New insights into the clinical and molecular spectrum of the novel CYFIP2-related neurodevelopmental disorder and impairment of the WRC-mediated actin dynamics

Abstract

Purpose: A few de novo missense variants in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene have recently been described as a novel cause of severe intellectual disability, seizures, and hypotonia in 18 individuals, with p.Arg87 substitutions in the majority.

Methods: We assembled data from 19 newly identified and all 18 previously published individuals with CYFIP2 variants. By structural modeling and investigation of WAVE-regulatory complex (WRC)-mediated actin polymerization in six patient fibroblast lines we assessed the impact of CYFIP2 variants on the WRC.

Results: Sixteen of 19 individuals harbor two previously described and 11 novel (likely) disease-associated missense variants. We report p.Asp724 as second mutational hotspot (4/19 cases). Genotype-phenotype correlation confirms a consistently severe phenotype in p.Arg87 patients but a more variable phenotype in p.Asp724 and other substitutions. Three individuals with milder phenotypes carry putative loss-of-function variants, which remain of unclear pathogenicity. Structural modeling predicted missense variants to disturb interactions within the WRC or impair CYFIP2 stability. Consistent with its role in WRC-mediated actin polymerization we substantiate aberrant regulation of the actin cytoskeleton in patient fibroblasts.

Conclusion: Our study expands the clinical and molecular spectrum of CYFIP2-related neurodevelopmental disorder and provides evidence for aberrant WRC-mediated actin dynamics as contributing cellular pathomechanism.

Keywords: CYFIP2; WASF; WAVE-regulatory complex (WRC); epilepsy; intellectual disability.

Fig. 1. CYFIP2 variants and morphological features of affected individuals.

(a) Schematic drawing of the CYFIP2 gene in chromosomal region 5q33.3 with known and novel variants identified. (Likely) pathogenic variants are shown above and putative loss-of-function variants are depicted below the gene scheme. Red labeling represents recurrent variants, and bold letters indicate variants detected in the present study. All variants were shown to have occurred de novo except those marked with # (not maternal), § (not paternal), or $ (segregation unknown). Variant nomenclature and gene structure according to NM_001291722.1 with exons numbered from 1 to 32 consecutively and NC_000005.10 for intronic sequences. Regions coding for the conserved protein domains DUF1394 and FragX_IP are depicted in light green and light blue, respectively (according to the National Center for Biotechnology Information [NCBI] conserved domain database). (b) Morphological features of individuals with likely disease-associated variants in CYFIP2. Note a shared but not clearly recognizable facial gestalt including high, narrow forehead; apparent hypertelorism; depressed nasal bridge; bulbous nasal tip; full cheeks; everted lip vermilion; and long tapered fingers in several individuals. Composite facial appearance in younger and older individuals is shown in F2GY and F2GO, respectively (created by Face2Gene [FDNA Inc., Boston, MA, USA] with ten photos of nine individuals from this study and Zweier et al. each).

Fig. 2. Structural location of CYFIP2 missense variants.

(a) Structure of the WAVE-regulatory complex. The individual protein components are shown in ribbon presentation and colored as follows: CYFIP2 (orange), NCKAP1 (cyan), WASF1 (green), BRICK1 (yellow), and ABI2 (red). Residues for which missense variants were detected are shown in space-filled presentation and are colored according to the atom type. Black and gray labels denote residues identified in the present and in previous studies, respectively. The same coloring scheme is also used for (b–f), which show enlargements of structurally relevant regions. (b) Arg87, Ile664, Glu665, Asp724, and Gln725 (all colored according to their atom types) are located in the interface with WASF1 (green space-filled presentation). (c) Ala455, Met456, and Tyr690 are all located in the interface with NCKAP1 (cyan space-filled presentation). (d) Glu468 is located in the interface with BRICK1 (yellow space-filled presentation). (e) Met311 is buried in the CYFIP2 structure. All atoms closer than 5 Å to Met311 are shown in orange space-filled presentation. (f) Phe888 is buried in the CYFIP2 structure (orange) close to the interaction site of NCKAP1 (cyan ribbon). All atoms closer than 5 Å to Phe888 are shown in space-filled presentation.

Fig. 3. Phenotype in primary fibroblasts of affected individuals heterozygous for pathogenic CYFIP2 missense variants and unaffected controls.

(a) Representative images of PDGF-stimulated primary fibroblasts with phalloidin staining of F-actin. Fewer dorsal ruffles (indicated with white arrows) were observed in patient cells compared with unaffected control cells. (b) Quantification of dorsal ruffle formation in patient and control fibroblasts. The number of counted dorsal ruffles was normalized to cell number. Three independent experiments are shown, and at least 200 cells were analyzed in each. (c) Representative images of wound healing assays with fibroblasts carrying the p.(Tyr108His) variant and a control line at time points 0 hours and 24 hours. (d,e) Wound healing assay analysis of (d) time to half gap and (e) migration speed of patient and control fibroblasts. Three independent experiments with two technical replicates each were analyzed. Only fibroblasts harboring the p.(Tyr108His) variant showed a significant impairment of migration compared with controls. Mean and standard deviation are displayed, and statistical significance was evaluated using one-way analysis of variance (ANOVA) with p values indicated as follows: *p < 0.05, **p < 0.01, p**** < 0.0001.