De novo mutations in the GTP/GDP-binding region of RALA, a RAS-like small GTPase, cause intellectual disability and developmental delay

Abstract

Mutations that alter signaling of RAS/MAPK-family proteins give rise to a group of Mendelian diseases known as RASopathies. However, among RASopathies, the matrix of genotype-phenotype relationships is still incomplete, in part because there are many RAS-related proteins and in part because the phenotypic consequences may be variable and/or pleiotropic. Here, we describe a cohort of ten cases, drawn from six clinical sites and over 16,000 sequenced probands, with de novo protein-altering variation in RALA, a RAS-like small GTPase. All probands present with speech and motor delays, and most have intellectual disability, low weight, short stature, and facial dysmorphism. The observed rate of de novo RALA variants in affected probands is significantly higher (p = 4.93 x 10(-11)) than expected from the estimated random mutation rate. Further, all de novo variants described here affect residues within the GTP/GDP-binding region of RALA; in fact, six alleles arose at only two codons, Val25 and Lys128. The affected residues are highly conserved across both RAL- and RAS-family genes, are devoid of variation in large human population datasets, and several are homologous to positions at which disease-associated variants have been observed in other GTPase genes. We directly assayed GTP hydrolysis and RALA effector-protein binding of the observed variants, and found that all but one tested variant significantly reduced both activities compared to wild-type. The one exception, S157A, reduced GTP hydrolysis but significantly increased RALA-effector binding, an observation similar to that seen for oncogenic RAS variants. These results show the power of data sharing for the interpretation and analysis of rare variation, expand the spectrum of molecular causes of developmental disability to include RALA, and provide additional insight into the pathogenesis of human disease caused by mutations in small GTPases.

Fig 1. Facial features of individuals with variation in RALA.

Overlapping features include a broad, prominent forehead, horizontal eyebrows, epicanthus, mild ptosis, slightly anteverted nares, wide nasal bridge, short philtrum, thin upper lip vermillion with an exaggerated Cupid’s bow, pointed chin, and low-set ears with increased posterior angulation.

Fig 2. Variation observed in RALA clusters in GTP/GDP-binding regions.

A. Linear model of RALA, including GTP/GDP-binding regions (depicted in yellow, as defined by molecular modeling data) and the CAAX motif (CCIL in the case of RALA; depicted in green). Positions of amino acid residues that form the GTP/GDP-binding region are listed below the model, and residues within those regions are listed above the model. Residues affected by variation observed here are shown in red. The predicted protein changes for described variation are shown above the affected amino acid residues. B. Positions of RALA amino acid residues affected by variation relative to the GDP molecule. C. A zoomed in view of the variation observed within the GTP/GDP-binding region. GDP is shown in a licorice representation in orange. The RALA protein is shown in a cartoon representation in green, with the mutated residues in licorice representation. V25 is in yellow, K128 in blue, D130 in red, S157 in magenta, and A158 in black. Hydrogen bonds between the side chains of these amino acids and GDP are shown as black dashed lines. See S1–S5 and S8 Figs for consequences of individual variants on the protein structure.

Fig 3. Missense variation in RALA affects GTPase activity and RALA effector binding.

A. GTPase activity of purified recombinant RALA proteins was assessed using a luminescence assay. Raw luminescence values (measuring remaining free GTP) were subtracted from 100 to calculate activity, and were then normalized to a no template control (NTC). WT, wild-type RALA. G23D, predicted constitutively active mutant (not from a proband). ** indicates p-value = 0.0015 compared to WT, *** indicates p-value = 0.0003, and **** indicates p-value < 0.0001 compared to WT. Mean values of one experiment performed in triplicate are shown. B. Binding of purified recombinant RALA proteins to an effector molecule was assessed using an ELISA-based assay. Absorbances were normalized to a no template control (NTC). Mean values of one experiment performed in triplicate are shown. WT, wild-type RALA. **** indicates p-value < 0.0001 compared to WT. #### indicates p-value < 0.0001 compared to NTC. ### indicates p-value = 0.0001 compared to NTC.