Fragile X syndrome due to a missense mutation

Abstract

Fragile X syndrome is a common inherited form of intellectual disability and autism spectrum disorder. Most patients exhibit a massive CGG-repeat expansion mutation in the FMR1 gene that silences the locus. In over two decades since the discovery of FMR1, only a single missense mutation (p.(Ile304Asn)) has been reported as causing fragile X syndrome. Here we describe a 16-year-old male presenting with fragile X syndrome but without the repeat expansion mutation. Rather, we find a missense mutation, c.797G>A, that replaces glycine 266 with glutamic acid (p.(Gly266Glu)). The Gly266Glu FMR protein abolished many functional properties of the protein. This patient highlights the diagnostic utility of FMR1 sequencing.

Figure 1

Identification of a patient with a novel FMR1 missense mutation. (a) Patient's characteristic facial features that are consistent with Fragile X Syndrome, including tall forehead, elongated face, and large ears. (b) DNA chromatogram of the wild-type and patient alleles showing the single nucleotide substitution (NM_002024.5:c.797G>A) that replaces the glycine at residue 266 with glutamic acid (p.(Gly266Glu)). (c) ClustalW alignment across multiple species of FMRP amino acids 247–296. FMRP at residue 266 is highly conserved from human through Drosophila.

Figure 2

Functional analysis of mutant G266E-FMRP. (a) Constitutive AMPA receptor assay showing that G266E-FMRP is unable to rescue exaggerated AMPA receptor internalization in KO neurons. Hippocampal neurons from Fmr1 KO mice were cultured for 18 days, infected with either WT or G266E-FMRP, and the percentage of internalized to total AMPA receptors was calculated from individual dendrites. G266E-FMRP-infected neurons were statistically different from WT-FMRP-infected neurons (one-way ANOVA; n=30; F=609.92, P<0.001, Tukey post hoc analysis: ^***P<0.001 for all pairwise comparisons except WT versus KO+WT-FMRP P=0.42). As the variance in uninfected KO neurons was so low, G266E-infected neurons were still statistically different from KO neurons even though the mutant protein clearly does not rescue AMPA receptor internalization like WT-FMRP. Data are represented as boxplot with whiskers from minimum to maximum. (b) Polyribosome assay showing that G266E-FMRP does not associate with polyribosome fractions. The top graph is a representative A254 absorbance profile from Fmr1 KO MEF cells infected with either WT or G266E-FMRP, and the monosome (80S) and polyribosome peaks are indicated. Below is the distribution of FMRP by western blot analysis for each fraction corresponding to the same region of the linear sucrose gradient above. S6 ribosomal protein is also shown to verify sample loading in each well. These are representative blots from n=3 experiments. (c) RNA co-immunoprecipitation showing that G266E-FMRP does not bind three well-validated FMRP targets using qPCR analysis of the relative mRNA enrichment of Map1B, PSD95, and CamKII mRNAs after FMRP immunoprecipitation. Cortical neurons from Fmr1 KO mice were cultured for 10 days, infected with GFP, WT-FMRP, or G266E-FMRP lentivirus, and then processed for FMRP-RNA co-immunoprecipitation. The relative mRNA level for each primer set was normalized to each sample's β-actin mRNA and also relative FMRP expression level as determined by western blot densitometry. When mRNA enrichment (IP:input) for WT-FMRP is set to equal 1.0, G266E-FMRP mRNA enrichment drops by twofold to the same levels as GFP-infected neurons (paired Student's t-test; n=4; t=16.92, ^***P<0.001 for Map1B; t=12.544, ^**P=0.001 for PSD95; t=12.919, ^**P=0.001 for CamKII). Data are represented as mean±SD.

Figure 3

Structural analysis of mutant G266E-FMRP. (a) Ribbon representation of the FMRP KH1 (blue) and KH2 (green) domains from Protein Data Bank code 2QND. The three β-strands (1, 2, and 3) and three α-helices (αA, αB, and αC) of KH1 are labeled, and the position of Gly266 is highlighted in pink as indicated by the arrow. (b) Stick representation of residues 266–268 showing the close proximity of three negatively charged amino acids when residue 266 is converted from glycine (neutral) to glutamic acid (negative). Arrow points to residue 266. (c) Sphere representations of glycine (left) or glutamic acid (right) at residue 266. There is high probability for glutamic acid to crash into residues V250 and A271 due to the space constraints predicted by this structural model.