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. 2025 Jan 9;12(5):101522.
doi: 10.1016/j.gendis.2025.101522. eCollection 2025 Sep.

Clinical-molecular profiling of atypical GNAO1 patients: Novel pathogenic variants, unusual manifestations, and severe molecular dysfunction

Gonzalo P Solis  1 , Federica Rachele Danti  2 , Yonika A Larasati  1 , Federica Graziola  2 , Carolina Croci  3 , Elisa Osanni  4 , Alexey Koval  1 , Giovanna Zorzi  2 , Vladimir L Katanaev  1
Affiliations

Clinical-molecular profiling of atypical GNAO1 patients: Novel pathogenic variants, unusual manifestations, and severe molecular dysfunction

Gonzalo P Solis et al. Genes Dis. .
No abstract available

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Conflict of interest statement

The authors declared no competing interests.

Figures

Figure 1
Figure 1
Biochemical, cellular, and structural characterization of the GNAO1 variants c.751T > C; p.Phe251Leu (F251L) and c.791C > T; p.Ser264Phe (S264F). (A–H) The curves of the BODIPY-GTPγS uptake (A, E) and BODIPY-GTP hydrolysis (C, G) of recombinant His6-tagged Gαo wild-type together with the pathogenic F251L (A, C) or S264F (E, G) mutants, and quantification of the corresponding binding rate constants (kbind; n = 3 or 4) (B, F) and hydrolysis rate constants (khydr; n = 3 or 4) (D, H). (I, J) The effect of increasing ZnCl2 concentrations on BODIPY-GTPγS binding (I) and BODIPY-GTP hydrolysis (J) by Gαo F251L. (K) Confocal images of N2a cells co-expressing Gαo wild-type or the pathogenic F251L and S264F mutants alongside a GFP-fusion of the Golgi marker mannosidase II (MannII-GFP). Cells were immunostained against Gαo and stained with DAPI in blue for nuclei. Scale bar, 10 μm. (L) HEK293T cells were co-transfected with GFP-tagged Gβ1 and Gγ3, and Gαo wild-type, F251L, S264F, or the constitutively active (non-pathogenic) Q205L mutant used as control. Immunoprecipitation (IP) of GFP-Gβ1γ3 was done using a nanobody against GFP, and the co-IP of Gαo proteins was analyzed by western blotting and immunodetection using antibodies against Gαo and GFP. (M) Quantification of the interaction between Gβ1γ3 and Gαo variants (n = 4). (N) An illustration of the BRET-based M2 muscarinic acetylcholine receptor (M2R)-coupling assay. M2R tagged with nano-luciferase (M2R-NLuc) excites the GFP-fusion of Gαo (Gαo-GFP). The steady-state low BRET signal increased upon acetylcholine (Ach) treatment (ΔBRET). (O) Quantification of the ΔBRET for Gαo wild-type, F251L, S264F, and Q205L (n = 3–6). (P) HEK293T cells were co-transfected with a GFP-tagged RGS19 construct, and Gαo wild-type, F251L, S264F, or the active Q205L control. The IP GFP-RGS19 and immunodetection were done as in (L). (Q) Quantification of the co-IP of Gαo constructs with RGS19 (n = 5). (R) Confocal images of N2a cells co-expressing a GFP-fusion of Ric8A (GFP-Ric8A) with Gαo wild-type or the pathogenic F251L and S264F variants. Cells were immunostained against Gαo and nuclei were stained in blue with DAPI. Scale bar, 10 μm. (S–V) HEK293T cells were co-transfected with the GFP-tagged Ric8A (S) or Ric8B (U) with Gαo wild-type, F251L, S264F, or the DEE17-linked G203R mutant used as control. IP and immunodetection were done as in (L). Quantification of the co-IP of Gαo variants and Ric8A (T) or Ric8B (V) (n = 4 or 5). Measurements are displayed as mean ± standard error of the mean. Data in (B, D, F, H) were analyzed by a two-sided unpaired t-test; for the remaining data, a one-way ANOVA followed by Dunnett's multiple comparisons test was used. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
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References

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