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.

Arnold Palmer Hospital Division of Genetics Orlando FL United States of America

Centre de Référence Déficiences Intellectuelles de Causes Rares Paris France Assistance Publique Hôpitaux de Paris service de Génétique Hôpital Necker Enfants Malades Paris France

Department of Biology and Medical Genetics Charles University 2nd Faculty of Medicine and University Hospital Motol Prague Czech Republic

Department of Emerging Genetic Medicine Ambry Genetics Aliso Viejo CA United States of America

Department of Genetics La Pitié Salpêtrière Hospital Assistance Publique Hôpitaux de Paris Paris France

Department of Genetics La Pitié Salpêtrière Hospital Assistance Publique Hôpitaux de Paris Paris France Centre de Référence Déficiences Intellectuelles de Causes Rares Paris France Groupe de Recherche Clinique UPMC Déficience Intellectuelle et Autisme Paris France

Department of Genetics McMaster Children's Hospital Hamilton Ontario Canada

Department of Genetics University of Alabama at Birmingham Birmingham AL United States of America

Department of Pediatrics and Adolescent Medicine Diagnostic and Research Unit for Rare Diseases Charles University 1st Faculty of Medicine and General University Hospital Prague Czech Republic

Department of Pediatrics and Human Development Michigan State University East Lansing MI United States of America

Division of Clinical Genetics Department of Pediatrics Columbia University Medical Center New York NY United States of America

Division of Clinical Genetics Louisiana State University Health Sciences Center New Orleans LA United States of America Department of Genetics Children's Hospital New Orleans LA United States of America

Division of Pediatric Neurology University of Tennessee Health Science Center Neuroscience Institute and Le Bonheur Comprehensive Epilepsy Program Memphis TN United States of America Le Bonheur Children's Hospital Memphis TN United States of America

GeneDx Gaithersburg MD United States of America

HudsonAlpha Institute for Biotechnology Huntsville AL United States of America

HudsonAlpha Institute for Biotechnology Huntsville AL United States of America Department of Genetics University of Alabama at Birmingham Birmingham AL United States of America

Institute for Genomic Medicine Columbia University Medical Center New York NY United States of America

Kaiser Permanente Hawaii Honolulu HI United States of America

Laboratory of NMR Spectroscopy University of Chemistry and Technology Prague Czech Republic

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$a De novo mutations in the GTP/GDP-binding region of RALA, a RAS-like small GTPase, cause intellectual disability and developmental delay / $c SM. Hiatt, MB. Neu, RC. Ramaker, AA. Hardigan, JW. Prokop, M. Hancarova, D. Prchalova, M. Havlovicova, J. Prchal, V. Stranecky, DKC. Yim, Z. Powis, B. Keren, C. Nava, C. Mignot, M. Rio, A. Revah-Politi, P. Hemati, N. Stong, AD. Iglesias, SF. Suchy, R. Willaert, IM. Wentzensen, PG. Wheeler, L. Brick, M. Kozenko, ACE. Hurst, JW. Wheless, Y. Lacassie, RM. Myers, GS. Barsh, Z. Sedlacek, GM. Cooper,

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$a 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.

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