*Department of Clinical Research, National Saigata Hospital

Ohgata-machi, Niigata 949-3193, JAPAN

Correspondence to: Dr. Masayuki Ikeda

Department of Clinical Research, National Saigata Hospital, Ohgata-machi, Niigata 949-3193, JAPAN, Phone +81-255-34-3131, Fax +81-255-34-6734 (e-mail: massie@saigata-nh.go.jp)

Abstract

To test the hypothesis that iron overload predisposes to epilepsy, I studied transferrin saturation in 130 patients with epilepsy and sex- and age-matched 128 control subjects without epilepsy. I found that transferrin saturation was significantly higher in the epilepsy group (39.9ｱ19.6 %: mean ｱ SD) than in the control group (29.1ｱ14.9 %). Abnormally high transferrin saturations (men: > 60%, women: >50%) were found in 10 patients with epilepsy but in only one subject without epilepsy. Antiepileptic drugs did not affect the transferrin saturation. Of the 11 with abnormally high transferrin saturation, two with epilepsy were heterozygotic for H63D in the haemochromatosis gene but no patient had the C282Y mutation. These results indicate that iron overload other than the C282Y mutation underlies epilepsy.

Keywords: Epilepsy, Haemochromatosis, Iron Overload

Iron accumulation results in the formation of free radicals and subsequent brain injury.[1] Neurological diseases associated with iron overload vary: asymptomatic deposition of iron in the basal ganglia,[2] psychiatric diseases,[3] mental retardation,[4] parkinsonism,[5][6] dementia, ataxia and myoclonic jerks.[7] Siderosis in the brain is associated with epilepsy.[8][9] Animal studies suggest that iron accumulation may underlie the pathophysiology of epilepsy.[10][11]

The aim of my study was to test the iron metabolism of epileptic patients. I measured transferrin saturation as an index of iron overload in patients with epilepsy and age- and sex-matched control subjects. In patients with high transferrin saturations, I also examined mutations in the haemochromatosis gene (HFE ).

Patients and Methods

Patients

I studied 258 subjects, 130 patients with epilepsy (63 men, 67 women, aged 38.7 ｱ 10.3 years: mean ｱ SD) and 128 sex- and age-matched (63 men, 65 women, aged 40.8 ｱ 10.3 years) control subjects without epilepsy. I excluded subjects with pica, those receiving iron-containing drugs, blood transfusions or alcohol. None of the subjects studied got haematologic diseases or active liver diseases. All subjects, whether epileptic or not, were mentally retarded and cared for by the nursing staff of Ranzan Institute in Saitama, Japan. All of them could eat and did not receive forced nutrition. Although I did not make a quantitative comparison, I found no obvious difference between the two groups in daily activities.

Methods

Measurement of serum iron, transferrin and ferritin

Serum samples after an overnight fast were obtained from each subject. I measured serum iron by standard spectrophotometry. Serum transferrin levels were determined by rate immunoturbidimetry on an automated analyser (model TBA-20FR, Toshiba Medical, Tokyo, Japan). Serum ferritin levels were measured by chemiluminescence immunoassay (Eiken Chemical Co.,Ltd., Tokyo) in patients with high transferrin saturation (men: > 60%, women: >50%).

Identification of the C282Y and H63D mutations in HFE

I also examined HFE mutations in 11 patients with abnormally high transferrin saturation. The mutation study was approved by the ethics committee at Ranzan institute. Since the subjects could not understand the explanation of the study due to mental retardation, I obtained written informed consent from their parents or legal guardians.

HFE contains two common missense mutations.[12] One mutation (guanine to adenine at nucleotide 845) in HFE results in the substitution of tyrosine for cysteine at amino acid 282 and is termed the C282Y mutation. The other mutation (cytosine to guanine at nucleotide 187) in HFE results in the substitution of aspartate for histidine at amino acid 63 and is termed the H63D mutation.

PCR amplification of the regions containing the missense mutations was performed with the primer sequences of Feder et al..[12] The C282Y and the H63D mutations were identified with allele-specific oligonucleotide hybridisation.[13]

Ethical considerations

After detailed explanations of the study, written informed consent was obtained from the family or legal guardian of each patient. I performed this study after the approval by the committee for human investigations of Ranzan Institute.

Statistical analysis

All values are presented as means ｱ SD. Differences between means were analysed by a two-tailed Studentﾕs t-test or Mann-Whitneyﾕs U-test.

Results

The serum iron was significantly higher ( P < 0.01 ) in the epilepsy group (106 ｱ 8 ｵg/dL) than in the control group (88 ｱ 8 ｵg/dL) while the unsaturated iron binding capacity was significantly lower ( P < 0.01 ) in the epilepsy group (173ｱ78 ｵg/dL) than in the control group (228 ｱ 76 ｵg/dL). Thus, the transferrin saturation was significantly higher ( P < 0.01 ) in the epilepsy group (39.9ｱ19.6 %) than in the control group (29.1ｱ14.9 %). On the assumption that some antiepileptics may affect iron metabolism, I compared the degrees of transferrin saturation in the subgroups within the epilepsy group according to the prescribed drugs (Table 1). There was no significant difference in transferrin saturation between the subjects who were taking one of the four antiepileptic drugs and those who were not.

Table 2 shows that an abnormal increase in transferrin saturation (men: > 60%, women: >50%) was found in 11 patients (5 men and 6 women) consisting of 10 patients with epilepsy and only one in the control group. I found no cause of secondary iron overload in these patients. Serum ferritin was not increased in any of them. Among the 11 patients, two were heterozygous for H63D. Both of them were epileptic. No C282Y mutation was found in any of the 11 patients with abnormally high transferrin saturations.

Discussion

I found that the transferrin saturation was significantly higher in patients with epilepsy than in those without epilepsy. Moreover, abnormally high transferrin saturations were found in 10 patients in the epilepsy group but only in one in the control group. These data indicate iron overload in the patients with epilepsy. Factors which cause secondary iron overload, including diet, blood transfusions, alcohol, liver injury and haematologic diseases, were ruled out. Antiepileptic drugs cannot explain the iron overload in epileptic patients, either. Since phenytoin is an iron-chelator,[14] it would reduce iron load rather than increase it. Previous studies on rats and mice showed that administration of phenytoin, phenobarbital or primidone does not change the iron concentration in the serum or brain.[15][16] My data, showing that none of the antiepileptics affected the transferrin saturation, also provide evidence that the higher transferrin saturation in the epilepsy group is not due to antiepileptics.

I then studied mutations in HFE because haemochromatosis is the most common disease of primary iron overload. I found two patients hetetozygotic for the H63D mutation, but no patient with the C282Y mutation. Haemochromatosis is thought to be uncommon in Japanese,[17] but the frequency is unknown. Merryweather-Clarke and others [18] reported that the C282Y mutation was most frequent in northern European populations and absent from 484 Asian chromosomes. The positive predictive value of the transferrin saturation test, i. e. , the possibility that a patient with a positive result actually has haemochromatosis, is unknown in Japanese.

I do not assume that heterozygosity for H63D affects iron metabolism in epileptic patients, because its high frequency in control populations, ranging from 16 to 23%,[19] makes the heterozygosity for H63D unlikely to be pathogenic. H63D is probably deleterious only in compound heterozygotes (heterozygous for both C282Y and H63D).[20] To determine the cause of iron overload in patients with epilepsy, I continue to look at other genes regulating iron metabolism in these patients.

Acknowledgements

I acknowledge the invaluable co-operation of Professor Ernest Beutler in HFE mutation analysis. I am grateful to Drs Akiko Takaki, Shunji Takaki and Kenji Kuroda at Ranzan Institute for obtaining the informed consent of the patients and to Dr Toshiyuki Himi at Tokyo Medical and Dental University for the DNA extraction.

References

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