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A de novo missense mutation of GABRB2 causes early myoclonic encephalopathy

View Article: PubMed Central - PubMed

ABSTRACT

Background: Early myoclonic encephalopathy (EME), a disease with a devastating prognosis, is characterised by neonatal onset of seizures and massive myoclonus accompanied by a continuous suppression-burst EEG pattern. Three genes are associated with EMEs that have metabolic features. Here, we report a pathogenic mutation of an ion channel as a cause of EME for the first time.

Methods: Sequencing was performed for 214 patients with epileptic seizures using a gene panel with 109 genes that are known or suspected to cause epileptic seizures. Functional assessments were demonstrated by using electrophysiological experiments and immunostaining for mutant γ-aminobutyric acid-A (GABAA) receptor subunits in HEK293T cells.

Results: We discovered a de novo heterozygous missense mutation (c.859A>C [p.Thr287Pro]) in the GABRB2-encoded β2 subunit of the GABAA receptor in an infant with EME. No GABRB2 mutations were found in three other EME cases or in 166 patients with infantile spasms. GABAA receptors bearing the mutant β2 subunit were poorly trafficked to the cell membrane and prevented γ2 subunits from trafficking to the cell surface. The peak amplitudes of currents from GABAA receptors containing only mutant β2 subunits were smaller than that of those from receptors containing only wild-type β2 subunits. The decrease in peak current amplitude (96.4% reduction) associated with the mutant GABAA receptor was greater than expected, based on the degree to which cell surface expression was reduced (66% reduction).

Conclusion: This mutation has complex functional effects on GABAA receptors, including reduction of cell surface expression and attenuation of channel function, which would significantly perturb GABAergic inhibition in the brain.

No MeSH data available.


Related in: MedlinePlus

Expression of mutant β2 (p.Thr287Pro) subunits reduces the peak current amplitudes of γ-aminobutyric acid-A (GABAA) channels. (A) Representative GABA current traces obtained following rapid application of 1 mM GABA for 4 s to lifted HEK293T cells voltage-clamped at −20 mV. The current traces from GABAA receptors containing the mutant β2(T287P) was compared with their respective wild-type (wt) α1β2γ2s current traces. (B) Bar graph shows the average peak current from cells expressing wt and mutant GABAA receptors. Values represent mean±SEM (n=10 patches). Statistical differences were determined using unpaired t-test; **** indicates p<0.0001 compared with the wt condition.
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JMEDGENET2016104083F6: Expression of mutant β2 (p.Thr287Pro) subunits reduces the peak current amplitudes of γ-aminobutyric acid-A (GABAA) channels. (A) Representative GABA current traces obtained following rapid application of 1 mM GABA for 4 s to lifted HEK293T cells voltage-clamped at −20 mV. The current traces from GABAA receptors containing the mutant β2(T287P) was compared with their respective wild-type (wt) α1β2γ2s current traces. (B) Bar graph shows the average peak current from cells expressing wt and mutant GABAA receptors. Values represent mean±SEM (n=10 patches). Statistical differences were determined using unpaired t-test; **** indicates p<0.0001 compared with the wt condition.

Mentions: Lower surface β2 subunit content may reduce receptor channel current amplitude, because this subunit is required for α1β2 or α1β2γ2 receptor assembly.19 We then measured the current amplitudes of α1β2γ2 receptors or α1β2(Thr287Pro)γ2 using patch-clamp whole-cell recording. We recorded currents evoked by 4-s applications of GABA from HEK293T cells co-transfected with α1 and γ2S subunits and either wild-type or mutant β2 subunits (figure 6A). The peak amplitude of the α1β2(Thr287Pro)γ2S receptor currents (0.26±0.10 nA, n=10) were smaller than that of wild-type α1β2γ2S receptor currents (7.3±0.51 nA, n=10) (figure 6B). This is consistent with the reduced surface expression of mutant β2(Thr287Pro). Furthermore, the decreased peak amplitude associated with the α1β2(Thr287Pro)γ2S receptor (96.4% reduction) was much more pronounced than would have been expected based on the degree to which surface expression was reduced (66% reduction), indicating that the mutation engenders Cl− channel dysfunction in addition to its deleterious effects on trafficking by reducing surface subunit expression (figure 4D).


A de novo missense mutation of GABRB2 causes early myoclonic encephalopathy
Expression of mutant β2 (p.Thr287Pro) subunits reduces the peak current amplitudes of γ-aminobutyric acid-A (GABAA) channels. (A) Representative GABA current traces obtained following rapid application of 1 mM GABA for 4 s to lifted HEK293T cells voltage-clamped at −20 mV. The current traces from GABAA receptors containing the mutant β2(T287P) was compared with their respective wild-type (wt) α1β2γ2s current traces. (B) Bar graph shows the average peak current from cells expressing wt and mutant GABAA receptors. Values represent mean±SEM (n=10 patches). Statistical differences were determined using unpaired t-test; **** indicates p<0.0001 compared with the wt condition.
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Related In: Results  -  Collection

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JMEDGENET2016104083F6: Expression of mutant β2 (p.Thr287Pro) subunits reduces the peak current amplitudes of γ-aminobutyric acid-A (GABAA) channels. (A) Representative GABA current traces obtained following rapid application of 1 mM GABA for 4 s to lifted HEK293T cells voltage-clamped at −20 mV. The current traces from GABAA receptors containing the mutant β2(T287P) was compared with their respective wild-type (wt) α1β2γ2s current traces. (B) Bar graph shows the average peak current from cells expressing wt and mutant GABAA receptors. Values represent mean±SEM (n=10 patches). Statistical differences were determined using unpaired t-test; **** indicates p<0.0001 compared with the wt condition.
Mentions: Lower surface β2 subunit content may reduce receptor channel current amplitude, because this subunit is required for α1β2 or α1β2γ2 receptor assembly.19 We then measured the current amplitudes of α1β2γ2 receptors or α1β2(Thr287Pro)γ2 using patch-clamp whole-cell recording. We recorded currents evoked by 4-s applications of GABA from HEK293T cells co-transfected with α1 and γ2S subunits and either wild-type or mutant β2 subunits (figure 6A). The peak amplitude of the α1β2(Thr287Pro)γ2S receptor currents (0.26±0.10 nA, n=10) were smaller than that of wild-type α1β2γ2S receptor currents (7.3±0.51 nA, n=10) (figure 6B). This is consistent with the reduced surface expression of mutant β2(Thr287Pro). Furthermore, the decreased peak amplitude associated with the α1β2(Thr287Pro)γ2S receptor (96.4% reduction) was much more pronounced than would have been expected based on the degree to which surface expression was reduced (66% reduction), indicating that the mutation engenders Cl− channel dysfunction in addition to its deleterious effects on trafficking by reducing surface subunit expression (figure 4D).

View Article: PubMed Central - PubMed

ABSTRACT

Background: Early myoclonic encephalopathy (EME), a disease with a devastating prognosis, is characterised by neonatal onset of seizures and massive myoclonus accompanied by a continuous suppression-burst EEG pattern. Three genes are associated with EMEs that have metabolic features. Here, we report a pathogenic mutation of an ion channel as a cause of EME for the first time.

Methods: Sequencing was performed for 214 patients with epileptic seizures using a gene panel with 109 genes that are known or suspected to cause epileptic seizures. Functional assessments were demonstrated by using electrophysiological experiments and immunostaining for mutant &gamma;-aminobutyric acid-A (GABAA) receptor subunits in HEK293T cells.

Results: We discovered a de novo heterozygous missense mutation (c.859A&gt;C [p.Thr287Pro]) in the GABRB2-encoded &beta;2 subunit of the GABAA receptor in an infant with EME. No GABRB2 mutations were found in three other EME cases or in 166 patients with infantile spasms. GABAA receptors bearing the mutant &beta;2 subunit were poorly trafficked to the cell membrane and prevented &gamma;2 subunits from trafficking to the cell surface. The peak amplitudes of currents from GABAA receptors containing only mutant &beta;2 subunits were smaller than that of those from receptors containing only wild-type &beta;2 subunits. The decrease in peak current amplitude (96.4% reduction) associated with the mutant GABAA receptor was greater than expected, based on the degree to which cell surface expression was reduced (66% reduction).

Conclusion: This mutation has complex functional effects on GABAA receptors, including reduction of cell surface expression and attenuation of channel function, which would significantly perturb GABAergic inhibition in the brain.

No MeSH data available.


Related in: MedlinePlus