Limits...
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 protein expression of Gβ5 and RGS9-2 complex. aCartoon 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. bCartoon 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, fWestern blot analysis of GNB5 expression in patient lymphoblastoid cells compared to normal control. gImmunoblot 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5037613&req=5

Fig3: Effect of S81L mutation on protein expression of Gβ5 and RGS9-2 complex. aCartoon 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. bCartoon 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, fWestern blot analysis of GNB5 expression in patient lymphoblastoid cells compared to normal control. gImmunoblot 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

Mentions: We next sought to determine the mechanisms by which S81L mutation affects Gβ5 function. In silico prediction suggests that the S81L variant likely has deleterious structural effects with three algorithms concurring on very high pathogenicity scores (1.0 on PolyPhen, 0.0 on SIFT, and 34 on CADD). To obtain structural insights into the impact of the S81L mutation on Gβ5 at the atomic level, we modeled the consequences of this substitution using crystal structure of RGS9-Gβ5 complex (Fig. 3a, b) [40]. S81 is buried inside the β-strand S2β2 of WD1 repeat close to central axis of β-propeller fold. The S81 is involved in side-chain–main-chain type of hydrogen bond with V108 (Fig. 3c) and such interactions are known to be crucial for maintaining stable structure of the protein [41, 42]. Our modeling suggests that substituting Ser81 with hydrophobic leucine would abolish hydrogen bond formation with V108 and bulkier side chain of leucine at this position would not fit into the tightly packed antiparallel β-sheet of WD1 repeat resulting in a steric clash with neighboring residues (V87 on WD1; V108, C111, and C122 on WD2 (Fig. 3c). Thus, S81L substitution is predicted to compromise Gβ5 folding and/or stability. To test these predictions, we analyzed the expression of Gβ5 in patient-derived lymphoblasts by immunoblotting. Indeed, we detected a modest but consistent reduction of Gβ5 protein levels in the two available lymphoblastoid lines derived from affected patients compared to healthy controls (Fig. 3d, e). In order to rule out the possibility that the apparent reduction in GNB5 protein may have originated at the transcript level, qRT-PCR using patient and control RNA revealed equivalent levels of GNB5 transcripts (Fig. 3).Fig. 3


GNB5 mutation causes a novel neuropsychiatric disorder featuring attention deficit hyperactivity disorder, severely impaired language development and normal cognition
Effect of S81L mutation on protein expression of Gβ5 and RGS9-2 complex. aCartoon 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. bCartoon 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, fWestern blot analysis of GNB5 expression in patient lymphoblastoid cells compared to normal control. gImmunoblot 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037613&req=5

Fig3: Effect of S81L mutation on protein expression of Gβ5 and RGS9-2 complex. aCartoon 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. bCartoon 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, fWestern blot analysis of GNB5 expression in patient lymphoblastoid cells compared to normal control. gImmunoblot 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
Mentions: We next sought to determine the mechanisms by which S81L mutation affects Gβ5 function. In silico prediction suggests that the S81L variant likely has deleterious structural effects with three algorithms concurring on very high pathogenicity scores (1.0 on PolyPhen, 0.0 on SIFT, and 34 on CADD). To obtain structural insights into the impact of the S81L mutation on Gβ5 at the atomic level, we modeled the consequences of this substitution using crystal structure of RGS9-Gβ5 complex (Fig. 3a, b) [40]. S81 is buried inside the β-strand S2β2 of WD1 repeat close to central axis of β-propeller fold. The S81 is involved in side-chain–main-chain type of hydrogen bond with V108 (Fig. 3c) and such interactions are known to be crucial for maintaining stable structure of the protein [41, 42]. Our modeling suggests that substituting Ser81 with hydrophobic leucine would abolish hydrogen bond formation with V108 and bulkier side chain of leucine at this position would not fit into the tightly packed antiparallel β-sheet of WD1 repeat resulting in a steric clash with neighboring residues (V87 on WD1; V108, C111, and C122 on WD2 (Fig. 3c). Thus, S81L substitution is predicted to compromise Gβ5 folding and/or stability. To test these predictions, we analyzed the expression of Gβ5 in patient-derived lymphoblasts by immunoblotting. Indeed, we detected a modest but consistent reduction of Gβ5 protein levels in the two available lymphoblastoid lines derived from affected patients compared to healthy controls (Fig. 3d, e). In order to rule out the possibility that the apparent reduction in GNB5 protein may have originated at the transcript level, qRT-PCR using patient and control RNA revealed equivalent levels of GNB5 transcripts (Fig. 3).Fig. 3

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