Spatially clustering de novo variants in CYFIP2, encoding the cytoplasmic FMRP interacting protein 2, cause intellectual disability and seizures

Abstract

CYFIP2, encoding the evolutionary highly conserved cytoplasmic FMRP interacting protein 2, has previously been proposed as a candidate gene for intellectual disability and autism because of its important role linking FMRP-dependent transcription regulation and actin polymerization via the WAVE regulatory complex (WRC). Recently, de novo variants affecting the amino acid p.Arg87 of CYFIP2 were reported in four individuals with epileptic encephalopathy. We here report 12 independent patients harboring a variety of de novo variants in CYFIP2 broadening the molecular and clinical spectrum of a novel CYFIP2-related neurodevelopmental disorder. Using trio whole-exome or -genome sequencing, we identified 12 independent patients carrying a total of eight distinct de novo variants in CYFIP2 with a shared phenotype of intellectual disability, seizures, and muscular hypotonia. We detected seven different missense variants, of which two occurred recurrently (p.(Arg87Cys) and p.(Ile664Met)), and a splice donor variant in the last intron for which we showed exon skipping in the transcript. The latter is expected to escape nonsense-mediated mRNA decay resulting in a truncated protein. Despite the large spacing in the primary structure, the variants spatially cluster in the tertiary structure and are all predicted to weaken the interaction with WAVE1 or NCKAP1 of the actin polymerization regulating WRC-complex. Preliminary genotype-phenotype correlation indicates a profound phenotype in p.Arg87 substitutions and a more variable phenotype in other alterations. This study evidenced a variety of de novo variants in CYFIP2 as a novel cause of mostly severe intellectual disability with seizures and muscular hypotonia.

Fig. 1

Gene and protein structure of CYFIP2 with the eight variants identified in twelve patients. a Schematic drawing of the CYFIP2 gene as well as CYFIP2 protein with its conserved domains. The position of the eight variants identified in our twelve patients (indicated in black) as well as the recently published variants (Nakashima et al. [22], indicated in grey and with †) are depicted. All variants occurred de novo and have been detected in heterozygous state (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, protein structure according to NP_001278651.1 and NCBI's conserved domain database [51]). b, c Splice variant c.3669+1G>T affecting the conserved splice donor site after exon 31 induces skipping of exon 31 in P2. RT-PCR on RNA from peripheral blood leukocytes with primers located in the exons 29 and 32 (indicated as arrows) resulted in an additional aberrant product of 381 bp in the patient (lane 1), whereas the amplification in six controls (lanes 2–7) resulted in the expected 529 bp fragment only (S, size standard; lane 8, genomic DNA control; lane 9, no template control). Exon skipping in the patient was verified by the sequencing of amplified products (Supplemental Figure S1) and is predicted to result in a frameshift followed by a premature stop codon after 3 altered amino acids (NM_001291722.1:r.3522_3669del, p.(Glu1174Aspfs*3))

Fig. 2

a Structure of the WAVE regulatory complex (WRC) indicating the sites of variants. CYFIP is shown in orange and the C-terminus, which is absent in the p.(Glu1174Aspfs*3) splice variant, is highlighted in red. The C-terminus forms tight interactions to the NCKAP1 protein (shown in cyan space-filled presentation). Residues affected by missense variants are shown in space-filled presentation and colored by atom types. Residues Arg87, Ile664, Glu665, Asp724 and Gln725 interact with WAVE1 (shown in green space-filled presentation) and Ala455 interacts with NCKAP1. Tyr108 is located in close vicinity to the CYFIP-WAVE1 interface. b Close-up view on the CYFIP2-WAVE1 interaction site disturbed by most of the variants observed. WAVE1 residues 151-173 comprising helix α6 (important for VCA domain stabilization) and residues 531-543 corresponding to the C-helix (part of VCA domain) are shown in light green and dark green, respectively. The interacting CYFIP residues Arg87, Ile664, Glu665, Asp724, and Gln725 are shown in space-filled presentation and colored by atom types. c Close-up view of the CYFIP2-WAVE1 interface in the vicinity of Tyr108. Residues 133-142 of WAVE1 are shown in green and residues 100-114 of CYFIP2 are shown in orange (ribbon representation). Key residues discussed in text are shown in space-filled presentation and Tyr108 is colored by atom type. d Close-up view on the CYFIP2-NCKAP1 interface in the vicinity of Ala455. NCKAP1 is shown in cyan and residues 444-469 of CYFIP2 are shown as orange ribbon with Ala455 in space-filled presentation

Fig. 3

Facial appearances of eight patients (P1, P2, P3, P5, P6, P9, P11, and P12) with de novo variants in CYFIP2 and hands of four patients (P1, P3, P11, and P12)

Fig. 4

Schematic structural organization of the WAVE regulatory complex (WRC) with its members WAVE1, CYFIP2, NCKAP1, ABI2, and HSPC300 in inactivated condition (left part) and after activation of WAVE function by RAC1-GTP binding (upper right part) or aberrant WAVE activation by CYFIP2 mutation as suggested by our data (lower right part)