GABRG2 Variants Associated with Febrile Seizures
Abstract
:1. Introduction
2. Materials and Methods
2.1. Complementary DNA Constructs
2.2. Cell Culture and Transfection of Human GABAA Receptors
2.3. Determination of GABA-Elicited Responses by Automated Patch Clamp
2.4. Western Blot and Surface Biotinylation
2.5. Structural Simulation of GABAA Receptor γ2 Subunit Mutations
2.6. Mutation Tolerance of the GABAA Receptor γ2 Subunit
2.7. Statistical Analysis
3. Results
3.1. GABRG2 Variants and Proband Phenotypes
3.2. GABRG2 Variants Were Mapped to Structural Cassettes of the γ2 Subunits That Govern the GABAA Receptor Function
3.3. Mutant γ2 Subunits Altered Structural Domains Related to GABAA Receptor Gating
3.4. Mutant γ2 Subunits Alter GABAA Receptor Kinetics
3.5. Mutant γ2 Subunits Decreased GABAA Receptor Function
3.6. Mutant γ2 Subunits Changed GABAA Receptor Composition
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Patient 1 | Patient 2 | Patient 3 | Patient 4 | Patient 5 | Patient 6 | Patient 7 | Patient 8 | Patient 9 | |
---|---|---|---|---|---|---|---|---|---|
Inheritance | Father | De Novo | Father | Mother | De Novo | Mother | De Novo | Mother | Father |
Genomic position * | Chr5:161580337 | Chr5:161580316 | Chr5:161530907 | Chr5:161580349 | Chr5:161580349 | Chr5:161580169 | Chr5:161576165 | Chr5:161524801 | Chr5:161530933 |
Gene | GABRG2 | GABRG2 | GABRG2 | GABRG2 | GABRG2 | GABRG2 | GABRG2 | GABRG2 | GABRG2 |
Nucleotide change | c.1391T > C | c.1370T > C | c.644G > A | c.1403A > G | c.1403A > G | c.1223G > A | c.974C > T | c.485G > A | c.670C > T |
Amino acid change | p.Phe464 Ser | p.Phe457 Ser | p.Arg215 His | p.Tyr468 Cys | p.Tyr468 Cys | p.Arg408Lys | p.Ser325 Leu | p.Trp162 X | p.Arg224 X |
Predictions Polyphen2 | Damaging | Damaging | Damaging | Damaging | Damaging | Benign | Damaging | Damaging | Damaging |
Predictions SIFT | Deleterious | Deleterious | Deleterious | Deleterious | Deleterious | Benign | Deleterious | Deleterious | Deleterious |
Sex | Male | Female | Male | Male | Male | Female | Female | Female | Female |
Diagnosis | FS | FS | FS | GEFS+/FS | FS | FS | FS | FS | MAE |
Current age | Two years one month | Five years nine months | One year one month | Four years seven months | Three years three months | Four years three months | One year five months | Two years seven months | Seven years |
Age at onset | One year three months | One year three months | One year | Eleven months | One year six months | One year six months | One year five months | One year | N/D |
EEG | The bilateral posterior has a slightly higher quantity of slow waves in the waking stage; bilateral central area episodic spikes and wave complex during sleep. | Background activity lower frequency. Right central vertex spikes and slow-wave complex discharges; widespread slow-wave paroxysm during sleep. | Normal | A few diffuse spike-wave and slow spike-wave discharges during sleep. Posterior head (three years); normal EEG (three years nine months). | Normal | Normal | N/D | Normal | Epileptiform discharges: synchronous bilateral spike–slow wave and bifrontal maximum consistent (3.8 yrs.); generalized 2.5 Hz spike-wave complex discharge associated with epileptic spasms and slow rhythms (four years); generalized spike-wave discharges in stage II sleep (seven years). |
Seizure types | Tonic-clonic seizures | Tonic-clonic seizures | Tonic seizures | Tonic seizures | Tonic seizures | Tonic-clonic seizures | Tonic-clonic seizures | Tonic seizures | Complex partial seizures; frontal lobe epilepsy; absence and myoclonic seizures. |
AED response | N/A | N/A | N/A | LEV was only used as a prophylactic drug in febrile conditions. | CZP was only used as a prophylactic drug in febrile conditions. | N/A | N/A | N/A | Responded to ketogenic diet therapy; and TPM. |
Seizure outcome | Six times per year | Twice and seizure-free for three years | Two times total, within two months | Seizure-free for two years and nine months | Two times per year | Three times per year | Two times total, within one month | Five times per year | At 4 years old, 3 times per mon.; at 6.5 years old, seizure-free for a while, then returned. |
MRI findings | CT: normal | N/D | Normal | Normal | Normal | Normal | CT: normal | Normal | Within normal limits. |
Neurological exam | Normal | Normal | Normal | Normal | Normal | Normal | Normal | Normal | Shaking extremities; epileptic spasms showed spells consisting of a head nod, arms abduction, and recovery within 3–4 s associated with spike-wave discharges on EEG (4 years). |
Motor development | Normal | Normal | Normal | Normal | Normal | Normal | Normal | Normal | N/D |
Cognitive outcome | Intellectual disability | Normal | Normal | Normal | Normal | Normal | Normal | Normal | Mild intellectual developmental delay. |
Others | Sleep disorders | Neonatal hypoxic-ischemic encephalopathy | Has family history, brother. | Has family history, six cases out of ten family members, including three males and three females. | N/A | N/A | Neonatal amniotic fluid choking cough. | Has family history, mother. | Headaches (3–4 times a month) in the last 2.5 years. |
Deactivation (ms) | Rise Time (ms) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Mean | S.D. | n | Summary | Adjusted p Value 1 | Mean | S.D. | n | Summary | Adjusted p Value 1 | |
WT | 199.9 | 43.88 | 8 | 13.67 | 3.065 | 7 | ||||
W162X | 533.8 | 124.6 | 4 | **** | <0.0001 | 23.51 | 7.964 | 6 | ns | 0.3086 |
R215H | 263 | 64.65 | 7 | ns | 0.7903 | 31.68 | 6.894 | 5 | * | 0.0123 |
R224X | 350.1 | 126.4 | 7 | * | 0.0414 | 72.64 | 7.629 | 5 | **** | <0.0001 |
S325L | 307.7 | 75.67 | 9 | ns | 0.1888 | 68.01 | 15.44 | 4 | **** | <0.0001 |
Y468C | 433.9 | 180.7 | 8 | *** | 0.0002 | 26.52 | 5.408 | 4 | ns | 0.1744 |
F464S | 332.6 | 79.92 | 8 | ns | 0.0748 | 29.22 | 7.173 | 5 | * | 0.0398 |
F457S | 224.8 | 48.26 | 4 | ns | 0.9994 | 60.51 | 12.64 | 4 | **** | <0.0001 |
R408K | 165.4 | 84.56 | 6 | ns | 0.9924 | 40.23 | 14.13 | 4 | *** | 0.0003 |
IMAX (nA) | IMAX variant/IMAX WT (%) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Mean | S.E.M. | n | Summary | Adjusted p Value 1 | Mean | S.D. | n | Summary | Adjusted p Value 1 | |
WT | 2.00 | 0.38 | 8 | 100.00 | 2.95 | 8 | ||||
W162X | 0.48 | 0.13 | 8 | **** | <0.0001 | 24.07 | 3.87 | 8 | **** | <0.0001 |
R215H | 0.61 | 0.14 | 11 | **** | <0.0001 | 30.44 | 3.10 | 11 | **** | <0.0001 |
R224X | 0.85 | 0.14 | 8 | *** | 0.0007 | 42.52 | 1.70 | 8 | **** | <0.0001 |
S325L | 0.78 | 0.14 | 13 | **** | <0.0001 | 39.07 | 2.13 | 13 | **** | <0.0001 |
Y468C | 0.80 | 0.22 | 8 | *** | 0.0004 | 40.06 | 2.39 | 8 | **** | <0.0001 |
F464S | 0.96 | 0.18 | 8 | ** | 0.0025 | 47.93 | 1.58 | 8 | **** | <0.0001 |
F457S | 0.44 | 0.15 | 6 | **** | <0.0001 | 21.96 | 4.60 | 6 | **** | <0.0001 |
R408K | 0.22 | 0.06 | 7 | **** | <0.0001 | 11.10 | 7.98 | 7 | **** | <0.0001 |
pEC50 | S.D. | n | Summary | Adjusted p Value 1 | |
---|---|---|---|---|---|
WT | 5.92 | 0.27 | 5–10 | ||
W162X | 6.10 | 0.54 | 5–14 | ns | 0.2353 |
R215H | 4.60 | 0.21 | 5–16 | **** | <0.0001 |
R224X | 6.02 | 0.27 | 5–11 | ns | 0.8233 |
S325L | 5.39 | 0.35 | 5–13 | **** | <0.0001 |
Y468C | 4.56 | 0.64 | 5–12 | **** | <0.0001 |
F464S | 4.72 | 0.34 | 5–10 | **** | <0.0001 |
F457S | 4.35 | 0.68 | 6–13 | **** | <0.0001 |
R408K | 4.81 | 0.22 | 5–10 | **** | <0.0001 |
Surface | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
α1 | β3 | γ2 | |||||||||||||
Mean | S.E.M. | n | Summary | Adjusted p Value 1 | Mean | S.E.M. | n | Summary | Adjusted p Value 1 | Mean | S.E.M. | n | Summary | Adjusted p Value 1 | |
WT | 0.998 | 0.003 | 4 | 0.998 | 0.003 | 4 | 0.997 | 0.002 | 4 | ||||||
W162X | 1.373 | 0.110 | 4 | * | 0.0249 | 1.481 | 0.232 | 4 | ** | 0.0018 | 0.177 | 0.049 | 4 | **** | <0.0001 |
R215H | 0.901 | 0.030 | 4 | ns | 0.9688 | 0.809 | 0.044 | 4 | ns | 0.5488 | 0.701 | 0.044 | 4 | ns | 0.1179 |
R224X | 1.992 | 0.158 | 4 | **** | <0.0001 | 1.528 | 0.076 | 4 | *** | 0.0005 | 0.000 | 0.000 | 4 | **** | <0.0001 |
S325L | 0.987 | 0.047 | 4 | ns | 0.9999 | 0.652 | 0.127 | 4 | * | 0.0464 | 0.513 | 0.066 | 4 | ** | 0.0018 |
Y468C | 0.871 | 0.004 | 4 | ns | 0.8831 | 0.680 | 0.060 | 4 | ns | 0.0805 | 0.118 | 0.003 | 4 | **** | <0.0001 |
F464S | 1.416 | 0.114 | 4 | ** | 0.0094 | 1.107 | 0.094 | 4 | ns | 0.9413 | 0.152 | 0.018 | 4 | **** | <0.0001 |
F457S | 1.669 | 0.163 | 4 | **** | <0.0001 | 1.110 | 0.162 | 4 | ns | 0.9331 | 0.200 | 0.038 | 4 | **** | <0.0001 |
R408K | 0.936 | 0.043 | 4 | ns | 0.9972 | 0.841 | 0.079 | 4 | ns | 0.7358 | 0.198 | 0.017 | 4 | **** | <0.0001 |
Total | |||||||||||||||
α1 | β3 | γ2 | |||||||||||||
Mean | S.E.M. | n | Summary | Adjusted p Value 1 | Mean | S.E.M. | n | Summary | Adjusted p Value 1 | Mean | S.E.M. | n | Summary | Adjusted p Value 1 | |
WT | 0.997 | 0.002 | 4 | 0.995 | 0.003 | 4 | 0.998 | 0.003 | 4 | ||||||
W162X | 1.143 | 0.056 | 4 | ns | 0.1496 | 2.099 | 0.100 | 4 | **** | <0.0001 | 0.152 | 0.019 | 4 | **** | <0.0001 |
R215H | 0.630 | 0.038 | 4 | **** | <0.0001 | 0.561 | 0.029 | 4 | **** | <0.0001 | 0.622 | 0.018 | 4 | **** | <0.0001 |
R224X | 1.012 | 0.108 | 4 | ns | 0.9997 | 0.923 | 0.083 | 4 | ns | 0.8255 | 0.417 | 0.018 | 4 | **** | <0.0001 |
S325L | 0.934 | 0.011 | 4 | ns | 0.8971 | 0.855 | 0.052 | 4 | ns | 0.1798 | 0.661 | 0.011 | 4 | **** | <0.0001 |
Y468C | 0.759 | 0.068 | 4 | ** | 0.0031 | 0.669 | 0.033 | 4 | **** | <0.0001 | 0.251 | 0.008 | 4 | **** | <0.0001 |
F464S | 0.932 | 0.040 | 4 | ns | 0.8822 | 0.763 | 0.071 | 4 | ** | 0.0042 | 0.164 | 0.002 | 4 | **** | <0.0001 |
F457S | 0.728 | 0.013 | 4 | *** | 0.0006 | 0.757 | 0.054 | 4 | ** | 0.0031 | 0.226 | 0.012 | 4 | **** | <0.0001 |
R408K | 0.902 | 0.015 | 4 | ns | 0.5654 | 0.904 | 0.053 | 4 | ns | 0.6213 | 0.416 | 0.019 | 4 | **** | <0.0001 |
Surface/Total | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
α1 | β3 | γ2 | |||||||||||||
Mean | S.E.M. | n | Summary | Adjusted p Value 1 | Mean | S.E.M. | n | Summary | Adjusted p Value 1 | Mean | S.E.M. | n | Summary | Adjusted p Value 1 | |
WT | 0.9997 | 0.0002 | 4 | 0.9995 | 0.0003 | 4 | 0.9997 | 0.0002 | 4 | ||||||
W162X | 1.2186 | 0.1474 | 4 | ns | 0.7537 | 0.6958 | 0.0822 | 4 | ns | 0.4171 | 0.0861 | 0.0861 | 4 | **** | <0.0001 |
R215H | 1.4511 | 0.1230 | 4 | ns | 0.0832 | 1.4422 | 0.0371 | 4 | ns | 0.0931 | 1.1348 | 0.0998 | 4 | ns | 0.9731 |
R224X | 1.9858 | 0.0804 | 4 | **** | <0.0001 | 1.7130 | 0.2173 | 4 | ** | 0.0012 | 0.0000 | 0.0000 | 4 | **** | <0.0001 |
S325L | 1.0567 | 0.0504 | 4 | ns | 0.9996 | 0.7890 | 0.1730 | 4 | ns | 0.7855 | 0.7743 | 0.0963 | 4 | ns | 0.7279 |
Y468C | 1.1723 | 0.0907 | 4 | ns | 0.9051 | 1.0146 | 0.0715 | 4 | ns | 0.9999 | 0.4699 | 0.0037 | 4 | * | 0.0277 |
F464S | 1.5324 | 0.1621 | 4 | * | 0.0265 | 1.4955 | 0.1905 | 4 | * | 0.0453 | 0.9243 | 0.1031 | 4 | ns | 0.9994 |
F457S | 2.2951 | 0.2244 | 4 | **** | <0.0001 | 1.5292 | 0.2986 | 4 | * | 0.0277 | 0.9053 | 0.1843 | 4 | ns | 0.9968 |
R408K | 1.0362 | 0.0309 | 4 | ns | 0.9997 | 0.9543 | 0.1379 | 4 | ns | 0.9996 | 0.4772 | 0.0377 | 4 | * | 0.0308 |
GABRG2 Variant | Variant Category 1 | Phenotype 2 | Location 3 | Reference | gnomAD 4 | Other SNPs 4 |
---|---|---|---|---|---|---|
R82Q | LP/P | CAE | ECD | [54,56,57] | yes | R82W 4 |
R82Q | LP/P | FS | ECD | [54,56,57] | yes | - |
K328M | LP/P | GEFS+ | TMD | [58] | no | - |
R177G | LP/P | FS | ECD | [59] | no | R177Q 5 |
N79S | LB/B | GTCS | ECD | [15,60,61] | yes | - |
P83S | LP/P | FS | ECD | [15,62] | no | P83L 6 |
P83S | LP/P | DEE | ECD | [63] | no | - |
Y274C | LP/P | MAE | TMD | [55] | no | - |
R323Q | LP/P | DEE | TMD | [21,24] | yes | - |
R323Q | LP/P | MAE | TMD | [20] | yes | - |
A106T | LP/P | DEE | ECD | [24,64] | yes | A106P 5 |
I107T | LP/P | DEE | ECD | [24] | no | - |
P282S | LP/P | DEE | TMD | [24] | no | - |
R323W | LP/P | DEE | TMD | [24] | yes | - |
F343L | LP/P | DEE | TMD | [24] | no | - |
P282T | LP/P | DEE | TMD | [63] | no | - |
S306F | LP/P | DEE | TMD | [63] | no | - |
G257R | LP/P | DEE | ECD | [21] | no | - |
I218S | LP/P | DEE | ECD | [65] | no | - |
M199V | LP/P | GEFS+ | ECD | [25] | yes | M199K/T 7 |
P302L | LP/P | DEE | TMD | [22] | no | - |
F464S | LP/P | FS | TMD | Current study | no | - |
F457S | LP/P | FS | TMD | Current study | no | F457L 5 |
R215H | LP/P | FS | ECD | Current study | no | R215P/L 4 |
Y468C | LP/P | GEFS+ | TMD | Current study | yes | - |
Y468C | LP/P | GEFS+ | TMD | Current study | yes | -- |
R408K | LB/B | FS | CD | Current study | no | R408T 5 |
S325L | LP/P | FS | TMD | Current study | no | - |
W162X | LP/P | FS | ECD | Current study | no | - |
R224X | LP/P | MAE | ECD | Current study | no | R224Q 5,7 |
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© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Hernandez, C.C.; Shen, Y.; Hu, N.; Shen, W.; Narayanan, V.; Ramsey, K.; He, W.; Zou, L.; Macdonald, R.L. GABRG2 Variants Associated with Febrile Seizures. Biomolecules 2023, 13, 414. https://doi.org/10.3390/biom13030414
Hernandez CC, Shen Y, Hu N, Shen W, Narayanan V, Ramsey K, He W, Zou L, Macdonald RL. GABRG2 Variants Associated with Febrile Seizures. Biomolecules. 2023; 13(3):414. https://doi.org/10.3390/biom13030414
Chicago/Turabian StyleHernandez, Ciria C., Yanwen Shen, Ningning Hu, Wangzhen Shen, Vinodh Narayanan, Keri Ramsey, Wen He, Liping Zou, and Robert L. Macdonald. 2023. "GABRG2 Variants Associated with Febrile Seizures" Biomolecules 13, no. 3: 414. https://doi.org/10.3390/biom13030414