University of Groningen Genotyping and phenotyping epilepsies of childhood Vlaskamp, Danique

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Genotyping and phenotyping epilepsies of childhood

Vlaskamp, Danique

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Publication date: 2018

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Vlaskamp, D. (2018). Genotyping and phenotyping epilepsies of childhood. Rijksuniversiteit Groningen.

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Chapter 7

+DSORLQVXIÀFLHQF\RIWKHSTX1B gene

is associated with myoclonic astatic

epilepsy

Published as: DRM Vlaskamp, P Rump, PMC Callenbach, YJ Vos, B Sikkema-Raddatz, CMA van Ravenswaaij-Arts, OF Brouwer. Haploinsuﬃ ciency of the STX1B gene is associated with myoclonic

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Danique R M Vlaskamp,1,2 _{Patrick Rump,}2_{Petra M C Callenbach,}1_{Yvonne J Vos,}2_Birgit

Sikkema-Raddatz,2_{Conny M A van Ravenswaaij,}2_{Oebele F Brouwer}1

1_{University of Groningen, University Medical Center Groningen, Department of Neurology,}

Groningen, The Netherlands. 2_{University of Groningen, University Medical Center Groningen,}

Department of Genetics, Groningen, The Netherlands.

Acknowledgements. P.M.C. Callenbach received an unrestricted research grant from UCB

Pharma BV, the Netherlands. UCB Pharma BV had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the parents of the patient for giving their informed consent to report his case, and J. Senior and K. Mc Intyre for editing the manuscript.

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ABSTRACT

We describe an 18-year-old male patient with myoclonic astatic epilepsy (MAE), moderate to severe intellectual disability, behavioral problems, several dysmorphisms and a 1.2-Mb de novo deletion on chromosome 16p11.2. This deletion results in haploinsufficiency of STX1B and other genes. Recently, variants in the STX1B gene have been associated with a wide spectrum of fever-related epilepsies ranging from single febrile seizures to severe epileptic encephalopathies. Two previously reported patients with a STX1B missense variant or deletion were diagnosed with MAE. Our observation of a STX1B deletion in a third patient with MAE therefore supports that STX1B gene variants or deletions can be involved in the aetiology of MAE. Furthermore, STX1B encodes for syntaxin-1B, of which interaction with the protein encoded by the STXBP1 gene is essential for the regulation of the synaptic transmission of neurotransmitters. STXBP1 gene variants have been identified in patients with many different types of epilepsy, including Dravet syndrome and epileptic encephalopathies, suggesting STX1B plays a similar role. We recommend that analysis of

STX1B should be considered in the diagnostic work-up of individuals with MAE.

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INTRODUCTION

Myoclonic Astatic Epilepsy (MAE), or Doose syndrome, is classiﬁed as an idiopathic generalized epilepsy.1_{Although likely pathogenic sequence variants in the SCN1A, SCN1B, SLC2A1 and GABRG2}

genes have been identiﬁed in some patients with MAE, the cause of MAE remains unknown in most patients.2

Recently, sequence variants in the STX1B gene (MIM 601485) have been shown to cause a broad spectrum of fever-associated epilepsy syndromes, ranging from simple febrile seizures to severe epileptic encephalopathies.3_{Two patients in this study with a de novo missense sequence variant}

or a de novo 0.8-Mb deletion including STX1B had MAE.3_{Here, we present a new patient with MAE}

who has a 1.2-Mb de novo 16p11.2 deletion that only has a 160-kb overlap with the previously published deletion, yet it also resulted in the loss of the STX1B gene.

CASE STUDY

This 18-year-old male was born after a normal pregnancy and delivery, as the ﬁrst child of healthy Dutch parents. At one year of age, he had two generalized clonic seizures followed, a few days later, by some focal motor seizures. After having been seizure-free on valproic acid until the age of 20 months, his seizures re-occurred and resembled infantile spasms. An EEG showed high-amplitude polyspike waves and he was treated with vigabatrin and prednisone.

At two years of age, he presented with several seizure types including staring and head nodding, myoclonic, myoclonic-astatic and astatic seizures, as well as focal seizures with epigastric uprising and impairment of consciousness. He became seizure-free on a combination of valproic acid, lamotrigine and clobazam, but diurnal atonic seizures re-occurred after six months. An EEG showed slow polyspike-wave paroxysms with normal background activity. Based on these seizure types and the EEG ﬁndings, his epilepsy was classiﬁed as MAE.

From age two and a half to eight years, he only experienced some generalized seizures during fever. Both lamotrigine and clobazam were successfully withdrawn. During tapering oﬀ of the valproic acid at the age of 11 years, tonic-clonic seizures re-occurred. Currently, his epilepsy is well controlled with valproic acid monotherapy.

Since one year old, developmental delay became apparent. Formal neuropsychological testing at age 23 months showed delays in communication, social behavior, adaptation, fine motor skills (developmental age 14 months) and gross motor skills (developmental age 15 months). Later, at five years and three months old, his developmental age was between two and two and a half years. He attended a special school for children with severe learning difficulties. Over this time,

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Processed on: 29-10-2018 PDF page: 149PDF page: 149PDF page: 149PDF page: 149 behavioral problems became manifest with temper tantrums, attention diﬃculties, and sleeping

problems. During puberty, he showed compulsive behavior and frequent episodes of aggression with screaming, beating and kicking. Several behavioral therapies were ineﬀective.

At the age of 17 years, he has a normal height, weight and head circumference. His appearance is characterized by dark curly hair, prominent eyebrows with a mild synophrys, mild hypertelorism, short alae nasi, overfolded helices of both ears, retrognathia with a dental overbite, wide-spaced teeth and a primary failure of tooth eruption with only a few elements of his permanent dentition visible. Additionally, he has a pectus carinatum, scoliosis, pes plano valgus and a hyperpigmented macula on his left ankle. He also has a rotational nystagmus, but good visual acuity. In his family, one cousin (the son of a maternal uncle) had epilepsy between 18 months and 16 years of age and an attention deﬁcit hyperactivity disorder, but normal development. Further information about the epilepsy in this cousin was not available. The family history was otherwise unremarkable. Initial diagnostic investigations, including a brain CT-scan at age one year, a metabolic screen, a muscle biopsy to evaluate respiratory chain defects and mitochondrial DNA sequence variants, standard karyotyping, and DNA analysis for fragile-X syndrome (MIM 300624) did not reveal the underlying cause of his epilepsy, developmental delay and behavioral problems. At the age of 17 years, targeted next-generation sequencing was performed using an epilepsy gene panel (Agilent Sure Select Design 0501411, Agilent Technologies Inc., Santa Clara, CA, USA) including 94 genes associated with early infantile and other epileptic encephalopathies, syndromes with epilepsy and intellectual disability (ID) and fever-associated epilepsies (see Supplemental Table 1 for the genes included in this panel). Sanger sequencing of the PTHR1 gene (MIM 168468, associated with primary failure of tooth eruption) showed no likely pathogenic variants. Microarray analysis (Illumina Omni Express 12-V1.0, Illumina, San Diego, CA, USA) showed a de novo deletion of chromosome 16p11.2 (arr[hg19]16p11.2 (30,943,951-32,151,753)x1) in the patient that included the

STX1B gene (Figure 1).

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F

igur

e 1.

The 16p11.2 deletions obser

ved in our patient, in the patients r

epor ted by Schuber t et al . 3 and in the t w o DECIPHER patients . Th e c o o rdinat es are b as ed o n N C B I B u ild 3 7 [ h g1 9] an d th e small es t re gi o n o f over lap is in di ca te d by th e hi ghli gh te d zo n e. All th e p rot ein -c o din g g en es are sh ow n . < < an d > > in di ca te that th e b reak p o in ts o f th e d el eti o n s are o u ts id e th e re gi o n p re sen te d h ere .

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DISCUSSION

We report a 18-year-old patient with MAE, moderate to severe ID, behavioral problems and several dysmorphic characteristics, in whom we found a 1.2-Mb de novo 16p11.2 deletion that includes the STX1B gene. Involvement of the STX1B gene in the aetiology of MAE was previously reported by Schubert et al.3_{They described two patients with MAE with a STX1B deletion and}

a de novo missense variant, respectively. Furthermore, atonic seizures, which are considered to be part of the MAE phenotype, were also reported by the same authors in six relatives in two other families with either a nonsense sequence variant or a complex insertion/deletion sequence variant in the STX1B gene.3_{Lastly, seven (24%) of the 29 individuals reported with STX1B sequence}

variants or deletions had no febrile seizures and three (10%) others experienced afebrile seizures at the onset of their epilepsy and later also developed seizures during fever. Our patient did not have febrile seizures prior to epilepsy onset. Together, these ﬁndings support the hypothesis that

STX1B is not only involved in the aetiology of fever-associated epilepsies, but also in the aetiology

of non-fever-related epilepsies, including MAE.

The STX1B gene encodes for the syntaxin-1B protein (STX1B). Syntaxins are cellular receptors for vesicle transport. In rats, the stx1b protein is involved in the fusion of presynaptic vesicle membranes to release their neurotransmitters. STX1B consists of an α-helical domain, which can be unfolded (‘open’ conformation) or folded (‘closed’ conformation), a soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) motif and a transmembrane region.4_‘Open’

stx1b is able to bind other SNARE proteins and syntaxin binding protein-1 (stxbp1) resulting in presynaptic vesicle exocytosis in rats.5, 6_{‘Closed’ stx1b can also bind to stxbp1, which is essential for}

the regulation of presynaptic vesicle release.4, 7_{Stxbp1 thus executes and mediates the synaptic}

transmission by binding to the ‘open’ and ‘closed’ conformations of stx1b, respectively.7_Stxbp1

is encoded by the STXBP1 gene (MIM 602926). STXBP1 sequence variants have been associated with a wide spectrum of epilepsies, including fever-related Dravet syndrome, focal epilepsies in children with ID, and early onset epileptic encephalopathies like Ohtahara syndrome and infantile spasms.8-12_{As the interaction between STX1B and STXBP1 is essential for the synaptic}

transmission of neurotransmitters, it is understandable that alterations in one of these genes can lead to epilepsy. The diversity of the related epilepsy syndromes, however, has not yet been explained.

Remarkably, STXBP1 sequence variants have been associated with infantile spasms, which were not observed in the previously reported patients with STX1B sequence variants or deletions.12

The patient presented here had seizures resembling infantile spasms at a relatively old age (20 months). Unfortunately, original EEGs were no longer available to conﬁrm the presence of hypsarrhythmia or allow the diagnosis of West syndrome.

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Processed on: 29-10-2018 PDF page: 152PDF page: 152PDF page: 152PDF page: 152 The DECIPHER database includes two other patients with STX1B deletions but no reported

epilepsy. Patient 291666 (chr16:26,746,122-31,394,579x1) had ID and a double outlet of the right ventricle. Patient 291768 (chr16:29,159,416-33,286,258x1) had ID, aplasia or hypoplasia of the corpus callosum, a high-arched palate, hypertelorism, low-set ears and micrognathia. The deletions of these DECIPHER patients are larger – 4.6 and 4.1 Mb, respectively – but include the region of overlap between the deletions in our and Schubert’s patient (see Figure 1).3

Besides epilepsy, a developmental delay, mainly related to speech, was observed in some members of the reported families with STX1B sequence variants.3_{A more severe and global}

developmental delay was observed in our patient and Schubert’s patient with STX1B deletions.3

Furthermore, both patients and one DECIPHER patient had facial dysmorphisms, which were not reported in patients with STX1B sequence variants. Possibly, one or more of the other deleted genes in the region of overlap might be associated with these additional phenotypes (Figure 1). For example, the STX4 gene (MIM 186591) encodes syntaxin-4 (STX4), another protein of the syntaxin protein family. Stx4 has been shown to be expressed in rat brains and to be involved in the recycling of endosomes in dendritic spines.9_{No clinical phenotype has been associated with}

STX4 yet, but we speculate that haploinsuﬃciency of this gene could have contributed to the

patients’ developmental problems.

Recently, pathogenic variants in the SLC6A1 gene have also been found in some individuals with MAE.13_{As the SLC6A1 gene was not included in our targeted next-generation sequencing epilepsy}

gene panel, we cannot exclude the possibility of an additional variant in the SLC6A1 gene in our patient.

In conclusion, we report a 18-year-old male patient with MAE and a 16p11.2 deletion that includes the STX1B gene. This gene has recently been associated with fever-related epilepsies and with MAE in two other patients. Our observation of a STX1B deletion in a third patient supports that MAE is indeed part of the STX1B-related epilepsy spectrum. We therefore suggest considering analysis of STX1B in the diagnostic work-up of MAE patients. An association between STX1B and infantile spasms is still doubtful and needs further investigation.

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REFERENCES

1. Engel J,Jr, International League Against Epilepsy (ILAE). A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE Task Force on Classiﬁcation and Terminology. Epilepsia 2001; 42: 796-803.

2. Tang S, Pal DK. Dissecting the genetic basis of myoclonic-astatic epilepsy. Epilepsia 2012; 53: 1303-1313. 3. Schubert J, Siekierska A, Langlois M, et al. Mutations

in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes. Nat Genet 2014; 46: 1327-1332.

4. Dulubova I, Sugita S, Hill S, et al. A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J 1999; 18: 4372-4382.

5. Jahn R, Scheller RH. SNAREs--engines for membrane fusion. Nat Rev Mol Cell Biol 2006; 7: 631-643.

6. Dulubova I, Khvotchev M, Liu S, Huryeva I, Sudhof TC, Rizo J. Munc18-1 binds directly to the neuronal SNARE complex. Proc Natl Acad Sci U S A 2007; 104: 2697-2702. 7. Gerber SH, Rah JC, Min SW, et al. Conformational switch

of syntaxin-1 controls synaptic vesicle fusion. Science 2008; 321: 1507-1510.

8. Saitsu H, Kato M, Mizuguchi T, et al. De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy. Nat Genet 2008; 40: 782-788.

9. Kennedy MJ, Davison IG, Robinson CG, Ehlers MD. Syntaxin-4 deﬁnes a domain for activity-dependent exocytosis in dendritic spines. Cell 2010; 141: 524-535. 10. Barcia G, Chemaly N, Gobin S, et al. Early epileptic

encephalopathies associated with STXBP1 mutations: Could we better delineate the phenotype? Eur J Med Genet 2014; 57: 15-20.

11. Carvill GL, Weckhuysen S, McMahon JM, et al. GABRA1 and STXBP1: novel genetic causes of Dravet syndrome. Neurology 2014; 82: 1245-1253.

12. Boutry-Kryza N, Labalme A, Ville D, et al. Molecular characterization of a cohort of 73 patients with infantile spasms syndrome. Eur J Med Genet 2015; 58: 51-58. 13. Carvill GL, McMahon JM, Schneider A, et al. Mutations

in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures. Am J Hum Genet 2015; 96: 808-815.

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SUPPLEMENTAL DATA

Supplemental Table 1: Genes analysed in our patient with MAE using a targeted next generation sequencing gene panel

Genes included in the panel (n = 94)

ALDH7A GAMT MED12 RAB39B SLC25A22

ARHGEF9 GATM MEF2C RAI1 SMC1A

ARX GPC3 MOCS1 RANBP2 SMS

ATP6AP2 GPHN MOCS2 RNASEH2A SPTAN1

ATRX GPR98 NRXN1 RNASEH2B SRGAP2

AUTS2 GRIA3 NSDHL RNASEH2C SRPX2

CASK GRIN2A OFD1 ROGDI ST3GAL3

CDKL5 GRIN2B OPHN1 RPS6KA3 STXBP1

CLCN2 HPRT PAK3 SAMHD1 SYN1

CNKSR2 HSD17B10 PCDH19 SCN1A SYNGAP1

CNTNAP2 IQSEC2 PHF6 SCN1B SYP

CUL4B KCNJ10 PHGDH SCN2A TBC1D24

DCX KCNQ2 PLCB1 SCN8A TBCE

DYRK1A KCNT1 PLP1 SCN9A TCF4

FGD1 KDM5C PNKP SLC6A8 TREX1

FLNA MAGI2 PQBP1 SLC9A1 UBE2A

FOXG1 MAPK10 PRPS1 SLC9A6 UBE3A

GABRD MBD5 PSAT1 SLC16A2 ZEB2

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