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Mosaicism of a missense SCN1A mutation and Dravet syndrome in a Roma/Gypsy family

Epileptic Disorders. Volume 12, Numéro 2, 117-24, June 2010, Original article

DOI : 10.1684/epd.2010.0311

Summary

Auteur(s) : Dimitar N Azmanov, Sashka Zhelyazkova, Petya S Dimova, Melania Radionova, Veneta Bojinova, Laura Florez, Shelagh J Smith, Ivailo Tournev, Assen Jablensky, John Mulley, Ingrid Scheffer, Luba Kalaydjieva, Josemir W Sander , Laboratory for Molecular Genetics, Centre for Medical Research and Western Australian Institute for Medical Research, The University of Western Australia, Perth, Australia, Department of Neurology, Medical University, Sofia, Bulgaria, Clinic of Child Neurology, St. Naum University Hospital of Neurology and Psychiatry, Medical University, Sofia, Bulgaria, Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom, Department of Cognitive Science and Psychology, New Bulgarian University, Sofia, Bulgaria, School of Psychiatry and Clinical Neurosciences, The University of Western Australia, Perth, Australia, Epilepsy Research Program, Women's and Children's Hospital, Adelaide, Australia, Department of Medicine, The University of Melbourne, Austin Health, Heidelberg, Victoria, Australia, Department of Paediatrics, The University of Melbourne, Royal Children's Hospital, Melbourne, Australia, SEIN – Epilepsy Institute of the Netherlands Foundation, Heemstede, The Netherlands.

Illustrations

ARTICLE

Auteur(s) : Dimitar N Azmanov¹, Sashka Zhelyazkova², Petya S Dimova³, Melania Radionova², Veneta Bojinova³, Laura Florez¹, Shelagh J Smith⁴, Ivailo Tournev^2,5, Assen Jablensky⁶, John Mulley⁷, Ingrid Scheffer^8,9, Luba Kalaydjieva¹, Josemir W Sander^4,10

¹Laboratory for Molecular Genetics, Centre for Medical Research and Western Australian Institute for Medical Research, The University of Western Australia, Perth, Australia
²Department of Neurology, Medical University, Sofia, Bulgaria
³Clinic of Child Neurology, St. Naum University Hospital of Neurology and Psychiatry, Medical University, Sofia, Bulgaria
⁴Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom
⁵Department of Cognitive Science and Psychology, New Bulgarian University, Sofia, Bulgaria
⁶School of Psychiatry and Clinical Neurosciences, The University of Western Australia, Perth, Australia
⁷Epilepsy Research Program, Women's and Children's Hospital, Adelaide, Australia
⁸Department of Medicine, The University of Melbourne, Austin Health, Heidelberg, Victoria, Australia
⁹Department of Paediatrics, The University of Melbourne, Royal Children's Hospital, Melbourne, Australia
¹⁰SEIN – Epilepsy Institute of the Netherlands Foundation, Heemstede, The Netherlands

Article reçu le 23 Novembre 2009, accepté le 18 Mars 2010

Mutations in the SCN1A gene, encoding the sodium channel alpha 1 subunit, account for 70-80% of patients with Dravet syndrome, also referred to as severe myoclonic epilepsy of infancy (SMEI) (Depienne et al., 2009; Harkin et al., 2007; Marini et al., 2007). Dravet syndrome is a severe infantile-onset epilepsy, characterised by multiple seizure types, cognitive decline and poor outcome (Dravet et al., 2005). Approximately 95% of SCN1A mutations in Dravet syndrome originate de novo (Harkin et al., 2007; Marini et al., 2009), while the remainder are inherited from a parent either with a phenotype corresponding to the less severe end of the GEFS+ spectrum or who is totally asymptomatic (Claes et al., 2009; Lossin, 2009; Mulley et al., 2005).

SCN1A mutations are also identified in around 10% of families with the genetic (formerly generalised) epilepsy with febrile seizures plus syndrome (GEFS+) (Scheffer and Berkovic, 1997; Scheffer et al., 2009). Whereas the spectrum of SCN1A mutations in Dravet syndrome ranges from missense mutations to gene rearrangements, missense mutations represent the only type of molecular defect in SCN1A occurring in GEFS+ families (Claes et al., 2009; Lossin, 2009; Mulley et al., 2005). The wide range of phenotypes associated with SCN1A missense mutations in GEFS+ families (Scheffer and Berkovic, 1997; Scheffer et al., 2009) and among the SCN1A -related infantile epileptic encephalopathies (Harkin et al., 2007), poses challenges for genetic counselling, disease prognosis and treatment.

Genetic background and modifying factors are invoked to explain the phenotypic variation in large multiplex families with GEFS+ and SCN1A mutations. In contrast, parental mosaicism has been demonstrated as the underlying mechanism in two-generation families where a parent of a child (or children) with Dravet syndrome was mildly affected or asymptomatic (Gennaro et al., 2006; Marini et al., 2009). The documented cases of parental mosaicism are caused by truncating mutations and major rearrangements in SCN1A (see appendix 1). In this study of a Roma/Gypsy family, we have identified a missense SCN1A mutation in a proband with Dravet syndrome and mosaicism of this mutation in the father with a mild GEFS+ phenotype. This SCN1A missense mutation may therefore be added to the spectrum of mutations in Dravet syndrome which are explained by parental mosaicism.

Subjects and methods

Subjects

The proband III-1 from Gypsy family M1 (figure 1A) was characterised by the Paediatric Neurology Clinic of the Medical University, Sofia, Bulgaria, during multiple admissions for intractable seizures. Further examinations, detailed interviews with the parents, paternal grandparents and other relatives, and EEG recordings were conducted during two home visits by the research team. The population control samples consisted of 118 subjects of Gypsy ethnicity, affected by different forms of epilepsy (Angelicheva et al., 2009) and a panel of 546 control individuals representing diverse Gypsy sub-isolates (Kalaydjieva et al., 2005; Morar et al., 2004).

Genetic analyses

SCN1A mutation analysis in the proband was performed on the entire coding sequence by dHPLC, as previously described (Harkin et al., 2007). Subsequent analysis of exon 4 was performed by DNA sequencing using primers F-CACTGATGGAGTGATAAGAAA and R-ATTCTACAGGTAAAGCAAACC (Genbank accession number AB093548). Analysis of the remaining members of the family was performed by direct sequencing. The population control samples were genotyped for the p.D194N mutation with a custom-designed TaqMan assay (primers and probes available on request), following the manufacturer's protocol (Applied Biosystems).

Sequence homology of SCN1A paralogues and orthologues was analysed using the online Multalign tool (http://bioinfo.genotoul.fr/multalin/ multalin.html).

Inherited Dravet syndrome SCN1A mutations were reviewed using the SCN1A variant database at http://www.molgen.ua.ac.be/SCN1AMutations/Home/Default.cfm (Claes et al., 2009) and corresponding original articles. Overall, the nature of the mutations, phenotype descriptions, information on parental mosaicism, family structure, and overlap between mutations reported in inherited Dravet syndrome and in multiplex GEFS+ families were examined.

Results

Clinical findings

The proband, III-1, was a 12-year-old boy of Gypsy ethnicity. The pregnancy was complicated by a chest infection and antibiotic treatment in the first trimester and bleeding and recurrent contractions in the last trimester. The delivery was at term and uneventful. Early development was delayed, with talking at 24 months and walking initially on tiptoes at about the same age.

His first seizure comprised convulsive status epilepticus at age 4.5 months, a few hours after his diphtheria/tetanus/pertussis (DTP) immunisation. In the following months, he developed brief generalised seizures, occurring several times per day despite starting phenobarbital treatment at six months. At one year, he had a prolonged febrile convulsive seizure with head and eye deviation to the left. Thereafter, he had up to four generalised tonic-clonic seizures per month, both febrile and afebrile, lasting 15-20 minutes with alternating lateralisation in terms of head and eye deviation.

Seizure control was not achieved by carbamazepine and valproate which were introduced at two years of age. Topiramate reduced the frequency to 1-2 seizures per month, but was discontinued because the mother encountered problems with her son's hyperactive, reckless and aggressive behaviour. Subsequently, the generalised seizures persisted despite treatment regimens including valproate, phenobarbitone, oxcarbazepine and levetiracetam. In addition to the convulsive attacks, from one year the patient developed brief partial seizures, sometimes with secondary generalisation. His aura consisted of a poorly described sensation “of an oncoming seizure” and visual phenomena (“hit by sunlight”).

Yearly EEG studies from age six years showed diffuse background slowing, with generalised spike-wave discharges upon hyperventilation (figure 2) and multi-focal epileptiform activity emanating from the frontal, temporal and parieto-temporal regions. A sleep EEG at 12 years showed no abnormalities. MRI scans at six and 12 years were normal.

Psychological assessment showed moderate intellectual disability (IQ 50), mildly reduced attention span and concentration, and difficulties with visuospatial function.

His father, II-1, was a 40-year-old man of normal intelligence who owned a small farm. He experienced a simple febrile seizure at age 5-6 months. The information about subsequent seizure semiology was uncertain but suggested generalised convulsions and partial seizures with secondary generalisation. His attacks were often triggered by stress and febrile illnesses, and occurred during both wakefulness and sleep. He was never treated, and his seizures ceased spontaneously at 12 years. An EEG in 2008 was normal.

Mutation analysis

SCN1A analysis in the proband identified the missense mutation c.580 G>A, leading to a substitution of the conserved aspartic acid residue at position 194 by asparagine (p.D194N) in the S3 segment of the DI protein domain (see appendix 2). Mutation analysis of the members of the expanded family showed that the p.D194N mutation was also present in the father but not in the paternal grandparents or in other relatives (figure 1A). Inspection of the father's sequencing chromatogram pattern suggested mosaicism in his peripheral blood cells (figure 1B). This was supported by the results of the TaqMan assay, where quantification of the mutant over the normal allele using the ΔΔCt calculation (Livak and Schmittgen, 2001) suggested that 60-70% of the paternal cells carried the mutation (figure 1C). An additional estimate of the proportion of mutant cells was obtained by mixing the proband's DNA (all cells carrying the G>A substitution) with DNA from the mutation-negative mother in a 2:1 and 1:2 ratio. The sequencing chromatogram of the 2:1 mix closely resembled that of the father, supporting the presence of the mutation in 60-70% of the paternal cells (figure 1B).

The mutation was not detected in any other epilepsy patients and families of the same ethnic background. Screening of the Gypsy population control samples was also negative.

A review of published inherited mutations associated with Dravet syndrome

Our review of the SCN1A database and published original studies identified 26 inherited SCN1A mutations identified in patients with Dravet syndrome (see appendix 1). These were classified into three groups, based on evidence of parental mosaicism:

– well characterised two-generation families with proven or highly probable (gonadal) mosaicism;
– mosaicism not investigated but suggested by mild parental phenotypes and mutation inheritance;
– limited information with speculative mosaicism based on the presence of mutation in a parent with unspecified phenotype. In addition, we found a small group of five mutations identified in individuals with Dravet syndrome and GEFS+ (see appendix 1), either in the same multiplex families with variable phenotypes, or unrelated. In these latter cases, parental mosaicism is irrelevant and additional modifying factors are necessary to account for the phenotypic variation.

Truncated mutations or major gene rearrangements account for all nine previously reported cases of group 1 (see appendix 1). The p.D194N substitution identified in this study is the first missense mutation associated with parental mosaicism and Dravet syndrome, indicating that this genetic mechanism may be more common than previously thought.

Discussion

The p.D194N mutation in SCN1A appears to be a rare recurrent defect associated with the severe epileptic encephalopathy of Dravet syndrome. The scenario that p.D194N is a founder mutation in the general Gypsy population requiring additional modifying/susceptibility genes for the disease phenotype to manifest, was ruled out by negative screening of the p.D194N mutation in the remainder of the family, nearly 1,100 normal alleles in our control panel and the affected Gypsy families. The limited segregation of the mutation in the family is consistent with its de novo appearance in the proband's mosaic father. The same missense mutation has been described previously in two unrelated patients of different ethnic backgrounds: a Dravet syndrome case (Mancardi et al., 2006), where the mutation occurred de novo and a patient diagnosed as borderline SMEI (Harkin et al., 2007), where inheritance was not determined.

Our proband was also classified as borderline SMEI, with atypical features including evidence of abnormal early development and lack of myoclonic seizures. As the contribution of SCN1A mutations is similar between borderline and classic cases of Dravet syndrome, we previously suggested that both should be referred to as Dravet syndrome (Harkin et al., 2007). The finding of the p.D194N mutation in both classical and borderline forms supports this notion. The p.D194N mutation is most likely to be specific to Dravet syndrome, and the milder GEFS+ phenotype in the transmitting parent in our family attributed to somatic mosaicism, rather than variable gene expression.

Although seizure occurrence in the proband followed DTP immunisation, detection of an SCN1A mutation excluded vaccine encephalopathy, consistent with previous observations (Berkovic et al., 2006).

Genotype-phenotype correlations in epilepsy are notoriously difficult (Dibbens et al., 2009; Scheffer et al., 2009) with broad variation in severity often observed within a family (Singh et al., 2009). At present, the presumed modifying factors remain unidentified. Parental mosaicism is a rare exception, where phenotypic variation can be attributed to a specific mechanism, thus facilitating prognosis and genetic counselling. Our finding of a missense mutation transmitted by a mosaic parent implies that the mechanism is likely to be more common than currently recognised.

Acknowledgments

We are grateful to all the participants in this study and to Xenia Iona who detected the mutation by dHPLC screening and characterised the variant as p.D194N. The study was funded by grant 458736 and Training Fellowship 634551 of the National Health and Medical Research Council of Australia. The authors declare no conflict of interests.

This study has been approved by the Ethics Committees of the Medical University, Sofia, the University of Western Australia, and University College London. Written informed consent has been provided by all participants in the study.

Financial support.

The study was funded by grant 458736 and Training Fellowship 634551 of the National Health and Medical Research Council of Australia.

Disclosure.

None of the authors has any conflict of interest to disclose.

Genetic mechanisms potentially contributing to the phenotypic variation associated with SCN1A mutations in Dravet syndrome

A) Unique SCN1A mutations in small 2-generation families with Dravet syndrome offspring
Mosaicism in mutation-positive parent	Mutation	Mutational mechanism	Protein segment1	Protein domain1	Phenotype of proband	Phenotype of mutation-positive siblings	Inheritance	Phenotype of mutation-positive parent	Independently observed in multiplex GEFS+ families	Reference
Proven or highly probable	c.580G>A	p.D194N	S 3	D I	Dravet	Nil	Paternal	GEFS+	NO	This study2
	c.602+1G>A	splice	S 3	D I	Dravet	Dravet	Paternal3	FS	NO	(Depienne et al., 2009 ; Marini et al., 2006 ; Marini et al., 2007)
	c.[730G>T; 735_736delGAinsTT]	p.[V244L; K245N; L246X]	S 4-5	D I	Dravet	Dravet	Maternal	Unaffected	NO	(Morimoto et al., 2006)
	c.965-2A>C	splice	S 5-6	D I	Dravet	Dravet	Paternal	Unaffected	NO	(Depienne et al., 2006 ; Depienne et al., 2009)
	c.1624C>T	p.R542X	-	D I-II	Dravet	Dravet	Maternal	Unaffected	NO	(Depienne et al., 2006 ; Depienne et al., 2009)
	c.3985C>T	p.R1329X	S 4	D III	Dravet	Dravet	Maternal	Migrane	NO	(Selmer et al., 2009)
	c.5240A[3]	p.N1747KfsX33	S 5-6	D IV	Dravet	Dravet	Maternal	FS	NO	(Gennaro et al., 2003, Gennaro et al., 2006)
	dup SCN1A exon 8-16		-	-	Dravet	Dravet	Maternal	FS	NO	(Marini et al., 2009)
	del SCN1A exon 1-22		-	-	Dravet	Dravet	Both parents mutation- negative	Both parents unaffected	NO	(Marini et al., 2009)
	c.3550+1G>A	splice	-	D II-III	Dravet	Dravet	Both parents mutation- negative	Both parents unaffected	NO	(Gennaro et al., 2006)

Possible (not stated)	c.865G>T	p.E289X	S 5-6	D I	Dravet		Maternal	Mildly affected	NO	(Mancardi et al., 2006 ; Nabbout et al., 2003)
	c.890C>T	p.T297I	S 5-6	D I	Dravet		Maternal	Unaffected	NO	(Nabbout et al., 2003)
	c.3878delA	p.D1293VfsX7	S 3	D III	Dravet		Paternal	FS	NO	(Depienne et al., 2009 ; Marini et al., 2007)
	c.5075T>C	p.F1692S	S 5	D IV	Dravet		Paternal	FS	NO	(Fukuma et al., 2004)
	c.5138G>A	p.S1713N	S 5-6	D IV	Dravet	Dravet	Paternal	FS	NO	(Kimura et al., 2005)

No data on mosaicism Insufficient information on phenotypes	c.371T>A	p.I124N	S 1	D I	Dravet		Maternal	Unknown	NO	(Depienne et al., 2009)
	c.571A>T	p.N191Y	S 3	D I	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.1066A>G	p.R356G	S 5-6	D I	Dravet		Unspecified	Unknown	NO	(Marini et al., 2007)
	c.1377+1G>A	splice	-	D I-II	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.4101T>A	p.D1367K	S 5	D III	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.4834G>A	p.V1612I	S 3	D IV	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.4888G>A	p.V1630M	S 3-4	D IV	Dravet		Unspecified	Unknown	NO	(Marini et al., 2007)
	c.4973C>T	p.T1658M	S 4-5	D IV	Dravet		Maternal	Unknown	NO	(Depienne et al., 2009)
	c.4991T>A	p.M1664K	S 4-5	D IV	Dravet		Maternal	Unknown	NO	(Depienne et al., 2009)
	c.5346C>G	p.I1782M	S 6	D IV	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.5734C>T	p.R1912X	-	C-terminal	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)

A) Unique SCN1A mutations in small 2-generation families with Dravet syndrome offspring
Mosaicism in mutation-positive parent	Mutation	Mutational mechanism	Protein segment1	Protein domain1	Phenotype of proband	Phenotype of mutation-positive siblings	Inheritance	Phenotype of mutation-positive parent	Independently observed in multiplex GEFS+ families	Reference
Proven or highly probable	c.580G>A	p.D194N	S 3	D I	Dravet	Nil	Paternal	GEFS+	NO	This study2
	c.602+1G>A	splice	S 3	D I	Dravet	Dravet	Paternal3	FS	NO	(Depienne et al., 2009 ; Marini et al., 2006 ; Marini et al., 2007)
	c.[730G>T; 735_736delGAinsTT]	p.[V244L; K245N; L246X]	S 4-5	D I	Dravet	Dravet	Maternal	Unaffected	NO	(Morimoto et al., 2006)
	c.965-2A>C	splice	S 5-6	D I	Dravet	Dravet	Paternal	Unaffected	NO	(Depienne et al., 2006 ; Depienne et al., 2009)
	c.1624C>T	p.R542X	-	D I-II	Dravet	Dravet	Maternal	Unaffected	NO	(Depienne et al., 2006 ; Depienne et al., 2009)
	c.3985C>T	p.R1329X	S 4	D III	Dravet	Dravet	Maternal	Migrane	NO	(Selmer et al., 2009)
	c.5240A[3]	p.N1747KfsX33	S 5-6	D IV	Dravet	Dravet	Maternal	FS	NO	(Gennaro et al., 2003, Gennaro et al., 2006)
	dup SCN1A exon 8-16		-	-	Dravet	Dravet	Maternal	FS	NO	(Marini et al., 2009)
	del SCN1A exon 1-22		-	-	Dravet	Dravet	Both parents mutation- negative	Both parents unaffected	NO	(Marini et al., 2009)
	c.3550+1G>A	splice	-	D II-III	Dravet	Dravet	Both parents mutation- negative	Both parents unaffected	NO	(Gennaro et al., 2006)

Possible (not stated)	c.865G>T	p.E289X	S 5-6	D I	Dravet		Maternal	Mildly affected	NO	(Mancardi et al., 2006 ; Nabbout et al., 2003)
	c.890C>T	p.T297I	S 5-6	D I	Dravet		Maternal	Unaffected	NO	(Nabbout et al., 2003)
	c.3878delA	p.D1293VfsX7	S 3	D III	Dravet		Paternal	FS	NO	(Depienne et al., 2009 ; Marini et al., 2007)
	c.5075T>C	p.F1692S	S 5	D IV	Dravet		Paternal	FS	NO	(Fukuma et al., 2004)
	c.5138G>A	p.S1713N	S 5-6	D IV	Dravet	Dravet	Paternal	FS	NO	(Kimura et al., 2005)

No data on mosaicism Insufficient information on phenotypes	c.371T>A	p.I124N	S 1	D I	Dravet		Maternal	Unknown	NO	(Depienne et al., 2009)
	c.571A>T	p.N191Y	S 3	D I	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.1066A>G	p.R356G	S 5-6	D I	Dravet		Unspecified	Unknown	NO	(Marini et al., 2007)
	c.1377+1G>A	splice	-	D I-II	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.4101T>A	p.D1367K	S 5	D III	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.4834G>A	p.V1612I	S 3	D IV	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.4888G>A	p.V1630M	S 3-4	D IV	Dravet		Unspecified	Unknown	NO	(Marini et al., 2007)
	c.4973C>T	p.T1658M	S 4-5	D IV	Dravet		Maternal	Unknown	NO	(Depienne et al., 2009)
	c.4991T>A	p.M1664K	S 4-5	D IV	Dravet		Maternal	Unknown	NO	(Depienne et al., 2009)
	c.5346C>G	p.I1782M	S 6	D IV	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)
	c.5734C>T	p.R1912X	-	C-terminal	Dravet		Paternal	Unknown	NO	(Depienne et al., 2009)

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Marini C, Scheffer IE, Nabbout R, et al. SCN1A duplications and deletions detected in Dravet syndrome: Implications for molecular diagnosis. Epilepsia 2009; 50: 1670-8.

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Appendix 2 Conservation of the aspartic acid residue at position 194 of human SCN1A

NP_008851: human SCN1A; NP_001035232: human SCN2A; NP_008853: human SCN3A; NP_002967: human SCN7A; NP_002968: human SCN9A; vertebrate SCN1A: XP_001154158: Pan troglodytes; XP_001100928: Macaca mulatta; NP_061203: Mus musculus; NP_110502: Rattus norvegicus; XP_001252710: Bos taurus; XP_422021: Gallus gallus; CAQ13572: Danio rerio; * invariant residue.

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