A novel ATP1A2 gene mutation in familial hemiplegic migraine and ...

Brief Report

A novel ATP1A2 gene mutation in familial hemiplegic migraine and epilepsy

Cephalalgia 2014, Vol 34(1) 68–72 ! International Headache Society 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0333102413498941 cep.sagepub.com

Cinzia Costa1,2, Paolo Prontera3,4, Paola Sarchielli1, Alessandra Tonelli5, Maria Teresa Bassi5, Letizia Maria Cupini6, Stefano Caproni1, Sabrina Siliquini1, Emilio Donti3 and Paolo Calabresi1,2 Abstract Background: Familial hemiplegic migraine (FHM) is a rare autosomal dominant migraine subtype, characterized by fully reversible motor weakness as a specific symptom of aura. Mutations in the ion transportation coding genes CACNA1A, ATP1A2 and SCN1A are responsible for the FHM phenotype. Moreover, some mutations in ATP1A2 or SCN1A also may lead to epilepsy. Case: Here we report on a three-generation family with five patients having a novel ATP1A2 mutation on exon 19, causing guanine-to-adenine substitution (c.2620G>A, p.Gly874Ser) that co-segregated in the five living relatives with migraine, four of whom had hemiplegic migraine. Moreover, three patients presented with epilepsy, one of whom had generalized epilepsy with febrile seizures plus (GEFSþ). Conclusions: The present study provides further evidence on the involvement of ATP1A2 mutations in both migraine and epilepsy, underlying the relevance of genetic analysis in families with a comorbidity of both disorders. Keywords FHM2, ATP1A2 gene, GEFSþ, migraine, epilepsy Date received: 13 December 2012; revised: 25 June 2013; accepted: 26 June 2013

Introduction There is strong evidence of comorbidity between migraine and epilepsy (1). Migraine attacks and epileptic seizures may, in fact, be triggered by excessive neocortical cellular excitability and share common genetic bases (2–4). Specifically, it is known that epilepsy and migraine can co-occur in relatives with familial hemiplegic migraine (FHM) (2). FHM is a rare autosomal dominant subtype of migraine with aura, characterized by a fully reversible motor weakness as a specific symptom of aura. Mutations in the ion transportation genes CACNA1A, ATP1A2 and SCN1A are all responsible for the FHM phenotype, thus indicating a genetic heterogeneity for this disorder (3). The familial forms of hemiplegic migraine, caused by these mutations, are referred to as FHM1, FHM2 and FHM3, respectively (5–7). Moreover, epileptic seizures, presenting independently from migraine attacks, have been reported in some families with FHM, mainly with FHM2. In about 20% of families with an ATP1A2 mutation, some members experienced epileptic seizures (7), and

FHM partially co-segregated with benign familial infantile convulsions (BFIC) (2). In this study, we describe a novel ATP1A2 mutation in an Italian family that co-segregates various phenotypes: migraine, hemiplegic migraine, and epilepsy, including a single 1

Clinica Neurologica, Universita` di Perugia, Ospedale S. Maria della Misericordia, Italy 2 Fondazione Santa Lucia, IRCCS, Italy 3 Sezione di Genetica Medica, Dipartimento di Medicina Clinica e Sperimentale, Universita` di Perugia, Ospedale S. Maria della Misericordia, Italy 4 Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita` di Perugia, Italy 5 IRCCS E. Medea, Laboratorio di Biologia Molecolare, Italy 6 Centro Cefalee, UOC Neurologia, Ospedale S Eugenio, Italy C.C. and P.P. contributed equally to this work. E.D. and P.C. contributed as co-senior authors. Corresponding author: Cinzia Costa, Clinica Neurologica, Universita` di Perugia, Ospedale S. Maria della Misericordia, S. Andrea delle Fratte, 06156 Perugia, Italy. Email: [email protected]

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Costa et al. case of generalized epilepsy with febrile seizures plus (GEFSþ).

Patients and methods Clinical data The proband belongs to a four-generation family including 14 subjects, five of whom were affected by hemiplegic migraine, and one by migraine without aura (Figure 1). The maternal great-grandmother of the proband (Figure 1, I.1), died at 65 years of unspecified reasons. Family members reported that she had suffered from migraine with aura (visual, sensory and motor symptoms lasting one to two days) as well as generalized epilepsy. With the exception of this relative, we were able to directly interview and clinically evaluate all the other five affected family members. Their neurological examinations were unremarkable. Diagnoses of migraine and epilepsy were made according to standardized criteria (International Headache Society second edition criteria, and the International League Against Epilepsy criteria of 1981). Histories, physical and neurological examinations, as well as brain imaging, did not suggest the presence of any underlying disorders.

Molecular analysis After obtaining written informed consent, blood was drawn from the proband and all family members. DNA was extracted from peripheral blood

(Nucleospin Blood Kit, Macherey-Nagel, Duren, Germany), and mutation analyses of CACNA1A (GenBank accession no. NM_001127221) and ATP1A2 (GenBank accession no. NM_000702.3) were performed by direct sequencing of all exons and flanking splice sites. Genomic sequences of these genes are available at the Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway). Mutation analyses were performed by direct sequencing of polymerase chain reaction products obtained with a Big Terminator Sequencing Kit (version 3.1; Applied Biosystems) in an automated sequencer (ABI 3130 XL Applied Biosystems). Additionally, genomic DNA from 225 unrelated healthy Italian individuals (450 alleles) was employed as a control. Prediction of potential pathogenicities of the novel missense change was determined by using the following programs: MutPred, SNPs&GO, SNAP, PhD-SNP, Pmut and Panther.

Results Clinical data The proband (Figure 1, IV.1) was a 22-year-old male who had febrile seizures from 2–6 years of age. Specifically, the first febrile convulsion occurred at 2 years of age, while between 3 and 5 years of age brief seizures were experienced, mostly associated with fever. At 6 years and 11 months, the proband had a febrile seizure. GEFSþ was suspected on the basis of the clinical information available.

Negative ATP1A2 genetic test

I.

Positive ATP1A2 genetic test

2

1

Migraine without aura Hemiplegic migraine Hemiplegic migraine and seizures

II. 1

III.

2

1

2

3

IV. 1

2

3

4

5

6

7

Figure 1. Family pedigree: The black arrow indicates the proband; the legend for the symbols is at the top of the figure.

70 At the ages of 19 and 22, the proband had head traumas without concussion that were accompanied by headache attacks with vomiting. These attacks were preceded by aura symptoms, including visual impairment (black spots), one-sided sensory symptoms, weakness in the right limbs and speech disturbances lasting 20 to 30 minutes. The attack durations were four to six hours. In both events, the patient was immediately taken to the emergency department where he underwent head computed tomography, brain magnetic resonance imaging, epiaortic vessel duplex ultrasonography and laboratory tests, all of which were negative. However, in both events, an electroencephalographic (EEG) examination showed slowing over the left parieto-temporo-occipital lobe. Neurological assessments, performed after the resolutions of the attacks, were negative. After 48 hours a second EEG was normal for both events. Family history revealed that the mother of the proband (44 years old) (Figure 1, III.1) had suffered from migraine without aura since the age of 24, and the maternal grandmother (70 years old) (Figure 1, II.1) had from youth, several attacks with headache, vomiting, visual symptoms (black spots), speech difficulties (aphasia), sensory symptoms afflicting one arm and weakness in the limbs and face, lasting one to two days. A 47-year-old maternal uncle (Figure 1, III.2) had since the age of 14, two to four attacks of aura per year, without headache, lasting for 15 minutes. During these attacks, he referred to black spots, sensory symptoms in the tongue, speech difficulties and weakness in one hand. Moreover, at the ages of 11 and 12, he presented complex partial seizures and therefore was treated with phenobarbital for two years. A 25-year-old maternal male cousin (Figure 1, IV.3) from the age of 15 had three attacks with headache, vomiting, visual symptoms (black spots), speech difficulties, confusion, sensory symptoms in one arm and weaknesses in an arm and the face. These attacks lasted one to two days. One of the three episodes was misdiagnosed as a transient ischemic attack.

Genetic analyses Molecular analysis of the CACNA1A gene was normal, while the ATP1A2 gene sequencing revealed a novel heterozygous nucleotide transition (c.2620G > A) in the exon 19 (Figure 2a), that was present in all living relatives affected by migraine and epilepsy, but not in healthy members of the family (Figure 1). Such a variation leads to the amino acid change p.Gly874Ser, which was not found in any of the 450 control alleles analyzed, not even in the Single Nucleotide Polymorphism database (dbSNP, http://www.ncbi.nlm.nih.gov/snp). The mutant Gly874 residue is located in the extracellular loop between M7 and M8 (aa 871-916, Figure 1b),

Cephalalgia 34(1) and is highly evolutionarily conserved among alpha subunits coming from several species (Supplementary Table 1). The novel variant is predicted to be pathogenic by different programs, as shown in Table 1.

Discussion We observed a novel missense mutation in the Naþ/KþATPase gene ATP1A2 from a family having a variable association between hemiplegic migraine and epilepsy. Several findings support the pathogenetic role of this mutation. First of all, it was present only in the affected members of the family, and was not found in healthy relatives or in the other 450 control alleles coming from the same population (Central Italy), as well as in the dbSNP or Ensembl databases. The mutant residue Gly874 was located in the extracellular loop between M7 and M8, a domain responsible for binding the betasubunit, which is essential for Naþ, Kþ pump activity (8). Four mutations are located within the highly evolutionarily conserved M7-M8 loop (Figure 2b and Supplementary Table 2). Functional data indicate a putative pathogenic mechanism for the p.W887R mutation, most likely triggered by the loss of function of one of the two ATP1A2 alleles. A similar mechanism could be involved in the novel mutation described in this manuscript, as also suggested by bioinformatic analysis (Table 1). In the family studied here, the only completely penetrant feature was migraine, while aura (motor, visual and sensory) and seizures were present in five (83%) and three (50%) patients, respectively. Both migraine aura and seizure attacks could have been the result of the same ATP1A2 mutation here described, because this mutation diminishes the capacity to maintain the neuronal resting membrane potential, so that neurons can be more easily brought to threshold and excited (2). FHM2 frequently occurs with epilepsy; however, the percentage of patients with seizures in the family studied was higher than that reported for other FHM2 families (7), suggesting that the mutation c.2620G > A is capable of inducing not only cortical spreading depression but also abnormal neuronal discharges. ATP1A2 mutations have been associated with BFIC (2), but never with GEFSþ. Therefore, the co-occurrence of FHM2 with GEFSþ in the proband could be a coincidental finding, considering that febrile seizures are very common in 2- to 6-year-olds (3%–8%) (9); however, our clinical and genetic findings suggest a link between GEFSþ and ATP1A2 mutation. The etiology of hereditary epileptic syndrome GEFSþ is widely heterogeneous, and five genes have been identified: SCN1A, SCN1B, SCN2A, GABRG2 and GABRD (10). Moreover, a feature of GEFSþ is a wide range of intrafamilial variability, in terms of seizure

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c.2620G>A (a)

Proband

aa 871

p.R908Q

p.E902K

p.G900R

p.W887R

p.l883N

p.G874S

p.R879Q. p.R879W

Control

AENGFLPSRLLGIRLDWDDRTMNDLEDSYGQEWTYEQRKVVEFTCH 916

(b)

M7-M8 loop

Extracellular

M 1

M 2

M 3

M 4

M 5

M 6

M 7

M 8

M 9

M 10

COOH NH2

Intracellular

Figure 2. (a) Electropherogram of the sequence encompassing the mutation in the proband, compared to the control, is shown. (b) Schematic representation of the ATP1A2 protein with the topology of the extracellular loop between M7 and M8 and the corresponding sequence, the novel mutation identified indicated with the big brake; the known mutations (black arrowhead), the known variation (gray arrowhead) and the known polymorphism (white arrowhead), are shown.

Table 1. Prediction of pathogenicity for the novel mutation p.Gly874Ser. Method

Prediction

Result

Website

MutPred SNPs&GO SNAP

Probability: 0.906 Reliability Index: 9 Reliability Index: 3, Expected Accuracy: 78% Reliability Index: 8 Prediction: 0.6307, Reliability: 2 subPSEC: -4.74732, P: 0.85161

Deleterious Disease Non-neutral

http://mutpred.mutdb.org/ http://snps-and-go.biocomp.unibo.it/snps-and-go/ http://rostlab.org/services/snap/

Disease Pathological Deleterious

http://gpcr2.biocomp.unibo.it/cgi/predictors/PhD-SNP/PhD-SNP.cgi http://mmb2.pcb. ub.es:8080/PMut/ http://www.pantherdb.org/tools/csnpScoreForm.jsp

PhD-SNP Pmut Panther

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type and severity, as in family members sharing the same SCN1A mutation (11,12). Since allelic mutations in the SCN1A gene lead to FHM3 or GEFSþ alone, our observation may suggest that the ATP1A2 gene is a good candidate for familial GEFSþ, even without migraine. In conclusion, the present study provides further evidence of the involvement of ATP1A2 mutations in both migraine and epilepsy, therein underlying the value of genetic analysis in families with a comorbidity of both disorders. Specifically, genetic analysis could contribute to better treatment choices, particularly in cases where migraine and epilepsy are both present. As well, genetic analysis can provide greater insight into the potential causes of both disorders, and for this could be useful for pathogenetic purposes.

Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest P.C. receives/received research support from Bayer Schering, Biogen-Dompe´, Boehringer Ingelheim, Eisai, Lundbeck, Merck Serono, Novartis, Sanofi Aventis, Sigma-Tau and UCB Pharma. He also receives/received support from Ricerca Corrente IRCCS, Ricerca Finalizzata IRCCS, European Community Grant REPLACES (restorative plasticity at corticostriatal excitatory synapses), the Italian Minister of Health and AIFA (Agenzia Italiana del Farmaco); C.C., P.P., P.S., A.T., M.T.B., L.M.C., S.C., S.S. and E.D. report no disclosures.

Clinical implications . The present study identified a new ATP1A2 mutation responsible for familial hemiplegic migraine 2 (FHM2), thus providing relevant information for the molecular interpretation of laboratory results and the genotype-phenotype correlation. . The present study provides further evidence of the involvement of ATP1A2 mutations in both migraine and epilepsy, therein highlighting the importance of genetic analysis in families with a comorbidity of both disorders. . Genetic analysis could contribute to better treatment choices, particularly in cases where both migraine and epilepsy are present. As well, genetic analysis can provide greater insight into the potential causes of both disorders, and for this could be useful for pathogenetic purposes. . This is the first report linking generalized epilepsy with febrile seizures plus (GEFSþ) to ATP1A2 mutation. This observed association, even in the absence of a statistical significance, may suggest that the ATP1A2 gene is a good candidate for familial GEFSþ, even without migraine. This is supported by the analogies with mutations in SCN1A that are responsible for FHM3 or GEFSþ. References 1. Haut SR, Bigal ME and Lipton RB. Chronic disorders with episodic manifestations: Focus on epilepsy and migraine. Lancet Neurol 2006; 5: 148–157. 2. Vanmolkot KR, Kors EE, Hottenga JJ, et al. Novel mutations in the Naþ, Kþ-ATPase pump gene ATP1A2 associated with familial hemiplegic migraine and benign familial infantile convulsions. Ann Neurol 2003; 54: 360–366. 3. Rogawski MA. Common pathophysiologic mechanisms in migraine and epilepsy. Arch Neurol 2008; 65: 709–714. 4. Parisi P, Piccioli M, Villa MP, et al. Hypothesis on neurophysiopathological mechanisms linking epilepsy and headache. Med Hypotheses 2008; 70: 1150–1154. 5. Deprez L, Weckhuysen S, Peeters K, et al. Epilepsy as part of the phenotype associated with ATP1A2 mutations. Epilepsia 2008; 49: 500–508. 6. Pierelli F, Grieco GS, Pauri F, et al. A novel ATP1A2 mutation in a family with FHM type II. Cephalalgia 2006; 26: 324–328.

7. Russell MB and Ducros A. Sporadic and familial hemiplegic migraine: Pathophysiological mechanisms, clinical characteristics, diagnosis, and management. Lancet Neurol 2011; 10: 457–470. 8. Lutsenko S and Kaplan JH. An essential role for the extracellular domain of the Na,K-ATPase b-subunit in cation occlusion. Biochemistry 1993; 32: 6737–6743. 9. Sadleir LG and Scheffer IE. Febrile seizures. BMJ 2007; 334: 307–311. 10. Nakayama J. Progress in searching for the febrile seizure susceptibility genes. Brain Dev 2009; 5: 359–365. 11. Wallace RH, Scheffer IE, Barnett S, et al. Neuronal sodium-channel alpha1-subunit mutations in generalized epilepsy with febrile seizures plus. Am J Hum Genet 2001; 68: 859–865. 12. Fujiwara T. Clinical spectrum of mutations in SCN1A gene: Severe myoclonic epilepsy in infancy and related epilepsies. Epilepsy Res 2006; 70S1: S223–S230.