Clinical characterization of GABRA1/GABRG2 microdeletion patient

Clinical Score: 0.950 Price: $0.50 epilepsy with optic atrophy human patient Status: proposed

What This Experiment Tests

Clinical experiment designed to assess clinical efficacy targeting GABRA1, GABRG2 in human patient. Primary outcome: clinical phenotype characterization

Description

Clinical evaluation of a human patient with microdeletion of GABRA1 and GABRG2 genes presenting with intractable epilepsy and blindness due to optic atrophy. The study included electroencephalography recordings to characterize seizure activity and fundus photography to evaluate bilateral optic nerve atrophy. This represents the first reported case of haploinsufficiency of both GABR genes causing this phenotype combination.

TARGET GENE

GABRA1, GABRG2

MODEL SYSTEM

human patient

ESTIMATED COST

TIMELINE

0 months

PATHWAY

GABAergic neurotransmission

SOURCE

extracted_from_pmid_37703949

PRIMARY OUTCOME

clinical phenotype characterization

Scoring Dimensions

📖 Wiki Pages

GABRA1 Genegene GABRG2 — GABA-A Receptor Gamma2 Subunitgene GABRG2 Protein — GABA-A Receptor Gamma2 Subunitprotein GABRA1 Proteinprotein Epilepsydisease Epilepsydisease Fastigial Nucleus (Fastigial Cerebellar Nucleus) Ncell Serotonergic Dysfunction in Neurodegenerationmechanism GABA-A Gamma2 Neuronscell GABA-A Alpha1 Neuronscell Parvalbumin Neurons (PV+)cell Nucleus of the Lateral Lemniscus (LLL) Neuronscell Inferior Temporal Cortex Neurons in Prosopagnosiacell GABAergic Signaling Pathway in Neurodegenerationmechanism

Protocol

Phase 1: Initial Clinical Assessment (Day 0)

Objective: Establish baseline phenotype and confirm molecular diagnosis

Neurological examination: Complete motor, sensory, reflex, cranial nerve assessment
Ophthalmological evaluation: Visual acuity (Snellen), fundoscopy, OCT retinal nerve fiber layer (RNFL) thickness, visual field perimetry (Humphrey 24-2)
EEG: 32-channel EEG (International 10-20 system), 60-minute recording including sleep
MRI Brain: 3T MRI with T1-weighted MPRAGE, T2-FLAIR, diffusion tensor imaging (DTI), susceptibility-weighted imaging (SWI), optimized for optic nerve visualization

Reagents/Equipment:

MRI: Siemens Prisma 3T
EEG: Nihon Kohden JE-120 amplifier
OCT: Spectralis HRA+OCT (Heidelberg Engineering)

...

Phase 1: Initial Clinical Assessment (Day 0)

Objective: Establish baseline phenotype and confirm molecular diagnosis

Neurological examination: Complete motor, sensory, reflex, cranial nerve assessment
Ophthalmological evaluation: Visual acuity (Snellen), fundoscopy, OCT retinal nerve fiber layer (RNFL) thickness, visual field perimetry (Humphrey 24-2)
EEG: 32-channel EEG (International 10-20 system), 60-minute recording including sleep
MRI Brain: 3T MRI with T1-weighted MPRAGE, T2-FLAIR, diffusion tensor imaging (DTI), susceptibility-weighted imaging (SWI), optimized for optic nerve visualization

Reagents/Equipment:

MRI: Siemens Prisma 3T
EEG: Nihon Kohden JE-120 amplifier
OCT: Spectralis HRA+OCT (Heidelberg Engineering)

Controls: Age/sex-matched historical control EEG/MRI data from literature

Phase 2: Molecular Characterization (Days 1-7)

Objective: Confirm microdeletion breakpoints and characterize transcriptional consequences

DNA extraction: Peripheral blood mononuclear cells (PBMCs) using QIAamp DNA Blood Mini Kit (Qiagen)
aCGH (Array Comparative Genomic Hybridization): Agilent 180K oligonucleotide array for precise breakpoint mapping
WES (Whole Exome Sequencing): Illumina NovaSeq 6000, 150bp paired-end, 100x coverage minimum
cDNA analysis: RNA extracted from patient-derived lymphoblastoid cell lines (transformed with EBV), subject to:
RT-qPCR for GABRA1 and GABRG2 expression: TaqMan assays (Thermo Fisher Hs00161575_m1, Hs00181333_m1)
Western blot: Primary antibodies anti-GABRA1 (Abcam ab1545, 1:500), anti-GABRG2 (Abcam ab157436, 1:500), anti-β-actin (Sigma A5441, 1:5000) as loading control
Secondary: HRP-conjugated anti-rabbit IgG (Cell Signaling 7074, 1:5000)
ECL detection: SuperSignal West Femto (Thermo Fisher 34096)

Controls:

Patient baseline (pre-symptom if available)
Age-matched healthy donor PBMCs
Public expression data: GTEx database for matched tissue comparisons

Phase 3: Neurophysiological Profiling (Days 8-14)

Objective: Define seizure characteristics and establish electrophysiological biomarkers

Long-term video-EEG monitoring: 72-hour continuous monitoring to capture ≥3 spontaneous seizures
MEG (Magnetoencephalography): 306-channel MEG ( Elekta Neuromag TRIUX) for interictal spike localization
Evoked potentials: Visual evoked potentials (VEP) using pattern-reversal checkerboard (1° check, 2 Hz reversal), pattern electroretinography (PERG)
Transcranial magnetic stimulation (TMS): Single and paired-pulse TMS for cortical excitability assessment (Magstim Rapid2)

Equipment:

EEG: Nihon Kohden EEG-1200
MEG: Elekta Neuromag TRIUX
TMS: Magstim Rapid2 with figure-of-eight coil

Controls: Historical cohort data from 20 age/sex-matched epilepsy patients with focal seizures

Phase 4: Advanced Imaging (Days 15-21)

Objective: Quantify structural and functional brain alterations

7T MRI: Ultra-high field MRI for subcortical structure visualization
T2*-weighted imaging for iron deposition in basal ganglia
MR spectroscopy: TE=30ms, PRESS sequence, voxels placed in occipital cortex and thalamus
Quantitative susceptibility mapping (QSM) for optic nerve iron quantification
FDG-PET: 18F-fluorodeoxyglucose PET/CT for regional glucose metabolism
OCT-Angiography: Quantify retinal microvasculature and ganglion cell complex (GCC) thickness

Controls: Published normative 7T MRI values from age-matched controls (n≥30)

Phase 5: Longitudinal Monitoring (Months 1, 3, 6, 12)

Objective: Track disease progression and treatment response

Monthly assessments: Seizure diary review, adverse event recording, Vineland Adaptive Behavior Scales (VABS-III)
Quarterly: OCT-RNFL measurement, video-EEG (24hr), plasma biomarker panel (NFL, tau, GFAP via Simoa analyzer)
Annual: Comprehensive neuropsychological battery (WAIS-IV, WMS-IV, Trails A/B, Stroop)

Biomarker assays:

Neurofilament light (NFL): NFL Simoa assay (UBio 102-153)
Total tau: Tau kit (Fujirebio HK-Tau)
GFAP: GFAP kit (LU-HSF10)

Controls: Published normative values for age-matched population; historical data from Dravet syndrome cohort (closest genetic epilepsy comparator)

Phase 6: Functional Validation (Months 1-6, parallel to Phase 5)

Objective: Establish cellular phenotype of patient mutation

Patient-derived iPSCs: Reprogramming of PBMCs using CytoTune-iPS 2.0 Sendai kit (Thermo Fisher)
Neuronal differentiation: Dual-SMAD inhibition protocol, 35-day differentiation to cortical excitatory neurons
Electrophysiology:
Whole-cell patch clamp: Record GABA-A receptor currents (EPC-10 amplifier, HEKA)
Drug responses: Picrotoxin (100 μM), Diazepam (1 μM), THIP (10 μM) application
Miniature excitatory postsynaptic currents (mEPSCs): Recorded at -70mV, filtered at 2kHz

Controls:

Isogenic correction line (CRISPR-Cas9)
Age/sex-matched control iPSC line
Scrambled siRNA (Qiagen SI00154213) for knockdown validation

Expected Outcomes

aCGH reveals exact breakpoints: Microdeletion spans 142-158kb on chromosome 5q34, encompassing GABRA1 (ENST00000406563) and GABRG2 (ENST00000329208) with no other RefSeq genes disrupted

GABRA1/GABRG2 mRNA levels reduced to 23% ± 4% of control (mean ± SD, n=3 technical replicates, p<0.001, two-tailed t-test) as measured by RT-qPCR

Western blot quantification: GABRA1 protein at 31% ± 7% of control; GABRG2 at 28% ± 5% of control (densitometry, ImageJ normalization to β-actin)

...

Success Criteria

Molecular confirmation: aCGH identifies microdeletion encompassing GABRA1/GABRG2 with breakpoint precision ±5kb, confirmed by orthogonal methods (ddPCR or MLPA)
Protein reduction threshold: GABA-A receptor subunit proteins reduced to <40% of age-matched control levels on ≥2 independent Western blot experiments (p<0.01, effect size Cohen's d >1.5)
Clinical phenotype documentation: Complete neurological, ophthalmological, and neuropsychological assessment performed per protocol with <5% missing data

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Related Hypotheses (1)

Biorhythmic Interference via Controlled Sleep Oscillations0.661

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