A Progeroid Syndrome Caused by RAF1 deficiency Underscores the importance of RTK signaling for Human Development

Somatic and germline gain-of-function point mutations in RAF, the first oncogene to be discovered in humans, delineate a group of tumor-prone syndromes known as RASopathies. In this study, we document the first human phenotype resulting from the germline loss of function of the proto-oncogene RAF1 ( a.k.a. CRAF) . In a consanguineous family, we uncovered a homozygous p.Thr543Met mutation segregating with a neonatal lethal progeroid syndrome with cutaneous, craniofacial, cardiac and limb anomalies. Structure-based prediction and functional tests using human knock-in cells showed that threonine 543 is essential to: 1) ensure RAF1’s stability and phosphorylation, 2) maintain its kinase activity towards substrates of the MAPK pathway and 3) protect from stress-induced apoptosis. When injected in Xenopus embryos mutant RAF1T543M failed to phenocopy the effects of overactive FGF/MAPK signaling confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel segmental progeroid syndrome which highlights the importance of RTK signaling for human development and homeostasis. Short summary A germline homozygous recessive loss-of-function mutation p.T453M in RAF1 causes a neonatal lethal progeroid syndrome. In vitro and in vivo tests demonstrate that Thr543 is necessary for RAF1’s protein stability, to transduce signaling to the MAPK pathway and to respond to stress-induced apoptosis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by a Strategic Positioning Fund for Genetic Orphan Diseases, an inaugural A*STAR Investigatorship from the Agency for Science, Technology and Research in Singapore. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All human studies were reviewed and approved by the Turkish and Singaporean institutional review board (A#x002A;STAR IRB #2019-087 and Koc University 2015.120.IRB2.047). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. * CNS : Central nervous system DCM : Dilated cardiomyopathy ECHO : Echocardiogram EGF : Epidermal growth factor FGF(R) : Fibroblast growth factor (receptor) GoF : Gain-of-function HCM : Hypertrophic cardiomyopathy HDR : Homology-directed repair LoF : Loss-of-function OFC : Occipital frontal circumference PDA : Patent ductus arteriosus PFO : Patent foramen ovale TRK : receptor tyrosine kinases USG : Ultrasonography VSD : Ventricular septal defect