case_images: PMC8504539_01
case_images: Sanger sequencing confirms a de novo GNB1 variant. (A) Genogram of the proband with the respective results for GNB1 sequencing of the patient and each parent. The c.217G>A heterozygous mutation was detected in the proband but not in the parents, indicating a de novo occurring variant. Results from Sanger sequencing were aligned with the genomic GNB1 sequence (NG_047052.1) using the MAFFT 7 algorithm. The gDNA lane represents the genomic GNB1 sequence; the protein lane represents the translation of amino acids from GNB1 exon 6; the sequencing lane represents the results from Sanger sequencing of each patient, with a c.217G>A variant in the proband. (B) Mutation map of all GNB1 variants implicated in GNB1 encephalopathy to the date of the study. Numbers inside each box represent the translated exons of GNB1 (exons 1, 2, and 12 are not translated). Whenever possible, the mutation is represented by the change in protein structure. The variant detected in the patient is highlighted in purple.
case_images: fneur-12-735549-g0001.jpg
case_images: PMC8504539_01_fneur-12-735549-g0001.jpg
case_images: F1
case_images: Finally, the GNB1 variant was confirmed by Sanger sequencing in the patient and shown to occur de novo, since it was not detected in either parent (Figure 1A). Interestingly, this variant maps to exon 6, one of the two GNB1 exons with mutational hotspots (Figure 1B).
case_images: The A73T mutation might compromise a WD40 repeat from the WD40 domain of GNB1. (A) Sequence alignment of GNB1 protein from different species demonstrating that the amino acid sequence is almost unchanged across evolution. The alignment was performed using Jalview (Version 2) software. (B) Three-dimensional structure of GNB1 (pdb -1gp2, chain B). A73 (red sticks) is located in a core beta-sheet from an WD repeat (boxed) that composes the circularized beta-propeller WD40 domain of GNB1. Represented in green sticks are the residues that distance less than 4A of A73. (C) Three-dimensional structure detail of the WD repeat encompassing A73 residue (left) and simulated A73T mutagenesis (right). Residues that are within 4A distance that might be stabilized by A73 are represented as green sticks. Upon A73T mutagenesis, using the highest probable side chain orientations for threonine, an additional residue (T102) from an adjacent WD40 repeat appears within 4A distance, indicating that A73T might have an impact on this region. (D) Three-dimensional structure detail of the Gbetagamma dimer, showing that A73 is unlikely to be required for dimer formation. (E) Three-dimensional structure detail of the Galphabetagamma trimer. The inset represents the interaction between Galpha D26 and Gbeta K78 that is required for trimer assembly, which is potentially affected by the A73T mutation. Structural modeling was performed using PyMOL.
case_images: fneur-12-735549-g0002.jpg
case_images: PMC8504539_01_fneur-12-735549-g0002.jpg
case_images: F2
case_images: In fact, the amino acid sequence is identical between humans and other mammals, such as mouse, rat and bovine, and differs only by 4 residues from zebrafish (Figure 2A). WD40 repeat motif that is part of the circularized beta-propeller WD40 domain of the GNB1 protein (Figure 2B). WD40 domains are mainly involved in the assembly of large molecular complexes and the WD motifs act as protein interaction scaffolds. S72, S74, K78, C79, V100 and C103 (Figure 2C, left). Substitution of the non-polar A73 for the polar hydroxyl residue threonine (A73T, Figure 2C, right) might locally impact the structure. Moreover, simulation of the A73T substitution, using the highest probable side chain orientations for threonine, revealed an additional residue (T102, Figure 2C, right) in the proximity (within 4A) that belongs to an adjacent WD40 motif. Gbetagamma dimer (Figure 2D), K78 establishes a polar contact with D26 from Galpha (Figure 2E), helping in the stabilization of the Galpha N-terminal helix near the beta-propeller domain of Gbeta. A destabilization of this region might therefore impact the Galpha-Gbetagamma interaction, with important functional consequences. Given the position of A73 within the WD40 motif and the functionality of the WD40 domain, a mutation in this residue could also impact G protein-effector interactions.