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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

p.Thr287Pro mutation in the β2 subunit and γ-aminobutyric acid-A (GABAA) receptor is shown. (A) Cartoon representation of the location of the p.Thr287Pro mutation of β2 subunit of the GABAA receptor. (B) Three-dimensional structural model of the GABAA receptor that is composed predominantly of two α1 (blue ribbons), two β2 (red ribbons) and one γ2 (grey ribbon) subunits in the mammalian central nervous system. β2 subunits have four transmembrane domains (TM1 to TM4). The GABRB2 de novo p.Thr287Pro mutation is mapped onto the β2 subunit in black at the second transmembrane domain (TM2). (C) Extracellular view of the transmembrane domain in a structural model of pentameric αβγ GABAA receptor (The N-terminal domain was removed for clarity) displaying the GABRB2 mutations (in black) on β (red ribbons) subunits. TM2 domains of five subunits form the Cl− ion pore (dashed black circle) and the p.Thr287Pro mutations are within the pore region.
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JMEDGENET2016104083F3: p.Thr287Pro mutation in the β2 subunit and γ-aminobutyric acid-A (GABAA) receptor is shown. (A) Cartoon representation of the location of the p.Thr287Pro mutation of β2 subunit of the GABAA receptor. (B) Three-dimensional structural model of the GABAA receptor that is composed predominantly of two α1 (blue ribbons), two β2 (red ribbons) and one γ2 (grey ribbon) subunits in the mammalian central nervous system. β2 subunits have four transmembrane domains (TM1 to TM4). The GABRB2 de novo p.Thr287Pro mutation is mapped onto the β2 subunit in black at the second transmembrane domain (TM2). (C) Extracellular view of the transmembrane domain in a structural model of pentameric αβγ GABAA receptor (The N-terminal domain was removed for clarity) displaying the GABRB2 mutations (in black) on β (red ribbons) subunits. TM2 domains of five subunits form the Cl− ion pore (dashed black circle) and the p.Thr287Pro mutations are within the pore region.

Mentions: The β2 subunit of GABAA receptor consists of four transmembrane domains (TM1 to TM4) connected by loops and extracellular N and C termini (figure 3A). TM2, where the mutation resides, forms Cl− ion pore of GABAA receptors (figure 3B, C). The GABAA receptors in the central nervous system are pentamers consisting of two each of the α1 and β2 subunits and one γ2 subunit, which are encoded by GABRA1, GABRB2 and GABRG2, respectively (figure 3B). We asked whether the p.Thr287Pro mutation might impair Cl− ion channel function of the GABAA receptors, which might in turn hamper GABAergic neuronal inhibition. Furthermore, since we have demonstrated that trafficking deficiency is a major defect caused by GABRG2 mutations,917 we also examined trafficking of the mutant β2 (Thr287Pro) subunits. HEK293T cells were co-transfected with α1 and γ2 subunits and either wild-type β2 only, mixed wild-type β2 and the mutant β2 (Thr287Pro) subunits or the mutant β2 (Thr287Pro) subunits only (mut). Total and surface protein expression of the wild-type and mutant β2 subunits were then compared. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to determine total β2 subunits (figure 4A). Compared with the total β2 subunits in the ‘wild type receptor only’ condition, both the mixed and the mutant β2 subunits (0.61±0.02 for mixed, 0.35±0.04 for mutant β2 (Thr287Pro) subunits vs 1 for wild type, n=4) were reduced when co-expressed with α1 and γ2 subunits (figure 4B). Reduced total β2 subunit expression could result in the reduced expression of surface β2 subunit. We next determined surface protein expression of the mutant β2 (Thr287Pro) subunit. We used the high-throughput flow cytometry to quantify the amount of surface wild-type or mutant β2 subunit when co-expressed with the α1 and γ2 subunits; this is because pre-assembled pentameric receptors are trafficked to the cell surface (figure 4C). Similar to the total protein expression levels, surface β2 subunits for both the mixed and mutant receptors were reduced (0.54±0.045 for mixed, 0.34±0.042 for mutant β2 (Thr287Pro) subunits vs 1 for wild type, n=4) (figure 4D).


A de novo missense mutation of GABRB2 causes early myoclonic encephalopathy
p.Thr287Pro mutation in the β2 subunit and γ-aminobutyric acid-A (GABAA) receptor is shown. (A) Cartoon representation of the location of the p.Thr287Pro mutation of β2 subunit of the GABAA receptor. (B) Three-dimensional structural model of the GABAA receptor that is composed predominantly of two α1 (blue ribbons), two β2 (red ribbons) and one γ2 (grey ribbon) subunits in the mammalian central nervous system. β2 subunits have four transmembrane domains (TM1 to TM4). The GABRB2 de novo p.Thr287Pro mutation is mapped onto the β2 subunit in black at the second transmembrane domain (TM2). (C) Extracellular view of the transmembrane domain in a structural model of pentameric αβγ GABAA receptor (The N-terminal domain was removed for clarity) displaying the GABRB2 mutations (in black) on β (red ribbons) subunits. TM2 domains of five subunits form the Cl− ion pore (dashed black circle) and the p.Thr287Pro mutations are within the pore region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384423&req=5

JMEDGENET2016104083F3: p.Thr287Pro mutation in the β2 subunit and γ-aminobutyric acid-A (GABAA) receptor is shown. (A) Cartoon representation of the location of the p.Thr287Pro mutation of β2 subunit of the GABAA receptor. (B) Three-dimensional structural model of the GABAA receptor that is composed predominantly of two α1 (blue ribbons), two β2 (red ribbons) and one γ2 (grey ribbon) subunits in the mammalian central nervous system. β2 subunits have four transmembrane domains (TM1 to TM4). The GABRB2 de novo p.Thr287Pro mutation is mapped onto the β2 subunit in black at the second transmembrane domain (TM2). (C) Extracellular view of the transmembrane domain in a structural model of pentameric αβγ GABAA receptor (The N-terminal domain was removed for clarity) displaying the GABRB2 mutations (in black) on β (red ribbons) subunits. TM2 domains of five subunits form the Cl− ion pore (dashed black circle) and the p.Thr287Pro mutations are within the pore region.
Mentions: The β2 subunit of GABAA receptor consists of four transmembrane domains (TM1 to TM4) connected by loops and extracellular N and C termini (figure 3A). TM2, where the mutation resides, forms Cl− ion pore of GABAA receptors (figure 3B, C). The GABAA receptors in the central nervous system are pentamers consisting of two each of the α1 and β2 subunits and one γ2 subunit, which are encoded by GABRA1, GABRB2 and GABRG2, respectively (figure 3B). We asked whether the p.Thr287Pro mutation might impair Cl− ion channel function of the GABAA receptors, which might in turn hamper GABAergic neuronal inhibition. Furthermore, since we have demonstrated that trafficking deficiency is a major defect caused by GABRG2 mutations,917 we also examined trafficking of the mutant β2 (Thr287Pro) subunits. HEK293T cells were co-transfected with α1 and γ2 subunits and either wild-type β2 only, mixed wild-type β2 and the mutant β2 (Thr287Pro) subunits or the mutant β2 (Thr287Pro) subunits only (mut). Total and surface protein expression of the wild-type and mutant β2 subunits were then compared. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to determine total β2 subunits (figure 4A). Compared with the total β2 subunits in the ‘wild type receptor only’ condition, both the mixed and the mutant β2 subunits (0.61±0.02 for mixed, 0.35±0.04 for mutant β2 (Thr287Pro) subunits vs 1 for wild type, n=4) were reduced when co-expressed with α1 and γ2 subunits (figure 4B). Reduced total β2 subunit expression could result in the reduced expression of surface β2 subunit. We next determined surface protein expression of the mutant β2 (Thr287Pro) subunit. We used the high-throughput flow cytometry to quantify the amount of surface wild-type or mutant β2 subunit when co-expressed with the α1 and γ2 subunits; this is because pre-assembled pentameric receptors are trafficked to the cell surface (figure 4C). Similar to the total protein expression levels, surface β2 subunits for both the mixed and mutant receptors were reduced (0.54±0.045 for mixed, 0.34±0.042 for mutant β2 (Thr287Pro) subunits vs 1 for wild type, n=4) (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 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