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PRRT2 Mutations Cause Benign FamilialInfantile Epilepsy and Infantile Convulsionswith Choreoathetosis Syndrome

Sarah E. Heron,1 Bronwyn E. Grinton,2 Sara Kivity,3 Zaid Afawi,4 Sameer M. Zuberi,5 James N. Hughes,6

Clair Pridmore,7 Bree L. Hodgson,1 Xenia Iona,1 Lynette G. Sadleir,8 James Pelekanos,2,9

Eric Herlenius,10 Hadassa Goldberg-Stern,3 Haim Bassan,11 Eric Haan,12 Amos D. Korczyn,4

Alison E. Gardner,13 Mark A. Corbett,13 Jozef Gecz,6,13,14 Paul Q. Thomas,6 John C. Mulley,6,14,15

Samuel F. Berkovic,2,* Ingrid E. Scheffer,2,16,17 and Leanne M. Dibbens1,17

Benign familial infantile epilepsy (BFIE) is a self-limited seizure disorder that occurs in infancy and has autosomal-dominant inheritance.

We have identified heterozygous mutations in PRRT2, which encodes proline-rich transmembrane protein 2, in 14 of 17 families (82%)

affected by BFIE, indicating that PRRT2mutations are the most frequent cause of this disorder. We also report PRRT2mutations in five of

six (83%) families affected by infantile convulsions and choreoathetosis (ICCA) syndrome, a familial syndrome in which infantile

seizures and an adolescent-onset movement disorder, paroxysmal kinesigenic choreoathetosis (PKC), co-occur. These findings show

that mutations in PRRT2 cause both epilepsy and a movement disorder. Furthermore, PRRT2 mutations elicit pleiotropy in terms of

both age of expression (infancy versus later childhood) and anatomical substrate (cortex versus basal ganglia).

Benign familial infantile epilepsy (BFIE) (OMIM 605751) is

an autosomal-dominant seizure disorder that occurs in

infancy and in which seizure onset occurs at a mean age

of 6 months; seizure offset usually occurs by 2 years of

age. Genetic-linkage analyses of families affected by BFIE

have suggested that causative mutations occur in genes

residing at three different chromosomal loci. Guipponi

et al. and Li et al. have reported linkage to chromosomal

regions 19q12–13.11 and 1p362, respectively, in BFIE-

affected families. The vast majority of reported families

(approximately 50) affected by BFIE show linkage to the

pericentromeric region from 16p.11.2–16q12.1.3–7 This

region of chromosome 16 contains more than 150 genes,

and despite concerted efforts by many groups, there has

been no success in determining the underlying genetic

mutation that causes BFIE.

In infantile convulsions and choreoathetosis (ICCA)

syndrome (OMIM 602066), individual family members

can be afflicted with infantile seizures, the adolescent

onset movement disorder paroxysmal kinesigenic chor-

eoathetosis (PKC) (OMIM 128200), or both.When patients

are affected by both of these uncommon clinical pheno-

types, presentation might be separated by many years.

1Epilepsy Research Program, School of Pharmacy and Medical Sciences, Unive

Research Centre, Department of Medicine, University of Melbourne, Austin

Center of Israel, Petach Tikvah 49202, Israel; 4Tel-Aviv University, Tel-Aviv 6

Neurosciences Unit, Royal Hospital for Sick Children, Glasgow G3 8SJ, Unite

of Adelaide, Adelaide, South Australia 5005, Australia; 7Department of Neur

5006, Australia; 8Department of Paediatrics, University of Otago, Wellington

The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia

Astrid Lindgren Children’s Hospital, Karolinska Institutet, Stockholm SE-171 7

Hospital, Tel Aviv Sourasky Medical Center, Tel-Aviv 64239, Israel; 12South A

Hospital, North Adelaide, South Australia 5006, Australia; 13Neurogenetics P

South Australia 5006, Australia; 14School of Paediatrics and Reproductive He15Department of Genetic Medicine, SA Pathology, Women’s and Children’s H

ence Institutes and Department of Paediatrics, University of Melbourne, Roya17These authors contributed equally to this work

*Correspondence: [email protected]

DOI 10.1016/j.ajhg.2011.12.003. �2012 by The American Society of Human

152 The American Journal of Human Genetics 90, 152–160, January 1

Families affected by ICCA and families affected by auto-

somal-dominant PKC also show linkage to the large peri-

centromeric region of chromosome 16.8–10 The shared

linkage region and co-occurrence of these disorders in

families affected by ICCA have previously led to specula-

tion that BFIE, ICCA, and autosomal-dominant PKCmight

be allelic.8,9 Identification of the gene or genes in which

mutations cause these disorders is required for confirma-

tion of this hypothesis.

We studied 23 families affected by either BFIE (n¼ 17) or

ICCA (n ¼ 6). The study was approved by the Human

Research Ethics Committees of Austin Health and the

Women’s and Children’s Health Network. Informed

consent was obtained from all participants. Individuals

underwent detailed phenotyping involving a validated

seizure questionnaire.11 All previous medical records and

EEG and neuroimaging data were obtained where avail-

able. Australian control samples were obtained from anon-

ymous blood donors. Israeli control samples came from

unaffected, unrelated members of families recruited for

studies on the genetic causes of epilepsy.

We performed linkage analyses to identify families in

which the data were consistent with a causative mutation

rsity of South Australia, Adelaide, South Australia 5000, Australia; 2Epilepsy

Health, Heidelberg, Victoria 3084 Australia; 3Schneider Children’s Medical

9978, Israel; 5Paediatric Neurosciences Research Group, Fraser of Allander

d Kingdom; 6School of Molecular and Biomedical Science, the University

ology, Women’s and Children’s Hospital, North Adelaide, South Australia

6242, New Zealand; 9Academic Discipline of Paediatrics and Child Health,

; 10Neonatal Research Unit, Department of Women’s and Children’s Health,

7, Sweden; 11Pediatric Neurology and Development Unit, Dana Children’s

ustralian Clinical Genetics Service, SA Pathology, Women’s and Children’s

rogram, SA Pathology, Women’s and Children’s Hospital, North Adelaide,

alth, the University of Adelaide, Adelaide, South Australia 5005, Australia;

ospital, North Adelaide, South Australia 5006, Australia; 16Florey Neurosci-

l Children’s Hospital, Parkville, Victoria 3052, Australia

Genetics. All rights reserved.

3, 2012

mailto:[email protected]

http://dx.doi.org/10.1016/j.ajhg.2011.12.003

in the gene located in the chromosome 16 region. Microsa-

tellite markers that linked to the BFIE loci on chromosomes

1, 16, and 19 were genotyped by standard methods. We

calculated LOD scores for sufficiently sized families by

using FASTLINK.12 Maximum LOD scores of 3.27, 3.0,

and 2.71 for the chromosome 16 locus were obtained for

families 1, 2, and 5, respectively. We found that data on

families 1–9, 11, and 12 were consistent with linkage to

the chromosomal region 16p11.2–q12.1 (Table 1).

Previous strategies of intensive candidate-gene

sequencing and array comparative genome hybridization

have not revealed the chromosome 16 genetic aberration

that causes BFIE13 (S.E.H. and J.C.M., unpublished data).

We designed a sequence-capture array to extract coding

sequences, minimal promoter sequences, and microRNAs

in the 20.3 Mb linkage interval between the microsatellite

markers D16S3093 and D16S411 on chromosome 16.

Sequence capture, massively parallel sequencing (MPS),

and bioinformatic analysis were performed as described

previously.14,15

MPS was performed on one individual each from fami-

lies 1 and 5. Unique sequence variants were detected in

the brain-expressed genes A2LP, ARMC5, and BCKDK

in the individual from family 5 (Table S1, available online).

The variants in ARMC5 and BCKDK segregated with the

phenotype in this family, and these genes were screened

with Sanger sequencing in ten more BFIE-affected patients

from families for which data were consistent with linkage

to chromosome 16. Only one additional unique coding

variant was identified, and it was therefore concluded

that mutations in neither of these genes cause BFIE.

In a separate study, Chen et al. identified mutations in

PRRT2, located at chromosomal region 16p11.2, in eight

Chinese families affected by autosomal-dominant PKC

by using MPS analysis.16 In the study reported here, DNA

from the probands of 23 families affected by either BFIE

or ICCA was sequenced for the coding regions of PRRT2

(NM_145239.2) with direct Sanger sequencing. We have

now identified five different mutations in PRRT2 in 14 of

17 (82%) families affected by BFIE and in five of six

(83%) families affected by ICCA. Mutations were thus

identified in a total of 19 out of 23 (83%) families in which

some members had been clinically diagnosed with BFIE or

ICCA. The four families in which mutations were not

found were small—they comprised two or three affected

individuals—and were clinically indistinguishable from

the 19 PRRT2-mutation-positive families.

The PRRT2 mutations (NM_145239.2) comprise

two frameshifts (c.629_630insC [p.Ala211Serfs*14] and

c.649_650insC [p.Arg217Profs*8]), which are each pre-

dicted to cause protein truncation (Figure 1), two splice-

site mutations (c.879þ1G>T and c.879þ5G>A), and a

missense mutation (c.950G>A [p.Ser317Asn]). The first

of the splice-site mutations alters the consensus G of the

canonical donor splice site. The second splice-site muta-

tion does not alter an invariant nucleotide but signifi-

cantly reduces the splice-site score (splice-site score calcu-

The Americ

lator) from 8.3 to 4.9. Together with this reduced splice-

site score, the segregation of the mutation in a large

BFIE-affected family and its absence in controls strongly

suggest pathogenicity. The missense mutation alters an

amino acid residue in a PRRT2 transmembrane domain

that has been evolutionarily conserved from zebrafish to

humans (the PRRT2 protein is only found in vertebrates).

Pathogenicity of the p.Ser317Asn substitution is also

supported by PolyPhen-2 and SIFT, which predict the

substitution to be probably damaging and not tolerated,

respectively.

We analyzed family members and controls for the

c.629_630insC and c.649_650insC frameshift mutations

with direct sequencing. We screened family members and

controls for the c.879þ1 and c.879þ5 mutations with

high-resolution melting (HRM) analysis by using the

LightScanner (Idaho Technology, Salt Lake City, UT). We

screened the controls for the c.950G>A mutation with

LightScanner, and we screened family members for the

same mutation by using Sanger sequencing. The PRRT2

mutations segregated with either the BFIE or the ICCA

phenotype in each of the 19 mutation-positive families

(Figure 2, Table 1) and were not present in 92 controls, in

dbSNP, or in 1000 Genomes data. The primer sequences

and PCR conditions used for Sanger sequencing and

screening are available upon request.

In the 19 families in which the PRRT2 mutation segre-

gated, there were a total of 77 mutation-positive individ-

uals with BFIE or ICCA. In addition, there were 23 appar-

ently unaffected individuals with a PRRT2 mutation

(Figure 2). However, an accurate clinical history of the

occurrence of infantile seizures could not always be ob-

tained for older family members, making the precise pene-

trance of the mutations difficult to determine. Only one

individual (4-III-1) with infantile seizures lacked the

familial PRRT2 mutation and was therefore considered a

phenocopy. We also observed two nonsynonymous

PRRT2 sequence variants in the controls: c.647C>T

(p.Pro216Leu) (rs76335820) in 6 of 115 (5.2%) Australian

controls and one patient and c.644C>G (p.Pro215Arg) in

1 of 97 (1%) Sephardic-Jewish controls.

Our results demonstrate that PRRT2 mutations cause

BFIE. We have also shown that two distinct disorders,

BFIE and ICCA, are allelic—that is, are caused bymutations

in the same gene. Detection of PRRT2 mutations in 14 of

17 (82%) BFIE-affected and five of six (83%) ICCA-affected

families indicates that mutations in this gene are the most

common cause of these distinctive epilepsy syndromes.

Fifteen of the 19 mutation-positive families (79%) carry

the same mutation, c.649_650insC, which is seen in 12

BFIE-affected and three ICCA-affected families. It is most

likely that this mutation arose independently in at least

some of the families, given their diverse ethnic origins:

Australasian of Western-European heritage (12), Swedish

(1), and Israeli Sephardic-Jewish (2). Furthermore, genotyp-

ing of three microsatellite markers closely linked to PRRT2

in families 5–8, 11, 12, 14, and 16 (Australian), 9 and 10

an Journal of Human Genetics 90, 152–160, January 13, 2012 153

Table 1. Clinical and Genetic Details of Families with PRRT2 Mutations

Linkage Results

Family

Number ofMembers withBFIE or ICCA

Mean Age ofSeizure Onset(Range) [Data on n]a

Age Range ofSeizure Offset[Data on n] Phenotype Ethnic Origin Mutation Chromosome 1 Chromosome 16 Chromosome 19

1 9 9 m (7.5–11 m) [3] N/A BFIE Israeli, AshkenaziJewish

c.879þ5G>A excluded Yes. LODscore: 3.27

ND

2 15 6.7 m (3–12 m) [7] 12–25 m [7] ICCA Scottish c.629_630insC(p.Ala211Serfs*14)

ND Yes. LODscore: 3.0

ND

3 3 6 m (5–8 m) [3] 6 m–2 yr [3] BFIE Israeli, SephardicJewish

c.879þ1G>T excluded not excluded excluded

4 14 4.4 m (3–6 m) [8] 5 m–2 yr ICCA Australasian ofWestern-Europeanheritage

c.950G>A(p.Ser317Asn)

excluded not excluded excluded

5 9 5.2 m (3.5–7 m) [9] 5–14 m [9] ICCA Australasian ofWestern-Europeanheritage

c.649_650insC(p.Arg217Profs*8)

excluded Yes. LODscore: 2.71

ND

6 7 6.4 m (5–11 m) [6] 5–12 m [6] BFIE Australasian ofWestern-Europeanheritage





not excluded not excluded excluded

8 6 9.5 m (8–13 m) [4] 10 m–3yr [4] BFIE Australasian ofWestern-Europeanheritage


not excluded not excluded excluded

9 7 6.5 m (5–8 m) [6] 6 m–2yr [5] BFIE Israeli, SephardicJewish



10 3 6.8 m (6–8 m) <2.5 yr BFIE Israeli, SephardicJewish


ND ND ND

11 3 4.3 m (3–6 m) [3] 3 m–2 yr [3] BFIE Australasian ofWestern-Europeanheritage


not excluded not excluded not excluded

154

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nJournalofHumanGenetics

90,152–160,January

13,2012

Table 1. Continued

Linkage Results

Family

Number ofMembers withBFIE or ICCA

Mean Age ofSeizure Onset(Range) [Data on n]a

Age Range ofSeizure Offset[Data on n] Phenotype Ethnic Origin Mutation Chromosome 1 Chromosome 16 Chromosome 19

12 4 4.5 m (4–5 m) [2] 5 m–<1 yr [2] BFIE Australasian ofWestern-Europeanheritage


not excluded not excluded not excluded

13 3 4 m (4 m) [3] 6–16 m [3] BFIE Swedish c.649_650insC(p.Arg217Profs*8)

ND ND ND



ND ND ND

15 2 3.3 m (3–3.5 m) [2] 4 m [2] BFIE Australasian ofWestern-Europeanheritage


ND ND ND



ND ND ND



ND ND ND

18 4 6 m [1] ICCA Australasian ofWestern-Europeanheritage


ND ND ND

19 4 5.5 m (5–6 m) [2] 6 m [2] ICCA Australasian ofWestern-Europeanheritage


ND ND ND

Abbreviations are as follows: m, months; N/A, not available; and ND, not done. See text for remaining abbreviations.a Number of cases from which data is derived in each family.

TheAmerica

nJournalofHumanGenetics

90,152–160,January

13,2012

155

Figure 1. Locations of the Five Mutations in the PRRT2 Sequence(A) Sequence traces showing the five mutations identified in the 19 BFIE- and ICCA-affected families are compared to wild-type traces.(B) Gene structure of the coding exons of PRRT2 shows the locations of the five mutations in BFIE- and ICCA-affected families.(C) The structure of PRRT2 shows the locations of the five mutations. The transmembrane (TM) domains are indicated in purple. In (B)and (C), the frameshift mutations are marked in light blue, the splice-site mutations in dark blue, and the missense mutation in pink.The lines between (B) and (C) indicate which regions of the protein are coded by the three coding exons.

(Sephardic Jewish), and 13 (Swedish) did not show any

common haplotypes (Figure S2), indicating that the muta-

tions are the result of independent mutational events.

The PRRT2 c.649_650insC mutation clearly occurs at

a mutation ‘‘hot spot’’ because it was seen in 15 of the 19

mutation-positive familiesdescribedhere andsixof the eight

Chinese families affected by autosomal-dominant PKC.16

The high frequency of this frameshift mutation is probably

due to the sequence context inwhich it occurs. The insertion

of a cytosine (C) base occurs in a homopolymer of nine C

bases adjacent to four guanine (G) bases. ThisDNA sequence

has the potential to form a hairpin-loop structure, possibly

leading to DNA-polymerase slippage and the insertion of

an extra C base during DNA replication.

The mutations in families 1 and 5 were not readily de-

tected by MPS despite coverage of PRRT2 on the capture

array used for enrichment of sequences from the BFIE

region on chromosome 16. The percentage of reads con-

taining the common insertion mutation in family 5 was

below the threshold set for mutation calling (Figure S1B).

In addition, the reads for the homopolymer tract showed

a variable number of C bases. This illustrates that MPS is

not always a robust method for mutation detection, espe-

cially in ‘‘difficult’’ sequences such as homopolymer tracts

or GC-rich regions.


PRRT2 encodes a 340 amino acid, proline-rich trans-

membrane protein of unknown function. According to

mRNA-expression data (Ensembl) in human tissue,

PRRT2 is expressed primarily in the brain and is most

highly expressed in the cerebral cortex and basal ganglia

(GeneNote). This expression pattern is consistent with

the clinical expression of BFIE and PKC. To investigate

expression of the mouse ortholog, we performed in situ

hybridization on postnatal (P2, P21, and P46) mouse brain

tissue as described previously17 by using a 504 bp antisense

Prrt2 probe that was complementary to all known tran-

scripts. We generated the probe template by cloning

(pGEM-TEasy Vector System) a PCR fragment amplified

frommouse embryonic stem-cell cDNAwith the following

primers: 50-AGATGAAGGGGGTGGAAGAC-30 and 50-TCAGGACCTCTGTGGTAGGG-30.Prrt2waswidely expressed in the brain andwas readily de-

tected in regions implicated in BFIE and PKC pathology;

such regions included the cerebral cortex and the basal

ganglia, although expression in the latter was generally

less intense (Figure 3 and data not shown). Although the

molecular functionofPRRT2 isnot known,yeast two-hybrid

studies suggest that PRRT2 interacts with synaptosomal-

associated protein 25 kDa (SNAP25) (OMIM 600322).18

SNAP25 is a presynaptic plasma-membrane-bound protein

3, 2012

Figure 2. Pedigrees of Families with PRRT2 MutationsPedigrees of the 19 families affected by BFIE or ICCA show the segregation of the PRRT2 mutation within each family. Individuals witha PRRT2mutation are indicated bym/þ, and individuals tested for mutations and found to be negative are indicated byþ/þ. Individualsfor whom the presence of a mutation was inferred on the basis of its presence in relatives are indicated by (m/þ). Four additional familiesin which no mutation was detected are not shown.

The American Journal of Human Genetics 90, 152–160, January 13, 2012 157

Figure 3. Expression of Prrt2 in the Murine BrainPrrt2 is expressed in the murine brain after birth.(A–E) In situ hybridization analysis of Prrt2 expression in P21 sagittal (A and B) and P46 coronal (D and E) mouse brain sections. Robust,widespread expression was detected in the cerebral cortex (Cx) and caudate putamen (CPu) at P21 (A) and P46 (D). No signal was gener-ated by the sense control probe (B–E). Nissl staining of flanking sections shows cell bodies (C–F). The scale bar represents 0.5 mm.

involved in neurotransmitter release from synaptic vesicles.

Its putative binding partner PRRT2 might play a role in

this process.

The discovery of PRRT2 mutations associated with

BFIE reveals that PRRT2 plays a role in epilepsy. In ICCA,

three different familial mutations (c.629_630insC,

c.649_650insC, and c.950G>A) were detected, high-

lighting that ICCA is not caused by one particular

mutation. It remains unclear why one individual should

experience infantile seizures, PKC, or both of these pheno-

types within a family affected by ICCA—the pleiotropy is

remarkable in terms of both age of onset and anatomical

substrate. Possible explanations include genetic back-

ground or the influence of the wild-type PRRT2 allele,

which might modify the phenotypic expression of the

mutant allele; the mechanisms underlying such variable

expressivity are not yet understood.

Recently, a homozygous frameshift mutation in PRRT2

has been reported in a consanguineous Iranian family

affected by intellectual disability (ID).19 However, no infor-

mation was given regarding the occurrence of seizures in

either the individuals with ID or their heterozygous

parents. Phenotypic heterogeneity is not uncommon for

genes involved in epilepsy20,21 but is more unusual when

one considers both epilepsy and movement disorders,

which engage different neuronal networks.22 The genetic


overlap between epilepsy and movement disorders has

recently been recognized in glucose-transporter-1 defi-

ciency syndrome, in which both epilepsy and paroxysmal

exercise-induced dyskinesia co-occur in families and

individuals.23,24 The identification of PRRT2 significantly

extends our current knowledge of the molecular basis for

infantile epilepsies25 and continues to expand the impor-

tance of the role of non-ion-channel genes in the patho-

genesis of epilepsy. Although the molecular basis of the

BFIE and ICCA phenotypes has not yet been defined for

approximately 20% of the families affected by these disor-

ders, the identification of a BFIE-associated genetic muta-

tion will assist the classification of autosomal-dominant

infantile seizure syndromes.26 Our findings will also aid

the diagnosis, treatment, prognosis, and genetic coun-

seling of patients with BFIE.

Supplemental Data

Supplemental Data include two figures and one table and can be

found with this article online at http://www.ajhg.org.

Acknowledgments

We thank the patients and their families for their participation

and cooperation in our research. We would like to thank Marta

3, 2012

http://www.ajhg.org

Bayly and Bev Johns for technical assistance. This work was

supported by the National Health and Medical Research Council

of Australia (Program Grant 628952 to S.F.B., I.E.S., L.M.D.,

P.Q.T., and J.G., Australia Fellowship 466671 to S.F.B., Senior

Research Fellowship 508043 to J.G., Practitioner Fellowship

1006110 to I.E.S., and Training Fellowship 1016715 to S.E.H.)

and SA Pathology. P.Q.T. is a Pfizer Australia Research Fellow.

L.M.D. is an MS McLeod Research Fellow.

Received: September 29, 2011

Revised: November 23, 2011

Accepted: December 8, 2011

Published online: January 12, 2012

Web Resources

The URLs for data presented herein are as follows:

1000 Genomes browser, http://browser.1000genomes.org/index.

html

dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP

Ensembl, http://www.ensembl.org/index.html

GeneNote, http://bioinfo2.weizmann.ac.il/cgi-bin/genenote/

home_page.pl

Online Mendelian Inheritance in Man (OMIM), http://www.

omim.org

PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/index.shtml

SIFT, http://sift.jcvi.org/

Splice-Site Score Calculator, http://rulai.cshl.edu/new_alt_exon_

db2/HTML/score.html

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