Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome

Abstract

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Keywords: F-box protein; FBXW7; Neurodevelopment; brain malformation; epilepsy; gastrointestinal issues; global developmental delay; hypotonia; intellectual disability; macrocephaly.

Figure 1

Characteristics of FBXW7 neurodevelopmental syndrome (A) Genotype-phenotype matrix of clinical features of key phenotypes associated with FBXW7 neurodevelopmental variants. Each square represents an individual overlaid with variant class, and each row represents a clinical feature (affected—yellow; unaffected—blue). Variant types are depicted by dots: red (frameshift, stop, whole-gene deletion), blue (missense affecting WD40 domain), and gray (missense not in a WD40 domain). (B) Clinical features of affected individuals depicting phenotype by variant type: individual 1, aged 3 years, frontal and lateral, with arrow marking preauricular pit; individual 2, aged 3 years 2 months, frontal and lateral; individual 3, aged 14 years 9 months, from family 1, frontal and lateral; individual 4, aged 11 years 9 months, from family 1, frontal and lateral; individual 5, aged 6 years 3 months, from family 1, frontal and lateral; individual 6, aged 44 years, father of individuals 3–5 from family 1, frontal and lateral (note midface retrusion with class III malocclusion); individual 8, aged 5 years, frontal; individual 12 at 12 months, frontal, lateral, and at 26 months, frontal; mother of individual 12, aged 34 years, frontal and lateral; individual 15 at 3 years and 15 years; individual 19 at age 6 years, frontal, and with cutaneous Blaschkoid dyspigmentation suggestive of somatic mosaicism; individual 20 at 5 years, frontal and lateral; individual 21 at 3 years; individual 23 at 3 years; individual 24 at 5 years, frontal and lateral; individual 30 aged 15 years, frontal and lateral; individual 31 aged 15 years, frontal and lateral; individual 32 aged 2 years; individual 33 aged 10 years; individual 34 aged 1 year, frontal, and 12 years, frontal and lateral; individual 35 aged 3 years and 7 years, frontal. Deeply set eyes with upper eyelid fullness are evident in individuals 1, 2, 3, 5, 15, 21, 24, 32 (also in individual 21, not pictured). (C) Neuroradiological features of selected individuals; sagittal images of T1-weighted brain MRI scans of individuals 3, 20, 27, and 30 and T2-weighted brain scan of individual 24, displaying large cerebellar vermis with tonsillar ectopia (white arrowheads) and thick callosal genu (arrows)—note the generally thinned corpus callosum in individuals 24 and 30; axial T1-weighted brain MRI scans of individuals 20, 27, and 30 and T2-weighted brain scan of individual 24 displaying scattered subcortical white-matter hyperintensities and severely delayed myelination, equivalent to 7–10 months.

Figure 2

FBXW7 variants detected in this cohort cluster within the substrate-binding surface of the WD40 domain (A) The gene structure surrounding FBXW7 on chromosome 4 (GRCh37: 4q31–3q32.1) demonstrates the genomic position of two large genomic deletions identified in individuals 8 and 9 (thick red bars above chromosome). (B) Missense FBXW7 variants identified in this study cluster within the WD40-repeat domain. Frameshift, stop-gain, or splice-site (red) and missense (blue—within the WD40 domain; and gray—outside the WD40 domain) variants are shown above the protein. Recurrent non-familial (bold); recurrent familial (underlined); F-box domain (orange); and a WD40-repeat domain (beige) derived from DECIPHER. (C) Representation of the resolved structure of FBXW7 when in complex with a CYCLIN E1 degron (residues 360–390) demonstrating that the residues of FBXW7 that directly interact with CYCLIN E1 span similar residues as disease variants identified in this cohort. The positions of FBXW7 residues that directly interact with CYCLIN E1 are shown below the schematic depiction. F-box helices (rectangles H-1, H0, and H1–H3), linker α-helical domain (rectangles H4–H5), and the canonical eight-bladed β-propeller structure of the WD40 domain with each blade consisting of four antiparallel β strands (arrows [A–D] are shown, Amino acids in bold have also been shown to directly interact with DISC1. (D) FBXW7 variants associated with neurodevelopmental disorder are predominantly located at the substrate-binding surface of the WD40 repeat domain. The location of residues (in sticks with carbon atoms in purple, nitrogen atoms in blue, and oxygen atoms in red) impacted by mutations is shown on the tertiary structure of FBXW7 (cartoon) in configuration with CYCLIN E1 (surface in gray). The docking location of the conserved FBXW7 substrate-binding TPPXQ motif (cartoon in green) of CYCLIN E1 is demonstrated in close proximity to many of the impacted residues. Figure S1 provides an overlay of the variant residue with the wild-type residue for each individual variant, allowing identification of the change predicted in interaction for each missense variant.

Figure 3

Disease-associated variants impair the ability of FBXW7 to degrade substrates CYCLIN E1 and CYCLIN E2 (A) The majority of disease-associated FBXW7 variants do not impact steady-state protein amounts. Representative immunoblots of wild-type or mutant FBXW7 with and without inhibition of the ubiquitin proteasome system are shown. HEK293T cells exogenously expressing wild-type FBXW7 or mutant FBXW7 with a C-terminal Myc tag for 32 h were treated with 5 μM MG-132 for 16 h (four independent replicates). (B) Quantification of FBXW7:GAPDH from DMSO-treated samples of mutant FBXW7 protein in (A) relative to FBXW7^{wild type}; statistical support for altered steady-state amounts of the mutant FBXW7 protein was only evident for FBXW7^Arg689Gln (p = 0.007). (C) Quantification of FBXW7:GAPDH in MG-132-treated cells and versus their DMSO-treated counterpart in (A), demonstrating the change in steady-state mutant FBXW7 protein relative to FBXW7^{wild type} protein after UPS inhibition; statistical support for altered steady-state protein amount was only evident for FBXW7^Arg689Gln (p = 0.003). (D) Certain FBXW7 mutant proteins demonstrate impaired CYCLIN E1 substrate degradation. Representative immunoblots of wild-type or mutant FBXW7 co-expressed with the substrate CYCLIN E1 are shown. Whole-cell lysates extracted from HEK293T cells that exogenously expressed wild-type FBXW7 or mutant FBXW7 with a C-terminal Myc tag and CYCLIN E1 with a C-terminal V5 tag for 48 h are shown (nine independent replicates). (E) Quantification of CYCLIN E1:GAPDH in (D) for samples expressing mutant FBXW7 versus FBXW7^{wild type} protein; statistical support for altered steady-state protein amount of CYCLIN E1 was evident for FBXW7^Gly423Arg (p = 0.002), FBXW7^Asp480Gly (p = 0.002), and FBXW7^Val544Gly (p = 0.007). (F) Quantification of FBXW7:GAPDH in (D) for FBXW7 mutant proteins versus FBXW7^{wild type} protein when cells were co-transfected with CYCLIN E1. Statistical support for altered steady-state protein amount was evident only for FBXW7^Arg674Pro (p = 0.005). (G) The majority of FBXW7 mutant proteins demonstrate impaired CYCLIN E2 substrate degradation. Representative immunoblots of wild-type or mutant FBXW7 co-expressed with the substrate CYCLIN E2 are shown. Whole-cell lysates extracted from HEK293T cells that exogenously expressed wild-type FBXW7 or mutant FBXW7 with a C-terminal Myc tag and CYCLIN E2 with a C-terminal V5 tag for 48 h are shown (ten independent replicates). (H) Quantification of CYCLIN E2:GAPDH in (G) for samples expressing FBXW7 mutant protein versus FBXW7^{wild type} protein; statistical support for altered steady-state protein amount of CYCLIN E2 was evident for FBXW7^Gly423Arg (p = 0.00005), FBXW7^Asp480Gly (p = 0.02), FBXW7^Val544Gly (p = 0.000005), FBXW7^Ser640Arg (p = 0.000007), FBXW7^Arg674Trp (p = 0.005), and FBXW7^Arg674Pro (p = 0.02). (I) Quantification of FBXW7:GAPDH in (G) for FBXW7 mutant proteins versus FBXW7^{wild type} protein when cells were co-transfected with CYCLIN E2; statistical support for altered steady-state protein amount was evident for FBXW7^Arg674Pro (p = 0.04) and FBXW7^Arg674Pro (p = 0.02). All graphs present mean ± SEM. Student’s t test: ^∗p < 0.05; ^∗∗p < 0.01; and ^∗∗∗p < 0.001.

Figure 4

Knockdown of the FBXW7 Drosophila ortholog ago, specifically in neurons, can lead to deficits in habituation learning deficits and more severe neuronal dysfunction (A) Simplified scheme of the habituation assay, used for assessing the stimulus-induced escape response of individual flies upon repeated exposure. In controls, as depicted, the initial high jump response gradually wanes. Of note, in reality, the amplitude of jumps does not wane, but the frequency decreases in the tested population. B) Knockdown of ago with RNAi-1 and either elav^(I)-Gal4 or elav^(III)-Gal4 severely impairs jumping. Knockdown of ago with RNAi-2 driven by either driver is less detrimental, allowing assessment of habituation learning. (C) Neuronal knockdown of ago by elav^(III)-Gal4 and RNAi-2 reduces the ability of flies to habituate to the stimulus (in blue); in ccontrast to their genetic-background controls (in gray), they keep jumping with increased frequency throughout the course of the experiment. (D) Quantification of habituation according to mean trials to no-jump criterion (mTTC). Precise genotypes tested in (C) and (D): w/Y; 2xGMR-wIR/+; elav-Gal4^(III), UAS-Dicer-2/ UAS-RNAi-2 (in blue; n = 71, mTTC = 14.91, p = 0.0015). Genetic background control w/Y; 2xGMR-wIR/+; elav-Gal4^(III), UAS-Dicer-2/+ (in gray; n = 71, mTTC = 7.46). Statistical significance was assessed by a linear-model regression analysis on the log-transformed mTTC values; ^∗p = 0.05, ^∗∗p = 0.01, and ^∗∗∗p < 0.001.