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Keywords:

  • Benign epilepsy;
  • Channelopathies;
  • KCNQ2 ;
  • KCNQ3 ;
  • PRRT2 ;
  • SCN2A

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Purpose

To dissect the genetics of benign familial epilepsies of the first year of life and to assess the extent of the genetic overlap between benign familial neonatal seizures (BFNS), benign familial neonatal-infantile seizures (BFNIS), and benign familial infantile seizures (BFIS).

Methods

Families with at least two first-degree relatives affected by focal seizures starting within the first year of life and normal development before seizure onset were included. Families were classified as BFNS when all family members experienced neonatal seizures, BFNIS when the onset of seizures in family members was between 1 and 4 months of age or showed both neonatal and infantile seizures, and BFIS when the onset of seizures was after 4 months of age in all family members. SCN2A, KCNQ2, KCNQ3, PPRT2 point mutations were analyzed by direct sequencing of amplified genomic DNA. Genomic deletions involving KCNQ2 and KCNQ3 were analyzed by multiple-dependent probe amplification method.

Key Findings

A total of 46 families including 165 affected members were collected. Eight families were classified as BFNS, 9 as BFNIS, and 29 as BFIS. Genetic analysis led to the identification of 41 mutations, 14 affecting KCNQ2, 1 affecting KCNQ3, 5 affecting SCN2A, and 21 affecting PRRT2. The detection rate of mutations in the entire cohort was 89%. In BFNS, mutations specifically involve KCNQ2. In BFNIS two genes are involved (KCNQ2, six families; SCN2A, two families). BFIS families are the most genetically heterogeneous, with all four genes involved, although about 70% of them carry a PRRT2 mutation.

Significance

Our data highlight the important role of KCNQ2 in the entire spectrum of disorders, although progressively decreasing as the age of onset advances. The occurrence of afebrile seizures during follow-up is associated with KCNQ2 mutations and may represent a predictive factor. In addition, we showed that KCNQ3 mutations might be also involved in families with infantile seizures. Taken together our data indicate an important role of K-channel genes beyond the typical neonatal epilepsies. The identification of a novel SCN2A mutation in a family with infantile seizures with onset between 6 and 8 months provides further confirmation that this gene is not specifically associated with BFNIS and is also involved in families with a delayed age of onset. Our data indicate that PRRT2 mutations are clustered in families with BFIS. Paroxysmal kinesigenic dyskinesia emerges as a distinctive feature of PRRT2 families, although uncommon in our series. We showed that the age of onset of seizures is significantly correlated with underlying genetics, as about 90% of the typical BFNS families are linked to KCNQ2 compared to only 3% of the BFIS families, for which PRRT2 represents the major gene.

Afebrile seizures with onset within the first year of life are often associated with metabolic disorders, structural brain lesions, may cause epileptic encephalopathies, and usually have a sporadic occurrence (Matsumoto et al., 1983). However, seizures may manifest in newborns and infants with normal neurologic or metabolic status and family history of early onset seizures (Plouin & Anderson, 2002; Vigevano & Bureau, 2002). Three epileptic conditions have been so far delineated, accounting for predominant secondarily generalized focal seizures, benign outcome, and autosomal dominant mode of inheritance. These conditions are differentiated mainly for the age of onset: typically before the fifth day of life in benign familial neonatal seizures (BFNS) (Rett & Teubel, 1964), between 2 days and 6 months of age in benign familial neonatal-infantile seizures (BFNIS) (Kaplan & Lacey, 1983), and between 3 and 8 months of age in benign familial infantile seizures (BFIS) (Vigevano et al., 1992). At the clinical level, these disorders show analogous, usually brief, focal motor manifestations, typically head and eye deviation and subsequent tonic and clonic movements, staring, and apnea. Seizures may occur sporadically or in clusters of many per day with spontaneous remission within the first year of age (Deprez et al., 2009).

Genetic studies led to the identification of mutations in the voltage-gated potassium channel genes KCNQ2 and KCNQ3 in BFNS (Singh et al., 1998; Biervert et al., 1998; Charlier et al., 1998) and in the voltage-gated sodium channel alpha2 subunit gene SCN2A in BFNIS (Heron et al., 2002; Berkovic et al., 2004), and a recently identified PRRT2 gene on chromosome 16p12-q12 in BFIS families (Heron et al., 2012; Schubert et al., 2012). Although these three conditions show a significant clinical overlap, a comprehensive mutational screening of the candidate genes on a large set of families has been not yet exploited. In the present study we performed genetic analysis of 46 families, assessed the extent of the genetic overlap between BFNS, BFNIS, and BFIS, and determined the role of each gene in these group of disorders.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Patients

We included families with at least two first-degree relatives affected by focal seizures starting within the first year of life, normal development before seizure onset, and no underlying disorders or neurologic disabilities. Each proband underwent neurologic examination, at least one electroencephalography (EEG) recording, and, when possible, brain computerized tomography (CT) or magnetic resonance imaging (MRI). Information about seizure type, developmental milestones, and neurologic status of the patients' relatives was collected at home, by telephone, or from medical records. All investigated subjects signed informed consent to the clinical and genetic investigations. Seizures with onset within the first month of age were defined neonatal, infantile when above the first month of age. Families were classified as BFNS when all family members experienced neonatal seizures, BFNIS when the onset of seizures in family members was between 1 and 4 months of age or showed both neonatal and infantile seizures, and BFIS when the onset of seizures was after 4 months of age in all family members. BFIS families with affected individuals showing paroxysmal kinesigenic dyskinesia (PKD) later in life were subclassified as infantile convulsion and choreoathetosis (ICCA).

Genetic analysis

Genetic analysis was performed into a diagnostic settings from 2000 to 2012. Probands were initially screened for KCNQ2 and KCNQ3 point mutations by direct sequencing of amplified genomic DNA according to standard Sanger sequencing protocols, as described (Schubert et al., 2012; Striano et al., 2006a) and for genomic deletions involving KCNQ2 and KCNQ3 by MLPA (multiple-dependent probe amplification) using the SALSA MLPA kits P166 and 197 (MRC Holland, Amsterdam, The Netherlands). Negative patients were further screened for point mutations in SCN2A and then in PPRT2. Deletions encompassing the whole KCNQ2 or KCNQ3 genes were further followed by array comparative genomic hybridization (CGH) to define the extent of the genomic rearrangement. Array-CGH was performed using the Agilent Human Genome CGH Microarray Kit 44B or 244K. Labeling and hybridization were performed following the manufacturer's protocol. Variants were classified as mutations according to the following criteria: (1) absent in database of single nucleotide polymorphisms (dbSNP135) or showing a population frequency <1%; (2) cosegregating with epilepsy in families, (3) previously reported in familial early onset epilepsies and/or with clear-cut detrimental effect on protein structure (frameshift, nonsense, splice-site mutations, genomic deletions) or functionally damaging according to prediction software SIFT for missense mutations (SIFT score <0.05) (Kumar et al., 2009).

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

A total of 46 families were collected. The sample includes 165 affected members, with a range of 2–17 affected individuals per family (average 3.6) (Table 1) (Data S1). Eight families were classified as BFNS, 9 as BFNIS, and 29 as BFIS.

Table 1. Clinical and genetic features of 151 affected individuals carrying KCNQ2, KCNQ3, or SCN2A mutations
Family IDDiagnosisIndividual IDSex/age at last contactNeonatal/Infantile seizuresFebrile seizures (onset)Additional afebrile seizures (onset/year)Current therapy (outcome)Additional neurological symptomsGeneMutation
OnsetTypeClustersOffsetTreatment
  1. na, not available; PB, phenobarbital; PHT, phenytoin, VPA, valproate; GVG, vigabatrin; CBZ, carbamazepine; DZP, diazepam; PKD, paroxysmal kinesigenic dyskinesia.

  2. Probands are highlighted in bold font.

  3. a15 additional family members reported to be affected but not tested.

N1BFNSI:1M/81<1 yearnananananana
II:2 F/392 dayTonic–clonic Yes22 dayNoneTLE(6 year)TPM + LTG
II:3M/372 dayTonic–clonic Yes15 dayNoneNone (sf)––
II:5F/352 dayTonic–clonic Yes22 dayNoneTLE (17 year)PB
III:3F/72 dayTonic–clonic Yes22 dayNoneGTCS (16 year)None (sf)
III:8M/32 dayTonic–clonic Yes15 dayNoneNone (sf)––
N2BFNSI:3F/78neonatalTonic–clonicNanaNoneCPS (13 year)None (sf) KCNQ2 Deletion 470 kb
II:3F/344 dayTonic–clonicYes5 monthPBCPS (10 year)CBZ KCNQ2 Deletion 470 kb
III:1 M/68 dayTonic–clonicYes10 dayPBNone (sf) KCNQ2 Deletion 470 kb
N3BFNSII:2M/342 dayFocalYes6 monthPBNone (sf) KCNQ2 Deletion exons 13–17
III:1 M/62 dayTonic–clonic, focalYes5 monthPB, PHT20 monthCBZ KCNQ2 Deletion exons 13–17
N4BFNSII:2F/3910 dayFocal, tonic–clonic Yes1 month PBNone (sf) KCNQ2 c.1116+3A>G
III:1 M/61 dayFocal, clonicYes4 monthPBNone (sf) KCNQ2 c.1116+3A>G
N5BFNSII:3F/405 dayFocalYesna3 yearna KCNQ2 c.1639C>T, p.Arg547Trp
III:2 F/43 dayFocal Yes2 monthNoneNone (sf) KCNQ2 c.1639C>T, p.Arg547Trp
N6BFNSII:3F/37neonatalTonic–clonicYesnanana KCNQ2 Deletion exon 1
III:1 F/55 dayTonic–clonicYes3,5 monthPB, VPANone (sf) KCNQ2 Deletion exon 1
N7BFNSII:1F/151 dayFocal, tonic–clonicYes PB, GVG None (sf) KCNQ2 Deletion 187 kb
II:2 F/85 dayFocal, tonic–clonicYes PB, CBZNone (sf) KCNQ2 Deletion 187 kb
N8BFNSII:2F/353 dayTonic–clonic Yes6 yearPBNone (sf) KCNQ2 c.476G>A, p.Gly159Glu
II:3M/322 dayTonic–clonic Yes3 monthPB, VPANone (sf) KCNQ2 c.476G>A, p.Gly159Glu
III:3 M/44 dayFocal, tonic–clonicYes1 monthPBNone (sf) KCNQ2 c.476G>A, p.Gly159Glu
NI1BFNISI:1M/483,5 monthTonic–clonicSingle seizure4 monthNoneNone (sf) KCNQ2 c.587C>T, p.Ala196Val
II:1 M/113 monthFocal, clonicYes4 monthPB Rolandic Seizures (4 years)None (sf) KCNQ2 c.587C>T, p.Ala196Val
II:2M/63 dayFocal, tonic–clonicYesnaPB, GVG 19 monthEpileptic spasms (12 month)None (sf) KCNQ2 c.587C>T, p.Ala196Val
NI2BFNISII:1M/434 monthFocal, clonicSingle seizure4 monthNoneNone (sf) KCNQ2 c.1342C>T, Arg448X
III:1 M/63,5 monthFocal, tonic–clonicYes3,5 monthDZP, PBDZP KCNQ2 c.1342C>T, Arg448X
NI3BFNISI:1M/68<1 yearnaNananana KCNQ2 c.460T>G, p.Tyr154Asp
II:1M/382 monthnaYesnaPBNone (sf) KCNQ2 c.460T>G, p.Tyr154Asp
II:3M/38<1 yearnaNananana KCNQ2 c.460T>G, p.Tyr154Asp
III:1 M/143 dayFocal, tonic–clonicYes12 monthPBNone (sf) KCNQ2 c.460T>G, p.Tyr154Asp
III:2F/134 daynaYesnaPBNone (sf) KCNQ2 c.460T>G, p.Tyr154Asp
III:3M/7neonatalnaYesnaPB None (sf) KCNQ2 c.460T>G, p.Tyr154Asp
NI4BFNISII:5M/354 monthFocalYes5 monthPBNone (sf) KCNQ2 c.297-2A>G
III:1M/1117 dayFocalYes3 monthPBNone (sf) KCNQ2 c.297-2A>G
III:2F/63.5 monthFocal, tonic–clonicYes4 monthVPANone (sf) KCNQ2 c.297-2A>G
III:4 F/55 dayFocal, tonicYes2 dayNoneNone (sf) KCNQ2 c.297-2A>G
NI5BFNISIII:5F/3417 dayTonic–clonicNo1 monthVPANone (sf)
IV:2 F/62 monthFocalNo2 monthNoneNone (sf)
NI6aBFNISII:12F/363 monthFocal, tonic–clonicYes14 monthNoneNone (sf) SCN2A c.768 G>A, p.Arg223Gln
III:11 M/43.5 monthFocal, tonic–clonicYes12 monthNoneNone (sf) SCN2A c.768 G>A, p.Arg223Gln
NI7BFNISI:1M/725 month naNanananananana SCN2A c.3956 G>A, Arg1319Gln
II:2M/453.5 monthFocal, tonic–clonicYes3,5 monthPBNone (sf) SCN2A c.3956 G>A, Arg1319Gln
III:1 M/154 monthFocal, tonic–clonicYes4 monthPBNone (sf) SCN2A c.3956 G>A, Arg1319Gln
III:2F/185 month naNanananananana SCN2A c.3956 G>A, Arg1319Gln
NI8BFNISII:2F/342 monthTonic–clonic Single seizure2 monthNoneNone (sf) KCNQ2 c.592-594 delinsA, p.Arg198 fs
II:3M/321 monthTonic–clonicYes6 yearNoneNone (sf) KCNQ2 c.592-594 delinsA, p.Arg198 fs
III:1 M/72 monthFocal Yes4 monthPBNone (sf) KCNQ2 c.592-594 delinsA, p.Arg198 fs
NI9BFNISII:4F/356 monthFocal, tonic–clonicYes5 yearPBNone (sf) KCNQ2 c.1887+5G>A
III:2 M/82.5 monthFocal, clonicYes4 monthPB1 year 10 monthNone (sf) KCNQ2 c.1887+5G>A
I1BFISI:1F/416 monthnaYes7 monthNoneNone (sf) PRRT2 c.291delC
II:4M/206 monthFocalNo6 monthPBNone (sf) PRRT2 c.291delC
II:5M/187 monthFocalYes7 monthCBZNone (sf) PRRT2 c.291delC
II:6M/1514 monthNone (sf) PRRT2 c.291delC
II:7 M/125 monthFocal Yes6 monthCBZNone (sf) PRRT2 c.291delC
II:8M/84 monthFocalYes4 monthCBZNone (sf) PRRT2 c.291delC
I2BFISI:2F/44nanaNananananana PRRT2 c.291delC
II:1M/246 monthTonic–clonicNo6 monthPBNone (sf) PRRT2 c.291delC
II:2 M/146 monthTonic–clonicNo6 monthVPANone (sf) PRRT2 c.291delC
I3BFISI:2F/deceased nanananananananana PRRT2 c.649_650dupC
II:1M/71nanaNanananananana PRRT2 c.649_650dupC
III:1M/336 monthTonic–clonicYes7 monthNoneNone (sf) PRRT2 c.649_650dupC
IV:1 F/75 monthFocalYes5 monthPBNone (sf) PRRT2 c.649_650dupC
IV:2F/36 monthFocalYes6 monthPB,VPANone (sf) PRRT2 c.649_650dupC
I4BFISI:1M/375 monthTonic, clonicNo7 monthPBNone (sf) KCNQ3 c.2338C>T, p.Arg780Cys
II:1M/134.5 monthTonic–clonicSingle seizure4 monthNoneNone (sf) KCNQ3 c.2338C>T, p.Arg780Cys
II:2 M/84 monthFocal, tonic–clonicYes4,5PBNone (sf) KCNQ3 c.2338C>T, p.Arg780Cys
I5BFISI:2F/784 monthnaNanananananana PRRT2 c.649_650dupC
II:1M/465 monthFocalYes7 monthNoneNone (sf) PRRT2 c.649_650dupC
III 1 F/104 monthFocalYes4 monthPBNone (sf) PRRT2 c.649_650dupC
I6BFISII:2F/67<1 yearnaNananana PRRT2 c.649_650dupC
III:2F/43<1 yearnaNananana PRRT2 c.649_650dupC
IV:2 F/154 monthFocalYes5 monthPBNone (sf) PRRT2 c.649_650dupC
IV:3F/135 monthFocalYes6 monthVPAVPA PRRT2 c.649_650dupC
I7BFISIII:2F/38nanananananananana PRRT2 c.649_650dupC
III:3F/346 monthFocalYes8 monthNone None (sf) PRRT2 c.649_650dupC
IV:1 F/105 monthFocalYes5 monthPBNone (sf) PRRT2 c.649_650dupC
IV:2M/65 monthFocalYes7 monthPBNone (sf) PRRT2 c.649_650dupC
IV:3M/205 monthTonic–clonicNo7 monthNoneNone (sf) PRRT2 c.649_650dupC
I8BFISIII:1F/115 monthFocalNo5 monthPBNone (sf) PRRT2 c.649_650dupC
III:3 M/75 monthFocalYesnaNoneNone (sf) PRRT2 c.649_650dupC
III:4M/74 monthFocalYes6 monthNoneNone (sf) PRRT2 c.649_650dupC
I9BFIS/ICCAI:2F/43<1 yearnaNanaNoneNone (sf) PRRT2 c.649_650dupC
II:1 F/86 monthFocal, tonic–clonicYes8 monthPBNone (sf)PKD (3 year) PRRT2 c.649_650dupC
II:2M/76 monthFocal, tonic–clonicYes9 monthPBNone (sf)PKD (7 year) PRRT2 c.649_650dupC
I10BFIS II:1 M/156 monthFocalNo7 monthnaComplex partial seizures (5 year)na PRRT2 c.649_650dupC
II:2M/117 monthFocalYes7 monthVPANone (sf) PRRT2 c.649_650dupC
II:3M/118 monthFocalNo8 monthVPANone (sf) PRRT2 c.649_650dupC
I11BFISII:2F/304 month FocalNo8 monthnanaMigraine in adulthood SCN2A c.3956 G>A, Arg1319Gln
II:3F/287 monthTonic–clonicNo8 monthnananana SCN2A c.3956 G>A, Arg1319Gln
III:1M/18VPA7 monthVPA Choroid plexus papilloma SCN2A c.3956 G>A, Arg1319Gln
III:2 M/125.5 monthTonic–clonicYes6 month PB naNone (sf)na SCN2A c.3956 G>A, Arg1319Gln
I12BFISI:1M/425.5 monthFocal, tonic–clonic Yes6,5 monthPBNone (sf) SCN2A c.3003 T>A, Asn1001Lys
II:1 M/115 monthFocal, clonicYes5 monthPBNone (sf) SCN2A c.3003 T>A, Asn1001Lys
I13BFISII:3M/38<1 yearnaYes6 monthPBNone (sf) PRRT2 c.604_607delTCAC
III:3 M/66 monthFocalYes6 monthPBNone (sf) PRRT2 c.604_607delTCAC
I14BFISII:1M/65 monthFocalNo6 monthPBNone (sf) PRRT2 c.649_650dupC
II:2 F/55 monthFocalYes5 monthCBZNone (sf) PRRT2 c.649_650dupC
I15BFISII:2F/466 monthFocalYesnaPHTNone (sf) PRRT2 c.649_650dupC
III:3M/84 monthFocalYes4 monthCBZ None (sf) PRRT2 c.649_650dupC
III:5 F/106 monthTonic–clonicNo7 monthNone None (sf) PRRT2 c.649_650dupC
III:6M/75 monthTonic–clonicNo7 monthNoneNone (sf) PRRT2 c.649_650dupC
I16BFISI:1M/376 monthTonic–clonicNanaNoneNone (sf) KCNQ2 c.649A>G, p.Thr217Ala
II:1F/74 monthFocalSingle seizure4 monthNoneNone (sf) KCNQ2 c.649A>G, p.Thr217Ala
II:2 M/44 monthFocal, tonic–clonicYes6 monthPBNone (sf) KCNQ2 c.649A>G, p.Thr217Ala
I17BFISII:2F/61<1 yearnaNanananananana
III:2F/34<1 yearnaNanananananana
IV:1 M/67 monthTonic–clonicNo7 monthPBNone (sf)
I18BFISI:1M/736 monthnaYes12 monthNoneNone (sf) SCN2A c.4923A>C, Lys1641Asn
II:1M/416 monthnaYes8 monthNoneNone (sf) SCN2A c.4923A>C, Lys1641Asn
III:1 F/87.5 monthFocal, tonic Yes12 monthNoneNone (sf) SCN2A c.4923A>C, Lys1641Asn
I19BFIS/ICCAII:3M/69nanaNananananana PRRT2 c.649_650dupC
III:3F/43nanaNananananana PRRT2 c.649_650dupC
IV:2 F/87 monthFocalYes8 monthPBNone (sf) PRRT2 c.649_650dupC
I20BFISII:1F/356 monthFocalYesnanana PRRT2 c.649_650dupC
III:1 M/47 monthTonic–clonicNo8 monthnana PRRT2 c.649_650dupC
I21BFISI:1F/356 monthFocalYesnanana
II:1 M/47 monthTonic–clonicNo8 monthnana
I22BFIS/ICCAII:1M/71<1 yearnaNanaNoneNone (sf) PRRT2 c.649_650dupC
III:1M/37<1 yearnaYes<yearPBnaNone (sf)PKD (13 year) PRRT2 c.649_650dupC
IV:1 M/137 monthFocalYes8 monthPBnaNone (sf)PKD (11 year) PRRT2 c.649_650dupC
I23BFISII:2M/526 monthFocalYes6 monthPB None (sf) PRRT2 c.649_650dupC
II:4F/435 monthnaYesnaNoneNone (sf) PRRT2 c.649_650dupC
II:5M/41<1 yearnaNanaNoneNone (sf) PRRT2 c.649_650dupC
III:1M/236 monthFocalYesnaNoneNone (sf) PRRT2 c.649_650dupC
III:2M/105,5 monthTonic–clonicNo5 monthPHTNone (sf)congenital hemiparesis PRRT2 c.649_650dupC
III:7 M/86 monthFocalYes7 monthNoneNone (sf) PRRT2 c.649_650dupC
I24BFISI:2F/91nanaNanananananana PRRT2 c.649_650dupC
II:3F/59naTonicNonaNoneNone (sf) PRRT2 c.649_650dupC
III:1F/41naTonicNonaNoneNone (sf) PRRT2 c.649_650dupC
III:4F/346 monthTonicNo7 monthNoneNone (sf) PRRT2 c.649_650dupC
IV:1 F/138 monthFocalYes8 monthPBNone (sf) PRRT2 c.649_650dupC
I25BFISII:4F/92nanaNanananananana PRRT2 c.649_650dupC
III:1M/536 monthTonic–clonicNonaNone None (sf) PRRT2 c.649_650dupC
III:4F/366 monthFocalNonaNoneNone (sf) PRRT2 c.649_650dupC
IV:3F/248 monthTonic–clonicYes8 monthPBNone (sf) PRRT2 c.649_650dupC
IV:6F/325 monthTonic–clonicNo5 monthPBNone (sf) PRRT2 c.649_650dupC
V:1 F/86.5 monthTonic–clonicNo6 monthVPANone (sf) PRRT2 c.649_650dupC
I26BFISI:2F/354 monthnananaNonenanaNone (sf)na
II:1M/94 monthTonic–clonicNo4 monthPBNone (sf)
II:2 M/54 monthTonic–clonicNo4 monthPBNone (sf)
I27BFIS II:1 F/128 monthFocalYes8 monthNoneNone (sf) PRRT2 c.649_650dupC
II:2F/77 monthFocalYes8 monthNoneNone (sf) PRRT2 c.649_650dupC
I28BFISI:2F/32<1 yearnaNanananananana PRRT2 c.649_650dupC
II: 1M/76 monthFocalYes6 monthPBNone (sf) PRRT2 c.649_650dupC
II:3 F/36.5 monthFocalYes6 monthPBNone (sf) PRRT2 c.649_650dupC
II:5M/14 monthFocalYes4 monthPBNone (sf) PRRT2 c.649_650dupC
I29BFIS/ICCAI:2F/404 monthnaYes4 monthPBNone (sf)PKD (12 year) PRRT2 c.649_650dupC
II:1 M/125 monthFocalYes5 monthPBNone (sf) PRRT2 c.649_650dupC

Genetic analysis led to the identification of 41 mutations, 14 affecting KCNQ2, 1 affecting KCNQ3, 5 affecting SCN2A, and 21 affecting PRRT2 (Fig. 1). The detection rate of mutations in the entire cohort was 89%.

image

Figure 1. Detection rate of KCNQ2, KCNQ3, SCN2A, and PRRT2 mutations in 46 families with familial epilepsy of the first year of life.

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Families segregating KCNQ2 mutations

Among KCNQ2 mutations, five are missense (c.460T>G, p.Tyr154Asp; c.476G>A, p.Gly159Glu; c.587C>T, p.Ala196Val; c.649A>G, p.Thr217Ala; and c.1639C>T, p.Arg547Trp), one nonsense (c.1342C>T, Arg448X), one frameshift (c.592-594 delinsA, p.Arg198 fs), three affect a splice site (c.297-2A>G, c.1116+3A>G, c.1887+5G>A), and four are genomic deletion encompassing the entire or part of the KCNQ2 gene (exon1 deletion, exons 13–17 deletion, 470 kb deletion encompassing 16 genes, 187 kb deletion encompassing four genes).

The phenotype is consistent with typical BFNS in 7 of 14 KCNQ2 families (families N2–N8) with onset between 1 and 10 days of life in all affected family members (14 in total), clusters of focal or tonic–clonic seizures, and normal neurologic development at follow-up, including all those harboring genomic deletions involving KCNQ2 and additional flanking genes. In family N2 segregating a large genomic deletion including 16 genes, the proband manifested neonatal seizures only without further neurologic signs or symptoms. In addition to neonatal seizures, her mother showed complex focal seizures with bilateral temporal EEG abnormalities at age 10 years, and similar seizures were reported during adolescence in the affected grandmother (Table 1). In addition, simple febrile seizures were observed in the proband's mother of family N5 and in the proband of family N3. During the follow-up the proband of family N4 showed bilateral spikes and waves complexes over centrotemporal areas, but he never experienced further seizures. Interictal EEG was otherwise normal in all probands.

Six families carrying KCNQ2 mutations show BFNIS. Families NI1, NI3, NI4 include individuals with both neonatal and infantile onset, whereas the onset of seizures in families NI2, NI8, and NI9 is between 2 and 6 months of age. The electroclinical features of the seizures in BFNIS families do not differ from those reported in BFNS. However, 3 of 20 affected individuals manifested a unique isolated seizure, a feature that is not observed in any BFNS patient (Table 1).

The proband of family NI1 also experienced focal motor seizures that occurred at age 3 and 11 months after an initial remission. Seizures persisted until the age of 10 years, he was never treated, and currently seizures and centrotemporal spikes remitted spontaneously (Table 1). His sibling manifested neonatal seizures followed by epileptic spasms at the age of 12 months and two febrile seizures at age 19 months. He was treated with phenobarbital and vigabatrin after the onset of epileptic spasms. Currently he is not receiving antiepileptic drug (AED) treatment. Moreover, during the follow-up, the individual II:3 of family NI8 presented at the age of 6 years few convulsive seizures with facial clonia during sleep after typical clusters of focal seizures at age 1 month. In addition, one family segregating a KCNQ2 mutation displayed BFIS (I16). This family accounts for three affected individuals with onset between 4 and 6 months of life. All subjects presented with focal seizures with secondary generalization, characterized by head and/or eye deviation, diffuse hypertonus, cyanosis, and followed by bilateral clonia. The proband presented another cluster of seizures at the age 6 months, requiring treatment with phenobarbital. His sibling presented only one convulsive seizure at the age of 4 months (Table 1).

Families segregating KCNQ3 mutations

A single KCNQ3 mutation (c.2338C>T; p.Arg780Cys) was identified in a family with typical BFIS (I4). The proband of family I4 presented a single cluster of seizures characterized by staring, eye deviation, and hypertonus of brief duration at the age of 3 months and 25 days. His sibling presented with an isolated secondarily generalized focal seizure at the age of 4 months, and their father had two isolated convulsive seizures at the age of 5 and 7 months. Their EEG studies were all normal and none of them had seizure recurrence or febrile seizures during the follow-up (Table 1). At follow-up, none of the affected individuals displayed seizures, impairment in intellectual development, or other neurologic disease.

Families segregating SCN2A mutations

Four of five SCN2A mutant families were described previously (Berkovic et al., 2004; Striano et al., 2006b). Family NI6 and NI7 were diagnosed as BFNIS with multiple affected individuals affected by early onset infantile seizures and no neonatal seizures, whereas family I11 and I12 displayed BFIS.

Family I18 is an unreported family characterized by a missense mutation (c.4923A>C, Lys1641Asn) segregating in three members who presented, respectively, at the age of 6, 6, and 7.5 months focal seizures with secondary generalization. In particular, the proband presented two focal seizures characterized by stare, mild upper limbs hypertonus at the age of 7.5 months followed by isolated seizures at the age of 10 and 12 months. Seizures were isolated and brief in duration in all subjects (Table 1).

In all 31 individuals carrying SCN2A mutations, seizures remitted within the first year of life and additional seizures did not manifest later in life.

Families segregating PRRT2 mutations

We identified 21 families segregating a PRRT2 mutation. Among these, 15 have been previously reported (I2, I3, I5, I7, I8, I10, I14, I15, I19, I20, I22–I24, I26, I27) (Schubert et al., 2012). These families account for 78 affected individuals and were all diagnosed as BFIS. All individuals experienced afebrile seizures with onset between 4 and 8 months of age, but one who manifested two febrile seizures at age 14 months (Individual II:6 of family I1) (Table 1). The offset of seizure in most patients was within the first year of life with or without AED therapy. A single individual experienced complex partial seizures from the age of 5 years (Ind. II:1, family I10). Individual II:6 of family I1 did not show typical infantile seizures but experienced two febrile seizures at the age of 14 months. Five individuals of three families showed PKD in childhood or adolescence (Table 1).

Families negative to genetic analysis

BFNS

A single BFNS family was negative at mutational screening (N1). The family accounts for 10 affected individuals. The proband is a 52-year-old woman (Ind. II:2) who presented convulsions from the second to the 22nd day of life; thereafter seizures spontaneously subsided. At age 6 years, she started presenting complex partial seizures that persisted over the years despite different AED combinations. She is actually experiencing monthly seizures on topiramate 300 mg/day and lamotrigine 150 mg/day. Brain MRI showed right hippocampal atrophy. Her father also presented early onset but not further specified epileptic fits. Three of six siblings presented neonatal convulsions from the age of 2 days up to 15 to 22 days. Her 47-year-old sister (II-5) also presented late-onset temporal lobe seizures beginning at 17 years, controlled by phenobarbital 125 mg/day. Brain MRI was normal. In the next generation, two of eight subjects presented convulsions from the second to the 22nd day of life. In addition, the 23-year-old proband's daughter (III:8) also presented rare generalized tonic–clonic seizures during adolescence, but she is now seizure-free without antiepileptic treatment.

BFNIS

KCNQ2/KCNQ3/SCN2A mutations were not detected in one nuclear BFNIS family (NI5). This family includes two affected subjects. The proband is a 6-year-old girl who manifested a cluster of seizures at the age of 1 months and 3 weeks. Episodes were of brief duration, and were characterized by diffuse hypertonus followed by head and eye deviation. No additional seizure occurred at follow-up and no therapy was administered. Her mother presented two tonic–clonic seizures at the age of 17 and 18 days of life. She was successfully treated with valproate until the age of 4 years. No other seizures were reported.

BFIS

Three BFIS families were negative at genetic analysis. The proband of family I17 is a 6-year-old boy who experienced isolated apparently generalized seizures from the age of 7 months. Seizures lasted 60–80 s and were characterized by diffuse hypertonus and loss of consciousness, motor arrest, and eye deviation. At age 9 months he was treated with phenobarbital (PB) until the age of 3 years. No seizures or additional neurologic symptoms were reported at the last follow-up visit at age 6 years. The mother and the maternal grandmother reported similar isolated seizures during infancy before the first year of age. Neurologic history and neurologic examination at age 35 and 69 was negative for both of them.

In family I21 the proband started to have seizures in cluster at the age of 7 months. Successive clusters appeared at the age of 10 and 12 months. In addition, he presented isolated seizures at the age of 18 and 20 months. His mother experienced a single cluster at the age of 6 months. They do not experience further seizures.

Family I26 includes two affected brothers and possibly an affected mother. The onset of seizures in the 9-year-old brother was at 3 months and 25 days. Seizures were brief, isolated, and occurred for 15 days always during sleep before PB treatment was started. Semiology of seizures includes eye deviation and hypertonus and bilateral clonic manifestation without automatisms and focal motor symptoms. Therapy was withdrawn at the age of 3 years, and no additional epileptic or neurologic symptoms were reported. His 5-year-old brother experienced similar epileptic fits from the age of 4 months and 10 days. Seizures were brief, isolated, apparently generalized, and occurred only during sleep. PB was started and no additional seizures were reported during follow-up. The mother of the affected siblings reported seizures during infancy, but no clinical or anamnestic data were available.

Genotype–phenotype correlations

The age of onset represents the key parameter for syndromic classification within this group of disorders. Most families with onset after 6 months of age are associated to PRRT2 mutations (21/29, 72%); on the other hand, families with seizure onset before 6 months of age frequently segregate KCNQ2 mutations (13/16, 76%).

PKD is specifically found only in families segregating PRRT2 mutations, although in a minority of affected members (5/68, 7%). Afebrile seizures after the offset of neonatal/infantile seizures occur more frequently in families carrying KCNQ2 mutations, compared to families segregating KCNQ3, SCN2A, or PPRT2 mutations (4/39 vs 1/78, p = 0.042, Fisher's exact test). Seizures with delayed onset are usually focal (rolandic or complex type). Semiology of neonatal/infantile seizures is usually focal or tonic–clonic and does not differ among the different groups of families. Seizures occur more frequently in clusters, but a significant proportion of affected individuals carrying KCNQ2, KCNQ3, SCN2A, or PRRT2 mutations manifest isolated seizures only.

Five of 46 families do not show mutations in any of the candidate genes. Families NI5, I17, I21, and I25 do not show distinctive clinical features. In family BFNS N1, three of six affected members manifested epilepsy later in life, classified as temporal lobe epilepsy in two of them, an association has not been reported in other families.

Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

We aimed to establish the extent of genetic overlap between BFNS, BFNIS, and BFNS and to develop a rational approach for molecular testing. Our results showed that the overall detection rate reaches 89% when all genes are screened and does not vary among the different syndromes. However, the mutation pattern of each syndrome is peculiar.

In BFNS, mutations specifically involve KCNQ2. In BFNIS, two genes are involved (KCNQ2, six families; SCN2A, two families). BFIS families are the most genetically heterogeneous with all four genes involved, even though about 70% of them segregate a PRRT2 mutation (Fig. 1). Our data highlight the important role of KCNQ2 in the entire spectrum of disorders, although progressively decreasing as the age of onset advances. A KCNQ2 mutation was previously identified in a single family with seizure onset between 2 and 4 months of age, which may fulfill the diagnosis of BFNIS (Zhou et al., 2006). However, the common involvement of KCNQ2 in families with infantile onset represents a novel finding with significant implications for molecular testing. Notably, all genomic deletions encompassing KCNQ2 are clustered in families with neonatal onset, whereas KCNQ2-related families with delayed onset segregate point mutations (Heron et al., 2007; Kurahashi et al., 2009). This finding may link early neonatal seizures to more a pronounced loss-of function of KCNQ2. In addition, we showed that KCNQ3 mutations might be also involved in families with infantile seizures.

Taken together our data indicate an important role of K-channel genes beyond the typical neonatal epilepsies.

The identification of a novel SCN2A mutation in a family with infantile seizures with onset between 6 and 8 months provides further confirmation that this gene is not specifically associated to BFNIS and is also involved in families with a delayed age of onset (Striano et al., 2006b).

On the other hand, our data indicate that PRRT2 mutations are clustered in families with BFIS. The specific age-dependent expression of mutations in these different genes suggests the pivotal role of developmental mechanisms in this group of disorders.

Afebrile seizures during follow-up occur more frequently in individuals carrying KCNQ2 mutations. However, large studies are needed to establish a robust correlation. Late-onset seizures were reported to be typically focal, and more frequently of the rolandic type (Maihara et al., 1999; Lee et al., 2000; Coppola et al., 2003; Singh et al., 2003). The occurrence of complex focal seizures in two subjects of family N2 and of rolandic seizures in the proband of family NI1 confirms this observation. We also showed that large genomic deletions involving KCNQ2 and flanking genes do not necessarily cause afebrile or febrile seizures later in life, in accordance with earlier reports (Kurahashi et al., 2009).In addition, PKD emerges as a distinctive feature of PRRT2 families, although not observed in many affected individuals in our series. The variable interfamilial and intrafamilial expressivity of PRRT mutations is still unknown and likely due to individual genetic or acquired factors.

Recently, the current definition of three different conditions has been challenged in view of the clinical similarities observed in these patients, including the appearance of focal seizures usually in clusters in otherwise normal neonates or infants, the spontaneous remission of seizures usually before the first year of age, the normal long-term development, and a unique clinical entity characterized by seizures with onset within the first year of life and heterogeneous etiology has been proposed (Mulley et al., 2011).

Our study further highlights that BFNS, BFNIS, and BFIS share the significant clinical features and may have a common genetic etiology. However, we showed that the age of onset of seizures—although limited to few months within the first year of life—is significantly correlated to underlying genetics as, about 90% of the typical BFNS families are linked to KCNQ2 compared to only 3% of the BFIS families, for which PRRT2 represents the major gene. The identification of KCNQ2 mutations in the majority of BFNIS families indicates a closer relationship between BFNS and BFNIS. The cumulative analysis of KCNQ2, KCNQ3, SCN2A, and PRRT2 yields to a mutation rate of about 90% in this group of disorders.

In conclusion, our study indicates that mutational screening should be initially accomplished for KCNQ2 in both BFNS and BFNIS, and for PRRT2 in BFIS families. SCN2A and KCNQ3 mutations represent a rare cause of familial epilepsy of the first year of life, and genetic testing should be considered as last option for diagnostic purposes. It is also expected that the spectrum of epilepsies associated with mutations in these genes will expand in the next years. In fact, de novo mutations in KCNQ2 (Weckhuysen et al., 2012) and SCN2A (Ogiwara et al., 2009) have been identified in neonates with intractable early onset epileptic encephalopathy. The phenotypic variations among patients might be due to the significant difference in functional properties of mutant channels. However, further research will clarify whether KCNQ2/3 and SCN2A screening should be included in the diagnostic work-up of refractory neonatal seizures of unknown origin.

In the “exomic” era, integrated genome-wide tests are becoming available and will probably soon overcome the “cost” issue of multiple testing. On the other hand, a clear clinical perspective represents a mandatory requirement to properly interpret large amount of genetic data within a diagnostic setting.

Acknowledgments

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

We thank the Italian League Against Epilepsy for supporting the study.

Disclosure

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

The authors declare no conflicts of interest. 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.

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  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
epi12089-sup-0001-DataS1.pdfapplication/PDF85KData S1. Pedigrees and segregation analysis of PRRT2 mutations.

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