Rare De Novo Missense Variants in RNA Helicase DDX6 Cause Intellectual Disability and Dysmorphic Features and Lead to P-Body Defects and RNA Dysregulation

Abstract

The human RNA helicase DDX6 is an essential component of membrane-less organelles called processing bodies (PBs). PBs are involved in mRNA metabolic processes including translational repression via coordinated storage of mRNAs. Previous studies in human cell lines have implicated altered DDX6 in molecular and cellular dysfunction, but clinical consequences and pathogenesis in humans have yet to be described. Here, we report the identification of five rare de novo missense variants in DDX6 in probands presenting with intellectual disability, developmental delay, and similar dysmorphic features including telecanthus, epicanthus, arched eyebrows, and low-set ears. All five missense variants (p.His372Arg, p.Arg373Gln, p.Cys390Arg, p.Thr391Ile, and p.Thr391Pro) are located in two conserved motifs of the RecA-2 domain of DDX6 involved in RNA binding, helicase activity, and protein-partner binding. We use functional studies to demonstrate that the first variants identified (p.Arg373Gln and p.Cys390Arg) cause significant defects in PB assembly in primary fibroblast and model human cell lines. These variants' interactions with several protein partners were also disrupted in immunoprecipitation assays. Further investigation via complementation assays included the additional variants p.Thr391Ile and p.Thr391Pro, both of which, similarly to p.Arg373Gln and p.Cys390Arg, demonstrated significant defects in P-body assembly. Complementing these molecular findings, modeling of the variants on solved protein structures showed distinct spatial clustering near known protein binding regions. Collectively, our clinical and molecular data describe a neurodevelopmental syndrome associated with pathogenic missense variants in DDX6. Additionally, we suggest DDX6 join the DExD/H-box genes DDX3X and DHX30 in an emerging class of neurodevelopmental disorders involving RNA helicases.

Keywords: DDX6; DEAD-box; DExD/H-box; RNA helicase; RecA domain; helicase; intellectual disability; mRNA metabolism; missense variants; neurodevelopmental disorder; p-bodies; processing bodies.

Figure 1

Individuals with Intellectual Disability and Developmental Delay and Rare De Novo Missense Variants in DDX6 Pictures of individuals with rare de novo missense variants in DDX6 (Subjects S1, S2, S4, and S5).

Figure 2

Localization of the Different De Novo Missense Variants in DDX6 (A) Linear representation of the DDX6 protein (GenBank: NP_004388) with highlighted functional DExD/H-box motifs. De novo missense variants identified in individuals with ID are indicated by red dashed lines, and missense variants observed in the gnomAD population are plotted in orange with an indication of the number of individuals carrying them. Motif residue numbers and colors are as follows: motifs Q (115–123) and GG (201–203) in yellow, motifs I (140–146) and II (DEAD) (246–249) in dark green, motifs Ia (171–181) and Ib (222–225) in orange, motif IV (338–340) and V (395–399) in light green, and motifs III (277–279), QxxR (370–373), and VI (419–427) in gray. Motifs Q, I, II, V, and VI are involved in ATP binding, and motifs GG, QxxR, Ia, Ib, IV, and V are involved in RNA binding. Motif III is involved in intramolecular interactions. (B) A heatmap spanning the linear DDX6 protein representing in silico impact (effect) predictions of all possible amino acid substitution mutations in DDX6 from the SNAP2 classifier. Dark red indicates a strong signal for effect, white indicates a weak signal, and blue indicates a strong signal for neutral or no effect. (C) Mapping of missense variants identified in the gnomAD population (blue spheres) and in probands (red spheres) on a 3D representative structure of DDX6 in complex with the CNOT1 MIF4G (pink) domain and the 4E-T CHD (green) domain (PDB: 5ANR). The two RecA-like units of DDX6 are colored in gray (helicase ATP-binding domain) and yellow (helicase C-terminal domain). (D) A hydrophobicity surface model of DDX6 (PDB: 4CT5) created with Chimera. Kyte-Doolittle scale coloring was used; colors range from dodger blue for the most hydrophilic, to white at neutral, to orange-red for the most hydrophobic regions. Missense variants identified in probands have been highlighted in yellow.

Figure 3

Fibroblasts from Individuals S1 and S2 Contain a Low Number of PBs (A) Fibroblasts from an unrelated, age-matched control individual and from S2, who carried the DDX6-p.Cys390Arg variant, were immunostained with DDX6 and EDC4 antibodies. Nuclei were stained with DAPI. Arrows point to some selected PBs within cells. The scale bar represents 20 μm. (B) Fibroblasts from S1, who carried the DDX6-p.Arg373Gln variant, and her healthy parents were analyzed as in (A) except that cells were grown for 2 h at 30°C before fixation. The scale bar represents 20 μm. (C) Quantification of the fibroblasts with PBs. PB-containing cells were counted and plotted as a percentage of total cells (98 to 126 cells from three and four independent experiments for S2 and S1, respectively). Error bars, SD; t test: p < 0.0001 for both probands, as compared with their respective control.

Figure 4

Rare Missense Variants in DDX6 Affect its Interaction with Protein Partners and PB Formation (A–C) Mutated DDX6 proteins are defective for PB assembly. (A) Complementation assays. HeLa cells were depleted for endogenous DDX6 by transfection of a siRNA targeting the DDX6 3′UTR at the time of plating (control). After 24 h, cells were transfected or not with indicated FLAG-DDX6-HA plasmids. 40 h later, cells were analyzed by immunofluorescence using the indicated antibodies. The scale bar represents 10 μm. (B) Protein extracts from cells transfected or not with siRNA were analyzed by immunoblot with the indicated antibodies to verify the DDX6 depletion. The DDX6 signal, normalized using the ribosomal protein S6, is indicated below. (C) The number of PBs per cell was counted in three independent experiments (21–67 cells per experiment) and expressed as the relative percentage of PBs compared to the complementation with wild-type DDX6. FLAG-DDX6-p.Arg373Gln, -p.Cys390Arg, -p.Thr391Ile, and -p.Thr391Pro correspond to the DDX6 variants identified in subjects 1, 2, 3, and 4, respectively. Error bars, SD; t test: p < 0.0001 for all mutants, as compared to wild type. (D) DDX6 pathogenic variants impair ligand binding. HEK293 cells were depleted for endogenous DDX6 and transfected 24 h later with the indicated FLAG-DDX6-HA plasmids as in (A–C). 48 h later, proteins were extracted in the presence of RNaseA and immunoprecipitated with anti-FLAG M2 antibodies. 10% of the eluates were analyzed by immunoblot using the same antibody (bottom frame), whereas bound proteins were revealed from the remaining eluates by immunoblotting with the indicated antibodies (upper frame). The input corresponds to 30 μg of HEK293 proteins. Indicated under the panels are the percentages of binding proteins compared to immunoprecipitation with the wild-type DDX6, after normalization using the amount of each immunoprecipitated FLAG protein. Similar results were obtained in three to six independent immunoprecipitation experiments.

Figure 5

DDX6 Targets and mRNA-Encoding Proteins Involved in Translation Initiation Are Enriched Among Genes Upregulated in Individual S2 (A) A biotype distribution (according to Ensembl) of the differentially-expressed (DE) genes identified in S2. (B) Pathway enrichment analysis revealed a significant enrichment of the GO terms “Biological Process” and “Molecular Function” related to protein translation, ribosome and RNA processing; it also revealed enrichment for the KEGG pathway “ribosome” among the upregulated genes. (C) The mRNAs up- (orange boxes) and downregulated (green boxes) in subject 2 (S2) were analyzed for their enrichment in a DDX6 CLIP experiment (left panel) and their differential expression after DDX6 silencing (right panel), both in K562 cells. The analysis was performed with various p value thresholds for the differential expression in S2, as indicated. The distribution of the whole dataset (all) is given for comparison (gray boxes). The whiskers indicate the 10–90 percentiles. Upregulated mRNAs in S2 are preferential DDX6 targets and preferentially upregulated after DDX6 depletion in an erythroid cell line. (D) Upregulated mRNAs in S2 tend to be excluded from PBs. The mRNAs up- and downregulated in S2 were analyzed for their enrichment in purified PBs, as in (C). (E) Upregulated mRNAs in S2 tend to have a high GC content. In panels C–E, two-tail Mann-Whitney tests were performed with respect to all mRNAs: ^∗∗∗p < 0.0005 and ^∗∗p < 0.005.