Maternal transmission of a rare GABRB3 signal peptide variant is associated with autism

Abstract

Maternal 15q11-q13 duplication is the most common copy number variant in autism, accounting for ∼1-3% of cases. The 15q11-q13 region is subject to epigenetic regulation, and genomic copy number losses and gains cause genomic disorders in a parent-of-origin-specific manner. One 15q11-q13 locus encodes the GABA(A) receptor β3 subunit gene (GABRB3), which has been implicated by several studies in both autism and absence epilepsy, and the co-morbidity of epilepsy in autism is well established. We report that maternal transmission of a GABRB3 signal peptide variant (P11S), previously implicated in childhood absence epilepsy, is associated with autism. An analysis of wild-type and mutant β3 subunit-containing α1β3γ2 or α3β3γ2 GABA(A) receptors shows reduced whole-cell current and decreased β3 subunit protein on the cell surface due to impaired intracellular β3 subunit processing. We thus provide the first evidence of an association between a specific GABA(A) receptor defect and autism, direct evidence that this defect causes synaptic dysfunction that is autism relevant and the first maternal risk effect in the 15q11-q13 autism duplication region that is linked to a coding variant.

Figure 1. Pedigree structure of ASD families bearing the P11S variant

17 ASD families in total were identified to harbor the variant. Individuals carrying the variant are marked with asterisks. Individuals for whom DNA was not available are marked N/A. Our strict analyses considered individuals affected if they met criteria for autism on the ADI-R diagnostic algorithm (completely filled black). Our broad analyses considered these individuals and individuals who met the AGP criteria ASD1 or ASD2 as affected (half filled black) (see Risi et al. 2006 (25) for details); note no individuals met ASD2. Unfilled individuals are considered unknown. For individuals who did not meet broad criteria Social Responsiveness Scale (SRS) t-scores from teacher (/parent) report are provided when available. SRS scores ranging from 60-75 are considered mild to moderate range for ASD; children with high functioning autism may score in the t-score range of 55-59. AGR 80-4 had significant language delay and impairment per the ADI-R, but did not meet our broad criteria or have a SRS. Individuals with definite seizures per the ADI-R are marked SZ = 2; those with suspected seizures per the ADI-R are marked SZ = 1.

Figure 2. MDS plot of all AGRE parents carrying the S11 risk allele

Black dots represent parents with p11s mutation. Out of 22 AGRE samples with rare variation, 7 are founders with AGRE Affy 5.0 genome-wide data available. Classical multidimensional scaling (MDS) was conducted in PLINK using the total sample of AGRE parents in order to estimate dimensions of population genetic variation. These dimensions are estimated from genome-wide average proportion of alleles shared by state for each possible pair of individuals in the sample. Graphical representation of the first two dimensions is used to identify population substructure and ancestry clusters. Figure 3 shows the variant to be present in Caucasian parents. Here colors represent best race and ethnicity approximations while black represents parents with the P11S variant.

Figure 3. Mutant β3(S11) subunit harboring receptors had reduced current and subunit surface expression

(A) Human GABA_A receptor currents were obtained from HEK 293T cells co-transfected with α3 and γ2S subunit cDNAs and wild-type β3(P11) and the mutant β3(S11) subunit for wild-type (wt α3β3γ2S 1:1:1 cDNA ratio, black), mixed of the wild-type β3(P11) and mutant β3(S11) (1:0.5:0.5:1, mix, green) or for mutant (mut α3β3(S11)γ2S) and evoked with 1 mM GABA for 6 sec (A). In A arrows indicates the peak of each actual trace. (B) The mean peak amplitude of each group was plotted (n = 10 for wt, n = 15 for mix, n = 13 for mutant from three different transfections). (C) HEK 293T cells co-transfected with α1, β3^HA (wt) or β3(P11S) ^HA (mut) and γ2S subunit cDNAs. Equal amounts of membrane-bound protein from live cells cell biotinylation, were pulled down with immobilized streptavidin, eluted with 1× NEB glycoprotein protein denaturing buffer (5% SDS, 0.4 M DTT) at room temperature for 30 min. The eluted products were then incubated in absence (U) or presence of PNGAse F (F) for 1hr at 37°C before fractionated by 10% SDS-PAGE and probed with monoclonal anti-HA antibody. (D) The relative amount of surface β3 ^HA subunit protein of wild-type and mutant receptors from C was plotted (n = 4). In B and D, the data ere plotted as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs. wild-type).

Figure 4. Mutant β3(P11S) subunit protein had impaired intracellular processing

(A) HEK 293T cells co-transfected with α1 and γ2S subunits with HA tagged β3(P11)^HA (Wt) or β3(S11) ^HA (mut) subunit cDNAs. Equal amounts of total lysates protein were analyzed by 10% SDS-PAGE and probed with monoclonal anti-HA and with monoclonal anti-Na⁺ K⁺ ATPase antibody as internal loading control. (B) The relative amount of surface β3^HA subunit protein versus loading control of wild-type and mutant receptors from A was plotted (n = 7) (C) Equal amount of total cell lysates from A were undigested (U) or PNGase-F (F) at 37°C for 3 hr. (DE) HEK 293T cells containing pulse-chase ³⁵S methionine radio-labeled wild-type β3^FLAG (W) and mutant β3(S11)^FLAG (M) subunits were pulse-labeled for a series of time points. The cells were lysed and the same amount of protein for each sample was used for immunopurification and SDS-PAGE (C). The relative ratio of radioactivity of the upper versus lower band is plotted at each time point for either the wild-type or mutant subunits (D, n = 4).