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. 2015 May 7;96(5):816-25.
doi: 10.1016/j.ajhg.2015.03.001. Epub 2015 Apr 9.

Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies

Marcello Niceta  1 , Emilia Stellacci  2 , Karen W Gripp  3 , Giuseppe Zampino  4 , Maria Kousi  5 , Massimiliano Anselmi  6 , Alice Traversa  7 , Andrea Ciolfi  2 , Deborah Stabley  8 , Alessandro Bruselles  2 , Viviana Caputo  9 , Serena Cecchetti  10 , Sabrina Prudente  11 , Maria T Fiorenza  12 , Carla Boitani  13 , Nicole Philip  14 , Dmitriy Niyazov  15 , Chiara Leoni  4 , Takaya Nakane  16 , Kim Keppler-Noreuil  17 , Stephen R Braddock  18 , Gabriele Gillessen-Kaesbach  19 , Antonio Palleschi  6 , Philippe M Campeau  20 , Brendan H L Lee  21 , Celio Pouponnot  22 , Lorenzo Stella  6 , Gianfranco Bocchinfuso  6 , Nicholas Katsanis  5 , Katia Sol-Church  8 , Marco Tartaglia  23
Affiliations

Mutations Impairing GSK3-Mediated MAF Phosphorylation Cause Cataract, Deafness, Intellectual Disability, Seizures, and a Down Syndrome-like Facies

Marcello Niceta et al. Am J Hum Genet. .

Abstract

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.

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Figures

Figure 1
Figure 1
De Novo Heterozygous Missense Mutations Affecting Residues of the GSK3 Phosphorylation Motifs within the Transactivation Domain of MAF Cause Aymé-Gripp Syndrome (A) Clinical features of affected subjects. Note the distinctive flat face, brachycephaly, ptosis, short nasal tip, long philtrum, small mouth, low-set and posteriorly angulated ears, and nail dystrophy. Permission to publish photographs was provided for all subjects shown. (B) Scheme of the MAF domain structure, and location of MAF mutations causing human disease. MAF contains an N-terminal transactivation domain (yellow) with regulatory function, and a C-terminal DNA binding domain, the latter containing an “extended homology” (green), “basic motif” (light blue), and leucine-zipper (pink) regions. The region containing the four in tandem arranged phosphorylation sites recognized by GSK3 (orange) is located within the transactivation domain. Residues mutated in subjects with Aymé-Gripp syndrome (red) and previously reported isolated cataracts/eye defects (black) are shown. (C) Cartoon illustrating the GSK3 recognition motifs and location of residues affected in Aymé-Gripp syndrome. The GSK3 catalytic domain is depicted with its active site (red) and the site binding to the priming phosphorylated residue (green). To phosphorylate its substrates, GSK3 requires a priming phosphorylation on the substrate four amino acids downstream the residue to be phosphorylated. The serine/threonine residues sequentially targeted by GSK3 are shown (red). Upon phosphorylation, they act as priming residues (green) to allow the subsequent phosphorylation of the upstream Ser/Thr. The kinase phosphorylating Ser70 has not been characterized yet. The residues affected by Aymé-Gripp syndrome-causing mutations (Ser54, Thr58, Pro59, Ser62, and Pro69) are indicated in bold.
Figure 2
Figure 2
Molecular Dynamics Simulations of the GSK3/MAF Decapeptide Complexes (A) Structural effects of the p.Pro59Leu and p.Pro59His changes. In both mutants, the conformation of the trimer comprised between the target and primed residues is considerably rearranged. Representative conformations are reported for wild-type MAF (left) and the p.Pro59Leu mutant (middle). In both mutants, larger and more variable distances are observed between the hydroxyl of Thr58, which is a GSK3 target residue, and the γ-phosphate of ATP (right, top plot) or the carboxyl group of the catalytic residue Asp181 (right, bottom plot). The distribution obtained in the simulations of the wild-type MAF sequence (black) and those referred to the peptides containing the p.Pro59His (red) and p.Pro59Leu (blue) substitutions are shown. (B) Effect of the p.Pro69Arg change. In the simulations, a stable interaction between pSer70 of the wild-type peptide and the priming site was observed (left), while a displacement of that residue from the site was documented for the peptide carrying the p.Pro69Arg change (middle). Such structural rearrangements are quantified by the distance occurring between the P atom of pSer70 and the ω-carbon atom in the side chain of the GSK3 priming site residue, Arg180 (wild-type peptide, black; p.Pro69Arg peptide, green) (right). In the left and middle panels, the surface of GSK is colored in brown, except for the catalytic residue Asp181 (red), and the priming site residues, Arg96, Arg180, and Lys205 (blue). ATP is shown in pink and the MAF backbone in yellow. The side chains of priming, target, and mutated MAF residues are shown in sticks representation.
Figure 3
Figure 3
Impact of Disease-Causing Mutations on MAF Function (A) Protein and phosphorylation levels of wild-type and disease-causing mutant MAF proteins in transiently transfected COS1 cells (upper panel). COS1 cells were maintained in high glucose DMEM, plus 10% FBS and supplements, and were transiently transfected to express wild-type MAF or each of the disease-causing alleles (FuGENE 6 [Promega]). To assess ubiquitination, we probed immunoprecipitated MAF with an anti-ubiquitin antibody (#8017, Santa Cruz Biotechnology) (middle panel). Whole-cell extracts were blotted with anti-MAF polyclonal (#7866, Santa Cruz Biotechnology), and anti-β-actin monoclonal (#A5441, Sigma-Aldrich) antibodies. Western blots are from a representative experiment of three performed. (B) Protein stability and proteasome-dependent degradation were assessed in COS1 cells transfected with the indicated constructs. Twenty-four hours after transfection, cells were treated with 20 μg/ml cycloheximide (CHX) or 20 μM MG132 for the indicated times. MAF protein levels were detected by immunoblotting with anti-MAF antibody. Western blots of a representative experiment of three performed are shown. (C) Confocal laser scanning microscopy analysis performed in COS1 cells transiently expressing wild-type MAF or one of three disease-causing alleles, without (upper panels) or with (lower panels) treatment with CSK buffer prior fixation. Cells were stained with anti-MAF polyclonal antibody and Alexa Fluor 488 goat anti-rabbit secondary antibody (green). Nuclei are DAPI stained (blue). Images are representative of 450 analyzed cells (Table S7). Experiments were performed as previously reported. (D) Transactivation assays were performed in COS1 cells transiently cotransfected with the IL4 promoter cloned into pGL3 vector reporter construct (kindly provided by Michael Lohoff, University of Marburg, Marburg, Germany) alone (black bar) or together with wild-type MAF (white bar) or each of the disease-causing MAF mutants (blue and red bars) (1:1 ratio), and 1:10 of Renilla luciferase control vector DNA (pRL-Act Renilla). After transfection (24 hr), firefly and Renilla luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega). Normalized luciferase activity (mean ± SD) of six experiments performed is reported as fold increase relative to cells not expressing exogenous MAF. p values were calculated using two-tailed Student’s t test. ∗, ∗∗, and ∗∗∗ indicate p < 0.05, p < 0.01, and p < 0.001, respectively. Protein levels of wild-type and disease-causing mutant MAF proteins were evaluated by immunoblotting with anti-MAF and anti-actin antibodies (lower panels).
Figure 4
Figure 4
In Vivo Impact of Aymé-Gripp Syndrome- and Isolated Cataract-Causing MAF Mutations on the Integrity of the Central Nervous System Using a Zebrafish Model (A) Dorsal views of uninjected zebrafish embryos (left), and embryos injected with the Aymé-Gripp syndrome-causing mutant (c.161C>T; p.Ser54Leu) (middle) and wild-type (right) MAF capped mRNA (100 pg) at 3 days after fertilization (dpf). Embryos were whole-mount stained using a primary antibody against acetylated tubulin (1:1000, T7451 [Sigma-Aldrich]) that marks neuronal axons, and an Alexa Fluor goat anti-mouse IgG secondary antibody (1:1000, A21207, Invitrogen). The circle highlights the area of the optic tectum that was measured. (B) Overexpression of wild-type MAF or the congenital cataracts-causing (c.863G>C; p.Arg288Pro) allele do not induce a significant reduction in the size of the optic tectum. By contrast, overexpression of each of the Aymé-Gripp syndrome-causing alleles results in a statistically significantly reduction of the size of the optic tectum (p < 0.0001). Bars indicate SE, and AU denotes arbitrary units. Statistical analysis was performed using two-tailed Student’s t test. For the measurements performed, we scored 86 control embryos, 61 embryos injected with wild-type MAF mRNA, and 58–70 embryos with each of the Aymé-Gripp syndrome-causing alleles’ mRNA. All experiments were performed blind to injection cocktail in duplicate.
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