Description of the pedigree.
The pedigree shown in contains 4 generations of individuals with FS, FS+, or epilepsy and shows a clear autosomal dominant pattern of inheritance. The family phenotype is highly consistent with GEFS+.
Figure 1 Four-generation pedigree with GEFS+, with symbols denoting each individual’s phenotype
Fifteen individuals were designated as affected (). None of them had a history of any illness or CNS insult likely to be related to epilepsy (e.g., severe head injury, stroke, brain tumor, brain surgery, brain infection), and all except III:1 (described below) were intellectually normal. Six individuals had FS: II:10, III:3, III:5, III:9, IV:1, and IV:3. All had onset of FS between ages 8 and 12 months, and resolution before age 4 years in those for whom that information was available; none had afebrile seizures.
Table 2 Clinical characteristics of affected individuals in GEFS+ family
Two subjects, II:5 and II:14, had FS and later epilepsy. Individual II:5 had afebrile seizures described as activity arrest with impairment of speech production, the perception that words that were read were repeating themselves, and fear. His seizures were typically induced by reading, and he had a single afebrile secondarily generalized convulsion. He was classified as FS and localization-related epilepsy. Individual II:14 had recurrent afebrile generalized tonic-clonic seizures (GTCs) with no aura or localizing symptoms reported at the onset and therefore was classified as FS and epilepsy with undetermined localization-related vs generalized onset.
Individuals III:8 and III:13 had FS+, in III:8 on the basis of FS accompanied by afebrile GTCs and in III:13 because FS persisted until age 7 years. Individual III:8 also had afebrile convulsive seizures with semiology consistent with focal onset, described as nervousness (screaming, hyperactivity, or calling her mother) and hypersalivation at the onset.
Subject III:1 had onset of FS at 18 months and had a total of 20 FS. He had afebrile GTCs sometimes as often as 4 per month between ages 3 and 6 years, myoclonic jerks reported at age 9 years, brief staring episodes diagnosed clinically as absence seizures, and complex partial seizures described as paroxysmal periods of altered responsiveness lasting up to 3 minutes between ages 5 and 10 years. Developmental milestones were normal in the first year of life, but he went on to have marked cognitive and behavioral difficulties. MRI of the brain was normal at age 7 years. EEG was normal at age 4 years; later EEGs, performed between ages 6 and 13 years, showed left- or right-posterior spike-wave complexes with generalization, bifrontal spike-wave complexes, and irregular generalized spike-wave complexes. This individual’s history satisfied criteria for FS+ and was also suggestive of borderline severe myoclonic epilepsy of infancy (SMEB) because of the mixture of seizure types and developmental delay after onset of recurrent FS in a previously normal child. However, the clinical picture also differed from SMEB in several aspects: onset after 1 year, normal EEG at age 4 years, and lack of evidence of status epilepticus. Thus we designated this person as FS+ and severe epilepsy with developmental delay.
Individuals I:1, II:2, and III:2 had epilepsy comprised of recurrent afebrile GTCs with no aura or localizing symptoms at the onset, therefore of undetermined onset. Individual II:4 had only an isolated unprovoked seizure but also had a child affected with epilepsy. In our analyses, we considered individual II:4 to be affected.
Linkage analyses and identification of a critical region.
lists the maximum multipoint lod scores for markers in the CIDR panel located closest to the known GEFS+ genes (SCN1B, SCN1A, SCN2A, GABRG2, and GABRD) and at the GEFS+ and familial FS loci. There was no evidence for linkage to any of these previously delineated loci, and most could effectively be excluded (lod <−2.0).
In the initial genome-wide microsatellite marker screen using the CIDR data, the maximum 2-point lod score was 3.21 for D6S474 (chromosome 6q21), and the multipoint lod score was 3.66 in the same region. This was the only region associated with a lod score greater than 2.05. Reanalysis of all available marker data on chromosome 6, including the 29 newly typed markers, yielded a maximum 2-point and multipoint lod score of 4.68 at D6S1706 () assuming 90% penetrance. In the model-free analysis, the maximum PPL for the 2-point and multipoint methods was 0.89 at marker D6S1706 (), indicating an 89% posterior probability of linkage, consistent with the results of the model-based linkage analysis.
Figure 2 Results of linkage analysis using model-based and PPL methods
Haplotype analysis using SIMWALK2 indicated that all affected individuals share a haplotype of alleles at 7 markers between D6S1021 and D6S304 (). The haplotype is delimited by 2 key recombination events: the first between markers D6S962 and D6S1021 in individual III:5 and his father II:7 (an obligate carrier), and the second between markers D6S304 and D6S287 in individual III:13. These data place the minimal genetic region (MGR) between markers D6S962 and D6S287 (i.e., the markers flanking the haplotype), an 18.1-megabase (Mb) region at 6q16.3-22.31 that spans from 107.15 to 119.55 cM on the deCODE map.
Two unaffected subjects, III:6 and III:12, also have recombinant haplotypes in this region (). Subject III:6 carries the portion of the disease haplotype centromeric to D6S1698, and subject III:12 carries the disease haplotype centromeric to D6S941. The evidence from subject III:6 suggests that the gene is likely to be telomeric to D6S941, which would reduce the MGR slightly, to the region between D6S941 and D6S287. Because of incomplete penetrance, the evidence from unaffected individuals is less definitive than that from affected individuals; thus, we have conservatively defined the MGR based on the data from affected individuals only. However, assuming 90% penetrance, it is unlikely that both III:6 and III:12 are nonpenetrant carriers; hence the disease locus is likely to be telomeric to D6S941.
Mutation screening of candidate genes in the critical region.
In the region delimited by microsatellite markers D6S962 and D6S287, we found 137 entries for genes, complementary DNAs, and messenger RNAs in the UCSC Genome Bioinformatics 2006 genome assembly. Of these, 19 were duplicate or overlapping entries, leaving 118 unique entries. We considered 42 of these entries to be poor candidates because they represented either hypothetical proteins or poorly characterized entries. We considered the following 16 genes to be very good candidates based on protein function (e.g., membrane and transmembrane proteins, solute carrier proteins, proteins involved in intracellular trafficking) and/or reported high levels of brain expression: MAN1A1, NUS1, GOPC, DCBLD1, PIST, GPR6, GPRC6A, KPNA5, FAM26E, DSE, HDAC2, SLC16A10, SLC22A16, SLC35F1, SNX3, and NR2E1.
Sequencing did not reveal any sequence changes that were predicted to alter the amino acid sequence or splicing in the exons or exon-intron junctions of these 16 candidate genes. We did observe some synonymous sequence variants, including previously reported single nucleotide polymorphisms.
Copy number assessment.
When we used an oligonucleotide array of chromosome 6 to screen for deletions or duplications, we did not observe any copy number abnormalities in either of the 2 affected subjects or 1 unaffected subject screened (data available on request).