Dominant SCN2A Mutation Causes Familial Episodic

Original Article

Dominant SCN2A Mutation Causes Familial Episodic Ataxia and Impairment of Speech Development Walid Fazeli1,2,
Kerstin Becker1,3 Peter Herkenrath1 Christoph Düchting1 Friederike Körber4 Pablo Landgraf5 Peter Nürnberg6 Janine Altmüller6 Holger Thiele6 Anne Koy1 Max C. Liebau3,7 Thorsten Simon5 Jörg Dötsch7 Sebahattin Cirak1,3,7,
1 Division of Pediatric Neurology, Department of Pediatrics, University

Hospital Cologne, Cologne, Germany 2 Institute for Molecular and Behavioral Neuroscience, University of Cologne, Cologne, Germany 3 Center for Molecular Medicine, University of Cologne, Cologne, Germany 4 Department of Pediatric Radiology, University Hospital Cologne, Cologne, Germany 5 Department of Pediatric Oncology and Hematology, University Hospital Cologne, Cologne, Germany 6 Cologne Center for Genomics, University of Cologne, Cologne, Germany 7 Department of Pediatrics, University Hospital Cologne, Cologne, Germany

Address for correspondence Walid Fazeli, MD, Division of Pediatric Neurology, Department of Pediatrics, University Children’s Hospital Cologne, Kerpener Strasse 62, 50937 Cologne, Germany (e-mail: [email protected]). Sebahattin Cirak, MD, Uniklinik KölnKlinik und Poliklinik für Kinderund Jugendmedizin, Gebäude 26, Kerpener Straße 62, 50937 Köln, Germany (e-mail: [email protected]).

Neuropediatrics

Abstract

Keywords

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ataxia exome sequencing ion channel NaV1.2 SCN2A vaccinations

Mutations in SCN2A are associated with a heterogeneous clinical spectrum including epilepsy and autism. Here, we have identified a peculiar phenotype associated with vaccination related exacerbations of ataxia. We report the first family with three individuals affected by SCN2A-associated episodic ataxia (EA) with impaired speech development. The index patient manifested his first episode of subacute cerebellar ataxia at the age of 12 months, 3 weeks after vaccinations for measles, mumps, rubella, and varicella. Cranial magnetic resonance imaging showed a lesion of the left cerebellar hemisphere, which was first considered as a potential cause of the ataxia. The patient fully recovered within 3 weeks, but developed three very similar episodes of transient ataxia within the following 24 months. Whole exome sequencing of the index patient revealed a heterozygous autosomal-dominant mutation in SCN2A (NM_021007, c.4949T > C; p.L1650P), which was confirmed in the likewise affected mother, and was then also identified in the younger brother who developed the first episode of ataxia. We hereby extend the recently described spectrum of SCN2A-associated neurologic disorders, emphasizing that SCN2A mutations should also be considered in familial cases of EA. Coincidental imaging findings or other associated events such as immunizations should not protract genetic investigations.

Introduction The increasing use of next generation sequencing approaches has facilitated genetic diagnoses in patients with neurodevelopmental disorders and may elucidate cases


Both authors are equal corresponding authors.

received April 28, 2018 accepted after revision July 3, 2018

with unclear etiology. Pathogenic variants in numerous genes encoding for neuronal ion channels have been identified as potential cause of epilepsy, and/or intellectual disability. However, important challenges such as the difficulties in correlating genotypes to phenotypes have remained unsolved, a typical example being the heterogeneous clinical spectrum of SCN2A-associated disorders.

© Georg Thieme Verlag KG Stuttgart · New York

DOI https://doi.org/ 10.1055/s-0038-1668141. ISSN 0174-304X.

Ataxia and Dominant SCN2A Mutation

Fazeli et al.

SCN2A encodes the α subunit of the neuronal voltage-gated sodium channel NaV1.2, which is highly expressed at the axon initial segment of excitatory neurons1 and within the cerebellar molecular layer,2,3 where it plays a crucial role for action potential backpropagation.4 SCN2A mutations have been associated with a heterogeneous spectrum of neurologic disorders, ranging from benign (or according to the recent ILAE classification, self-limited and pharmacoresponsive5) familial neonatal–infantile seizures6,7 over intellectual disability8 to autism.9 Very recently, an analysis of over 200 patients harboring SCN2A mutations demonstrated its association with a broad range of epilepsies, ranging from epileptic encephalopathies to self-limited disease courses.10 In addition, a relevant number of patients showed signs of mental retardation and/or autism without epilepsy.10 The SCN2A-associated spectrum was recently extended by reports of a total of six patients with a well-defined phenotype characterized by neonatal-onset epilepsy, later-onset episodic ataxia (EA), and unimpaired/mildly impaired psychomotor development.3,11–13 In addition, a boy with a divergent phenotype consisting of SCN2A-associated EA, severe psychomotor impairment, autistic features, cerebellar atrophy, yet absence of epilepsy was described.14 We now extend the clinical spectrum of SCN2A-associated EA. We report the first three cases of inherited SCN2A mutation presenting with recurrent episodes of early childhoodonset EA and impairment of speech development. Of note, two of the (hitherto) four EA episodes in the index were temporally associated with vaccinations within 3 weeks prior to the onset of EA.

Methods Patients The study was approved by the institutional review board of the ethics committee of the University Hospital of Cologne. Written informed consent for research and publication was obtained from the family. All procedures were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Genetic Work-Up Our whole exome sequencing procedures have been previously described.15 In brief, genomic deoxyribonucleic acid (DNA) isolated from ethylenediaminetetraacetic acid (EDTA)-blood of the index patient was subjected to whole exome sequencing on an Illumina Hiseq 4000, using the SureSelect Human All Exon V6 enrichment kit and a paired-end 75bp sequencing protocol. Data analysis was performed with the VARBANK (v2.25) exome pipeline of the Cologne Center for Genomics,16 including alignment against the human reference genome and variant calling. Sequencing data were filtered for rare (minor allele frequency < 0.1%), heterozygous variants in accordance with either autosomal recessive or the expected autosomal dominant inheritance pattern. Dideoxy sequencing was performed for confirmation and co-segregation of variants. Neuropediatrics

Results Patient 1 The index patient was born at 35 þ 5 weeks of gestation (birth weight: 3220 g [P 83], length: 50 cm [P 68], head circumference: 37cm [P 98]; APGAR score 9/10/9) after an uneventful pregnancy to non-consanguineous parents. At the age of 12 months, the boy presented with his first episode of subacute cerebellar ataxia: within 2 days, he had developed an unstable posture when sitting, loss of head control and wobbling of his head, and an intention tremor making it impossible for him to grasp for objects. Signs suspicious of epileptic seizures were not observed. The muscle tone was mildly reduced. The patient had received a combined vaccine against mumps, measles, rubella (Priorix®), and varicella (Varivax®) 3 weeks prior to the onset of ataxia and had a mild upper respiratory tract infection 1 week before. A cranial magnetic resonance imaging (MRI) showed a focal T1 and fluidattenuated inversion recovery (FLAIR) hyperintense lesion of the left cerebellar hemisphere, 1.7  1.1 cm of size, with no diffusion impairment (►Fig. 1a). Atrophy of the cerebellar hemispheres and/or vermis—as previously reported in SCN2A-associated EA14—were not seen. Cerebrospinal fluid (CSF) analysis and regular serum values were normal. Considering post-infectious cerebellitis as potential cause, treatment with high-dose intravenous methylprednisolone (10 mg/kg body weight/day) had been given over 3 consecutive days. After an initial worsening of symptoms within the first 3 days after admission, the patient improved and fully recovered from ataxia 3 weeks later. Within the following 4 weeks, the patient had two febrile episodes, which were not associated with ataxia. Several cranial MRIs were performed over the next 14 months; the cerebellar lesion became progressively well circumscribed and did not gain in size (►Fig. 1b). No additional lesions were observed. To date, the etiology of the lesion is still unknown. A second episode occurred 8 months later, being milder than the first one. This episode was again temporally associated with a prior vaccination, i.e., symptoms started 1 day after meningococcal vaccination (NeisVac C®). Three-day intravenous dexamethasone treatment was performed; full recovery was again achieved only after 3 weeks. Finally, the third and fourth EA episodes occurred 6 and 16 months later, at 26 and 36 months of age, respectively. Both episodes were not associated with either vaccinations or (febrile) respiratory infections. Symptoms were very similar to the prior episodes. Treatment with corticosteroids was performed during the third, but not during the fourth (and hitherto last) episode, given that both the family and our staff were not convinced that corticosteroid treatment had shown any positive effect. The boy was extensively tested at different stages regarding his psychomotor development: at the age of 3 months, the patient started visiting an outpatient center for early support because of a mild delay in motor development. Repeated testing of his cognition during the first year of life showed normal development. At 18 months, he showed a delay of speech development, with cognitive and motor development in the lower normal range. At 29 months, he showed a severe

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Fig. 1 MRI finding of the cerebellar lesion. (a) MRI finding at the onset of the first ataxia episode. Left: transversal FLAIR sequence showing a blurry, diffusely circumscribed increase in the signal intensity within the left cerebellar hemisphere. Middle and right: Coronar T1 sequence showing a predominantly solid lesion demonstrating an uptake of contrast medium and a small cyst at the boarder of the lesion. (b) MRI finding 24 months later. Left: transversal FLAIR sequence showing a well circumscribed round lesion, slightly reduced size compared with initial finding. Middle and right: Coronar T1 sequence showing decreased portion of contrast medium uptake within the lesion (versus (a)), but increased size of cystic portion. The arrows are indicating the cerebellar lesion. FLAIR, fluid-attenuated inversion recovery; MRI, magnetic resonance imaging.

delay of speech development, while the cognitive and motor development was mildly retarded. Finally, he was recently tested at 3 years and 6 months of age, his vocabulary was reduced by 1 year, his use of grammar corresponded approximately to 2 years of age, he showed a moderate phonologic impairment, and his receptive speech development was delayed by 1 to 1.5 years. His cognitive development at 3 years and 2 months of age showed an intelligence quotient (IQ) within the lower normal range (88). Several further investigations including abdominal ultrasound, echocardiography, electrocardiography, and, importantly, two electroencephalography (EEG) recordings were all normal. The genetic origin of the EA in this boy became likely when his mother remembered having had similar episodes during childhood.

Patient 2 The index’ mother was born to healthy non-consanguineous parents and had two healthy siblings. Other cases of (episodic) ataxia, epilepsy, migraine, autism, and/or intellectual disability were not found in the family. The index’ father was healthy, and the medical history of his family unremarkable. Pregnancy, birth, and immediate postnatal adaptation of the mother were normal. During early childhood, she showed a mild-to-moderate impairment of cognitive development and moderate impairment of speech development. She received standard vaccinations according to guidelines. During childhood, she had three episodes of EA at the ages of 3.5, 5, and 12 years,

respectively. Similar to the EA episodes of her son, each episode lasted approximately 3 to 4 weeks. In addition, once to twice per year during childhood, she presented with deterioration of her motor skills and insecure gait, which lasted for approximately 1 week and seemed much less pronounced than the three above mentioned EA episodes. Temporal correlations to fever and/or regularly performed vaccinations were not noticed. Extensive metabolic testing did not find any abnormalities. Three consecutive cranial MRIs did not show any pathology. Her global development was first assessed at the age of 5.1 years revealing a psychomotor developmental age of 4 years, with a pronounced impairment of speech development. According to the medical reports, two EEGs during childhood showed identical pathological findings, i.e., a dysrhythmic basal activity, sharp waves over the left temporoparietal region, and paroxysmal dysrhythmia with abnormal theta rhythms, which was not associated with epileptic seizures. At the age of 18 years, she showed a fourth and hitherto last EA episode, which was milder and shorter (1–2 weeks) than those experienced during childhood. The origin of her EA and her global psychomotor delay remained unknown until we had tested the index patient. We recently tested the mother using the German adaptation of the ‘Wechsler Adult Intelligence Scale’ (WAIS-III). Her overall IQ was 71 being nearly two standard deviations below mean and corresponding to a borderline level between learning disability and mild mental retardation. Interestingly, she showed a score of 86 in the performance part of the test, i.e., within the lower normal range and approximately only one Neuropediatrics

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standard deviation below mean, while the verbal intelligence score was 63. This heterogeneous cognitive profile resembles the one of the index patient with a nearly normal nonverbal performance and a severely affected development of verbal thinking. Finally, she was also assessed by our speech therapists where she showed the typical symptoms of an adult patient having had a disorder of speech development during early childhood: her vocabulary was unspecific and imprecise, and the use of grammar was clearly limited and occasionally wrong, while the pronunciation seemed normal.

Patient 3 The index patient’s brother (currently 13 months of age) was born after an uneventful pregnancy. The post-natal period and psychomotor development during infancy were unremarkable. Very recently, he developed a mild ataxia that initially resembled very much the onset of symptoms in his brother. However, the ataxia disappeared within 5 days. In view of his normal psychomotor development, no further diagnostic investigations were performed. A systematic assessment of his psychomotor development (Bayley Scale) is planned once he is around 2 years of age. An EEG was performed and was normal. As the index patient and his mother showed a very similar phenotype, a dominant genetic trait was suspected. Due to the large genetic heterogeneity of ataxia and intellectual disability, we performed whole exome sequencing in the index patient to identify the genetic cause of the disease.

Genetic Analysis Whole exome sequencing of the index patient led to the discovery of a heterozygous variant in SCN2A, NM_021007, c.4949T > C, p.L1650P, that was absent from the Exome Aggregation Consortium (ExAC) and genomeAD datasets. The variant was predicted to be disease causing by Mutation Taster, PolyPhen-2, PROVEAN and SIFT. The variant was confirmed by dideoxy sequencing and co-segregated with the disease, being present in the index patient, the affected mother, as well as the affected younger brother, while being absent from the unaffected father (►Fig. 2). The parents of the index’ mother were not available for genetic testing. This mutation was previously reported in a patient with epileptic encephalopathy.17 Genetic testing was performed in the younger brother before occurrence of the first episode of ataxia. The parents asked us to perform this testing at a pre-symptomatic stage as they were concerned that vaccinations might have triggered ataxia in the index patient and thus did not want any vaccinations to be performed in his younger brother, unless he was ruled out as a carrier of the pathogenic SCN2A variant. Thus, Dideoxy sequencing was performed, which identified the familial SCN2A variant. By co-incidence, he developed his first EA episode just after receipt of the test result. Screening the exome data of the index patient for genes in which mutations are known to cause intellectual disability and/or epilepsy (493 gene in-house panel, list available upon request) revealed a noteworthy hemizygous missense variant in GRIA3 (NM_000828.4, c.670G > A, p.E224K, rs757586471). Neuropediatrics

This variant was inherited from the mother. It was not present in the ExAC or the ClinVar database. One heterozygous carrier of East Asian origin (1/178595) was reported in the genomeAD database. Mutations in GRIA3 have been reported to cause Xlinked recessive intellectual disability. The contribution of this novel functional GRIA3 variant to the speech delay and intellectual development of the index case cannot be fully estimated; thus, this variant is currently classified as variant of unknown significance.

Discussion This is the first report describing a family with three affected members showing SCN2A associated EA, two of which additionally presenting impaired speech development. While six patients have previously been described with early-onset epilepsy, later-onset EA, and normal or mildly impaired psychomotor development,3,11–13 a single report described a much more severe phenotype with EA and autistic features, yet with absence of epileptic seizures.14 Our report expands the recently characterized clinical spectrum of SCN2A-associated neurologic disorders.10 The pathophysiological mechanisms underlying this heterogeneity are poorly understood and need further investigations, using both clinical data and basic science approaches, e.g., SCN2A mouse models such as the one we have generated and recently described.18 The phenotype shows a considerable overlap between the index patient and his mother, with recurrent episodes of ataxia lasting around 3 weeks, impairment of speech development, and lack of epileptic seizures. Despite this overlap, it is not justified to suggest a strong genotype–phenotype correlation, because this same mutation (SCN2A, p.L1650P) was also reported in a patient with early-infantile epileptic encephalopathy (EIEE).17 Though we unfortunately lack clinical details regarding this EIEE patient, it obviously differs from the phenotype reported here, thus underlining the heterogeneity of SCN2A-associated disorders, and the challenges in genotype–phenotype correlation, potentially caused by divergent genetic modifiers. The limited validity of genotype–phenotype correlation in SCN2A is underscored by the fact that even though a possible hotspot for SCN2A-associated EA has been described—i.e., four of the seven previously published cases of SCN2A-associated EA were due to the same mutation (SCN2A p. A263V)—this specific amino acid exchange has also been described in patients with severe intractable epileptic encephalopathy and Ohtahara syndrome without EA.10 This limited set of now 10 cases of SCN2A-associated EA does not allow any reliable clinical subdivision, yet further clinical reports may support our hypothesis of (at least) two divergent phenotypes: Type 1 with neonatal-onset epilepsy and later-onset ataxia with normal or only mildly impaired development, and EA episodes in the range of minutes to hours3,11–13; Type 2 that is defined by EA episodes lasting days to weeks, moderate-tosevere developmental impairment, and lack of epileptic seizures that has previously been described by Leach et al14 and is now supported by our report. The cerebellar lesion in the index patient is most likely a coincidental finding because persistent structural defects are

Ataxia and Dominant SCN2A Mutation

Fazeli et al.

Fig. 2 Genetic findings. (a) Pedigree including the genotypes of the family members showing autosomal dominant inheritance. The mother (Patient 2), the index patient (Patient 1), and his brother (Patient 3) were heterozygous for SCN2A, NM_021007, c.4949T > C, p.L1650P, whereas the unaffected father was homozygous for the wild type allele. (b) Protein sequence alignment for SCN2A. The variant resulted in a change from Leucine to Proline at amino acid position 1650, a conserved position across species. It was done with Jalview 2.10.1 using MafftWS alignment of protein sequences retrieved from UniProt. (c) Domain structure of SCN2A showing the 24 transmembrane domains and the location of the missense mutation in a cytoplasmic loop of the protein. It was generated with the software tool Protter (http://wlab.ethz.ch/protter/start/). The red arrow indicated the position of the missense mutation p.Leu1650Pro.

unlikely to cause episodic symptoms. The lack of any cerebellar lesion in the mother supports our hypothesis that the lesion is not caused by the SCN2A mutation, yet we cannot fully exclude such a causal link. From a functional perspective, the SCN2A (c.4949T > C; p. L1650P) mutation affects an important region of the NaV1.2 α subunit, namely a cytoplasmic loop within the domain IV, which links the transmembrane segments S4—i.e., the voltage sensor of the α subunit—and S5 (http://www.uniprot. org/uniprot/Q99250) (►Fig. 2c). This region plays a crucial role in both the fast and slow inactivation of NaV1.2.19,20 Based on our data, and the only available previous report,17 we cannot determine whether this mutation has a gain- or loss-of-function effect. Based on a previous report, divergent alterations of channel properties can lead to SCN2A-asso-

ciated EA.12 A mutation at the exact same location within the α subunit of NaV1.6—a voltage-gated sodium channel encoded by SCN8A which differs from NaV1.2 in its developmental expression pattern1—has been associated with earlyonset epileptic encephalopathy,21 thereby underscoring the pathophysiological relevance of this linker region within voltage-gated sodium channels. Also, a mutation in SCN2A neighboring the mutation of our patients was associated with recurrent episodes of encephalopathy.22 Interestingly, two of the four EA episodes in the index patient were associated with vaccinations. We cannot fully rule out a causal link between the vaccination and the onset of the first ataxia episode in the index patient, given that febrile seizures occurring after simultaneous vaccination against mumps, measles, and rubella are associated with Neuropediatrics

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variants in SCN2A and SCN1A.23 The temporal correlation in our patient does of course not prove any causal relationships, yet it is an interesting observation. The GRIA3 variant found in the index patient and his mother was classified as variant of unknown significance. GRIA3 has been linked to X-linked recessive intellectual disability, while the index patient has an IQ within the lower normal range. As 16% of a total of 201 patients within the largest hitherto published cohort of SCN2A patients showed signs of intellectual disability and/or autism,10 we suggest that the impaired cognitive development of the index’ mother is due to the SCN2A mutation. It remains elusive whether the GRIA3 variant has an additional effect on her cognitive performance. To conclude, our report describes a peculiar phenotype characterized by familial EA and impairment of speech development and thereby extends the clinical spectrum of SCN2A associated disorders.

6 Berkovic SF, Heron SE, Giordano L, et al. Benign familial neonatal-

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Conflict of Interest The authors declare that they have no conflict of interest. Written informed consent for research and publication was obtained from the family.

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Acknowledgments We thank the family for participating in this study and for their consent for publication. This work was supported by the Deutsche Forschungsgemeinschaft Emmy Noether Grant (CI 218/1–1) to Dr. Sebahattin Cirak. We are grateful to the Cologne Center for Genomics for performing the Next Generation Sequencing.

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