PRRT2 Mutations Cause Benign Familial Infantile Epilepsy and Infantile Convulsions with Choreoathetosis Syndrome

REPORT PRRT2 Mutations Cause Benign Familial Infantile Epilepsy and Infantile Convulsions with 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 Ge´cz,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 PRRT2 mutations are the most frequent cause of this disorder. We also report PRRT2 mutations 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 BFIEaffected 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 choreoathetosis (PKC) (OMIM 128200), or both. When patients are affected by both of these uncommon clinical phenotypes, presentation might be separated by many years.

Families affected by ICCA and families affected by autosomal-dominant PKC also show linkage to the large pericentromeric 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 speculation that BFIE, ICCA, and autosomal-dominant PKC might be allelic.8,9 Identification of the gene or genes in which mutations cause these disorders is required for confirmation of this hypothesis. We studied 23 families affected by either BFIE (n ¼ 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 available. Australian control samples were obtained from anonymous 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

1 Epilepsy Research Program, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5000, Australia; 2Epilepsy Research Centre, Department of Medicine, University of Melbourne, Austin Health, Heidelberg, Victoria 3084 Australia; 3Schneider Children’s Medical Center of Israel, Petach Tikvah 49202, Israel; 4Tel-Aviv University, Tel-Aviv 69978, Israel; 5Paediatric Neurosciences Research Group, Fraser of Allander Neurosciences Unit, Royal Hospital for Sick Children, Glasgow G3 8SJ, United Kingdom; 6School of Molecular and Biomedical Science, the University of Adelaide, Adelaide, South Australia 5005, Australia; 7Department of Neurology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia; 8Department of Paediatrics, University of Otago, Wellington 6242, New Zealand; 9Academic Discipline of Paediatrics and Child Health, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia; 10Neonatal Research Unit, Department of Women’s and Children’s Health, Astrid Lindgren Children’s Hospital, Karolinska Institutet, Stockholm SE-171 77, Sweden; 11Pediatric Neurology and Development Unit, Dana Children’s Hospital, Tel Aviv Sourasky Medical Center, Tel-Aviv 64239, Israel; 12South Australian Clinical Genetics Service, SA Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia; 13Neurogenetics Program, SA Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia; 14School of Paediatrics and Reproductive Health, the University of Adelaide, Adelaide, South Australia 5005, Australia; 15 Department of Genetic Medicine, SA Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia; 16Florey Neuroscience Institutes and Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville, Victoria 3052, Australia 17 These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.ajhg.2011.12.003. Ó2012 by The American Society of Human Genetics. All rights reserved.

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

in the gene located in the chromosome 16 region. Microsatellite 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 families 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 predicted to cause protein truncation (Figure 1), two splicesite 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 mutation does not alter an invariant nucleotide but significantly reduces the splice-site score (splice-site score calcu-

lator) from 8.3 to 4.9. Together with this reduced splicesite 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 segregated, there were a total of 77 mutation-positive individuals with BFIE or ICCA. In addition, there were 23 apparently unaffected individuals with a PRRT2 mutation (Figure 2). However, an accurate clinical history of the occurrence of infantile seizures could not always be obtained for older family members, making the precise penetrance 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 by mutations 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, genotyping of three microsatellite markers closely linked to PRRT2 in families 5–8, 11, 12, 14, and 16 (Australian), 9 and 10

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

Table 1.

Clinical and Genetic Details of Families with PRRT2 Mutations Linkage Results

Family

Number of Members with BFIE or ICCA

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

Age Range of Seizure 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, Ashkenazi Jewish

c.879þ5G>A

excluded

Yes. LOD score: 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. LOD score: 3.0

ND

3

3

6 m (5–8 m) [3]

6 m–2 yr [3]

BFIE

Israeli, Sephardic Jewish

c.879þ1G>T

excluded

not excluded

excluded

4

14

4.4 m (3–6 m) [8]

5 m–2 yr

ICCA

Australasian of Western-European heritage

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 of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

excluded

Yes. LOD score: 2.71

ND

6

7

6.4 m (5–11 m) [6]

5–12 m [6]

BFIE

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

excluded

not excluded

excluded

7

6

8.2 m (5–10 m) [5]

10–23 m [5]

BFIE

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

not excluded

not excluded

excluded

8

6

9.5 m (8–13 m) [4]

10 m–3yr [4]

BFIE

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

not excluded

not excluded

excluded

9

7

6.5 m (5–8 m) [6]

6 m–2yr [5]

BFIE

Israeli, Sephardic Jewish

c.649_650insC (p.Arg217Profs*8)

excluded

not excluded

excluded

10

3

6.8 m (6–8 m)

<2.5 yr

BFIE

Israeli, Sephardic Jewish

c.649_650insC (p.Arg217Profs*8)

ND

ND

ND

11

3

4.3 m (3–6 m) [3]

3 m–2 yr [3]

BFIE

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

not excluded

not excluded

not excluded

Table 1.

Continued Linkage Results

The American Journal of Human Genetics 90, 152–160, January 13, 2012 155

Family

Number of Members with BFIE or ICCA

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

Age Range of Seizure 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 of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

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

14

3

4.3 m (4–5 m) [3]

5–24 m [3]

BFIE

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

ND

ND

ND

15

2

3.3 m (3–3.5 m) [2]

4 m [2]

BFIE

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

ND

ND

ND

16

3

10.7 m (5–18 m) [3]

7–33 m [3]

BFIE

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

ND

ND

ND

17

9

6.3 m (4–8 m) [4]

5–8 m [4]

BFIE

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

ND

ND

ND

18

4

6 m [1]

ICCA

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

ND

ND

ND

19

4

5.5 m (5–6 m) [2]

ICCA

Australasian of Western-European heritage

c.649_650insC (p.Arg217Profs*8)

ND

ND

ND

6 m [2]

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

a

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 mutations 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 families described here and six of the eight Chinese families affected by autosomal-dominant PKC.16 The high frequency of this frameshift mutation is probably due to the sequence context in which it occurs. The insertion of a cytosine (C) base occurs in a homopolymer of nine C bases adjacent to four guanine (G) bases. This DNA 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 detected 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 containing 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, especially in ‘‘difficult’’ sequences such as homopolymer tracts or GC-rich regions.

PRRT2 encodes a 340 amino acid, proline-rich transmembrane 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 transcripts. We generated the probe template by cloning (pGEM-TEasy Vector System) a PCR fragment amplified from mouse embryonic stem-cell cDNA with the following primers: 50 -AGATGAAGGGGGTGGAAGAC-30 and 50 -TCA GGACCTCTGTGGTAGGG-30 . Prrt2 was widely expressed in the brain and was readily detected 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 function of PRRT2 is not known, yeast two-hybrid studies suggest that PRRT2 interacts with synaptosomalassociated protein 25 kDa (SNAP25) (OMIM 600322).18 SNAP25 is a presynaptic plasma-membrane-bound protein

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

Figure 2. Pedigrees of Families with PRRT2 Mutations Pedigrees of the 19 families affected by BFIE or ICCA show the segregation of the PRRT2 mutation within each family. Individuals with a PRRT2 mutation are indicated by m/þ, and individuals tested for mutations and found to be negative are indicated by þ/þ. Individuals for whom the presence of a mutation was inferred on the basis of its presence in relatives are indicated by (m/þ). Four additional families in 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 Brain Prrt2 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 generated 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, highlighting that ICCA is not caused by one particular mutation. It remains unclear why one individual should experience infantile seizures, PKC, or both of these phenotypes within a family affected by ICCA—the pleiotropy is remarkable in terms of both age of onset and anatomical substrate. Possible explanations include genetic background 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 information 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 deficiency 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 importance of the role of non-ion-channel genes in the pathogenesis 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 disorders, the identification of a BFIE-associated genetic mutation will assist the classification of autosomal-dominant infantile seizure syndromes.26 Our findings will also aid the diagnosis, treatment, prognosis, and genetic counseling 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

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

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

7.

8.

9.

10.

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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12.

13.

14.

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