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Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype  Ideke J.C.

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Presentation on theme: "Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype  Ideke J.C."— Presentation transcript:

1 Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype  Ideke J.C. Lamers, Margot R.F. Reijnders, Hanka Venselaar, Alison Kraus, Sandra Jansen, Bert B.A. de Vries, Gunnar Houge, Gyri Aasland Gradek, Jieun Seo, Murim Choi, Jong-Hee Chae, Ineke van der Burgt, Rolph Pfundt, Stef J.F. Letteboer, Sylvia E.C. van Beersum, Simone Dusseljee, Han G. Brunner, Dan Doherty, Tjitske Kleefstra, Ronald Roepman  The American Journal of Human Genetics  Volume 101, Issue 5, Pages (November 2017) DOI: /j.ajhg Copyright © 2017 American Society of Human Genetics Terms and Conditions

2 Figure 1 RAB11B Mutations in Five Individuals with Severe ID
(A) cDNA composition of RAB11B and location of exons (dark gray) and identified mutations (green stars). (B) Amino acid sequence of exon 2 of RAB11B. Both identified variants (green) localize at one of the two GTP binding sites (red stripes) and p.Ala68Thr also involves the switch 2 region (black stripe). Both identified mutations affect highly conserved amino acid residues among different species (light gray box). The known inactive variant p.Ser25Asn and known active variant p.Gln70Leu are marked orange. (C–E) Structural characterization of mutations. In all panels, the overall Protein DataBank structure 2f9m of RAB11B14 is colored gray and shown in ribbon representation, GNP (phosphoaminophosphonic acid-guanylate ester, a non-hydrolyzable analog of GTP) is multicolored in a spacefill representation with the guanidine ring in blue shades and the phosphates in red and yellow. The magnesium molecule is represented as a purple sphere. The side chains of the affected amino acids in the affected individuals are green and for the GDP/GTP-locked mutants are orange. (C) Overview of RAB11B bound to GNP and magnesium and the affected amino acids are highlighted. (D) Close up of the p.Val22Met variant shows the side chains of valine at position 22 in green and the methionine in red. (E) Close up of the p.Ala68Thr variant shows the side chains of alanine at position 68 in green and threonine in red, and the mutated glutamine at position 70 which is mutated in the GTP-locked mutant is in orange. The American Journal of Human Genetics  , DOI: ( /j.ajhg ) Copyright © 2017 American Society of Human Genetics Terms and Conditions

3 Figure 2 Brain Imaging Features and Facial Photographs of Individuals with RAB11B-Related Intellectual Disability (A, F, G, J) Markedly decreased cortical white matter volume in individuals 1, 3, 4, and 5. (B, H, K) Markedly thin corpus callosum (arrows), mildly thin brainstem, and mildly small, atrophic-appearing cerebellar vermis (bracket) in individual 1, 4, and 5. (C) Increased T2/FLAIR signal in the cortical white matter (asterisks) in individual 1. (D and E) Atypical partial rhombencephalosynapsis in individual 1 (arrowheads). (A, D, F, G, and H) are T2-weighted axial images. (B and H) are T1-weighted sagittal images. (C) is a T2/FLAIR-weighted axial image. (E) is a reformatted inversion-recovery coronal image. (K) is a T2-weighted sagittal image. (I and L) Facial photographs show upward slanted palpebral fissures, periorbital fullness, full nasal tip, and hypotonic face in individual 4 and upward slanted palpebral fissures, deep set eyes, and short philtrum in individual 5. The American Journal of Human Genetics  , DOI: ( /j.ajhg ) Copyright © 2017 American Society of Human Genetics Terms and Conditions

4 Figure 3 Localization of Wild-Type and Mutant RAB11B in hTERT-RPE1 Cells Shown in green is the expression of recombinant 3xFLAG-RAB11B wild-type (A and G) and 3xFLAG-RAB11B variants p.Gln70Leu (B and H), p.Ser25Asn (C and I), p.Val22Met (D and J), p.Ala68Thr (E and K) in hTERT RPE1 cells, detected using anti-FLAG antibodies (rabbit polyclonal, #F7425, Sigma Aldrich; dilution 1:500 in PBS). N-terminal tagging was performed to not disturb the prenylation of the C terminus of RAB11B, which is required for membrane association.10,18 Recombinant protein expression, immunocytochemistry, and image capture was performed as described previously.44 All secondary antibodies were Alexa Fluor conjugates (Thermo Fisher Scientific). The following transfection efficiencies were obtained per 3xFLAG-RAB11B construct: wild-type, 14%; p.Gln70Leu, 19%; p.Ser25Asn, 9%; p.Val22Met, 10%; p.Ala68Thr, 14%. Staining of Pericentriolar Material 1 (PCM1) using anti-PCM1 antibodies (goat polyclonal, #SC-50164, Santa Cruz Biotechnology; dilution 1:250 in PBS) was used to mark the peri-centrosomal region (red) and magnifications are shown in the insets (A–E). Staining of GM130 using anti-GM130 antibodies (mouse monoclonal, #610822, BD Biosciences; dilution 1:250 in PBS) was used to mark the Golgi apparatus (in red; G-K). Co-localization with PCM1 (F) or GM130 (L) was quantified by calculating the Pearson’s correlation coefficient (PCC) using the JACoP plugin in ImageJ (N = 12 cells for each construct, mean in red and error bars represent the SD).45–47 The significance of difference with WT was calculated with a Student’s t test (ns: not significant (p > 0.05); ∗: p < 0.05; ∗∗: p < 0.005; ∗∗∗: p < ). Wild-type and GTP-bound active RAB11B (p.Gln70Leu) are showing punctuated localization with an enrichment at the peri-centrosomal region. Both identified variants p.Val22Met and p.Ala68Thr show similar localization as GDP-bound inactive RAB11B (p.Ser25Asn) with dispersed localization throughout the cytoplasm and enrichment at the Golgi apparatus. In all pictures, nuclei were stained with DAPI (blue). Scale bars represent 10 μm. The American Journal of Human Genetics  , DOI: ( /j.ajhg ) Copyright © 2017 American Society of Human Genetics Terms and Conditions

5 Figure 4 Identified Interactors of RAB11B Using Yeast Two-Hybrid cDNA Library Screening and Their Affinity for RAB11B Variants Co-transformations were performed in PJ69-4α yeast strains with the identified clones from the library screens fused to pAD together with WT or mutant RAB11B constructs fused to pBD. The clones are sorted according to the library in which they have been identified, as indicated in the far left column. The quantification of the selection of the growth of two-hybrid clones grown on medium lacking leucine, tryptophan and histidine with 3mM 3AT is quantified in the “growth” column. α-Galactosidase reporter gene activation (α-gal column) or β-galactosidase reporter gene activation (β-gal column) are quantified as well. Quantifications are on a scale from 0-3; with 0 for no reporter gene activation and 3 for highest reporter gene activation. Details of the identified clones in the cDNA library screens can be found in Table S2, including quantification of reporter gene activation on –LWHA medium. Original images are shown in Figure S2. The American Journal of Human Genetics  , DOI: ( /j.ajhg ) Copyright © 2017 American Society of Human Genetics Terms and Conditions

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