Homozygous c.649dupC mutation in PRRT2 worsens the BFIS/PKD phenotype with mental retardation, episodic ataxia, and absences
Address correspondence to Antonio Gambardella, Cattedra ed U.O. di Neurologia, Università degli Studi “Magna Graecia”, Campus Universitario Germaneto, Viale Europa, 88100 Catanzaro, Italy. E-mail: firstname.lastname@example.org
Heterozygous mutations of PRRT2, which encodes proline-rich transmembrane protein 2, are associated with heterogeneous phenotypes including benign familial infantile seizures (BFIS), or familial paroxysmal kinesigenic dystonia (PKD). We report a consanguineous Italian family with BFIS/PKD phenotype that contained 14 living members with 6 affected individuals (four men, ranging in age from 6–44 years). We identified the reported c.649dupC (p.Arg217ProfsX8) mutation of PRRT2 gene that cosegregated with the disease and was not observed in 100 controls of matched ancestry. Four patients with BFIS phenotype were heterozygous for this mutation, including the consanguineous parents of the two affected brothers with more severe phenotypes of BFIS/PKD—mental retardation, episodic ataxia, and absences—who were the only individuals to carry a homozygous c.649dupC mutation. This family provides strong evidence that homozygous PRRT2 mutations give rise to more severe clinical disease of mental retardation, episodic ataxia, and absences, and, thus, enlarges the clinical spectrum related to PRRT2 mutations. Moreover, it suggests an additive effect of double dose of the genetic mutation and underscores the complexity of the phenotypic consequences of mutations in this gene.
A recent discovery has identified heterozygous mutations in PRRT2, which encodes proline-rich transmembrane protein 2, in most families affected by benign familial infantile seizures (BFIS) (Heron et al., 2012). PRRT2 is also the major causative gene for familial paroxysmal kinesigenic dystonia (PKD) (Chen et al., 2011), a rare disorder characterized by the episodic occurrence of involuntary attacks of choreoathetosis or dystonia. Moreover, these studies illustrated that in a given family both disorders may be variably co-inherited as a single trait (Heron et al., 2012), corroborating the existence of familial infantile convulsions with paroxysmal choreoathetosis (ICCA) (Szepetowski et al., 1997).
We report a consanguineous Italian family exhibiting an ICCA phenotype, with two affected siblings carrying a homozygous PRRT2 mutation that led to a more severe clinical disease of mental retardation, episodic ataxia, and absences.
The family containing 14 living members was ascertained in Calabria southern Italy. The pedigree is shown in Fig. 1. There were six living affected individuals (four men, ranging in age 6–44 years) and all underwent detailed clinical, electroencephalography (EEG), and brain magnetic resonance imaging (MRI) study. Genealogic study supported an autosomal dominant inheritance with incomplete penetrance, as three unaffected individuals transmitted the disease. Mutations in the SCN2A, GLUT-1, and CACNA1A genes, which have been associated with familial neonatal-infantile seizures or paroxysmal movement disorders (Jouvenceau et al., 2001; Berkovic et al., 2004; Suls et al., 2008), were excluded in this family.
Informed consent was obtained from all subjects. The university ethical committee approved the study. DNA was extracted using standard conditions. We screened the whole sequences of gene PRRT2 (exon, intron, 500 bp upstream of 5′UTR and 500 bp downstream of 3′UTR) in this family. The polymerase chain reaction (PCR) primers were designed to amplify the entire exons and the intron–exon boundaries of PRRT2. PCR amplification was carried out using an Ep-gradient S PCR System (Eppendorf, Hamburg, Germany) using standard conditions. The purified PCR products were sequenced with an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, U.S.A.) using standard conditions. The sequencing products were purified and analyzed on an ABI 3130 XL-Genetic Analyzer (Applied Biosystems). The sequences we obtained were compared with the genomic DNA sequence of PRRT2, and nucleotide changes were numbered corresponding to their position in PRRT2 mRNA (Ensembl gene ID: ENST00000358758).
Clinical characteristics are summarized in the Table 1. In all patients, clinical presentation met the diagnostic criteria for BFIS or ICCA. Almost all cases of BFIS manifested as deviation of eyes to one side, an altered consciousness, and bilateral limb jerks with hypertonia. Seizure onset ranged from 3 to 7 months of life. The attacks occurred mainly in clusters, with three to 10 episodes per day, when the patient was awake or asleep. Seizures remitted before age 2 years in all cases, the interictal EEG recordings showed no epileptiform abnormalities between attacks; psychomotor development was normal in all but two affected brothers (IV:2, and IV:4) who had a more severe phenotype. Their parents are first cousins. The father had BFIS alone, whereas their mother had no seizures or movement disorder in childhood. MRI study was always normal.
Table 1. Major clinical findings in eight members of the family carrying the PRRT2 c.649dupC mutation
|II:4 (74 year, M)||–|| None||None|| Normal||Normal||Heterozygous|
|III:2 (44 year, F)||7 months|| BFIS||None|| Normal||Normal||Heterozygous|
|III:4 (47 year, M)||6 months|| BFIS||None|| Normal||Normal||Heterozygous|
|III:5 (44 year, F)||–|| None||None|| Normal||Normal||Heterozygous|
|III:6 (35 year, F)||5 months|| BFIS||None|| Normal||Normal||Heterozygous|
|IV:2 (16 year, M)||3 months|| BFIS||PKD; EA; absences|| MR||gSW||Homozygous|
|IV:3 (13 year, M)||4 months|| BFIS||None|| Normal||Normal||Heterozygous|
|IV:4 (7 year, M)||4 months|| BFIS||PKD; absences|| MR; CS||gSW||Homozygous|
The proband (IV:2) is a 16-year-old boy delivered at full term, but he had delayed motor and cognitive milestones with a full-scale IQ (FSIQ) of 60, as assessed by the Wechsler Intelligence Scales for Children (WISC). He had BFIS from age 3 months until 18 months. At the age of 4 years he developed PKD, the trunk and limbs on both sides were involved, with a brief duration of 10–20 s, no loss of consciousness during attack and good response to carbamazepine. In addition, from age 5 years, he developed rare attacks of gait unsteadiness, blurred vision, sometimes leading to falls without loss of consciousness. The attacks did not follow any identifiable precipitant, and lasted from 60 min up to 24–72 h. At age 9, he also developed daytime absences. Neurologic examination and brain MRI were normal; interictal EEG showed generalized polyspike-waves (Fig. S1). The attacks of ataxia remitted after acetazolamide (250 mg/day); the add-on of lamotrigine in substitution of carbamazepine stopped absences. Now, he is taking lamotrigine plus acetazolamide with no further episode of PKD, absences, or ataxia. The last EEG was normal.
The proband brother (IV:4) is a 7-year-old boy delivered at full term, with delayed motor and cognitive milestones. On WISC testing, FSIQ was 45. At the age of 3 months, he developed seizures that were initially indistinguishable from BFIS but eventually were clearly more severe than typical BFIS, as they consisted of multiple daily tonic seizures that persisted despite treatment with phenobarbital. At the age 9 months, their parents noticed daily episodes of staring during which he lost awareness and did not respond to stimuli. Interictal EEG revealed 2.5–3 Hz generalized epileptiform discharges. Valproate resolved all seizures. At age 26 months, he developed PKD that remitted after starting carbamazepine. Because the patient developed intolerance to both drugs, treatment was switched to acetazolamide (125 mg/day) plus lamotrigine (100 mg/day) with no further seizure or PKD. At last visit, neurologic examination showed moderate mental retardation with poor speech and motor skills, restricted interests and repetitive behavior, and cerebellar eye signs (gaze-evoked nystagmus and impaired suppression of the vestibuloocular reflex). Brain MRI and EEG were normal, as also extensive laboratory that included karyotype and array-based comparative genomic hybridization (CGH).
We identified the reported c.649dupC (p.Arg217ProfsX8) mutation of PRRT2 gene (Fig. S2), which cosegregated with the disease in this family (Fig. 1). Patients with typical BFIS phenotype were heterozygous for this mutation, including consanguineous parents of the two affected brothers with more severe phenotypes, who were the only individuals to carry a homozygous c.649dupC mutation. This mutation was not observed in 100 control subjects of matched ancestry.
Heterozygous PRRT2 mutations are associated with heterogeneous phenotypes including BFIS, ICCA, or PKD, which share a self-limited disorder and good prognosis. Our family provides good evidence that homozygous PRRT2 mutations give rise to more severe clinical disease, including mental retardation, episodic ataxia, and absences. The implications of this observation are relevant for the definition of the variable phenotypes associated with PRRT2 mutations and suggest an additive effect of double dose of the genetic mutation on brain pathology and function.
PRRT2 is a membrane protein with two trans-membrane domains, which is predicted to interact with the presynaptic protein synaptosomalassociated protein 25 kDa (SNAP25), which is known to play an important role in synaptic vesicle handling and neuronal exocytosis (Lee et al., 2012), and is involved in the etiology of attention-deficit/hyperactivity disorders (Gizer et al., 2009). Because the c.649dupC mutation introduces a premature stop codon that causes truncated polypeptide lacking a trans-membrane domain, it is probable that homozygous mutations demolish any binding ability to interactive proteins or ligands such as SNAP25, and would well explain the very severe phenotype, especially mental retardation. Accordingly, a homozygous mutation in PRRT2 has been reported in a consanguineous family affected by intellectual disability, but there was no information regarding the occurrence of seizures in affected individuals or their heterozygous parents (Najmabadi et al., 2011).
Another intriguing feature strictly related to the BIFS/PKD phenotype in this family was that heterozygous mutations were always associated with BFIS, whereas the more complex phenotype of ICCA was seen only in the homozygous state. This observation reinforces the belief that the wild-type PRRT2 allele might modify the phenotypic expression of the mutant allele and thus contribute to heterogeneity in affected BFIS/PKD families. Probably, additional genetic or acquired factors may modify the clinical expression of PRRT2 mutations, and explain the worst phenotype in the younger brother with the homozygous mutation. Moreover, the mutation seen in this family, c.649dupC, represents a hotspot mutation, as it occurred independently in different BFIS/PKD families of distinct ethnic origin (Chen et al., 2011; Cao et al., 2012; Heron et al., 2012; Schubert et al., 2012). This indicates that the nine cytosines represent a highly unstable DNA sequence, frequently leading to an insertion of an additional cytosine.
In conclusion, the present family provides novel evidence that homozygous PRRT2 mutations give rise to more severe BFIS/PKD disease and, thus, enlarges the clinical spectrum related to PRRT2 mutations. Moreover, it suggests an additive effect of double dose of the genetic mutation and underscores the complexity of the phenotypic consequences of mutations in this gene.
None of the authors has any conflict of interest 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.