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. 2016 Sep 27;17(1):195.
doi: 10.1186/s13059-016-1061-6.

GNB5 mutation causes a novel neuropsychiatric disorder featuring attention deficit hyperactivity disorder, severely impaired language development and normal cognition

Hanan E Shamseldin  1 , Ikuo Masuho  2 , Ahmed Alenizi  3 , Suad Alyamani  4 , Dipak N Patil  2 , Niema Ibrahim  1 , Kirill A Martemyanov  5 , Fowzan S Alkuraya  6   7
Affiliations

GNB5 mutation causes a novel neuropsychiatric disorder featuring attention deficit hyperactivity disorder, severely impaired language development and normal cognition

Hanan E Shamseldin et al. Genome Biol. .

Abstract

Background: Neuropsychiatric disorders are common forms of disability in humans. Despite recent progress in deciphering the genetics of these disorders, their phenotypic complexity continues to be a major challenge. Mendelian neuropsychiatric disorders are rare but their study has the potential to unravel novel mechanisms that are relevant to their complex counterparts.

Results: In an extended consanguineous family, we identified a novel neuropsychiatric phenotype characterized by severe speech impairment, variable expressivity of attention deficit hyperactivity disorder (ADHD), and motor delay. We identified the disease locus through linkage analysis on 15q21.2, and exome sequencing revealed a novel missense variant in GNB5. GNB5 encodes an atypical β subunit of the heterotrimeric GTP-binding proteins (Gβ5). Gβ5 is enriched in the central nervous system where it forms constitutive complexes with members of the regulator of G protein signaling family of proteins to modulate neurotransmitter signaling that affects a number of neurobehavioral outcomes. Here, we show that the S81L mutant form of Gβ5 has significantly impaired activity in terminating responses that are elicited by dopamine.

Conclusions: We demonstrate that these deficits originate from the impaired expression of the mutant Gβ5 protein, resulting in the decreased ability to stabilize regulator of G protein signaling complexes. Our data suggest that this novel neuropsychiatric phenotype is the human equivalent of Gnb5 deficiency in mice, which manifest motor deficits and hyperactivity, and highlight a critical role of Gβ5 in normal behavior as well as language and motor development in humans.

Keywords: Attention deficit hyperactivity disorder (ADHD); G protein coupled receptors (GPCR); Hippocampus; Linkage; Mendelian; Neuropsychiatric disorders; Striatum.

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Figures

Fig. 1
Fig. 1
A novel neuropsychiatric disorder is linked to GNB5 mutation. a Pedigree of the study family. b Ideogram showing a single autozygous interval on chr15 (47,051,884-57,799,765, demarcated by SNPs rs11854077 and rs1280355) that is exclusively shared by the affected members. c Genome-wide linkage analysis shows a single linkage peak on chr15 with LOD ~4 that corresponds to the single autozygous interval shown in (b). A screenshot from the UCSC Genome Browser is shown to highlight the gene content of the linkage peak (GNB5 is boxed in red). d Schematic of GNB5 (transcript NM_ 006578) with the sequence chromatogram of the mutation shown on top. e Schematic of Gβ5 and the location of the missense mutation indicated. f Strong cross-species conservation of the Ser81 residue denoted with a red asterisk (black asterisks in the bottom denote highly conserved residues)
Fig. 2
Fig. 2
Effect of S81L mutation on GAP activity of RGS9-2 complex. a Schematic of the assay design. Stimulation of dopamine D2 receptor (D2R) by dopamine results in the dissociation of GαoA from the heterotrimer. Released Venus-tagged Gβγ subunits become available for interaction with Nluc-tagged GRK3ct reporter, producing the BRET signal, which is determined by the change in the emission ratio at 535 nm and 480 nm. RGS9-2/Gβ5 complexes exert GTPase Activating Protein (GAP) activity and accelerate deactivation of G proteins. b Representative BRET response of cells reconstituted D2R-GoA signaling. Responses to sequential application of dopamine (100 μM) and haloperidol (100 μM) were recorded. Data are means of six replicates. c Trace lines represent the deactivation phase of D2R-GoA signaling after haloperidol application to cells transfected with different condition (left without R7BP and right with R7BP). Data are means of six replicates. d k GAP rate constants were calculated as an enzymatic activity of RGS9-2 complexes (for further details, see “Methods”) and plotted as a bar graph. The same color code was used in panel c and d. A single asterisk (*) indicates P <0.0001. One-way ANOVA followed by Dunett’s post-hoc test was conducted with GraphPad Prism Ver. 6. Results shown are representative of two independent experiments each performed with six replicates. Values represent means ± SEM
Fig. 3
Fig. 3
Effect of S81L mutation on protein expression of Gβ5 and RGS9-2 complex. a Cartoon representation of RGS9-Gβ5 complex crystal structure (PDB ID:2PBI) with S81L mutation shown in the red sphere. RGS9 and Gβ5 are shown in gray and cyan, respectively. b Cartoon representation of Gβ5 alone (PDB ID:2PBI) with S81 mutation shown in the red sphere. Residue S81 is present on β-strand S2β2 of WD1 repeat. WD1 and the neighboring WD2 repeats are represented in blue and orange, respectively. c The hydrogen bond formation of side chain of S81 (red) with backbone of V108 (orange) is shown as a dotted black line. The substituted residue L81 (green stick) will not be able to form a hydrogen bond; instead its bulkier side chain will have steric clashes with neighboring amino acids (V87, V108, C111, and C122 represented in stick). All structural representations are made using PyMOL software https://www.pymol.org. d Summary of three qRT-PCR experiments (each performed in triplicates) using patient and control LCL to determine the relative abundance of GNB5 in patient vs. controls showing no significant difference (two-tailed t-test p value 0.67). e, f Western blot analysis of GNB5 expression in patient lymphoblastoid cells compared to normal control. g Immunoblot analysis of protein expression in HEK293T/17 cells. RGS9-2, Gβ5, and R7BP were expressed in different combinations. The proteins extracted from the transfected cells used in BRET assay were subjected to immunoblot analysis using the indicated specific antibodies. Anti-GAPDH antibody was used as a loading control. Representative experiment out of three independent evaluations is shown
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