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. 2009 Aug 26;29(34):10764-78.
doi: 10.1523/JNEUROSCI.2475-09.2009.

A functional null mutation of SCN1B in a patient with Dravet syndrome

Gustavo A Patino  1 , Lieve R F Claes, Luis F Lopez-Santiago, Emily A Slat, Raja S R Dondeti, Chunling Chen, Heather A O'Malley, Charles B B Gray, Haruko Miyazaki, Nobuyuki Nukina, Fumitaka Oyama, Peter De Jonghe, Lori L Isom
Affiliations

A functional null mutation of SCN1B in a patient with Dravet syndrome

Gustavo A Patino et al. J Neurosci. .

Abstract

Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Na(v)1.1 alpha subunits. Sodium channels are modulated by beta1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Na(v)1.1 alpha subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of beta1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b(-/-) versus Scn1b(+/+) mice. Scn1b(-/-) CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a(+/-) model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b(-/-) mice seize spontaneously, the seizure susceptibility of Scn1b(+/-) mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.

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Figures

Figure 1.
Figure 1.
SCN1B homozygous mutation found in Dravet syndrome. A, Chromatogram showing the c.373C>T homozygous mutation in the SCN1B exon 3 amplicon in the proband (top lane), whereas both parents are heterozygous (bottom 2 lanes) for the mutation. The mutation results in a change of arginine (R) at position 125 to cysteine (C) in the amino acid sequence. B, Alignment of the corresponding region of the sodium channel β1 subunit amino acid sequence across multiple species showing the high conservation of p.R125. C, Topology of the sodium channel β1 subunit. The extracellular domain contains an Ig loop bound by a disulfide bridge (S–S). A mutation in the distal cysteine of the disulfide bridge (p.C121; red circle) has been reported in families with GEFS+, as have two other mutations (p.170-E74del and p.R85C/p.85H; red circles). The amino acid position of the mutation found in the proband (p.R125C) is marked by the yellow circle. Domains shared by both splice variants, β1 and β1B, are shown in blue and include only the extracellular region. β1B contains a novel domain encoded by a retained intron. The transmembrane domain (TM) of β1 is followed by a short intracellular domain. D, The genotyping of four STR markers around the SCN1B site confirms the ancestral haplotype.
Figure 2.
Figure 2.
p.R125C and β1WT have no effect on the properties of sodium current in HEKrNav1.1 cells. Cells stably expressing rat Nav1.1 in a HEK-293 background (HEKrNav1.1) were transiently cotransfected with GFP and either β1WT (filled circles) or p.R125C (open triangles). HEKrNav1.1 cells transfected only with EGFP (filled squares) were used as negative controls. Whole-cell patch-clamp recordings of sodium currents were performed as described in Materials and Methods. A, B, Sodium current density is unchanged in the presence and absence of β1 subunits (A), as is the voltage dependence of activation (B). C, D, A similar lack of effect from either transfected β1 subunit is observed for the voltage dependence of inactivation (C) and recovery from inactivation (D). Insets depict the protocol scheme. A and B were obtained using the same protocol. Data points represent mean ± SEM, and solid lines represent fit to the means. Biophysical properties are provided in Table 1.
Figure 3.
Figure 3.
p.R125C does not modulate sodium current expressed by Nav1.2 in SNaIIa cells. SNaIIa cells were stably transfected with either β1WT (filled circles) or p.R125C (open triangles) and used for whole-cell patch-clamp experiments as described in Materials and Methods. Untransfected cells (filled squares) were used as negative controls. β1WT increased the current density (A), negatively shifted the voltage dependence of activation (B) and inactivation (C), slowed the recovery from inactivation (D), and reduced the availability of sodium channels under high-frequency stimulation (inset shows the protocol scheme) compared with cells expressing α alone (E). In contrast, p.R125C did not modulate sodium current properties. Protocols for A–D are the same as in Figure 2. Biophysical properties can be found in Table 2.
Figure 4.
Figure 4.
p.R125C is poorly expressed at the cell surface at physiological temperatures. A, Comparison of total cellular expression of β1WT versus p.R125C in 1610 cells. Representative Western blot of 1610 cells stably transfected with V5-tagged p.R125C (lane 3), demonstrating that the expression of the mutant protein is comparable with β1WT (lane 2). Untransfected cells (UT; lane 1) were used as a negative control. Reprobing the blot with anti-α-tubulin confirmed the presence of protein in all lanes. B, Cell surface expression of β1WT versus p.R125C. HEKrNav1.1 or 1610 cells stably transfected with V5-tagged β1WT or p.R125C were surface biotinylated, and the biotinylated proteins were probed as described in Materials and Methods. Untransfected cells show no anti-V5 immunoreactivity (UT; lane 1). Cells transfected with β1WT show robust cell surface expression (lane 2). Faint or no cell surface expression was detected in multiple clones of cells transfected with the mutant p.R125C (HEKrNav1.1 cells, samples 1 and 2; 1610 cells, samples 3–5). For comparison, sample 4 is the same cell line used to detect total cellular expression in A, p.R125C. C, Box plots of band intensities measured using NIH ImageJ for clones of HEKrNav1.1 and 1610 cells transfected with β1WT and p.R125C and processed to detect surface biotinylated proteins as described in Materials and Methods. We calculated a significant difference (*p < 10−6, Mann–Whitney U test) between the level of cell surface expression of β1WT (n = 18 experiments) compared with p.R125C (n = 15 experiments). D, p.R125C is poorly expressed at the cell surface in the presence of human Nav1.1. Top, Comparison of total cellular expression of β1WT versus p.R125C in HEKhNav1.1 cells. Representative Western blot of HEKhNav1.1 cells stably transfected with V5-tagged p.R125C (lane 2), demonstrating that the expression of the mutant protein is comparable with β1WT (lane 1). Middle, Reprobing the blot with anti-α-tubulin confirmed equal loading of protein in both lanes. Bottom, Cell surface expression of β1WT versus p.R125C. HEKhNav1.1 cells transiently transfected with V5-tagged β1WT or p.R125C were surface biotinylated, and the biotinylated proteins were probed as described in Materials and Methods. Cells transfected with β1WT show robust cell surface expression (lane 1). Faint or no cell surface expression was detected in cells transfected with the mutant p.R125C (lane 2). The blot is representative of triplicate experimental repeats. Molecular weight markers are in kilodaltons.
Figure 5.
Figure 5.
Cell surface expression of p.R125C is rescued at low temperature. The 1610 cells stably transfected with V5-tagged p.R125C were incubated at 37°C or 27°C for 48 h and then surface biotinylated as described in Materials and Methods. The resulting Western blot was probed with anti-V5 antibody. Incubation at 27°C rescued the cell surface expression of p.R125C, resulting in the presence of β1-immunoreactive bands at 40 kDa and higher, likely representing various levels of avidin attachment, whereas no band is detectable for the cells incubated at 37°C. Molecular weight markers are in kilodaltons.
Figure 6.
Figure 6.
p.R125C modulates sodium currents expressed by Nav1.2 in Xenopus oocytes. Xenopus laevis oocytes were injected with the cRNA encoding Nav1.2 alone (filled squares), with β1WT (filled circles), or with p.R125C (open triangles). The p.R125C mRNA was also diluted 1:50 before injection (open diamonds). Neither β1WT nor p.R125C had any measurable effect on the voltage dependence of activation of Nav1.2. A–C, p.R125C (A) modulates the voltage dependence of inactivation (B) and rate of recovery from inactivation (C) of Nav1.2 currents similar to β1WT. Insets show protocol schemes. Data points represent mean ± SEM. Solid lines represent fits to the means. Biophysical properties are provided in Table 3.
Figure 7.
Figure 7.
Time to death attributable to status epilepticus of Scn1b+/− mice is similar to Scn1b+/+ mice. Mice with either one (Scn1b+/−) or two (Scn1b+/+) copies of Scn1b were injected with PTZ to induce seizures. After a dose of 80 mg/kg, the majority of mice died as a consequence of status epilepticus. Cumulative survival curves show no significant differences between Scn1b+/− mice (broken line; n = 8) and Scn1b+/+ mice (solid line; n = 8). p = 0.383, log-rank test.
Figure 8.
Figure 8.
β1 is expressed in hippocampal CA3 bipolar neurons. Acutely dissociated hippocampal CA3 bipolar neurons from P10 Scn1b+/+ mice were fixed with 4% paraformaldehyde and stained for β1 using anti- β1intra antibody. A, Bright-field images. B, Anti-β1intra, green; DAPI, blue. Scale bars, 20 μm.
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