A Novel ATP1A2 Mutation in a Family with FHM Type II

Abstract

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura with an autosomal dominant pattern of inheritance. Six FHM families underwent extensive clinical and genetic investigation. The authors identified a novel ATP1A2 mutation (E700K) in three patients from one family. In the patients, attacks were triggered by several factors including minor head trauma. In one subject a 3-day coma developed after a cerebral angiography. Overall, the phenotype of the patients closely resembles that of previously reported cases of FHM type II. The E700K variant might be regarded as the cause of the disease in this family, but this was not tested functionally.

Introduction

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura with an autosomal dominant pattern of inheritance (1). Recurrent hemiparetic/hemiplegic aura attacks may last days or weeks. Other reversible, focal neurological signs or symptoms, including visual disturbances, language impairment and paraesthesias can be associated. Moreover, approximately one-third of FHM families also present with progressive cerebellar ataxia (2).

The first FHM gene, CACNA1A (MIM ∗601011), is located on chromosome 19p13 (FHM1, MIM ♯141501) and encodes the pore-forming subunit Cav2.1 of P/Q-type calcium channels (3). These channels are expressed throughout the central and peripheral nervous system and mediate transmitter release from synaptic nerve terminals (4) by modulating intracellular Ca²⁺ homeostasis. However, mutations in CACNA1A are found in only 50–75% of FHM families (2, 5). A second FHM locus (FHM2, MIM ♯602481) was later identified on chromosome 1q21–q23 in three French families (6) and in one large family of German-Native American descent (7). Lower penetrance, presence of epileptic seizures and absence of cerebellar dysfunction seem to differentiate FHM2 from FHM1 patients (2, 6). Families not linked to the existing loci have also been identified, indicating further heterogeneity (2).

Recently, narrowing of the FHM2 locus to a 0.9-Mb region on chromosome 1q23 (8) allowed the identification of mutations in the ATP1A2 gene (MIM ∗182340) in two Italian families (9). Five subjects (three in one family, two in the other) presented relatively benign partial seizures. Shortly after, additional families were found to harbour ATP1A2 mutations (10–14). In one family, FHM and benign familial infantile convulsions (BFIC) partially cosegregated (10). Interestingly, a new ATP1A2 mutation was associated with alternating hemiplegia of childhood (15, 16) (AHC, MIM ♯104290), a rare syndrome characterized by early-onset episodic hemi- or quadriplegia (17) and often considered to be a migraine equivalent or an unusual form of epilepsy.

We have analysed six FHM pedigrees in which we had ruled out CACNA1A mutations by direct gene sequencing. Although the possibility of having missed heterozygous large-scale deletion or intronic mutations cannot be completely excluded, we also tested the hypothesis of mutations in the ATP1A2 gene. We present the clinical and molecular data relating to one kindred and describe a novel ATP1A2 mutation.

Patients and methods

The clinical diagnosis followed the International Headache Society (IHS) 2nd edition criteria (1). Having obtained the patients’ informed consent, we purified genomic DNA from blood and polymerase chain reaction (PCR)-amplified the 23 exons and 80 base pairs of the flanking intronic sequences of ATP1A2. PCR products were sequenced using BigDye labelling in an ABI 377 genetic analyser (Applied Biosystems, Foster City, CA, USA). To confirm the mutation in patients and to rule out its presence in control chromosomes, we amplified a mispairing 127-bp PCR fragment using oligonucleotide primers (5′-3′) atp15F-ctccccagcagaagcccatc (mismatched nucleotide is underlined) and atp15R-cgcctctcactccatgcttg. The PCR reaction was performed in a 50-μl volume containing 50 ng of genomic DNA with Hot Master Taq DNA polymerase (Eppendorf, Milan, Italy): initial denaturation step at 95°C for 5 min, followed by 30 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C, and a 7-min terminal elongation step at 72°C. About one-tenth of the amplicon was cleaved with the endonuclease BstX I (New England Biolabs, Ipswich, MA, USA), resolved on a 12% polyacrylamide-bisacrylamide gel, and stained with ethidium bromide. The amplicon is normally cleaved into two fragments, sized 104 and 23 bp. The presence of the E700K mutation abolishes the single site of cleavage and produces fragments sized 127, 104, and 23 bp in the patients (Fig. 1a). We a posteriori genotyped 1q markers D1S2343, D1S1153, D1S2635, D1S2707, D1S2705, D1S2844, and snp rs6695366 in available members to haplotype the family.

Figure 1

(a) Restriction fragment length polymorphism (RFLP) analysis employed to confirm the E700K mutation in ATP1A2. A mispairing 127-bp polymerase chain reaction (PCR)-amplified fragment is normally cleaved by the endonuclease BstX I into two fragments, sized 104 and 23 bp (not shown). The mutation abolishes the single site of cleavage and produces fragments sized 127 (bold, arrow), 104, and 23 bp (not shown). Individuals are as in the superimposed pedigree. U, Uncut PCR-amplified fragment; C, normal control; M, DNA molecular marker size. Black squares denote affected individuals. FHM2 locus haplotypes for markers D1S2343, D1S1153, D1S2635, D1S2707, rs6695366, D1S2705, D1S2844 (top to bottom) are indicated. Haplotype in affected patients is boxed. (b) Electropherogram of the ATP1A2 gene flanking the new missense mutation E700K. The sequence of DNA from a normal control (top) and patient I-02 (bottom) is shown. Sequencing was performed by means of a sense primer. The mutation is indicated. (c) The pedigree of the five FHM families in this study who tested negative for mutations in CACNA1A and in ATP1A2 by direct gene sequencing. Circle, Female; square, male; solid symbol, affected patient; dashed symbol, subjects suffering from migraine with or without aura. (d) Local amino acid sequence alignment of ATPases among different species. The region flanking residue E700 (grey) is illustrated. SwissProt accession numbers for cited proteins are as follow: Na⁺,K⁺-ATPases: ATP1A2 of human (P50993), rat (P06686), and mouse (AAH25807), human ATP1A1 (P05023), human ATP1A3 (P13637), human ATP1A4 (Q13733), mouse ATP1A4 (Q9WV27); H⁺,K⁺,ATPase: ATP4A of human (NP_000695) and rat (P09626); SERCA ATPase ATP2A3 of human (Q93084) and rat (NP_037046).

Results

We identified a mutation in the ATP1A2 gene in one of the six families. Three individuals in this family were found to be affected. The proposita (I-01 in Fig. 1a) is a 72-year-old woman who experienced her first headache at age 6. She reported four to five typical HM attacks per year, presenting with the same distinctive features: hemiparesis, hemianoptic blurred vision, tingling spreading from the upper arm to the mouth, and speech disturbances. Neurological symptoms, which usually persisted for about 60 min, were followed by stabbing temporal headache, lasting about 3 h, and accompanied by nausea, vomiting, photophobia, phonophobia, and a morbid fear of smells (osmophobia). Cold weather and stress were potent attack triggers. The patient reported no ascendant relatives affected by headaches or migraine. The elder of her two sons (II-01) is a 48-year-old man who has suffered from HM since the age of 10, when, shortly after a minor head trauma, he developed right-sided hemiparesis and paraesthesias, dysphasia, and hemianoptic blurring of vision. Within around 30 min, his clinical condition had been complicated by a throbbing aching in the left side of his skull, nausea, photo-, phono- and osmophobia. Symptoms completely disappeared in 1 day. The patient has experienced a number of further attacks since that first one (about two per year until the age of 41; only three in the past 7 years). There was no clinical evidence of cerebellar dysfunction during the attacks and magnetic resonance imaging (MRI) did not show cerebellar atrophy. Interictal EEG was also normal. This patient identified several triggers for his attacks, including physical or emotional stress, alcohol intake, cold, minor head trauma, and ‘roasted coffee’ smell. He has only once entered a confused state, during a particularly violent and prolonged HM attack. The other son (patient II-02), aged 47 years, experienced his first migraine attack at the age of 10, after a minor head trauma sustained while playing soccer. His HM attacks occurred around twice a year. With the exception of the visual disturbances, which disappeared rapidly, full neurological recovery took a few days. Patient II-02 also experienced severe attacks with confusion and drowsiness. During one attack, when aged 40, he was admitted to an intensive care unit and a cerebral angiography was performed to rule out a malformation. This examination showed normal findings; however, the procedure was followed by a coma state lasting 3 days. MRI did not show cerebellar atrophy. Patient II-02 has two children (III-01 and III-02) who had a normal neurological examination at 12 and 9 years of age, respectively.

Direct sequencing of the ATP1A2 gene identified a novel, heterozygous c.2098G→A base change in patient I-01 (Fig. 1b). Mutation nomenclature follows the format indicated in http://www.hgvs.org/mutnomen and refers to the published cDNA sequence (NM_000702) with nucleotide +1 corresponding to the A of the ATG translation initiation codon. The single base change lies in ATP1A2 exon 15 and replaces glutamic acid for lysine (E700K). A sensitive BstX I-RFLP method confirmed the mutation in the two affected sons (Fig. 1a) but not in III-01 and III-02 (the two asymptomatic grandchildren). The mutation was also absent in 400 control chromosomes. Haplotype analysis did not exclude involvement of locus 1q23.

As with the family mentioned above, the other five FHM families (Fig. 1c) did not show pathogenic mutations in CACNA1A. Direct gene sequencing was also negative for changes in ATP1A2. Nevertheless, heterozygous gene deletions and changes in the promoter region or other regulatory elements cannot be formally ruled out. All the propositi had an early onset (<20 years of age) of the disorder, apparently similar clinical severity, and they did not present signs of cerebellar dysfunction, alternating hemiplegia of childhood, or epileptic seizures. In family 2, patient I-01 presents migraine with motor symptoms during the aura without a canonical IHS diagnosis of FHM (1). However, previous authors have already emphasized the possibility of incomplete penetrance and heterogeneous phenotypes in FHM families (7).

Discussion

Fewer than 20 FHM2 families have thus far been identified, although in most cases the specific mutation is yet to be confirmed. Clinical features in E700K-positive patients were not substantially different from those observed in earlier pedigrees (6–13). Affected individuals showed a stereotypic pattern of migrainous headache associated with hemisensory and hemiparetic attacks. Attack frequency decreased with age and the overall course was relatively benign. Contrary to previously identified FHM2 pedigrees, none of our cases showed epileptic seizures. Interestingly, minor head trauma triggered attacks in childhood in two patients, thus restricting their leisure activities. A similar trigger has been reported in 83% of patients in the German-Native American FHM family (7).

ATP1A2 encodes the α2 subunit of the Na^+/K⁺ pump, a heterodimeric structure formed by the large catalytic α subunit and the ancillary β subunit. By using arguments adopted elsewhere (11), the novel change in ATP1A2 reported in this work might be regarded as the cause of the disease by considering that it segregated in three affected individuals with typical FHM2 and it was absent in two healthy relatives and in a large subset of Italian control chromosomes. Moreover, the E700K mutation affects a highly conserved residue among Na^+/K⁺ and H^+/K⁺ pumps (Fig. 1d), and probably produces a different charge in the intracellular loop between TM4-TM5. This region incorporates important regulatory domains for the ion transport system, including a phosphorylation site and nucleotide- and magnesium-binding domains (18).

We did not test the consequences of the E700K variant. Functional analyses are seldom applied in FHM2-related mutations (9, 16). However, we noticed that mutagenesis of highly conserved residues located in the TM4-TM5 loop of the α1 subunit has shown that this protein domain affects correct folding of the loop (19), interferes with the correct insertion of the other transmembrane regions, and ultimately hinders the correct biosynthesis of Na,K-ATPase (20). This might be pertinent to the reported D718N and the R763H changes (11, 13) located in the TM4-TM5 loop of the human protein, but it might also apply to the novel E700K variant. Although the precise consequences of gene mutations in FHM2 patients remain unknown, it has been speculated that loss-of-function variants act by impairing brain K⁺ concentration—causing an ensuing cortical depolarization—or by facilitating higher levels of intracellular Ca²⁺ and Na⁺ in neurons (9, 10, 21), or both.

No pathogenic mutations were found in the remaining five FHM pedigrees where no prior linkage information was obtained. At least one additional FHM locus is supposed to exist (6). Understanding the pathological mechanisms shared by CACNA1A and ATP1A2 is likely to help in the discovery of other aetiologies in FHM.

Identification of the genes responsible for the more common forms of migraine with (MA) and without aura (MoA) is still a challenge in migraine genetics. Mutation carriers in FHM1 families may experience attacks of typical MA or MoA (2, 22), but the involvement of CACNA1A in common subtypes of migraine remains controversial (23). Although preliminary data do not support this hypothesis (24), further studies will be needed to establish whether ATP1A2 variants are also relevant in other primary headache forms, including sporadic cases of hemiplegic migraine (25).