Functional Dysregulation of CDC42 Causes Diverse Developmental Phenotypes

Abstract

Exome sequencing has markedly enhanced the discovery of genes implicated in Mendelian disorders, particularly for individuals in whom a known clinical entity could not be assigned. This has led to the recognition that phenotypic heterogeneity resulting from allelic mutations occurs more commonly than previously appreciated. Here, we report that missense variants in CDC42, a gene encoding a small GTPase functioning as an intracellular signaling node, underlie a clinically heterogeneous group of phenotypes characterized by variable growth dysregulation, facial dysmorphism, and neurodevelopmental, immunological, and hematological anomalies, including a phenotype resembling Noonan syndrome, a developmental disorder caused by dysregulated RAS signaling. In silico, in vitro, and in vivo analyses demonstrate that mutations variably perturb CDC42 function by altering the switch between the active and inactive states of the GTPase and/or affecting CDC42 interaction with effectors, and differentially disturb cellular and developmental processes. These findings reveal the remarkably variable impact that dominantly acting CDC42 mutations have on cell function and development, creating challenges in syndrome definition, and exemplify the importance of functional profiling for syndrome recognition and delineation.

Keywords: Noonan syndrome; cardiac defects; developmental anomalies; exome sequencing; functional profiling; genotype-phenotype correlations; microcephaly; mutation spectrum; phenotypic heterogeneity; thrombocytopenia.

Figure 1

Location of Disease-Causing CDC42 Mutations and Their Structural Impact (A) Secondary structure elements (α helices and β strands), conserved motifs critical for tight guanine nucleotide binding and hydrolysis (G1–G5), and position of the identified disease-causing CDC42 mutations are illustrated. (B) Variant residues are assigned to three groups according to their position in the context of CDC42 structure (PDB: 2QRZ): group I or switch mutations (Tyr64, Arg66, and Arg68) are part of the switch II loop; group II or pocket mutations (Cys81, Ser83, and Ala159) are located in the vicinity of nucleotide binding pocket; and group III or CRIB mutations (Ile21, Tyr23, and Glu171) are far outside of the major interaction sites of CDC42 with GTP/GDP and involve exposed residues located in or close to regions of the protein mediating binding to effectors containing a CRIB motif. (C) The position of the mutant residues relative to CDC42 interactions is illustrated by overlaying three different crystal structures of CDC42 in complex with ARHGAP1 (p50^GAP) (PDB: 1GRN), ITSN (PDB: 1KI1), and WAS (WASP) (PDB: 1CEE). Residues in reciprocal vicinity up to 4 Å were considered as part of binding interface. Residues of CDC42 mediating these interactions are shown in yellow. (D) Group I mutations. Tyr64 and Arg66 are solvent-exposed residues and contribute to interactions with regulatory proteins and effectors (left). Interaction of both residues with ARHGAP18 (PDB: 5c2j) is shown as a representative for other interactions such as GEFs and effectors (middle). The disease-causing amino acid changes are predicted to affect this interaction. Arg68 participates in stabilizing the conformation of the switch II region via intramolecular interactions with Glu62 and Asp65 (right). The Arg-to-Gln change is predicted to destabilizes the switch II loop that is crucial for the interaction with signaling partners. (E) Group II mutations. Cys81, Ser83, and Ala159 are in close vicinity of the phosphates (G1) and guanine base (G5) of bound GTP/GDP. Their substitutions are predicted to directly or indirectly affect the nucleotide binding affinity and to shift the balance between inactive and active CDC42 toward the latter. (F) Group III mutations. Ile21, Tyr23, and Glu171 are part of a cavity on the CDC42 surface that accommodates the CRIB motif of bound effector proteins (e.g., WASP) (left). Ile21 and Tyr23 are critical for hydrophobic interactions (middle) with these type of proteins, while Glu171 contribute to binding mediating an electrostatic interaction (right).

Figure 2

Assessment of the GTPase Activity, Nucleotide Exchange, and Binding to Effectors of Disease-Causing CDC42 Mutants (A) Mean rate constants (k_obs values) of p50^GAP-stimulated GTP hydrolysis. Grey bars indicate non-significant differences compared to wild-type CDC42; blue bars indicate abolished/impaired GTP hydrolysis, which in turn results in an increased amount of active, GTP-bound CDC42 and thus enhanced signal flow. Data were obtained from >4 independent experiments. (B) Mean rate constants (k_obs values) of the GEF-catalyzed release of labeled GDP (mantGDP). Grey bars indicate non-significant differences compared to wild-type CDC42; blue and magenta bars indicate increased or abolished nucleotide exchange, respectively. The former is predicted to promote enhanced signaling, while the latter blocks CDC42 in its inactive state. Data were obtained from >4 independent experiments. (C) CDC42 mutants variably affect binding to effectors. Dissociation constants (K_d) obtained for the interaction of CDC42 proteins with PAK1, WASP, IQGAP1, and FMNL2 determined by fluorescence polarization. Data were collected from titration of increasing concentrations of the respective effectors. They were obtained from >4 independent experiments and are illustrated as bar charts. Grey bars indicate non-significant differences compared to wild-type CDC42; blue and magenta bars indicate increased or decreased binding affinity, respectively. (D) Scheme summarizing the functional dysregulation of disease-causing mutants on downstream signaling pathways and cellular processes. ITSN1 is a specific GEF for CDC42 promoting the active state of the GTPase by catalyzing GDP release. p50^GAP negatively controls CDC42 function by stimulating the GTP hydrolysis reaction. CDC42 interaction with PAK1, WASP, FMNL2, and IQGAP1 activates signaling pathways controlling different cellular processes. For each specific function, the blue and magenta arrows indicate the hyperactive or defective behavior, respectively.

Figure 3

In Vitro and In Vivo Functional Characterization of CDC42 Mutations (A) CDC42 mutations differentially impact polarized migration and cell proliferation. Wound-healing assays (above) and proliferation assays (below) were performed using NIH 3T3 cells transiently transfected to express wild-type CDC42 or each of the indicated mutants. Mean ± SD densitometry values of three independent experiments are shown. The wound was generated 24 hr after transfection, and migration in the wounded area was evaluated after 4 and 7 hr. Cells expressing exogenous wild-type CDC42 migrate more rapidly into the scratched area than cells transfected with the empty vector (EV). Mutants differentially perturb polarized migration, with CDC42^Ser83Pro and CDC42^Ala159Val overexpression variably enhancing the wound closure ability of transfected cells compared to the wild-type protein, whereas CDC42^Tyr23Cys, CDC42^Arg68Gln, and CDC42^Glu171Lys fail to do that, supporting a gain-of-function and a loss-of-function effect of these mutants, respectively. Cell proliferation was evaluated in transfected cells at the indicated time points and quantified by manual counting using a Neubauer hemocytometer. The trypan blue dye exclusion test was used to consider viable cells only. While the CDC42^Ala159Val and CDC42^Ser83Pro mutants variably enhance proliferation compared to cells expressing wild-type CDC42, no effect on proliferation (CDC42^Glu171Lys) and reduced proliferation (CDC42^Tyr23Cys and CDC42^Arg68Gln) is documented for the other mutants, indicating a loss-of-function and a dominant-negative effect, respectively. Asterisks indicate significant differences compared with wild-type CDC42 (^∗p < 0.05; ^∗∗p < 0.01; Student’s t test). (B) Consequences of CDC-42 expression on vulval development in C. elegans. Ectopic expression of wild-type CDC-42 at the L2/L3 stage elicits a multivulva (Muv) phenotype (left, upper panel), and CDC-42 overexpression in a LET-23/EGFR hypomorphic background reduces the penetrance of the vulvaless (Vul) phenotype (left, lower panel). Compared to animals expressing wild-type CDC-42, those expressing CDC-42^Ser83Pro and CDC-42^Ala159Val show higher prevalence of the Muv phenotype and lower prevalence of the Vul phenotype, indicating a gain-of-function role on LET-60/RAS signaling. Animals expressing the other tested CDC-42 mutants do not significantly differ from those expressing wild-type CDC-42. Ectopic expression of wild-type CDC-42 at the early L3 stage elicits a protruding vulva (Pvl) phenotype (right, upper panel). Animals expressing CDC-42^Ser83Pro and CDC-42^Ala159Val show a higher prevalence of the phenotype compared to worms expressing wild-type CDC-42, while a less penetrant phenotype was scored for animals expressing CDC-42^Tyr23Cys, CDC-42^Arg68Gln, or CDC-42^Glu171Lys mutants. RNA interference (RNAi) experiments show that the Pvl phenotype associated with overexpression of wild-type CDC-42 is mediated, in part, by WSP-1/WASP (right, lower panel). White and gray bars indicate the penetrance of Pvl in non-interfered and interfered animals, respectively. Error bars indicate SEM of four independent experiments, and asterisks specify significance differences between animals expressing CDC-42 mutants and those expressing wild-type CDC-42 or between interfered and non-interfered nematodes (^∗p < 0.05; ^∗∗p < 0.001; ^∗∗∗p < 0.0001; ^∗∗∗∗p < 0.00005; two-tailed Fisher’s exact test). Comparisons between worms expressing wild-type CDC-42 and control animals are also shown. RNAi was performed by feeding using HT115 E. coli bacteria expressing double stranded wsp-1 RNA (Ahringer’s C. elegans RNAi feeding library) and optimized to overcome lethality. As a control of the efficiency of the modified RNAi protocol, let-60 RNAi experiments were performed on animals carrying the let-60 gain-of-function allele n1046 (p.Gly13Glu), and the prevalence of the Muv phenotype was scored at a dissecting microscope (Table S4).

Figure 4

Facial Features of Individuals with Heterozygous CDC42 Mutations (A–C) Subject 3 (p.Tyr64Cys) at age 2 years and 6 months (A) and 15 years (B and C) showing upslanted palpebral fissures, smooth philtrum, flaring alae nasi, thin upper vermilion, and wide mouth with widely spaced teeth. (D) Subject 4 (p.Arg66Gly) at 15 years showing broad forehead and broad nasal bridge with bulbous nasal tip. (E and F) Subject 6 (p.Arg68Gln) at 24 months (E) and 4 years (F) showing a prominent broad forehead, hypertelorism, long philtrum, and thin upper vermilion. (G and H) Subject 9 (p.Ser83Pro) at age 2 (G) and 6 (H) years showing prominent forehead, hypertelorism, wide mouth with cupid’s bow, thin upper vermilion, and widely spaced teeth. (I and J) Subject 10 (p.Ser83Pro) at 13 (I) and 32 (J) years showing prominent forehead, wide nasal bridge, ptosis, flared nostrils, and wide mouth with widely spaced teeth. (K and L) Subject 11 (p.Ala159Val) at 2 years (K) and at 3 years and 7 months (L) showing very broad and prominent forehead, bulbous nasal tip, flared nostrils, cupid’s bow, and downturned corners of the mouth. (M–O) Subject 1 (p.Ile21Thr) at age 3 months (M), 2 years (N), and 10 years (O) showing synophrys, wide palpebral fissures, high and narrow nasal bridge, bulbous nasal tip, wide mouth with downturned corners, and mildly laterally prominent ears. (P) Subject 2 (p.Tyr23Cys) at 14 years showing wide palpebral fissures, high nasal bridge with elevated nasal tip, short philtrum, and long neck. (Q) Subject 12 (p.Glu171Lys) at 12 years showing typical facial features of Noonan syndrome, including broad forehead, hypertelorism, low-set ears, bulbous nasal tip, and flared nostrils. (R) Subject 13 (p.Glu171Lys) showing ptosis, broad neck, and pectus deformity. Note that individuals fitting the different mutation groups share some facial characteristics, and that intragroup variability is also observed.