haemochromatosis (HH), a disease associated with progressive iron overload (7).
The haemochromatosis gene (HFE) is located in 6p21.3 and encodes for a HLA class I-like molecule involved in iron regulation (8). HH is characterized by excessive absorption of dietary iron and progressive deposition of this metal in the parenchymal cells of liver, pancreas, heart, pituitary gland and other organs. Two principal polymorphisms in the HFE gene, C282Y and H63D, have been identified as the cause of HH when present in the homozygous state (9, 10). Heterozygosity for these polymorphisms is associated with increased iron plasma concentrations (11). Cellular iron mismanagement and oxidative stress are associated with a number of neurological diseases (12). HFE gene mutations have therefore been studied as candidate risk factors in several diseases such as Alzheimer's disease, Parkinson's disease, stroke and amyotrophic lateral sclerosis (13–19).
We hypothesized that HFE gene polymorphisms would modify the occurrence and clinical features of migraine. To test this hypothesis, we performed a case–control association study in a cohort of Italian migraine patients recruited from a university-based Headache Clinic and in healthy controls.
Methods
Study population
A total of 256 consecutive unrelated migraine patients [98 men, 158 women; mean age (±SD) 40.3 ± 9.4 years] were involved in the study. The diagnosis of migraine was made according to the International Classification of Headache Disorders (IHCD-II) criteria (20). Two hundred and twenty-five patients fulfilled the diagnostic criteria for migraine without aura (MoA) and 31 for migraine with aura (MA). Age at onset and duration of the disease were 18.8 ± 10.5 years and 20.4 ± 15.7 years. A standardized record of all the clinical characteristics of headache, suitable for computer analysis, was obtained. A group of 237 sex, age and geographically (Northern Italy) matched healthy subjects [95 men, 142 women, mean age (±SD) 41.5 ± 13.3 years] were used as controls. The controls were blood donors and were screened by a neurologist specialized in headaches in order to exclude migraine. Written informed consent was obtained from all participants and the study was approved by the Hospital Ethics Committee.
Genetic analysis
Genomic DNA was extracted from 200 µl of peripheral blood using the QIAamp DNA mini Kit (Qiagen, Milan, Italy). All polymorphisms were analysed by enzymatic dygestion. Polymerase chain reaction (PCR) reactions were performed in a final volume of 50 µl, 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. An initial denaturation was performed using the following conditions: 95°C 10 min, 35 cycles 95°C 1 min at specific temperature for each primer, 72°C 1 min and a final elongation to 72°C 5 min. PCR products were electrophoresed on a 2% agarose TBE 1× gel and stained with ethidium bromide. Table 1 shows PCR conditions, primers and products of digestion.
Polymerase chain reaction conditions, primers and products of digestion
Ta, Temperature of annealing.
Statistical analysis
Statistical analyses were performed using Genepop version 3.4 (http://wbiomed.curtin.edu.au/genepop) and SigmaStat version 1.0, 1994 (Jandel Corp., San Rafael, CA, USA). Fisher's exact test, χ2 test and
Results
The Hardy–Weinberg equilibrium was verified for all tested populations. We found no C282Y mutation in either migraine patients or healthy controls. The genotype (GF) and allele frequencies (AF) of the H63D polymorphism in cases and controls are shown in Table 2. In our control group, GF and AF of this polymorphism were remarkably similar to what has been reported in other Italian populations (22). No gender difference was found in either cases or controls. χ2 test for AF and GF comparisons showed no significant differences between migraine patients and controls (χ2 = 4.52, P = 0.10 and χ2 = 2.53, P = 0.28, respectively). No significant difference between MA and MoA patients was found (GF, χ2 = 1.52, P = 0.54; AF, χ2 = 0.04, P = 0.84, respectively).
Genotype and allele frequencies of the HFE H63D polymorphisms in migraine patients and controls
GF, Genotype frequencies; AF, allele frequencies.
Clinical characteristics of migraine patients according to H63D genotypes are shown in Table 3. Age at onset of the disease was significantly (P = 0.014) later in patients carrying the D63D genotype in comparison with the remaining genotypes. Patients carrying the same genotype, in comparison with the remaining genotypes, showed a significantly (P < 0.001) increased frequency of migraine attacks. No significant difference in the other clinical features was found.
Clinical characteristics of migraine according to HFE H63D genotypes
P < 0.05 and
P < 0.001 in comparison with both HH and HD patients.
Discussion
In this study of an Italian population, we found no evidence of genetic association between the HFE gene and migraine. Both genotype and allele frequencies of the examined polymorphism were similarly distributed between cases and controls. Furthermore, when the patients were divided into different clinical subgroups (MA and MoA), no significant differences in HFE gene polymorphism frequencies were found. So, it is unlikely that genetic variations within the HFE gene greatly contribute to migraine susceptibility.
To the best of our knowledge, this is the first study to evaluate the association between migraine and the HFE gene and some caution is needed when interpreting the results. Key issues to be addressed in any association study include the phenotypic definition of the disease, the selection of the polymorphisms, adequate sample size of cases and controls and population stratification (23). The diagnosis of migraine relies on the IHCD-II criteria that are unambiguous and precise. The frequencies of C282Y and H63D mutations of the HFE gene vary between populations. In Europe, C282Y frequency declines from North to South: the highest frequency was found in Ireland and the lowest in Italy (1.3%) (24). No C282Y mutation was found in the Balearic Islands (Spain) or in North Africa (25, 26). In contrast, the H63D mutation frequency does not show a North–South decline (24). In our study, we found no C282Y mutation in either cases or controls, so it has not been possible to evaluate the association of this polymorphism with migraine. In contrast, allelic and genotypic frequencies of the H63D polymorphism were similar to those reported in other Italian populations (22). Participants in our study were all White and originate from the same region of Italy. It is therefore likely that cases and controls arose from the same homogeneous population. Finally, we increased the statistical power of our study, setting the level of statistical significance at P < 0.01 (21). However, the replication of our findings in independent populations is warranted to confirm our data.
When we compared the clinical characteristics of migraine according to different HFE genotypes, we found that patients carrying the D63D genotype, in comparison with the other genotypes, showed a different age at onset of the disease and an increased frequency of migraine attacks. Even if the number of patients carrying this genotype is small, our results are in accord with a previous study of cluster headache patients showing that the D63D genotype of the HFE gene influences the clinical characteristics of the disease (27). Taken together, the results of our study suggest that the HFE gene may be regarded as a disease modifier gene in primary headaches. The biological mechanisms underlying our findings are unknown. The D63D genotype of the HFE gene is associated with increased iron absorption (28, 29). In addition, the same polymorphism may influence the binding of the HFE protein to transferrin receptor, thereby influencing the mechanism of iron transport across the blood–brain barrier (30). Therefore, this polymorphism may modulate iron deposition in the periacquedutal grey matter as well as in other centres of the pain-modulating network within the central nervous system.
Previous molecular genetic studies have suggested the presence of a major gene for migraine within the HLA region. A significant association between migraine (mainly MoA) and polymorphisms of Class III tumour necrosis factor (TNF)-α and TNF-β genes and Class II HLA-DRB1 gene was found (31–33). The HFE gene maps 4Mb telomeric to HLA-A and is now considered the most telomeric Class I-like sequence. In the present study we found no significant association between the HFE gene and migraine, whereas in the same group of patients we previously found a significant association with both TNF-α and HLA-DRB1 gene polymorphisms (31, 33). Our studies suggest, therefore, that the region including Class III and Class II HLA genes must be investigated further in order to isolate genetic risk factors for migraine.
In conclusion, our data do not support the hypothesis that the HFE gene is a genetic risk factor for migraine. However, we have shown that the H63D polymorphism of the HFE gene may modify the clinical characteristics of the disease. Additional studies are needed in order to evaluate the precise role of the haemochromatosis gene in migraine pathogenesis.
Acknowledgements
The study was supported by a 2004 grant from the ‘Ministero dell'Università e della Ricerca Scientifica (MURST)' and by a 2004 grant from Regione Piemonte (Italy).