Journal: Mammalian Genome

Article Title: Gabra2 is a genetic modifier of Dravet syndrome in mice

doi: 10.1007/s00335-021-09877-1

Figure Lengend Snippet: Editing of Gabra2 B6 allele normalizes expression relative to 129. A The B6 Gabra2 intronic deletion is located on Chr 5 at 71,014,638 bp (GRCm38.p6) (yellow). Sequence of the B6 allele (black) is compared to the Edited (grey) and 129S6/SvEvTac alleles (blue). B Breeding scheme for mice used in the study. For expression and electrophysiology experiments, isogenic B6. Gabra2 Edited mice were crossed with B6 to generate offspring with Edited/B6 (grey) or B6/B6 (black) alleles at Gabra2 . For expression and seizure experiments, B6. Gabra2 mice were crossed with isogenic 129. Scn1a +/− mice to generate F1 .Scn1a +/− or F1. Scn1a +/+ mice with Edited/129 (purple) or B6/129 (red) alleles at Gabra2 . C Relative expression of Gabra2 transcript assayed by quantitative RT-ddPCR on whole brain samples from mice with B6/B6, Edited/B6, B6/129, Edited/129 and 129/129 alleles at Gabra2 . Transcript expression differed between genotypes ( F 4, 35 = 104.5, p < 0.0001; one-way ANOVA). Relative to B6/B6 (0.85 ± 0.04), Gabra2 transcript expression was ~ 1.8-fold higher in Edited/B6 (1.78 ± 0.05) and B6/129 (2.00 ± 0.08), and ~ threefold higher in Edited/129 (3.11 ± 0.14) and 129/129 (2.85 ± 0.06). **** p < 0.0001, Dunnett’s post-hoc comparisons to B6/B6. Symbols represent samples from individual mice, horizontal lines represent group averages, and error bars represent SEM with 6–13 mice per genotype. D Quantification of GABRA2 protein expression determined from western blots of whole brain membrane protein from mice with B6/B6, Edited/B6, B6/129, Edited/129 and 129/129 alleles at Gabra2 assayed by western blot. Relative GABRA2 protein differed between genotypes ( F 4, 22 = 5.349, p = 0.0037; One-way ANOVA). Relative to B6/B6 (1.0 ± 0.1), GABRA2 protein expression was ~ 2.2-fold higher in Edited/B6 (2.2 ± 0.3) and B6/129 (2. ± 0.2), and ~ threefold higher in Edited/129 (2.4 ± 0.3) and 129/129 (2.9 ± 0.4). * p < 0.03, ** p < 0.01, *** p < 0.0009, Dunnett’s post-hoc comparisons to B6/B6. E Representative immunoblot probed for GABRA2 (green) and GPR75/Mortalin (red), which served as a loading control

Article Snippet: Blots were probed with rabbit polyclonal Gabra2 antibody (822-GA2CL, PhosphoSolutions; RRID:AB_2492101; 1:1000) and mouse monoclonal anti-mortalin/GRP75 antibody (NeuroMab N52A/42; RRID:2120479; 1 μg/mL), which served as a normalization control.

Techniques: Expressing, Sequencing, Western Blot, Membrane, Control

Journal: Mammalian Genome

Article Title: Gabra2 is a genetic modifier of Dravet syndrome in mice

doi: 10.1007/s00335-021-09877-1

Figure Lengend Snippet: Survival and seizure burden improved in F1. Scn1a +/− with Edited/129 versus B6/129 alleles at Gabra2 . A Kaplan Meier survival plot comparing 8 week survival of B6/129 and Edited/129 F1. Scn1a +/− mice. Survival was worse in B6/129 mice (43%) compared to Edited/129 (97%) with n = 30–33 per genotype. **** p < 0.0001, Logrank Mantel-Cox. B The proportion of mice exhibiting spontaneous GTCS and average GTCS frequency differed between F1. Scn1a +/− mice with B6/129 (81% with seizures; 3.4 ± 0.7 GTCS/day) versus Edited/129 (35% with seizures; 0.8 ± 0.4 GTCS/day) alleles at Gabra2 . Symbols represent samples from individual mice, horizontal lines represent the group average, and error bars represent SEM with n = 20–21 per genotype. *** p = 0.0008, Mann–Whitney. C Percentage of seizures in each category of an adapted modified Racine scale (colors defined below) differed between F1. Scn1a +/− mice with B6/129 or Edited/129 alleles at Gabra2 . The majority of seizures in Edited/129 mice scored ≤ 4 compared to 63% in B6/129 mice. B6/129 n = 246 seizures total; Edited/129 n = 94 seizures total. p < 0.0006, chi-square test. D Among mice with GTCS, the average proportion of GTCS that progressed to HLE differed between mice with B6/129 (0.50 ± 0.07) versus Edited/129 (0.16 ± 0.04) alleles at Gabra2 . Symbols represent samples from individual mice, and error bars represent SEM with n = 7–17 per genotype. *** p = 0.0007, Mann–Whitney. E Spontaneous GTCS diary plots for individual F1. Scn1a +/− mice with B6/129 or Edited/129 alleles at Gabra2 . Each row represents a single F1. Scn1a +/− mouse ( n = 20–21 per genotype) over the 336-h monitoring period or until occurrence of sudden, unexpected death indicated by a black triangle. Green circles represent subjects with no GTCS events. Tick marks represent individual seizure events with color indicating severity score using an adapted modified Racine scale. Colors used for the modified Racine scale adapted for Scn1a +/− mice in c and e are as follows: Green = 1, rearing and paddling, straub tail with no other movement; Orange = 2, rearing and paddling, straub tail, loss of posture, short bursts of movement, often backwards; Grey = 3, rearing and paddling with wild running and/or jumping without loss of posture; Teal = 4, rearing and paddling with wild running and/or jumping with loss of posture; Pink = 5, rearing and paddling with wild running and/or jumping with loss of posture progressing to tonic hindlimb extension (HLE); Black = 6, rearing and paddling with wild running and/or jumping with loss of posture progressing to tonic hindlimb extension (HLE) and death

Article Snippet: Blots were probed with rabbit polyclonal Gabra2 antibody (822-GA2CL, PhosphoSolutions; RRID:AB_2492101; 1:1000) and mouse monoclonal anti-mortalin/GRP75 antibody (NeuroMab N52A/42; RRID:2120479; 1 μg/mL), which served as a normalization control.

Techniques: MANN-WHITNEY, Modification

Journal: Mammalian Genome

Article Title: Gabra2 is a genetic modifier of Dravet syndrome in mice

doi: 10.1007/s00335-021-09877-1

Figure Lengend Snippet: Human GABRA2 eQTL. Genetic variation at SNP rs279829 (chr4:46,333,638 in GRCh38.p12) is associated with variation in GABRA2 transcript expression ( p = 0.0000025; n = 205 donor samples). Individuals that are homozygous for the minor allele have elevated expression relative to individuals that are heterozygous or homozygous for the major allele. Data used for this figure was obtained from: the GTEx Portal on 07/16/2020 (dbGaP Accession phs000424.v8.p2) [ https://www.gtexportal.org/home/gene/GABRA2#eQTLBlock ]

Article Snippet: Blots were probed with rabbit polyclonal Gabra2 antibody (822-GA2CL, PhosphoSolutions; RRID:AB_2492101; 1:1000) and mouse monoclonal anti-mortalin/GRP75 antibody (NeuroMab N52A/42; RRID:2120479; 1 μg/mL), which served as a normalization control.

Techniques: Expressing