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

View Article: PubMed Central - PubMed

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.

Electronic supplementary material: The online version of this article (doi:10.1186/s13059-016-1061-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Effect of S81L mutation on GAP activity of RGS9-2 complex. aSchematic 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. cTrace 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. dkGAP 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
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Fig2: Effect of S81L mutation on GAP activity of RGS9-2 complex. aSchematic 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. cTrace 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. dkGAP 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

Mentions: Given the central role of dopamine in a variety of neuropsychiatric conditions and documented role of R7 RGS-Gβ5 complexes in controlling signaling by the D2 dopamine receptors (D2R) [28], we have next evaluated the functional impact of S81L mutation in Gβ5 on termination of D2R responses, using a representative member of the R7 family, RGS9-2. We used optical means to record activation and deactivation of G protein Go by D2R in living cells by monitoring changes in BRET signal caused by dissociation of Go heterotrimer (Fig. 2a). In this assay, the addition of dopamine resulted in a rapid increase in BRET signal, which returns to the baseline upon the addition of the antagonist haloperidol (Fig. 2b). The speed of this termination phase is accelerated by RGS9, which in turn depends on Gβ5 for its activity (Fig. 2c). Thus, the functional activity of Gβ5 was determined by its ability to speed up D2R deactivation upon the addition of haloperidol in the presence of RGS9-2. Indeed, expressing wild-type Gβ5 substantially accelerated response termination (Fig. 2c, green versus blue traces in the left graph). In contrast, the response offset kinetics was substantially slower in the presence of S81L (Fig. 2c, blue versus red traces in the left graph). Calculating the catalytic efficiency of the reaction by single exponential analysis revealed significantly weaker activity of RGS complexes containing mutant Gβ5 (Fig. 2d). In addition to Gβ5, R7 RGS complexes in vivo contain membrane anchoring subunit R7BP, which further augments their catalytic activity and requires Gβ5for function [38]. Therefore, we next determined the effect of Gβ5 mutation in the presence of R7BP. Again, the addition of WT Gβ5 dramatically facilitated the activity of RGS9-2, but this effect was very modest when S81L Gβ5 was used instead. Therefore, we conclude that S81L results in severe but incomplete loss of function, detrimentally affecting the ability of R7 RGS proteins to deactivate D2R-mediated signaling.Fig. 2


GNB5 mutation causes a novel neuropsychiatric disorder featuring attention deficit hyperactivity disorder, severely impaired language development and normal cognition
Effect of S81L mutation on GAP activity of RGS9-2 complex. aSchematic 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. cTrace 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. dkGAP 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
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Fig2: Effect of S81L mutation on GAP activity of RGS9-2 complex. aSchematic 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. cTrace 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. dkGAP 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
Mentions: Given the central role of dopamine in a variety of neuropsychiatric conditions and documented role of R7 RGS-Gβ5 complexes in controlling signaling by the D2 dopamine receptors (D2R) [28], we have next evaluated the functional impact of S81L mutation in Gβ5 on termination of D2R responses, using a representative member of the R7 family, RGS9-2. We used optical means to record activation and deactivation of G protein Go by D2R in living cells by monitoring changes in BRET signal caused by dissociation of Go heterotrimer (Fig. 2a). In this assay, the addition of dopamine resulted in a rapid increase in BRET signal, which returns to the baseline upon the addition of the antagonist haloperidol (Fig. 2b). The speed of this termination phase is accelerated by RGS9, which in turn depends on Gβ5 for its activity (Fig. 2c). Thus, the functional activity of Gβ5 was determined by its ability to speed up D2R deactivation upon the addition of haloperidol in the presence of RGS9-2. Indeed, expressing wild-type Gβ5 substantially accelerated response termination (Fig. 2c, green versus blue traces in the left graph). In contrast, the response offset kinetics was substantially slower in the presence of S81L (Fig. 2c, blue versus red traces in the left graph). Calculating the catalytic efficiency of the reaction by single exponential analysis revealed significantly weaker activity of RGS complexes containing mutant Gβ5 (Fig. 2d). In addition to Gβ5, R7 RGS complexes in vivo contain membrane anchoring subunit R7BP, which further augments their catalytic activity and requires Gβ5for function [38]. Therefore, we next determined the effect of Gβ5 mutation in the presence of R7BP. Again, the addition of WT Gβ5 dramatically facilitated the activity of RGS9-2, but this effect was very modest when S81L Gβ5 was used instead. Therefore, we conclude that S81L results in severe but incomplete loss of function, detrimentally affecting the ability of R7 RGS proteins to deactivate D2R-mediated signaling.Fig. 2

View Article: PubMed Central - PubMed

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 &beta; subunit of the heterotrimeric GTP-binding proteins (G&beta;5). G&beta;5 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 G&beta;5 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 G&beta;5 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 G&beta;5 in normal behavior as well as language and motor development in humans.

Electronic supplementary material: The online version of this article (doi:10.1186/s13059-016-1061-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus