PRRT2 Mutations Cause Benign Familial Infantile Epilepsy and Infantile Convulsions with Choreoathetosis Syndrome
2012; Elsevier BV; Volume: 90; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2011.12.003
ISSN1537-6605
AutoresSarah E. Heron, Bronwyn E. Grinton, Sara Kivity, Zaid Afawi, Sameer M. Zuberi, James N. Hughes, Clair Pridmore, Bree Hodgson, Xenia Iona, Lynette G. Sadleir, James T. Pelekanos, Eric Herlenius, Hadassa Goldberg‐Stern, Haim Bassan, Eric Haan, Amos D. Korczyn, Alison Gardner, Mark Corbett, Jozef Gécz, Paul Q. Thomas, John C. Mulley, Samuel F. Berkovic, Ingrid E. Scheffer, Leanne M. Dibbens,
Tópico(s)Epilepsy research and treatment
ResumoBenign 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) 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 19q12–13.11Guipponi M. Rivier F. Vigevano F. Beck C. Crespel A. Echenne B. Lucchini P. Sebastianelli R. Baldy-Moulinier M. Malafosse A. Linkage mapping of benign familial infantile convulsions (BFIC) to chromosome 19q.Hum. Mol. Genet. 1997; 6: 473-477Crossref PubMed Scopus (171) Google Scholar and 1p362Li H.Y. Li N. Jiang H. Shen L. Guo J.F. Zhang R.X. Xia K. Pan Q. Zi X.H. Tang B.S. A novel genetic locus for benign familial infantile seizures maps to chromosome 1p36.12-p35.1.Clin. Genet. 2008; 74: 490-492Crossref PubMed Scopus (11) Google Scholar, respectively, in BFIE-affected families. The vast majority of reported families (approximately 50) affected by BFIE show linkage to the pericentromeric region from 16p.11.2–16q12.1.3Caraballo R. Pavek S. Lemainque A. Gastaldi M. Echenne B. Motte J. Genton P. Cersósimo R. Humbertclaude V. Fejerman N. et al.Linkage of benign familial infantile convulsions to chromosome 16p12-q12 suggests allelism to the infantile convulsions and choreoathetosis syndrome.Am. J. Hum. Genet. 2001; 68: 788-794Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 4Weber Y.G. Berger A. Bebek N. Maier S. Karafyllakes S. Meyer N. Fukuyama Y. Halbach A. Hikel C. Kurlemann G. et al.Benign familial infantile convulsions: Linkage to chromosome 16p12-q12 in 14 families.Epilepsia. 2004; 45: 601-609Crossref PubMed Scopus (41) Google Scholar, 5Callenbach P.M. van den Boogerd E.H. de Coo R.F. ten Houten R. Oosterwijk J.C. Hageman G. Frants R.R. Brouwer O.F. van den Maagdenberg A.M. Refinement of the chromosome 16 locus for benign familial infantile convulsions.Clin. Genet. 2005; 67: 517-525Crossref PubMed Scopus (24) Google Scholar, 6Striano P. Lispi M.L. Gennaro E. Madia F. Traverso M. Bordo L. Aridon P. Boneschi F.M. Barone B. dalla Bernardina B. et al.Linkage analysis and disease models in benign familial infantile seizures: A study of 16 families.Epilepsia. 2006; 47: 1029-1034Crossref PubMed Scopus (24) Google Scholar, 7Weber Y.G. Jacob M. Weber G. Lerche H. A BFIS-like syndrome with late onset and febrile seizures: Suggestive linkage to chromosome 16p11.2-16q12.1.Epilepsia. 2008; 49: 1959-1964Crossref PubMed Scopus (26) Google Scholar 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 16.8Szepetowski P. Rochette J. Berquin P. Piussan C. Lathrop G.M. Monaco A.P. 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: 889-898Abstract Full Text PDF PubMed Scopus (293) Google Scholar, 9Rochette J. Roll P. Szepetowski P. Genetics of infantile seizures with paroxysmal dyskinesia: the infantile convulsions and choreoathetosis (ICCA) and ICCA-related syndromes.J. Med. Genet. 2008; 45: 773-779Crossref PubMed Scopus (29) Google Scholar, 10Rochette J. Roll P. Fu Y.H. Lemoing A.G. Royer B. Roubertie A. Berquin P. Motte J. Wong S.W. Hunter A. et al.Novel familial cases of ICCA (infantile convulsions with paroxysmal choreoathetosis) syndrome.Epileptic Disord. 2010; 3: 199-204Google Scholar 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.8Szepetowski P. Rochette J. Berquin P. Piussan C. Lathrop G.M. Monaco A.P. 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: 889-898Abstract Full Text PDF PubMed Scopus (293) Google Scholar, 9Rochette J. Roll P. Szepetowski P. Genetics of infantile seizures with paroxysmal dyskinesia: the infantile convulsions and choreoathetosis (ICCA) and ICCA-related syndromes.J. Med. Genet. 2008; 45: 773-779Crossref PubMed Scopus (29) Google Scholar 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 = 17) or ICCA (n = 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.11Reutens D.C. Howell R.A. Gebert K.E. Berkovic S.F. Validation of a questionnaire for clinical seizure diagnosis.Epilepsia. 1992; 33: 1065-1071Crossref PubMed Scopus (114) Google Scholar 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 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.12Lathrop G.M. Lalouel J.M. Easy calculations of lod scores and genetic risks on small computers.Am. J. Hum. Genet. 1984; 36: 460-465PubMed Google Scholar 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).Table 1Clinical and Genetic Details of Families with PRRT2 MutationsLinkage ResultsFamilyNumber of Members with BFIE or ICCAMean Age of Seizure Onset (Range) [Data on n]aNumber of cases from which data is derived in each family.Age Range of Seizure Offset [Data on n]PhenotypeEthnic OriginMutationChromosome 1Chromosome 16Chromosome 19199 m (7.5–11 m) [3]N/ABFIEIsraeli, Ashkenazi Jewishc.879+5G>AexcludedYes. LOD score: 3.27ND2156.7 m (3–12 m) [7]12–25 m [7]ICCAScottishc.629_630insC (p.Ala211Serfs∗14)NDYes. LOD score: 3.0ND336 m (5–8 m) [3]6 m–2 yr [3]BFIEIsraeli, Sephardic Jewishc.879+1G>Texcludednot excludedexcluded4144.4 m (3–6 m) [8]5 m–2 yrICCAAustralasian of Western-European heritagec.950G>A (p.Ser317Asn)excludednot excludedexcluded595.2 m (3.5–7 m) [9]5–14 m [9]ICCAAustralasian of Western-European heritagec.649_650insC (p.Arg217Profs∗8)excludedYes. LOD score: 2.71ND676.4 m (5–11 m) [6]5–12 m [6]BFIEAustralasian of Western-European heritagec.649_650insC (p.Arg217Profs∗8)excludednot excludedexcluded768.2 m (5–10 m) [5]10–23 m [5]BFIEAustralasian of Western-European heritagec.649_650insC (p.Arg217Profs∗8)not excludednot excludedexcluded869.5 m (8–13 m) [4]10 m–3yr [4]BFIEAustralasian of Western-European heritagec.649_650insC (p.Arg217Profs∗8)not excludednot excludedexcluded976.5 m (5–8 m) [6]6 m–2yr [5]BFIEIsraeli, Sephardic Jewishc.649_650insC (p.Arg217Profs∗8)excludednot excludedexcluded1036.8 m (6–8 m)<2.5 yrBFIEIsraeli, Sephardic Jewishc.649_650insC (p.Arg217Profs∗8)NDNDND1134.3 m (3–6 m) [3]3 m–2 yr [3]BFIEAustralasian of Western-European heritagec.649_650insC (p.Arg217Profs∗8)not excludednot excludednot excluded1244.5 m (4–5 m) [2]5 m– T and c.879+5G>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 calculator) 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.879+1 and c.879+5 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 (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.16Chen W.J. Lin Y. Xiong Z.Q. Wei W. Ni W. Tan G.H. Guo S.L. He J. Chen Y.F. Zhang Q.J. et al.Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia.Nat. Genet. 2011; 43: 1252-1255Crossref PubMed Scopus (361) Google Scholar 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 previously17Dibbens L.M. Tarpey P.S. Hynes K. Bayly M.A. Scheffer I.E. Smith R. Bomar J. Sutton E. Vandeleur L. Shoubridge C. et al.X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment.Nat. Genet. 2008; 40: 776-781Crossref PubMed Scopus (331) Google Scholar 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: 5′-AGATGAAGGGGGTGGAAGAC-3′ and 5′-TCAGGACCTCTGTGGTAGGG-3′. 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 synaptosomal-associated protein 25 kDa (SNAP25) (OMIM 600322).18Stelzl U. Worm U. Lalowski M. Haenig C. Brembeck F.H. Goehler H. Stroedicke M. Zenkner M. Schoenherr A. Koeppen S. et al.A human protein-protein interaction network: A resource for annotating the proteome.Cell. 2005; 122: 957-968Abstract Full Text Full Text PDF PubMed Scopus (1873) Google Scholar SNAP25 is a presynaptic plasma-membrane-bound protein 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).19Najmabadi H. Hu H. Garshasbi M. Zemojtel T. Abedini S.S. Chen W. Hosseini M. Behjati F. Haas S. Jamali P. et al.Deep sequencing reveals 50 novel genes for recessive cognitive disorders.Nature. 2011; 478: 57-63Crossref PubMed Scopus (690) Google Scholar 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 epilepsy20Wallace R.H. Wang D.W. Singh R. Scheffer I.E. George Jr., A.L. Phillips H.A. Saar K. Reis A. Johnson E.W. Sutherland G.R. et al.Febrile seizures and generalized epilepsy associated with a mutation in the Na+-channel β1 subunit gene SCN1B.Nat. Genet. 1998; 19: 366-370Crossref PubMed Scopus (94) Google Scholar, 21Wallace R.H. Marini C. Petrou S. Harkin L.A. Bowser D.N. Panchal R.G. Williams D.A. Sutherland G.R. Mulley J.C. Scheffer I.E. Berkovic S.F. Mutant GABA(A) receptor γ2-subunit in childhood absence epilepsy and febrile seizures.Nat. Genet. 2001; 28: 49-52Crossref PubMed Google Scholar but is more unusual when one considers both epilepsy and movement disorders, which engage different neuronal networks.22Crompton D.E. Berkovic S.F. The borderland of epilepsy: Clinical and molecular features of phenomena that mimic epileptic seizures.Lancet Neurol. 2009; 8: 370-381Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar 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.23Suls A. Dedeken P. Goffin K. Van Esch H. Dupont P. Cassiman D. Kempfle J. Wuttke T.V. Weber Y. Lerche H. et al.Paroxysmal exercise-induced dyskinesia and epilepsy is due to mutations in SLC2A1, encoding the glucose transporter GLUT1.Brain. 2008; 131: 1831-1844Crossref PubMed Scopus (289) Google Scholar, 24Mullen S.A. Suls A. De Jonghe P. Berkovic S.F. Scheffer I.E. Absence epilepsies with widely variable onset are a key feature of familial GLUT1 deficiency.Neurology. 2010; 75: 432-440Crossref PubMed Scopus (135) Google Scholar The identification of PRRT2 significantly extends our current knowledge of the molecular basis for infantile epilepsies25Heron S.E. Mulley J.C. The molecular genetics of benign epilepsies of infancy.in: Afawi Z. Clinical and Genetic Aspects of Epilepsy. Intech Open, Rijeka, Croatia2011: 95-112Google Scholar 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.26Mulley J.C. Heron S.E. Dibbens L.M. Proposed genetic classification of the “benign” familial neonatal and infantile epilepsies.Epilepsia. 2011; 52: 649-650Crossref PubMed Scopus (9) Google Scholar Our findings will also aid the diagnosis, treatment, prognosis, and genetic counseling of patients with BFIE. We thank the patients and their families for their participation and cooperation in our research. We would like to thank Marta 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. Download .pdf (.64 MB) Help with pdf files Document S1. Figures S1 and S2 and Table S1 The URLs for data presented herein are as follows:1000 Genomes browser, http://browser.1000genomes.org/index.htmldbSNP, http://www.ncbi.nlm.nih.gov/projects/SNPEnsembl, http://www.ensembl.org/index.htmlGeneNote, http://bioinfo2.weizmann.ac.il/cgi-bin/genenote/home_page.plOnline Mendelian Inheritance in Man (OMIM), http://www.omim.orgPolyPhen-2, http://genetics.bwh.harvard.edu/pph2/index.shtmlSIFT, http://sift.jcvi.org/Splice-Site Score Calculator, http://rulai.cshl.edu/new_alt_exon_db2/HTML/score.html
Referência(s)