Abstract
Introduction
Alternating hemiplegia of childhood (AHC) is a rare disorder mainly characterized by attacks of hemiplegia and mental retardation. It has been often associated with migraine. The CACNA1A gene on chromosome 19 is involved in familial hemiplegic migraine and other episodic cerebral disorders, but also with progressive neuronal damage.
Methods
We performed mutation analysis in this gene in four AHC patients, using single strand conformation polymorphism analysis.
Results
We found nine polymorphisms, but no mutations in any of the 47 exons.
Conclusions
Other cerebral ion channel genes remain candidate genes for AHC.
Introduction
Alternating hemiplegia of childhood (AHC) is a rare brain disorder characterized by:
repeated bouts of hemiplegia involving either side of the body at least in some attacks;
episodes of bilateral hemiplegia or quadriplegia starting either as generalization of a hemiplegia episode or bilateral from the start;
other paroxysmal phenomena, including tonic/dystonic attacks, choreoathetotic movements, nystagmus, strabismus, dyspnoea and autonomic phenomena, occurring during hemiplegic attacks or in isolation.
immediate disappearance of all symptoms on going to sleep, with recurrence 10–20 min after awakening in long-lasting bouts;
evidence of developmental delay, mental retardation and permanent neurological abnormalities, including choreoathetosis, dystonia or ataxia. Age at onset is in general before 18 months (1, 2).
The cause of AHC is unknown. It has often been regarded as a subtype of complicated migraine and some of the first reports included patients with familial hemiplegic migraine (3). Arguments in favour of an association between AHC and migraine are the clinical similarity between attacks of AHC and (hemiplegic) migraine, the paroxysmal nature of the disorders and the similar effect of sleep on the attacks. This is further supported by the reported occurrence of migraine-headache during some attacks of AHC, a positive family history of migraine with aura in 30% of reported AHC patients, and the co-occurrence of migraine with aura and AHC in some patients (1). Some of the aspects of AHC, however, are clearly distinct from migraine, including choreoathetosis and dystonic posturing, and a progressive course associated with mental deterioration (1). Although most patients appear to be sporadic cases, AHC has also been described in families (4, 5). In one AHC family, a balanced reciprocal translocation t(3;9)(p26; q34) was found in four patients in three generations (4, 6).
Familial hemiplegic migraine (FHM) is an autosomal dominant subtype of migraine with aura. Attacks are characterized by hemiplegia occurring before or during the headache (7). In some families, FHM is accompanied by progressive cerebellar ataxia with cerebellar atrophy. Approximately 50% of the FHM families, including all families with ataxia, are linked to chromosome 19 (7). A second locus is located on chromosome 1 and as some families could not be linked to either of these loci, there must be at least a third, as yet unknown, locus (8, 9). In 1996, we found missense mutations in the CACNA1A gene on chromosome 19, encoding the α1A subunit of a P/Q type calcium channel in FHM (10). In addition, truncating mutations in this gene were found responsible for episodic ataxia type 2 (EA2), mainly characterized by attacks of generalized ataxia which can be prevented by treatment with acetazolamide (10). Finally, a (stable) trinucleotide repeat expansion in the CACNA1A gene has been identified as the cause of spinocerebellar ataxia type 6 (SCA6), a progressive form of pure cerebellar ataxia (11). These findings show that mutations in the CACNA1A gene can cause both paroxysmal and chronic progressive cerebral dysfunction.
We considered the CACNA1A gene a good candidate gene for AHC, as mutations in this gene could explain the episodic hemiplegia of AHC, the progressive permanent cerebral damage and its association with migraine. In addition, AHC can lead to progressive ataxia with cerebellar atrophy (12) and can also be associated with episodic ataxia (13). Therefore, a mutation screening of all exons of the CACNA1A gene was performed in four AHC patients.
Patients
Patient 1
At the age of 3 months, this 4-year-old boy had an attack of stiffening for a few seconds, followed by head turning to the right, with the left eye in midline and the right eye deviated to the right. He was generally floppy throughout the attack, which lasted 10 min. Afterwards he fell asleep for a few minutes and was normal on awakening. Similar episodes have occurred frequently since. At present, he has attacks every 7–14 days. There is unilateral facial grimacing, left lateral eye deviation, and flexion of an arm with abduction at the shoulder in a dystonic posture. Weakness on the contralateral side may be present. The weakness may change from one side to another during the same attack with occasional bilateral episodes. There are no indications of headache during the attacks. He has a delay in his development: he was unable to sit unsupported before the age of 18 months, he is still unable to walk independently, and speaks only a few words. There was no improvement upon administration of phenytoin, carbamazepine or flunarazine. Acetazolamide has not been tried. Repeated EEGs, cerebral MRI, and metabolic investigation including ammonia, CSF and plasma lactate, urinary organic acids, plasma and urinary aminoacids, were normal. Family history is unremarkable.
Patient 2
This 14-year-old girl was born after a normal pregnancy. She smiled at 6 weeks, sat unsupported at 8 months, stood at 12 months, and walked independently at 15 months (though she was still unstable). At the same time she started talking. At the age of 2 years she started to have attacks of predominantly right-sided hemiplegia, lasting a few minutes to several hours, always without headache. As she got older, nominal aphasia and choreoathetotic movements of the limbs accompanied the attacks. From the age of 5 years, her walking detoriated. At this time, episodes occurred daily and seemed to be precipitated by physical exercise and excitement. Afterwards, she appeared pale and tired. With time, cognitive delay became apparent. At present, she is functioning at the level of a 7-year-old. EEGs (both ictal and interictal) and cerebral CT-scans were normal. Neurological examination revealed no abnormalities. The initial diagnosis was hemiplegic migraine, but this was later changed to AHC. Administration of phenytoin, flunarazine and nimodipine was ineffective. Acetazolamide has not been tried. The father of the patient suffers from migraine with visual aura. Her mother's cousin also has experienced similar attacks of weakness starting at the age of 2, but he is now developmentally and intellectually at a normal level.
Patient 3
This 6-year-old boy was delivered at term, having a low birth weight (2.8 kg) and small head circumference (34 cm; 25th percentile). Problems in the neonatal period included a temperature of 38°C, hypoglycaemia, hyponatremia, tense fontanel, probable inappropriate ADH secretion and vomiting. At day 4 he started having attacks of abnormal movements with nystagmus and was commenced on phenobarbitone. A cerebral CT scan on day 10 showed blood on the tentorium and in the interhemispheric fissure, but not intraparenchymally. The attacks persisted and treatment with phenytoin was started. At 6 weeks the attacks disappeared on carbamazepine, but at the age of 9 months they returned. The attacks started with eye flickering, followed by weakness on either side of the body and occasionally bilateral, lasting for as long as 10 days. In this period, the weakness improved after sleep, but returned after 15 min. Recently, the attacks have been associated with hypoventilation and apnoeas and focal twitching on the left side.
The patient is developmentally delayed. He is unable to walk unaided. His language is limited to a few words. Interictal neurological examination revealed an intermittent nystagmus. Repeated cerebral CT-scans, EEGs and cerebral MRI-scans were normal, as was a metabolic screen, including amino acids, organic acids, ammonia, magnesium, calcium, TSH, urate, biotinidase, very long chain fatty acids, and CSF and plasma lactate. The patient is currently on sodium valproate and flunarizine, without evident effect. Phenytoin, phenobarbitone, clonazepam, diazepam and paraldehyde were tried without effect. Acetazolamide has not been tried. Several family members suffer from migraine with aura.
Patient 4
This 12-year-old girl started having episodes of alternating hemiplegia and sometimes quadriplegia 10 days after birth. The episodes lasted from several hours to 8 days, in a frequency of 1–3 times per month. There was no headache during the attacks. Various anticonvulsants were ineffective. Flunarizine gave slight improvement. Since she started taking acetazolamide the attacks have disappeared. Neurological examination between episodes is normal, except for a slight mental retardation. The mother of the patient suffers from migraine without aura.
Methods
Genomic DNA samples
Blood samples of all patients were collected and genomic DNA was isolated from leucocytes as described by Miller et al. (14).
SSCP analysis
PCR products of all 47 exons of the CACNA1A gene were screened for sequence aberrations by SSCP analysis (15). The exons were amplified using exon-specific primer sets and PCR conditions previously described by Ophoff et al. (10). Essentially, for SSCP analysis, amplification was performed in two consecutive rounds, in such a way that PCR products were radioactively labelled by the incorporation of 32P-dCTP in the second round of amplification. Subsequently, samples were denatured in formamide buffer and subjected to SSCP analysis on an 8% polyacrylamide (19:1) gel containing 10% of glycerol. Electrophoresis was carried out at room temperature at a constant power of 28 W. Finally, SSCP gels were dried and exposed for autoradiography. Gels were visually inspected for the presence of aberrant banding patterns.
(CAG) n-repeat analysis
Determination of the number of CAG repeats at the 3' end of CACNA1A has been described previously (11). In brief, PCR products were electrophoresed on denaturing polyacrylamide gels and were compared with a sequencing ladder for size determination.
Results
IIn SSCP analysis, we found nine polymorphisms in four AHC patients, of which four had not been previously reported. The polymorphisms are shown in Table 1. No mutation was identified in any of the 47 exons of the CACNA1A gene in the four patients studied. Also, no patient had a CAG repeat length greater than 13 (normal < 17).
CACNA1A polymorphisms detected in AHC patients. The nucleotide change, amino acid involved, and location in the CACNA1A gene of each polymorphism are indicated. All nucleotide positions are corresponding to the sequence of Ophoff et al. (1996) (Ac nr. X99897) (10), except for the polymorphism in exon 47. As this is present in the long isoform of CACNA1A, and not in the X99897 sequence, the position is given in respect to the sequence by Hans et al. 1999 (Ac nr. AF004884) (20). Furthermore, it is indicated which of the AHC patients are heterozygous for the polymorphisms as well as the frequency of the polymorphisms in the general population. The new polymorphisms, presented in this article, were tested in a set of 96 randomly picked individuals from the Dutch population
Discussion
Given the episodic nature of AHC, its underlying pathophysiology might very well be an ionchannel dysfunction. Channelopathies are characterized as being unpredictable, episodic and paroxysmal, often precipitated by external conditions that are generally regarded as normal (16, 17). Considering the clinical similarities between AHC, FHM and EA2, the α1A calcium channel subunit gene CACNA1A seemed to be a good candidate gene for AHC. Interestingly, patient number 4 responded well on acetazolamide, a well-known treatment for EA2.
We found nine polymorphisms, of which four have not been previously reported (see Table 1). None of these, however, are expected to be causative for the disease. We failed to identify any mutation in the CACNA1A gene in four patients with AHC. There are several possible explanations for this. First, we used SSCP analysis, which is a reasonably sensitive (70–90%) method for screening genes for point mutations as long as the fragments are not longer than 200 bases (18). However, it is possible that micro- or macro-deletions in the gene, which are undetectable by SSCP, are the underlying cause of AHC. To investigate this, other techniques such as Southern blotting and/or pulsed-field gel electrophoresis should be used in the search for mutations. The genomic size of the CACNA1A gene, 350kb, however, currently makes it very hard to apply these alternative methods in a standard (and rapid) mutation screen.
Second, AHC may be caused by mutations in another (ion-channel) gene. Mutations in different ion-channel genes can cause a combination of paroxysmal and permanent cerebral dysfunction. Brain-specific ion-channel genes on chromosome 3 and 9 would be of specific interest, as a balanced reciprocal translocation t(3, 9)(p26;q34) was found in an AHC family (4). Therefore another interesting candidate gene is the CACNA1B gene, encoding an α1B subunit of a N-type calcium channel, located on 9q34 (19).
Third, AHC might not have a genetic basis at all. Except for two AHC families, the majority of AHC patients seem to be sporadic cases.
Because of the low frequency of the disorder and the fact that patients often are sporadic cases, genetic research in AHC will be problematic, as classical linkage studies are hardly feasible. Therefore, further evaluation of good candidate genes for AHC remains a useful step towards elucidating its pathogenesis (17).
Footnotes
Acknowledgements
We thank Professor R.D. Robinson, Department of Paediatric Neurology, and Dr S. Mohammed, Department of Genetics, Guy's Hospital, London, United Kingdom, for their assistance and Simone Houtman, Department of Human and Clinical Genetics, LUMC, Leiden, the Netherlands, for technical assistance in some of the work. This work was supported by the Netherlands Organization for Scientific Research (NWO) (nr. 903–52–291) and Migraine Trust.