e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e5
Official Journal of the European Paediatric Neurology Society
Case study
Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families Andreas Brunklaus a,c,1, Rachael Ellis a,b,1, Helen Stewart d, Sarah Aylett c, Eleanor Reavey a,b, Ros Jefferson e, Rakesh Jain f, Supratik Chakraborty g, Sandeep Jayawant f, Sameer M. Zuberi a,h,* a
The Paediatric Neurosciences Research Group, Royal Hospital for Sick Children, Glasgow, UK Molecular Diagnostics, West of Scotland Genetic Services, Southern General Hospital, Glasgow, UK c Developmental Neurosciences Programme at UCL-ICH, Great Ormond Street Hospital for Sick Children, London, UK d Department of Clinical Genetics, Oxford Radcliffe Hospitals NHS Trust, Oxford, UK e Royal Berkshire NHS Foundation Trust, Reading, UK f Department of Paediatric Neurology, Children's Hospital, Oxford, UK g Coventry and Warwickshire Partnership NHS Trust, Coventry, UK h School of Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, UK b
article info
abstract
Article history:
Background: Mutations in the gene encoding the alpha subunit of the voltage-gated sodium
Received 12 October 2014
channel SCN1A are associated with several epilepsy syndromes. These range from severe
Accepted 9 February 2015
phenotypes including Dravet syndrome to milder phenotypes such as genetic epilepsy with febrile seizures plus (GEFS+). To date the sequence variants identified have been hetero-
Keywords:
zygous in nature as one would expect for a disorder that occurs de novo or is dominantly
SCN1A
inherited.
Dravet syndrome
Methods and Results: We report the association of two novel homozygous missense muta-
Severe myoclonic epilepsy of in-
tions of the SCN1A gene in four children with infantile epilepsies from two consanguineous
fancy
pedigrees. We suggest that the nature and location of the identified amino acid changes
GEFSþ
allows heterozygous carriers to remain unaffected. However, having such changes on both
Febrile seizures
alleles may have a cumulative and detrimental effect. Conclusion: The presented cases illustrate how better understanding of the nature and location of SCN1A missense mutations may aid the interpretation of genotypeephenotype associations. SCN1A related epilepsies should be considered in children with infantile onset epilepsies even when an autosomal recessive neurological disorder is suspected. © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. The Paediatric Neurosciences Research Group, Royal Hospital for Sick Children, Glasgow G3 8SJ, United Kingdom. Tel.: þ44 141 201 9269; fax: þ44 141 201 9270. E-mail address: [email protected] (S.M. Zuberi). 1 Common first authors: A Brunklaus and R Ellis contributed equally to the manuscript. http://dx.doi.org/10.1016/j.ejpn.2015.02.001 1090-3798/© 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
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e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 5 ) 1 e5
Introduction
Mutations in the gene encoding the alpha subunit of the voltage-gated sodium channel SCN1A are associated with several epilepsy syndromes. These range from severe phenotypes including Dravet syndrome to milder phenotypes in families with genetic epilepsy with febrile seizures plus (GEFSþ) and febrile seizures (FS). Dravet syndrome typically presents within the first year of life in previously well children with prolonged febrile and afebrile generalized clonic, or hemiclonic epileptic seizures. Beyond the first year the phenotype evolves to include myoclonic, atypical absence and focal seizures. Seizures are difficult to control and children usually develop an epileptic encephalopathy.1 Claes et al.2 reported in 2001 that de novo heterozygous SCN1A mutations cause Dravet syndrome and over the past 13 years more than 1000 sequence variants of the SCN1A gene have been identified confirming that it is currently the most clinically relevant epilepsy gene. To date the SCN1A sequence variants identified in Dravet syndrome and other GEFSþ phenotypes have been heterozygous in nature as one would expect for a disorder that occurs de novo or is dominantly inherited. Here we report the finding of two novel homozygous missense mutations of the SCN1A gene in four children from two consanguineous pedigrees.
2.
Case studies
2.1.
Cases one & two
Patient one is a 14 year old female. Development was normal until five months when she presented with her first febrile seizure (10 min). She had recurrent and prolonged, febrile, hemiclonic and generalised tonic clonic seizures (GTCS) exacerbated by Carbamazepine and responding to Sodium Valproate. She had status epilepticus at eight months and recurrent prolonged (15e30 min) often febrile, seizures and atypical absences up to the age of two years after which seizures occurred mainly in association with fever. An EEG at 19 months showed a background which was diffusely slow for age. By 11 years the EEG showed a slow background of low amplitude in the wake state. In sleep recurrent bursts of rhythmic sharp waves occurred. EEG age 14 years showed no evidence of interictal epileptiform activity. Development stalled at seizure onset and by eight months had regressed significantly. At the age of 14 she has a profound learning disability, cannot walk independently and has a dyskinetic movement disorder characterised by mixed spasticity and dystonia. MRI at 20 months showed a small corpus callosum with markedly delayed myelination and an atrophic cerebellum. She has three siblings, an older sister who is well, a younger brother with bilateral sensorineural hearing loss (no genetic diagnosis) and another younger brother (Case 2) who carries the same homozygous SCN1A mutation. A maternal first cousin had febrile seizures until age five. Case two had normal development prior to a prolonged (20 min) afebrile seizure at 15 months. He remained well until 3 years when he re-presented with prolonged febrile and
afebrile GTC seizures. He had an episode of febrile status epilepticus age 3 years and no seizures since then, now age 4.5 years. He has never been on regular medication. EEG and MRI brain age 15 months were normal. His current development is normal. Parents are first cousins (Family A, Fig. 1).
2.2.
Cases three & four
Case three, a 17 year old male, presented following normal early development with a febrile GTCS at six months of age (5 min). He had three similar events until 4 years and remained seizure free until age 6 when he presented with frequent short afebrile focal seizures. Age 12 he had nighttime seizures with frontal semiology. He was considered for epilepsy surgery but EEG and SPECT confirmed his epilepsy was multifocal. Medications included carbamazepine, lamotrigine, topiramate, levetiracetam and stiripentol, none of which exacerbated seizures. He currently takes sodium valproate and clobazam. Following seizure onset he was delayed in his motor and language development and spoke his first sentence at 3½ years. Age 17 years he has severe learning difficulties as well as balance and motor coordination problems. MRI brain scans age 8 and 12 years were normal. He has three siblings, an older sister with sensorineural hearing impairment due to a connexin 26 mutation, a younger brother who is well and a younger sister who carries the same homozygous SCN1A mutation. The younger sister (Case 4) presented from 13 months onwards with recurrent prolonged febrile seizures (up to 10 min). At three years she started to have focal seizures with impairment of awareness characterised by behavioural arrest followed by head turning and hemi-clonic jerking. An EEG at 14 months was unremarkable. A recording age 3 years showed bilateral bursts of 3e4 Hz spike and wave. There was no photosensitivity. After not responding to carbamazepine she started on sodium valproate which controlled the seizures. Her cognition at 5 years is normal. MRI scan at 4 years was normal. Parents are first cousins (Family B, Fig. 1).
Fig. 1 e Pedigrees of the 2 families with homozygous SCN1A mutations.
Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
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Table 1 e SCN1A homozygous mutation findings.
Case 1 & 2 Case 3 & 4
3.
Mutation identified
Protein location
Grantham distance
Parental carrier status
c.[1198A > G]; [1198A > G]; p.[(Met400Val)]; [(Met400Val)] c.[1852C > T]; [1852C > T]; p.[(Arg618Cys)]; [(Arg618Cys)]
DIS6 DI-DII intracellular linker
27 180
Heterozygous carriers Heterozygous carriers
Molecular analysis
Cases 1 and 3 were referred for SCN1A genetic testing and patient DNA samples were analysed according to standard procedures. Molecular analysis was performed on genomic DNA extracted from patients' and their parent's venous blood. All 26 exons of the SCN1A gene were amplified by PCR. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large scale rearrangements.3,4 Cases 2 and 4 were tested following the findings in their siblings. Parents consented for their children's anonymous patient information to be published as part of a case series.
4.
Discussion
We report two novel homozygous SCN1A missense mutations in four children from consanguineous pedigrees with varying clinical phenotypes. Homozygosity was confirmed by performing multiplex ligation-dependent probe amplification (MLPA) analysis to exclude the possibility of a deletion on the other allele (Table 1, Fig. 2). All cases have febrile seizures; a hallmark of SCN1A related epilepsies and the two families have the familial epilepsy syndrome GEFSþ known to be associated with heterozygous variants in SCN1A. Case one is a female with Dravet syndrome and her sibling (Case two) has a phenotype of FSþ. The sibling pair cases three & four have febrile seizures plus focal
epileptic seizures. We were unable to establish whether any of the unaffected siblings carried SCN1A mutations as it is not common practice in the UK to undertake genetic testing on asymptomatic children. Both families are of South Asian origin and demonstrate the importance of ordering investigations based on phenotype and not discounting genetic investigations typically associated with dominant or de novo disease when a recessive disorder is thought more likely. Cases 1 and 3 had undergone extensive biochemical and neuroimaging investigations prior to genetic testing. These cases illustrate that determination of the clinical significance of SCN1A missense mutations remains challenging. We recently described that assessment of the amino acid properties and protein location can be helpful in determining the pathogenicity of genetic findings.4 Missense mutations leading to disease are more likely to occur in the voltage sensor (S4) and pore region (S5eS6 segments) of the four homologous domains. Significant disease is more likely when there is substantial change in the physicochemical properties of the amino acid as measured by Grantham distance (GS).4,5 In cases one and two the missense mutation Met400Val is located in an area of functional importance; however has a low GS, as one nonpolar amino acid (methionine) is exchanged for another nonpolar amino acid (valine). This conservative change might allow the heterozygous parents to remain unaffected; however having such a change on both alleles may have a cumulative and detrimental effect on the homozygous children. The reverse is true for cases
Fig. 2 e Position of mutations across SCN1A protein. Homologous domains (D1-4); transmembrane segments 1e4 are voltage sensing domains (shown in green/yellow); segments 4 (yellow) are voltage sensors; segments 5 to 6 (pink) make up the pore-lining regions; NH3þ represents the N-terminal and CO2¡ the C-terminal. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
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three and four. Here the missense mutation Arg618Cys has a very high GS, as a charged amino acid (arginine) is exchanged for an uncharged amino acid with a sulfhydryl group (cysteine). However; this change is located within a less important non-functional area of the protein and might allow the heterozygous parents to remain unaffected; whereas the homozygous children who are carrying this change on both alleles might experience a cumulative and detrimental effect (Fig. 2). Examples of worsening effects of homozygous SCN1A mutations include the R1648H GEFS þ mouse model. Heterozygous R1648H mice have normal life spans with reduced thresholds to drug induced seizures and infrequent generalized seizures as adults whilst the homozygous model exhibits spontaneous generalized seizures with a reduced life span.6 Interestingly, Patino et al.7 reported a case of Dravet syndrome caused by a homozygous mutation of the SCN1B gene. Similar to the situation described here, the consanguineous parents were heterozygous carriers of the missense mutation. SCN1B encodes the sodium channel beta subunit which modulates channel voltage-dependence and gating as well as channel cell surface expression.8 Heterozygous mutations of SCN1B have previously been shown to cause GEFSþ.9,10 Patino and colleagues demonstrated, using functional assays and animal models, that the homozygous mutant alpha-1 subunit polypeptides were synthesised normally but were not transported to the cell surface in mammalian fibroblasts in vivo.7 Furthermore, they showed that mice homozygous for the SCN1B mutation would have spontaneous seizures, whilst heterozygous mice had seizure susceptibility that was similar to that of wild types. We observed a marked heterogeneity of the phenotype in the two presented sibling pairs. It has previously been shown that the same SCN1A mutation can show significant intra and inter-familial variability highlighting that environmental factors, epigenetic mechanisms and the genetic background of the individual play a significant role in the development and evolution of the epilepsy phenotype.11,12 Given the consanguinity of the presented cases it is not surprising to find other pathologies such as hearing loss segregating in these families. We regard this as an independent unrelated pathology. Indeed the fact that all of our patients were born of consanguineous parents increases the chances that they may have other rare homozygous variants at other genetic loci that could contribute in varying degrees to their individual phenotype. Whole exome sequencing would be a useful tool to determine potential genetic variants in these cases but was not available here. In particular identification of the genetic variation across all ion channels in each individual could go some way to explaining why they are, or are not affected by seizures. Pathogenic ion channel alleles in mouse models can form therapeutic combinations with effects that mask one another, reducing the penetrance and the expressivity of the clinical disorder.13 Variants in Scn2a, Scn8a and Kcnq2 have been shown to influence the phenotype of mice carrying the Scn1aR1684H mutation.14 More recently detailed assessment of 237 ion channel genes in patients with idiopathic epilepsy and normal controls showed that both rare and common coding variants in known excitability disease genes were present in both patients and controls.15
The four cases presented suggest that deleterious alleles can be modified by the unique combination of variants present in an individual's ion channel variation profile; illustrating how better understanding of the nature and location of SCN1A missense mutations may aid the interpretation of genotypeephenotype associations. SCN1A related epilepsies should be considered in children with infantile onset epilepsies even when an autosomal recessive neurological disorder is suspected.
Conflict of interest None.
Acknowledgements Dr Andreas Brunklaus reports no disclosures. Rachael Ellis reports no disclosures. Dr Helen Stewart reports no disclosures. Dr Sarah Aylett is a co-investigator for research funded by Epilepsy Action and Dravet Syndrome UK. Dr Eleanor Reavey reports no disclosures. Dr Ros Jefferson reports no disclosures. Dr Rakesh Jain reports no disclosures. Dr Supratik Chakraborty reports no disclosures. Dr Sandeep Jayawant reports no disclosures. Dr Sameer M Zuberi is an Associate Editor of The European Journal of Paediatric Neurology, has received honoraria for speaking at a UCB Pharma and Glaxo Wellcome sponsored educational meetings and received research funding from Epilepsy Research UK (AP1218) and Dravet Syndrome UK.
references
1. Dravet C, Bureau M, Oguni H, et al. Severe myoclonic epilepsy in infancy (Dravet syndrome). In: Roger J, Bureau M, Dravet C, Genton P, Tassinari CA, Wolf P, editors. Epileptic syndromes in infancy, childhood and adolescence. 4th ed. London: John Libbey; 2005. p. 89e113. 2. Claes L, Del-Favero J, Ceulemans B, et al. De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am J Hum Genet 2001;68:1327e32. 3. Brunklaus A, Ellis R, Reavey E, et al. Prognostic, clinical and demographic features in SCN1A mutation-positive Dravet syndrome. Brain 2012;135:2329e36. 4. Zuberi SM, Brunklaus A, Birch R, et al. Genotype-phenotype associations in SCN1A related epilepsies. Neurology 2011;76:594e600. 5. Grantham R. Amino acid difference formula to help explain protein evolution. Science 1974;185:862e4. 6. Martin MS, Dutt K, Papale LA, et al. Altered function of the SCN1A voltage-gated sodium channel leads to gamma aminobutyric acid-ergic (GABAergic) interneuron abnormalities. J Biol Chem 2010;285:9823e34. 7. Patino GA, Claes LRF, Lopez-Santiago LF, et al. A functional null mutation of SCN1B in a patient with Dravet syndrome. J Neurosci 2009;29:10764e78.
Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
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8. Isom LL, De Jongh KS, Patton DE, et al. Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel. Science 1992;256:839e42. 9. Scheffer IE, Harkin LA, Grinton BE, et al. Temporal lobe epilepsy and GEFSþ phenotypes associated with SCN1B mutations. Brain 2007;130:100e9. 10. Wallace RH, Wang DW, Sing R, et al. Febrile seizures and generalized epilepsy associated with a mutation in the Naþchannel beta1 subunit gene SCN1B. Nat Genet 1998;19:366e70. 11. Kimura K, Sugawara T, Mazaki-Miyazaki E, et al. A missense mutation in SCN1A in brothers with severe myoclonic
12. 13. 14.
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epilepsy in infancy (SMEI) inherited from a father with febrile seizures. Brain Dev 2005;27:424e30. Mulley JC, Scheffer IE, Petrou S, et al. SCN1A mutations and epilepsy. Hum Mutat 2005;25:535e42. Glasscock E, Qian J, Yoo JW, et al. Masking epilepsy by combining two epilepsy genes. Nat Neurosci 2007;10:1554e8. Hawkins NA, Martin MS, Frankel WN, et al. Neuronal voltagegated ion channels are genetic modifiers of generalized epilepsy with febrile seizures plus. Neurobiol Dis 2011;41:655e60. Klassen T, Davis C, Goldman A, et al. Exome sequencing of ion channel genes reveals complex profiles confounding personal risk assessment in epilepsy. Cell 2011;145:1036e48.
Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
Official Journal of the European Paediatric Neurology Society
Case study
Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families Andreas Brunklaus a,c,1, Rachael Ellis a,b,1, Helen Stewart d, Sarah Aylett c, Eleanor Reavey a,b, Ros Jefferson e, Rakesh Jain f, Supratik Chakraborty g, Sandeep Jayawant f, Sameer M. Zuberi a,h,* a
The Paediatric Neurosciences Research Group, Royal Hospital for Sick Children, Glasgow, UK Molecular Diagnostics, West of Scotland Genetic Services, Southern General Hospital, Glasgow, UK c Developmental Neurosciences Programme at UCL-ICH, Great Ormond Street Hospital for Sick Children, London, UK d Department of Clinical Genetics, Oxford Radcliffe Hospitals NHS Trust, Oxford, UK e Royal Berkshire NHS Foundation Trust, Reading, UK f Department of Paediatric Neurology, Children's Hospital, Oxford, UK g Coventry and Warwickshire Partnership NHS Trust, Coventry, UK h School of Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, UK b
article info
abstract
Article history:
Background: Mutations in the gene encoding the alpha subunit of the voltage-gated sodium
Received 12 October 2014
channel SCN1A are associated with several epilepsy syndromes. These range from severe
Accepted 9 February 2015
phenotypes including Dravet syndrome to milder phenotypes such as genetic epilepsy with febrile seizures plus (GEFS+). To date the sequence variants identified have been hetero-
Keywords:
zygous in nature as one would expect for a disorder that occurs de novo or is dominantly
SCN1A
inherited.
Dravet syndrome
Methods and Results: We report the association of two novel homozygous missense muta-
Severe myoclonic epilepsy of in-
tions of the SCN1A gene in four children with infantile epilepsies from two consanguineous
fancy
pedigrees. We suggest that the nature and location of the identified amino acid changes
GEFSþ
allows heterozygous carriers to remain unaffected. However, having such changes on both
Febrile seizures
alleles may have a cumulative and detrimental effect. Conclusion: The presented cases illustrate how better understanding of the nature and location of SCN1A missense mutations may aid the interpretation of genotypeephenotype associations. SCN1A related epilepsies should be considered in children with infantile onset epilepsies even when an autosomal recessive neurological disorder is suspected. © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. The Paediatric Neurosciences Research Group, Royal Hospital for Sick Children, Glasgow G3 8SJ, United Kingdom. Tel.: þ44 141 201 9269; fax: þ44 141 201 9270. E-mail address: [email protected] (S.M. Zuberi). 1 Common first authors: A Brunklaus and R Ellis contributed equally to the manuscript. http://dx.doi.org/10.1016/j.ejpn.2015.02.001 1090-3798/© 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
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Introduction
Mutations in the gene encoding the alpha subunit of the voltage-gated sodium channel SCN1A are associated with several epilepsy syndromes. These range from severe phenotypes including Dravet syndrome to milder phenotypes in families with genetic epilepsy with febrile seizures plus (GEFSþ) and febrile seizures (FS). Dravet syndrome typically presents within the first year of life in previously well children with prolonged febrile and afebrile generalized clonic, or hemiclonic epileptic seizures. Beyond the first year the phenotype evolves to include myoclonic, atypical absence and focal seizures. Seizures are difficult to control and children usually develop an epileptic encephalopathy.1 Claes et al.2 reported in 2001 that de novo heterozygous SCN1A mutations cause Dravet syndrome and over the past 13 years more than 1000 sequence variants of the SCN1A gene have been identified confirming that it is currently the most clinically relevant epilepsy gene. To date the SCN1A sequence variants identified in Dravet syndrome and other GEFSþ phenotypes have been heterozygous in nature as one would expect for a disorder that occurs de novo or is dominantly inherited. Here we report the finding of two novel homozygous missense mutations of the SCN1A gene in four children from two consanguineous pedigrees.
2.
Case studies
2.1.
Cases one & two
Patient one is a 14 year old female. Development was normal until five months when she presented with her first febrile seizure (10 min). She had recurrent and prolonged, febrile, hemiclonic and generalised tonic clonic seizures (GTCS) exacerbated by Carbamazepine and responding to Sodium Valproate. She had status epilepticus at eight months and recurrent prolonged (15e30 min) often febrile, seizures and atypical absences up to the age of two years after which seizures occurred mainly in association with fever. An EEG at 19 months showed a background which was diffusely slow for age. By 11 years the EEG showed a slow background of low amplitude in the wake state. In sleep recurrent bursts of rhythmic sharp waves occurred. EEG age 14 years showed no evidence of interictal epileptiform activity. Development stalled at seizure onset and by eight months had regressed significantly. At the age of 14 she has a profound learning disability, cannot walk independently and has a dyskinetic movement disorder characterised by mixed spasticity and dystonia. MRI at 20 months showed a small corpus callosum with markedly delayed myelination and an atrophic cerebellum. She has three siblings, an older sister who is well, a younger brother with bilateral sensorineural hearing loss (no genetic diagnosis) and another younger brother (Case 2) who carries the same homozygous SCN1A mutation. A maternal first cousin had febrile seizures until age five. Case two had normal development prior to a prolonged (20 min) afebrile seizure at 15 months. He remained well until 3 years when he re-presented with prolonged febrile and
afebrile GTC seizures. He had an episode of febrile status epilepticus age 3 years and no seizures since then, now age 4.5 years. He has never been on regular medication. EEG and MRI brain age 15 months were normal. His current development is normal. Parents are first cousins (Family A, Fig. 1).
2.2.
Cases three & four
Case three, a 17 year old male, presented following normal early development with a febrile GTCS at six months of age (5 min). He had three similar events until 4 years and remained seizure free until age 6 when he presented with frequent short afebrile focal seizures. Age 12 he had nighttime seizures with frontal semiology. He was considered for epilepsy surgery but EEG and SPECT confirmed his epilepsy was multifocal. Medications included carbamazepine, lamotrigine, topiramate, levetiracetam and stiripentol, none of which exacerbated seizures. He currently takes sodium valproate and clobazam. Following seizure onset he was delayed in his motor and language development and spoke his first sentence at 3½ years. Age 17 years he has severe learning difficulties as well as balance and motor coordination problems. MRI brain scans age 8 and 12 years were normal. He has three siblings, an older sister with sensorineural hearing impairment due to a connexin 26 mutation, a younger brother who is well and a younger sister who carries the same homozygous SCN1A mutation. The younger sister (Case 4) presented from 13 months onwards with recurrent prolonged febrile seizures (up to 10 min). At three years she started to have focal seizures with impairment of awareness characterised by behavioural arrest followed by head turning and hemi-clonic jerking. An EEG at 14 months was unremarkable. A recording age 3 years showed bilateral bursts of 3e4 Hz spike and wave. There was no photosensitivity. After not responding to carbamazepine she started on sodium valproate which controlled the seizures. Her cognition at 5 years is normal. MRI scan at 4 years was normal. Parents are first cousins (Family B, Fig. 1).
Fig. 1 e Pedigrees of the 2 families with homozygous SCN1A mutations.
Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
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Table 1 e SCN1A homozygous mutation findings.
Case 1 & 2 Case 3 & 4
3.
Mutation identified
Protein location
Grantham distance
Parental carrier status
c.[1198A > G]; [1198A > G]; p.[(Met400Val)]; [(Met400Val)] c.[1852C > T]; [1852C > T]; p.[(Arg618Cys)]; [(Arg618Cys)]
DIS6 DI-DII intracellular linker
27 180
Heterozygous carriers Heterozygous carriers
Molecular analysis
Cases 1 and 3 were referred for SCN1A genetic testing and patient DNA samples were analysed according to standard procedures. Molecular analysis was performed on genomic DNA extracted from patients' and their parent's venous blood. All 26 exons of the SCN1A gene were amplified by PCR. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large scale rearrangements.3,4 Cases 2 and 4 were tested following the findings in their siblings. Parents consented for their children's anonymous patient information to be published as part of a case series.
4.
Discussion
We report two novel homozygous SCN1A missense mutations in four children from consanguineous pedigrees with varying clinical phenotypes. Homozygosity was confirmed by performing multiplex ligation-dependent probe amplification (MLPA) analysis to exclude the possibility of a deletion on the other allele (Table 1, Fig. 2). All cases have febrile seizures; a hallmark of SCN1A related epilepsies and the two families have the familial epilepsy syndrome GEFSþ known to be associated with heterozygous variants in SCN1A. Case one is a female with Dravet syndrome and her sibling (Case two) has a phenotype of FSþ. The sibling pair cases three & four have febrile seizures plus focal
epileptic seizures. We were unable to establish whether any of the unaffected siblings carried SCN1A mutations as it is not common practice in the UK to undertake genetic testing on asymptomatic children. Both families are of South Asian origin and demonstrate the importance of ordering investigations based on phenotype and not discounting genetic investigations typically associated with dominant or de novo disease when a recessive disorder is thought more likely. Cases 1 and 3 had undergone extensive biochemical and neuroimaging investigations prior to genetic testing. These cases illustrate that determination of the clinical significance of SCN1A missense mutations remains challenging. We recently described that assessment of the amino acid properties and protein location can be helpful in determining the pathogenicity of genetic findings.4 Missense mutations leading to disease are more likely to occur in the voltage sensor (S4) and pore region (S5eS6 segments) of the four homologous domains. Significant disease is more likely when there is substantial change in the physicochemical properties of the amino acid as measured by Grantham distance (GS).4,5 In cases one and two the missense mutation Met400Val is located in an area of functional importance; however has a low GS, as one nonpolar amino acid (methionine) is exchanged for another nonpolar amino acid (valine). This conservative change might allow the heterozygous parents to remain unaffected; however having such a change on both alleles may have a cumulative and detrimental effect on the homozygous children. The reverse is true for cases
Fig. 2 e Position of mutations across SCN1A protein. Homologous domains (D1-4); transmembrane segments 1e4 are voltage sensing domains (shown in green/yellow); segments 4 (yellow) are voltage sensors; segments 5 to 6 (pink) make up the pore-lining regions; NH3þ represents the N-terminal and CO2¡ the C-terminal. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
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three and four. Here the missense mutation Arg618Cys has a very high GS, as a charged amino acid (arginine) is exchanged for an uncharged amino acid with a sulfhydryl group (cysteine). However; this change is located within a less important non-functional area of the protein and might allow the heterozygous parents to remain unaffected; whereas the homozygous children who are carrying this change on both alleles might experience a cumulative and detrimental effect (Fig. 2). Examples of worsening effects of homozygous SCN1A mutations include the R1648H GEFS þ mouse model. Heterozygous R1648H mice have normal life spans with reduced thresholds to drug induced seizures and infrequent generalized seizures as adults whilst the homozygous model exhibits spontaneous generalized seizures with a reduced life span.6 Interestingly, Patino et al.7 reported a case of Dravet syndrome caused by a homozygous mutation of the SCN1B gene. Similar to the situation described here, the consanguineous parents were heterozygous carriers of the missense mutation. SCN1B encodes the sodium channel beta subunit which modulates channel voltage-dependence and gating as well as channel cell surface expression.8 Heterozygous mutations of SCN1B have previously been shown to cause GEFSþ.9,10 Patino and colleagues demonstrated, using functional assays and animal models, that the homozygous mutant alpha-1 subunit polypeptides were synthesised normally but were not transported to the cell surface in mammalian fibroblasts in vivo.7 Furthermore, they showed that mice homozygous for the SCN1B mutation would have spontaneous seizures, whilst heterozygous mice had seizure susceptibility that was similar to that of wild types. We observed a marked heterogeneity of the phenotype in the two presented sibling pairs. It has previously been shown that the same SCN1A mutation can show significant intra and inter-familial variability highlighting that environmental factors, epigenetic mechanisms and the genetic background of the individual play a significant role in the development and evolution of the epilepsy phenotype.11,12 Given the consanguinity of the presented cases it is not surprising to find other pathologies such as hearing loss segregating in these families. We regard this as an independent unrelated pathology. Indeed the fact that all of our patients were born of consanguineous parents increases the chances that they may have other rare homozygous variants at other genetic loci that could contribute in varying degrees to their individual phenotype. Whole exome sequencing would be a useful tool to determine potential genetic variants in these cases but was not available here. In particular identification of the genetic variation across all ion channels in each individual could go some way to explaining why they are, or are not affected by seizures. Pathogenic ion channel alleles in mouse models can form therapeutic combinations with effects that mask one another, reducing the penetrance and the expressivity of the clinical disorder.13 Variants in Scn2a, Scn8a and Kcnq2 have been shown to influence the phenotype of mice carrying the Scn1aR1684H mutation.14 More recently detailed assessment of 237 ion channel genes in patients with idiopathic epilepsy and normal controls showed that both rare and common coding variants in known excitability disease genes were present in both patients and controls.15
The four cases presented suggest that deleterious alleles can be modified by the unique combination of variants present in an individual's ion channel variation profile; illustrating how better understanding of the nature and location of SCN1A missense mutations may aid the interpretation of genotypeephenotype associations. SCN1A related epilepsies should be considered in children with infantile onset epilepsies even when an autosomal recessive neurological disorder is suspected.
Conflict of interest None.
Acknowledgements Dr Andreas Brunklaus reports no disclosures. Rachael Ellis reports no disclosures. Dr Helen Stewart reports no disclosures. Dr Sarah Aylett is a co-investigator for research funded by Epilepsy Action and Dravet Syndrome UK. Dr Eleanor Reavey reports no disclosures. Dr Ros Jefferson reports no disclosures. Dr Rakesh Jain reports no disclosures. Dr Supratik Chakraborty reports no disclosures. Dr Sandeep Jayawant reports no disclosures. Dr Sameer M Zuberi is an Associate Editor of The European Journal of Paediatric Neurology, has received honoraria for speaking at a UCB Pharma and Glaxo Wellcome sponsored educational meetings and received research funding from Epilepsy Research UK (AP1218) and Dravet Syndrome UK.
references
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Please cite this article in press as: Brunklaus A, et al., Homozygous mutations in the SCN1A gene associated with genetic epilepsy with febrile seizures plus and Dravet syndrome in 2 families, European Journal of Paediatric Neurology (2015), http:// dx.doi.org/10.1016/j.ejpn.2015.02.001
