- Email: [email protected]
PRRT2 is mutated in familial and non-familial benign infantile seizures
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 1 7 ( 2 0 1 3 ) 7 7 e8 1
Official Journal of the European Paediatric Neurology Society
Original article
PRRT2 is mutated in familial and non-familial benign infantile seizures Nicola Specchio a,*, Alessandra Terracciano b, Marina Trivisano c, Simona Cappelletti d, Dianela Claps a, Lorena Travaglini b, Raffaella Cusmai a, Carlo Efisio Marras e, Federico Zara f, Lucia Fusco a, Enrico Bertini b, Federico Vigevano a a
Neurology Unit, Department of Neuroscience, Bambino Gesu` Children’s Hospital, IRCCS, P.zza S. Onofrio 4, 00165 Rome, Italy Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, Department of Neurosciences, Bambino Gesu` Children’s Hospital, IRCCS, Rome, Italy c Clinic for Nervous System Diseases, University of Foggia, Foggia, Italy d Unit of Clinical Psychology, Department of Neuroscience, Bambino Gesu` Children’s Hospital, IRCCS, Rome, Italy e Neurosurgery Unit, Department of Neuroscience, Bambino Gesu` Children’s Hospital, IRCCS, Rome, Italy f Department of Neuroscience, Istituto ‘G. Gaslini’, Genova, Italy b
article info
abstract
Article history:
Background: Mutations of protein-rich transmembrane protein 2 (PRRT2) were recently
Received 20 July 2012
associated to benign familial infantile seizures (BFIS) (MIM 605751) and paroxysmal
Received in revised form
kinesigenic dyskinesias (PKD) (MIM12800).
24 July 2012
Aims: To report mutations of PRRT2 in BFIS, infantile convulsions and choreoathetosis
Accepted 24 July 2012
(ICCA), and in sporadic cases affected by benign infantile epilepsy (BIE). Methods: A mutational screening of PRRT2 was performed in 5 families, and in 7 sporadic
Keywords:
cases affected by BIE. All clinical and neurophysiological details were reviewed.
Epilepsy
Results: Thirty-three members among 5 families were collected. Fifteen individuals had
Benign familial infantile seizures
infantile seizures and one had infantile seizures followed by paroxysmal kinesigenic
Choreoathetosis
dyskinesia (PKD). We found the c.649_650InsC PRRT2 mutation in all tested patients (13 out
Genetics
of 15). Age at onset ranged from 3.5 to 10 months. Focal seizures, with or without secondary
PRRT2
generalization, occurred mainly in cluster. One patient at the age of 11 years presented with PKD successfully treated with carbamazepine. All patients had a normal cognitive development. Two out of 7 non-familial cases (28.5%) carried a de novo PRRT2 mutation: the c.649_650InsC mutation in one with clustered seizures at the age of 5 months and an unreported c.718C-T p.R240X mutation in the other who, after cluster focal seizures at the age of 5 months, experienced absences at the age of 5 years. Conclusion: Our findings emphasize that PRRT2 mutations might be responsible of both BFIS and ICCA, but might be causative also for sporadic cases of benign infantile seizures. The phenotypic spectrum comprises BFIS, ICCA, and PKD. ª 2012 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ39 0668592645; fax: þ39 0668592463. E-mail address: [email protected] (N. Specchio). 1090-3798/$ e see front matter ª 2012 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejpn.2012.07.006
78
1.
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 1 7 ( 2 0 1 3 ) 7 7 e8 1
Introduction
Mutations of protein-rich transmembrane protein 2 (PRRT2) were recently associated to benign familial infantile seizures (BFIS) (MIM 605751) and paroxysmal kinesigenic dyskinesias (PKD) (MIM12800).1e3 PRRT2 is located at chromosomal region 16p11.2, and encodes a membrane protein that seems to interact with synaptosomal-associated protein 25 kDa (SNAP25). Since 1992 when we reported the first description of BFIS,4 several families have been published confirming the existence of this syndrome, which now has its own genetic background. BFIS is characterized by focal seizures within the first year of life (around the 6th month) usually in cluster with a benign outcome, and autosomal dominant inheritance.4 A similar condition in infants without familiar history of seizures was reported as Benign Infantile Epilepsy (BIE).5 BFIS might be associated with other neurological symptoms as paroxysmal choreoathetosis appearing later in life (ICCA).6 We describe 5 families, affected by either BFIS or ICCA, and 2 cases affected by BIE with PRRT2 mutations. Our findings widen the number of patients with PRRT2 mutations, which might be mutated also in non-familial cases, and confirm that it might be responsible of BFIS, and ICCA in the first year of life.
2.
Materials and methods
We sequenced PRRT2 in 5 families, 4 affected by BFIS and 1 by ICCA, and 7 cases affected by BIE. We selected all cases observed at Neurology Division of the Bambino Gesu` Children’s Hospital in Rome. In all patients and in their relatives in whom we detected pathogenic mutations, we reviewed medical records, seizure semiology, seizure frequency and duration (isolated, cluster), treatment, interictal and ictal EEG, brain imaging, cognitive and behavior assessment, presence of additional neurological symptoms. For older family members a precise clinical history regarding infantile seizures could not always be collected. Informed consent was obtained for all participants. Scientific Directorate of our Hospital according to the local regulations approved the study.
2.1.
Mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes using QUIAMP DNA Blood mini kit, according to the manufacturer’s protocol (QUIAGEN, Germany). We carried out mutation analysis of PRRT2 by direct sequencing after PCR amplification (primer sequences are available on request). Amplified fragments were directly sequenced using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and were run on an ABI PRISM 3130 l Genetic Analyzer (Applied Biosystems).
3.
Results
Table 1 and Fig. 1A and B show clinical and genetic findings of familial and non-familial cases included in the study.
3.1.
Familial cases
Among 5 families a total of 33 members were collected. Fifteen individuals had infantile seizures and one had infantile seizures followed by PKD later in life. We found PRRT2 mutations in all tested patients (13 out of 15) (Fig. 1A). In all families we identified the insertion mutation c.649_650InsC. Mean age at onset was 4.35 months (range 3.5e5.25) for probands and 5.8 months (range 4e10) for family members. All probands presented with focal seizures, 4 of them had also a secondary generalization (SG). Among relatives generalized toniceclonic (GTC) seizures were reported in 4 cases and focal seizures with SG in 5. Seizures occurred in cluster in all probands and relatives with the exception of one member (family 1, case II.2) who had a single seizure. Clusters resolved in all cases within 48 h. Interictal EEG was available in all probands and in case I.1 of Family 5 (Table 1): it was normal in all of them but in two cases (family 1: case III.1; family 4: case II.1) in which bilateral posterior slow waves were present soon after the cluster. In one case ictal EEG revealed a right occipital focal discharge. All probands received antiepileptic medications, Phenobarbital (PB) in 2 cases and Carbamazepine (CBZ) in 3; treatment was discontinued after a mean period of 20.5 months in all cases. Patient II.1 of family 4 at the age of 11 years presented with PKD involving mainly upper limbs. She carried the same c.649_650InsC mutation of BFIS families. She was successfully treated with CBZ (100 mg/day). Two patients had febrile seizures later in life. All patients had a normal cognitive development; a slight hyperactivity was reported in one of them. A final cognitive outcome is to be awaited during the follow-up as to exclude minor learning disabilities.
3.2.
Non-familial cases
Within 7 non-familial cases we found de novo PRRT2 mutations in 2 (28.5%): the c.649_650InsC mutation in one case (Case#1) and c.718C-T p.R240X mutation in the other (Case#2). This latter is a new nonsense mutation affecting an amino-acid residue of PRRT2 protein evolutionarily conserved from zebrafish to humans (Fig. 1C and D). Case#1 presented with clustered seizures at the age of 5 months and 15 days, he was treated with PB for one year, and he had a normal development and no seizure recurrence during the follow-up (3.5 years). Case#2, at the age of 5 months, presented a cluster of focal seizures. At the age of 9 and 20 months he experienced isolated afebrile seizures, and, at the age of 5 years and 6 months, typical absences responsive to valproate which is still ongoing.
4.
Discussion
PRRT2 mutations have been firstly identified as causative of PKD; its role was discovered through an exome sequencing analysis in two independent studies.7,8 Later on mutations in the same gene were reported in families affected by BFIS or ICCA: in these descriptions were included 68 families with BFIS and 6 families with ICCA.1,2,9
Table 1 e Clinical and genetic findings in all studied cases. Pts
Sample Age at number study/sex
Age at onset
Seizure’s type at onset
Cluster at onset
Interictal EEG
Ictal EEG
Treatment (duration)
58 y/M 33/F 30/M 17 m/F 46/M
<1 y 5m 5m 5m7d 4m
n.a. GTC F with SG F with SG F with SG
n.a. Single event Cluster (24 h) 5 szs in 2 d Cluster
n.a. n.a. n.a. n.a. n.a. n.a. Post bil SW n.a. n.a. n.a.
n.a. PB (18 m) PB (4 y) PB (6 m) n.a.
III.1 III.2 III.3
13 y/F 15 y/M 3 y 9 m/F
4m 5m 4m7d
F with SG F with SG F with SG
Single cluster Single cluster Cluster
n.a. n.a. n.a. n.a. Post bil SW n.a
No No PB (2 y 2 m)
Family 4 (ICCA)
I.1 II.3 III.3 I.1 II.1
dec/M 31/M 3 y 2 m/M 48 y/M 22 y/F
<1 y GTC 7m F with SG 3 m 22 d CPS 6m GTC 3 m 15 d F with SG
n.a. Cluster Cluster n.a. Cluster (6 szs/day)
n.a. n.a. Normal n.a. Normal
n.a. n.a. n.a. n.a. Focal onset (R Occ)
Family 5 (BFIS)
I.1 II.1
29 y/M 10 m 6 y 11 m/M 5 m
Cluster Cluster
Normal Normal
n.a. n.a.
Post bil SW Focal onset CBZ (1 y) (L T) Normal n.a. PB (3 y 6 m)
Family 2 (BFIS)
Family 3 (BFIS)
GTC F with SG
Case#1
4 y/F
5 m 15 d CPS, F with SG
Cluster
Case#2
7 y/M
5m7d
CPS, F with SG
Cluster (8 szs/24 h)
Case#3
1 y 9 m/F
10 m
CPS, F with SG
Case#4
2 y 7 m/M
2 m 15 d F with SG
Case#5
8 y 11 m/M 11 m
F with SG
Single sz, cluster after 20 d (5 szs in 3 d) 1st cluster (8 szs in 24 h), 2nd cluster after 1 w Cluster
Case#6
1 y 7 m/F
7m
GTC
Case#7
3 y 1 m/M
9m
Cognitive & Behavior
Additional symptoms
PRRT2 mutations
Normal Normal Normal GQ 121 Normal
No No No No No
c.649_650 c.649_650 c.649_650 c.649_650 c.649_650
Normal Normal GQ 114
No No FS at 17 m
n.g. c.649_650 insC c.649_650 insC
n.a. PB (2 y) CBZ (1 y) No CBZ (2 y)
n.a. No No No Isolated seizure at 1 y No No 2nd cluster 11 m, 3rd cluster 15 m n.a. No No Few clusters to 1 y 2 clusters to 1 y
Normal Normal Hyperactivity Normal Normal
c.649_650 c.649_650 c.649_650 c.649_650 c.649_650
n.a. CBZ (2 y 7 m)
No No
Normal Normal
No No No No CA (11 y) responsive to CBZ No FS (18 m, 20 m)
No
Normal
No
Isolated focal szs 9 m, 20 m
Normal
c.649_650 insC (de novo) c.718C-T p.R240X (de novo)
Negative
Normal
Focal onset VPA, MDZ (L T-Occ) CBZ (10 m)
No
GQ: 102
Normal
n.a.
No
Normal
No
Negative
L T SW
Focal onset PB (L T) Focal onset CBZ (1 y) (R F-T)
No
Normal
No
Negative
No
GQ: 86
No
Negative
Normal
No
Negative
FS, cluster R F-T SW afebrile after 1 w (4 szs in 24 h) GTC (febr/afeb) Cluster Normal
n.a.
VPA (2 y 4 m) Isolated seizure (12 m)
insC insC insC insC insC
c.649_650 insC c.649_650 insC
Absences (5 y 6 m) responsive to VPA No
PB, LEV
insC insC insC insC insC 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 1 7 ( 2 0 1 3 ) 7 7 e8 1
I.1 II.2 II.3 III.1 II.4
Family 1 (BFIS)
Seizure recurrence (Follow-up)
Pts: patients, M: male, F: female, d: days, w: week, m: months, y: years, dec: deceased, n.a.: not available, F: focal, GTC: generalized toniceclonic, SG: secondary generalization, CPS: complex partial seizures, febr: febrile, afeb: afebrile, szs: seizures, post: posterior, bil: bilateral, SW: slow waves, L: left, R: right, F: frontal, T: temporal, Occ: occipital, PB: phenobarbitale, CBZ: carbamazepine, VPA: valproate, MDZ: midazolam, LEV: levetiracetam, FS: febrile seizure, GQ: general quotient, CA: choreoathetosis.
79
80
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 1 7 ( 2 0 1 3 ) 7 7 e8 1
Fig. 1 e A. Pedigrees of the 5 families with benign familial infantile seizures (BFIS) and infantile convulsions with choreoathetosis (ICCA). n.g. indicates individuals not genotyped, D indicates individuals with PRRT2 mutations, L indicates individuals without PRRT2 mutations. B. Graphic representation of the PRRT2 protein with the identified mutations. The PRRT2 gene contains four exons that encode several domains in the PRRT2 protein, including two extracellular domains (EC), two transmembrane domains (TM) and one cytoplasmic domain (IC). The proline-rich domain falls in the N-terminal extracellular domain. C. Sequence traces showing the new mutation identified in the sporadic patient compared to wild-type trace. D. Multiple sequence alignments of human and hortologs of PRRT2 from other species generated by Jalview editor (http://www.jalview.org). Residues were colored according to their physico-chemical properties (Zappo color scheme). Black arrows indicate residue involved in nonsense substitution. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
PRRT2, located at chromosomal region 16p11.2, encodes prolin-rich trans-membrane protein mainly expressed in the brain, in the cerebral cortex and basal ganglia. The localization of PRRT in the brain might explain the clinical expression of infantile seizures and choreoathetosis. Its function is not yet known but it seems to interact with synaptosomalassociated protein 25 kDa (SNAP-25) involved in neurotransmitter release from synaptic vesicles.8 All families included in this study carried the c.649_650insC mutation both BFIS and ICCA, and one of the two mutated non-familial cases. This is the most commonly reported mutation: it is a hot spot mutation consisting of a cytosine (C) base insertion in a homopolymer of nine C bases adjacent to four guanine bases.10 Our results confirm that genotypeephenotype correlation is not univocal. Even within the ICCA family there is a high variable clinical expression: the proband’s father had only a cluster of infantile seizures and the proband in addition had PKD at the age of 11 years. A possible explanation for this wide variability is not yet known. It might be due to the influence of the wild type PRRT2 allele, which might modify the phenotypic expression of the mutant allele.2 Considering the occurrence of infantile seizures and choreoathetosis in the same family, it might be supposed for both neurological disorders, a common pathogenesis on ion-channels.
After twenty years from its first description,4 BFIS is now a well-known condition. Clinical criteria reported for BFIS should be now supported by PPRT2 molecular analysis. However it should be taken into account that single families have been reported with mutations in SCN2A and KCNQ2 genes. Therefore, in negative cases these other genes should be screened. Regarding non-familial cases it may be even more difficult to confirm the diagnosis and PRRT2 molecular analysis could be a valid method to support it. PPRT2 mutations were never reported in sporadic cases. Among the 7 cases we tested, 2 (28.5%) were mutated. Case#1 had benign infantile seizures related to the known mutation c.649_650insC, while Case#2 had a novel unreported mutation (718C-T p.R240X). This nonsense mutation, as the c.649_650insC common variant, cause a premature termination in the segment of the gene coding for proline-rich domain of PRRT2, results in a truncated protein that completely lose their transmembrane function. Unlike other both familial and non-familial cases, the patient with 718C-T p.R240X mutation presented with absence seizures at the age of 5 and half years. There are no reports of benign infantile seizures associated to absence seizures. Because no other similar cases have been described, we cannot conclude if this atypical phenotype is due to this novel mutation, or if other factors might play a role. The identification of
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 1 7 ( 2 0 1 3 ) 7 7 e8 1
PRRT2 mutations in sporadic cases, is a novel finding that could be helpful in reaching earlier diagnosis. No clear differences regarding age at onset, seizures semiology, EEG findings, and outcome are evident between familial and non-familial cases in the present series. The increasing reports of families carrying PRRT2 mutations will allow better phenotypeegenotype correlations. The phenotypic spectrum comprises BFIS, ICCA, and PKD and is likely wider than currently known, and more patients need to be screened before the whole picture is clarified. Our findings emphasize that PRRT2 mutations might be responsible of both BFIS and ICCA, but might be causative also for sporadic cases of benign infantile seizures.
2.
3.
4. 5.
6.
Acknowledgments 7.
The authors have nothing to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
8.
9.
references 10. 1. Ono S, Yoshiura K, Kinoshita A, et al. Mutations in PRRT2 responsible for paroxysmal kinesigenic dyskinesias also
81
cause benign familial infantile convulsions. J Hum Genet 2012;57:338e41. Heron SE, Grinton BE, Kivity S, et al. PRRT2 mutations cause benign familial infantile epilepsy and infantile convulsions with choreoathetosis syndrome. Am J Hum Genet 2012;90:152e60. Lee HY, Huang Y, Bruneau N, et al. Mutations in the novel protein PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions. Cell Rep 2012;1:2e12. Vigevano F, Fusco L, Di Capua M, et al. Benign infantile familial convulsions. Eur J Pediatr 1992;151:608e12. Berg AT, Berkovic SF, Brodie MJ, et al. Revised terminology and concepts for organization of seizures and epilepsies: report of the ILAE Commission on Classification and Terminology, 2005e2009. Epilepsia 2010;51:676e85. Szepetowski P, Rochette J, Berquin P, et al. Familial infantile convulsions and paroxysmal choreoathetosis: a new neurological syndrome linked to the pericentromeric region of human chromosome 16. Am J Hum Genet 1997;61:889e98. Chen WJ, Lin Y, Xiong ZQ, et al. Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia. Nat Genet 2011;43:1252e5. Wang JL, Cao L, Li XH, et al. Identification of PRRT2 as the causative gene of paroxysmal kinesigenic dyskinesias. Brain 2011;134:3493e501. Schubert J, Paravidino R, Becker F, et al. PRRT2 mutations are the major cause of benign familial infantile seizures (BFIS). Hum Mutat 2012. http://dx.doi.org/10.1002/humu.22126. Cao L, Huang XJ, Zheng L, et al. Identification of a novel PRRT2 mutation in patients with paroxysmal kinesigenic dyskinesias and c.649dupC as a mutation hot-spot. Parkinsonism Relat Disord 2012;18:704e6.
Official Journal of the European Paediatric Neurology Society
Original article
PRRT2 is mutated in familial and non-familial benign infantile seizures Nicola Specchio a,*, Alessandra Terracciano b, Marina Trivisano c, Simona Cappelletti d, Dianela Claps a, Lorena Travaglini b, Raffaella Cusmai a, Carlo Efisio Marras e, Federico Zara f, Lucia Fusco a, Enrico Bertini b, Federico Vigevano a a
Neurology Unit, Department of Neuroscience, Bambino Gesu` Children’s Hospital, IRCCS, P.zza S. Onofrio 4, 00165 Rome, Italy Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Diseases, Department of Neurosciences, Bambino Gesu` Children’s Hospital, IRCCS, Rome, Italy c Clinic for Nervous System Diseases, University of Foggia, Foggia, Italy d Unit of Clinical Psychology, Department of Neuroscience, Bambino Gesu` Children’s Hospital, IRCCS, Rome, Italy e Neurosurgery Unit, Department of Neuroscience, Bambino Gesu` Children’s Hospital, IRCCS, Rome, Italy f Department of Neuroscience, Istituto ‘G. Gaslini’, Genova, Italy b
article info
abstract
Article history:
Background: Mutations of protein-rich transmembrane protein 2 (PRRT2) were recently
Received 20 July 2012
associated to benign familial infantile seizures (BFIS) (MIM 605751) and paroxysmal
Received in revised form
kinesigenic dyskinesias (PKD) (MIM12800).
24 July 2012
Aims: To report mutations of PRRT2 in BFIS, infantile convulsions and choreoathetosis
Accepted 24 July 2012
(ICCA), and in sporadic cases affected by benign infantile epilepsy (BIE). Methods: A mutational screening of PRRT2 was performed in 5 families, and in 7 sporadic
Keywords:
cases affected by BIE. All clinical and neurophysiological details were reviewed.
Epilepsy
Results: Thirty-three members among 5 families were collected. Fifteen individuals had
Benign familial infantile seizures
infantile seizures and one had infantile seizures followed by paroxysmal kinesigenic
Choreoathetosis
dyskinesia (PKD). We found the c.649_650InsC PRRT2 mutation in all tested patients (13 out
Genetics
of 15). Age at onset ranged from 3.5 to 10 months. Focal seizures, with or without secondary
PRRT2
generalization, occurred mainly in cluster. One patient at the age of 11 years presented with PKD successfully treated with carbamazepine. All patients had a normal cognitive development. Two out of 7 non-familial cases (28.5%) carried a de novo PRRT2 mutation: the c.649_650InsC mutation in one with clustered seizures at the age of 5 months and an unreported c.718C-T p.R240X mutation in the other who, after cluster focal seizures at the age of 5 months, experienced absences at the age of 5 years. Conclusion: Our findings emphasize that PRRT2 mutations might be responsible of both BFIS and ICCA, but might be causative also for sporadic cases of benign infantile seizures. The phenotypic spectrum comprises BFIS, ICCA, and PKD. ª 2012 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ39 0668592645; fax: þ39 0668592463. E-mail address: [email protected] (N. Specchio). 1090-3798/$ e see front matter ª 2012 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejpn.2012.07.006
78
1.
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 1 7 ( 2 0 1 3 ) 7 7 e8 1
Introduction
Mutations of protein-rich transmembrane protein 2 (PRRT2) were recently associated to benign familial infantile seizures (BFIS) (MIM 605751) and paroxysmal kinesigenic dyskinesias (PKD) (MIM12800).1e3 PRRT2 is located at chromosomal region 16p11.2, and encodes a membrane protein that seems to interact with synaptosomal-associated protein 25 kDa (SNAP25). Since 1992 when we reported the first description of BFIS,4 several families have been published confirming the existence of this syndrome, which now has its own genetic background. BFIS is characterized by focal seizures within the first year of life (around the 6th month) usually in cluster with a benign outcome, and autosomal dominant inheritance.4 A similar condition in infants without familiar history of seizures was reported as Benign Infantile Epilepsy (BIE).5 BFIS might be associated with other neurological symptoms as paroxysmal choreoathetosis appearing later in life (ICCA).6 We describe 5 families, affected by either BFIS or ICCA, and 2 cases affected by BIE with PRRT2 mutations. Our findings widen the number of patients with PRRT2 mutations, which might be mutated also in non-familial cases, and confirm that it might be responsible of BFIS, and ICCA in the first year of life.
2.
Materials and methods
We sequenced PRRT2 in 5 families, 4 affected by BFIS and 1 by ICCA, and 7 cases affected by BIE. We selected all cases observed at Neurology Division of the Bambino Gesu` Children’s Hospital in Rome. In all patients and in their relatives in whom we detected pathogenic mutations, we reviewed medical records, seizure semiology, seizure frequency and duration (isolated, cluster), treatment, interictal and ictal EEG, brain imaging, cognitive and behavior assessment, presence of additional neurological symptoms. For older family members a precise clinical history regarding infantile seizures could not always be collected. Informed consent was obtained for all participants. Scientific Directorate of our Hospital according to the local regulations approved the study.
2.1.
Mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes using QUIAMP DNA Blood mini kit, according to the manufacturer’s protocol (QUIAGEN, Germany). We carried out mutation analysis of PRRT2 by direct sequencing after PCR amplification (primer sequences are available on request). Amplified fragments were directly sequenced using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and were run on an ABI PRISM 3130 l Genetic Analyzer (Applied Biosystems).
3.
Results
Table 1 and Fig. 1A and B show clinical and genetic findings of familial and non-familial cases included in the study.
3.1.
Familial cases
Among 5 families a total of 33 members were collected. Fifteen individuals had infantile seizures and one had infantile seizures followed by PKD later in life. We found PRRT2 mutations in all tested patients (13 out of 15) (Fig. 1A). In all families we identified the insertion mutation c.649_650InsC. Mean age at onset was 4.35 months (range 3.5e5.25) for probands and 5.8 months (range 4e10) for family members. All probands presented with focal seizures, 4 of them had also a secondary generalization (SG). Among relatives generalized toniceclonic (GTC) seizures were reported in 4 cases and focal seizures with SG in 5. Seizures occurred in cluster in all probands and relatives with the exception of one member (family 1, case II.2) who had a single seizure. Clusters resolved in all cases within 48 h. Interictal EEG was available in all probands and in case I.1 of Family 5 (Table 1): it was normal in all of them but in two cases (family 1: case III.1; family 4: case II.1) in which bilateral posterior slow waves were present soon after the cluster. In one case ictal EEG revealed a right occipital focal discharge. All probands received antiepileptic medications, Phenobarbital (PB) in 2 cases and Carbamazepine (CBZ) in 3; treatment was discontinued after a mean period of 20.5 months in all cases. Patient II.1 of family 4 at the age of 11 years presented with PKD involving mainly upper limbs. She carried the same c.649_650InsC mutation of BFIS families. She was successfully treated with CBZ (100 mg/day). Two patients had febrile seizures later in life. All patients had a normal cognitive development; a slight hyperactivity was reported in one of them. A final cognitive outcome is to be awaited during the follow-up as to exclude minor learning disabilities.
3.2.
Non-familial cases
Within 7 non-familial cases we found de novo PRRT2 mutations in 2 (28.5%): the c.649_650InsC mutation in one case (Case#1) and c.718C-T p.R240X mutation in the other (Case#2). This latter is a new nonsense mutation affecting an amino-acid residue of PRRT2 protein evolutionarily conserved from zebrafish to humans (Fig. 1C and D). Case#1 presented with clustered seizures at the age of 5 months and 15 days, he was treated with PB for one year, and he had a normal development and no seizure recurrence during the follow-up (3.5 years). Case#2, at the age of 5 months, presented a cluster of focal seizures. At the age of 9 and 20 months he experienced isolated afebrile seizures, and, at the age of 5 years and 6 months, typical absences responsive to valproate which is still ongoing.
4.
Discussion
PRRT2 mutations have been firstly identified as causative of PKD; its role was discovered through an exome sequencing analysis in two independent studies.7,8 Later on mutations in the same gene were reported in families affected by BFIS or ICCA: in these descriptions were included 68 families with BFIS and 6 families with ICCA.1,2,9
Table 1 e Clinical and genetic findings in all studied cases. Pts
Sample Age at number study/sex
Age at onset
Seizure’s type at onset
Cluster at onset
Interictal EEG
Ictal EEG
Treatment (duration)
58 y/M 33/F 30/M 17 m/F 46/M
<1 y 5m 5m 5m7d 4m
n.a. GTC F with SG F with SG F with SG
n.a. Single event Cluster (24 h) 5 szs in 2 d Cluster
n.a. n.a. n.a. n.a. n.a. n.a. Post bil SW n.a. n.a. n.a.
n.a. PB (18 m) PB (4 y) PB (6 m) n.a.
III.1 III.2 III.3
13 y/F 15 y/M 3 y 9 m/F
4m 5m 4m7d
F with SG F with SG F with SG
Single cluster Single cluster Cluster
n.a. n.a. n.a. n.a. Post bil SW n.a
No No PB (2 y 2 m)
Family 4 (ICCA)
I.1 II.3 III.3 I.1 II.1
dec/M 31/M 3 y 2 m/M 48 y/M 22 y/F
<1 y GTC 7m F with SG 3 m 22 d CPS 6m GTC 3 m 15 d F with SG
n.a. Cluster Cluster n.a. Cluster (6 szs/day)
n.a. n.a. Normal n.a. Normal
n.a. n.a. n.a. n.a. Focal onset (R Occ)
Family 5 (BFIS)
I.1 II.1
29 y/M 10 m 6 y 11 m/M 5 m
Cluster Cluster
Normal Normal
n.a. n.a.
Post bil SW Focal onset CBZ (1 y) (L T) Normal n.a. PB (3 y 6 m)
Family 2 (BFIS)
Family 3 (BFIS)
GTC F with SG
Case#1
4 y/F
5 m 15 d CPS, F with SG
Cluster
Case#2
7 y/M
5m7d
CPS, F with SG
Cluster (8 szs/24 h)
Case#3
1 y 9 m/F
10 m
CPS, F with SG
Case#4
2 y 7 m/M
2 m 15 d F with SG
Case#5
8 y 11 m/M 11 m
F with SG
Single sz, cluster after 20 d (5 szs in 3 d) 1st cluster (8 szs in 24 h), 2nd cluster after 1 w Cluster
Case#6
1 y 7 m/F
7m
GTC
Case#7
3 y 1 m/M
9m
Cognitive & Behavior
Additional symptoms
PRRT2 mutations
Normal Normal Normal GQ 121 Normal
No No No No No
c.649_650 c.649_650 c.649_650 c.649_650 c.649_650
Normal Normal GQ 114
No No FS at 17 m
n.g. c.649_650 insC c.649_650 insC
n.a. PB (2 y) CBZ (1 y) No CBZ (2 y)
n.a. No No No Isolated seizure at 1 y No No 2nd cluster 11 m, 3rd cluster 15 m n.a. No No Few clusters to 1 y 2 clusters to 1 y
Normal Normal Hyperactivity Normal Normal
c.649_650 c.649_650 c.649_650 c.649_650 c.649_650
n.a. CBZ (2 y 7 m)
No No
Normal Normal
No No No No CA (11 y) responsive to CBZ No FS (18 m, 20 m)
No
Normal
No
Isolated focal szs 9 m, 20 m
Normal
c.649_650 insC (de novo) c.718C-T p.R240X (de novo)
Negative
Normal
Focal onset VPA, MDZ (L T-Occ) CBZ (10 m)
No
GQ: 102
Normal
n.a.
No
Normal
No
Negative
L T SW
Focal onset PB (L T) Focal onset CBZ (1 y) (R F-T)
No
Normal
No
Negative
No
GQ: 86
No
Negative
Normal
No
Negative
FS, cluster R F-T SW afebrile after 1 w (4 szs in 24 h) GTC (febr/afeb) Cluster Normal
n.a.
VPA (2 y 4 m) Isolated seizure (12 m)
insC insC insC insC insC
c.649_650 insC c.649_650 insC
Absences (5 y 6 m) responsive to VPA No
PB, LEV
insC insC insC insC insC 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 1 7 ( 2 0 1 3 ) 7 7 e8 1
I.1 II.2 II.3 III.1 II.4
Family 1 (BFIS)
Seizure recurrence (Follow-up)
Pts: patients, M: male, F: female, d: days, w: week, m: months, y: years, dec: deceased, n.a.: not available, F: focal, GTC: generalized toniceclonic, SG: secondary generalization, CPS: complex partial seizures, febr: febrile, afeb: afebrile, szs: seizures, post: posterior, bil: bilateral, SW: slow waves, L: left, R: right, F: frontal, T: temporal, Occ: occipital, PB: phenobarbitale, CBZ: carbamazepine, VPA: valproate, MDZ: midazolam, LEV: levetiracetam, FS: febrile seizure, GQ: general quotient, CA: choreoathetosis.
79
80
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 1 7 ( 2 0 1 3 ) 7 7 e8 1
Fig. 1 e A. Pedigrees of the 5 families with benign familial infantile seizures (BFIS) and infantile convulsions with choreoathetosis (ICCA). n.g. indicates individuals not genotyped, D indicates individuals with PRRT2 mutations, L indicates individuals without PRRT2 mutations. B. Graphic representation of the PRRT2 protein with the identified mutations. The PRRT2 gene contains four exons that encode several domains in the PRRT2 protein, including two extracellular domains (EC), two transmembrane domains (TM) and one cytoplasmic domain (IC). The proline-rich domain falls in the N-terminal extracellular domain. C. Sequence traces showing the new mutation identified in the sporadic patient compared to wild-type trace. D. Multiple sequence alignments of human and hortologs of PRRT2 from other species generated by Jalview editor (http://www.jalview.org). Residues were colored according to their physico-chemical properties (Zappo color scheme). Black arrows indicate residue involved in nonsense substitution. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
PRRT2, located at chromosomal region 16p11.2, encodes prolin-rich trans-membrane protein mainly expressed in the brain, in the cerebral cortex and basal ganglia. The localization of PRRT in the brain might explain the clinical expression of infantile seizures and choreoathetosis. Its function is not yet known but it seems to interact with synaptosomalassociated protein 25 kDa (SNAP-25) involved in neurotransmitter release from synaptic vesicles.8 All families included in this study carried the c.649_650insC mutation both BFIS and ICCA, and one of the two mutated non-familial cases. This is the most commonly reported mutation: it is a hot spot mutation consisting of a cytosine (C) base insertion in a homopolymer of nine C bases adjacent to four guanine bases.10 Our results confirm that genotypeephenotype correlation is not univocal. Even within the ICCA family there is a high variable clinical expression: the proband’s father had only a cluster of infantile seizures and the proband in addition had PKD at the age of 11 years. A possible explanation for this wide variability is not yet known. It might be due to the influence of the wild type PRRT2 allele, which might modify the phenotypic expression of the mutant allele.2 Considering the occurrence of infantile seizures and choreoathetosis in the same family, it might be supposed for both neurological disorders, a common pathogenesis on ion-channels.
After twenty years from its first description,4 BFIS is now a well-known condition. Clinical criteria reported for BFIS should be now supported by PPRT2 molecular analysis. However it should be taken into account that single families have been reported with mutations in SCN2A and KCNQ2 genes. Therefore, in negative cases these other genes should be screened. Regarding non-familial cases it may be even more difficult to confirm the diagnosis and PRRT2 molecular analysis could be a valid method to support it. PPRT2 mutations were never reported in sporadic cases. Among the 7 cases we tested, 2 (28.5%) were mutated. Case#1 had benign infantile seizures related to the known mutation c.649_650insC, while Case#2 had a novel unreported mutation (718C-T p.R240X). This nonsense mutation, as the c.649_650insC common variant, cause a premature termination in the segment of the gene coding for proline-rich domain of PRRT2, results in a truncated protein that completely lose their transmembrane function. Unlike other both familial and non-familial cases, the patient with 718C-T p.R240X mutation presented with absence seizures at the age of 5 and half years. There are no reports of benign infantile seizures associated to absence seizures. Because no other similar cases have been described, we cannot conclude if this atypical phenotype is due to this novel mutation, or if other factors might play a role. The identification of
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 1 7 ( 2 0 1 3 ) 7 7 e8 1
PRRT2 mutations in sporadic cases, is a novel finding that could be helpful in reaching earlier diagnosis. No clear differences regarding age at onset, seizures semiology, EEG findings, and outcome are evident between familial and non-familial cases in the present series. The increasing reports of families carrying PRRT2 mutations will allow better phenotypeegenotype correlations. The phenotypic spectrum comprises BFIS, ICCA, and PKD and is likely wider than currently known, and more patients need to be screened before the whole picture is clarified. Our findings emphasize that PRRT2 mutations might be responsible of both BFIS and ICCA, but might be causative also for sporadic cases of benign infantile seizures.
2.
3.
4. 5.
6.
Acknowledgments 7.
The authors have nothing to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
8.
9.
references 10. 1. Ono S, Yoshiura K, Kinoshita A, et al. Mutations in PRRT2 responsible for paroxysmal kinesigenic dyskinesias also
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