Domain-Specific Phenotypic Profiles in RAF1-Related Noonan Syndrome

Domain-Specific Phenotypic Profiles in RAF1-Related Noonan Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Domain-Specific Phenotypic Profiles in RAF1-Related Noonan Syndrome Alessandro Mussa, Andrea Gazzin, Marta Calvo, Federico Rondot, and 30 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7696796/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jan, 2026 Read the published version in European Journal of Human Genetics → Version 1 posted 9 You are reading this latest preprint version Abstract Pathogenic variants in RAF1 are a common cause of Noonan syndrome (NS), accounting for approximately 5% of cases. Nonetheless, RAF1 -related NS is often associated with severe clinical features, particularly hypertrophic cardiomyopathy (HCM). Although initial studies highlighted the occurrence of genotype-phenotype correlations, a comprehensive analysis specifically focused on RAF1 variants is still lacking. We conducted a retrospective observational study combining newly collected cases of RAF1 -related NS from a national multicenter retrospective cohort (RC) with systematically reviewed cases from literature search (literature cohort, LC). Variants were classified by protein domain, while the most recurrent variant, p.Ser257Leu, was analyzed separately to assess variant- and domain-specific phenotype correlations. A total of 203 cases were included. Variants in the CR2 domain accounted for 83% of cases, with p.Ser257Leu alone representing 53%. HCM was observed in 80.1% of affected individuals confirming its role as the predominant cardiac manifestation in RAF1 -related NS; neurodevelopmental features were reported in 44.5% of patients. The prevalence of clinical features varied significantly according to variant location. HCM was markedly more frequently associated with CR2 variants (89.4%) and in subjects heterozygous for the p.Ser257Leu change (94.2%) compared with non-CR2 variants (37.1%). Conversely, neurodevelopmental features were more common in patients with non-CR2 variants (69.2%) than in those with CR2 variants (38.2%) or p.Ser257Leu (29.4%). CR2 and p.Ser257Leu variants were associated with earlier age at diagnosis and increased mortality. Our findings confirm and document more comprehensively domain- and variant-specific phenotypes in RAF1 -related NS, emphasizing the importance of variant-level interpretation in clinical management and genetic counseling. Biological sciences/Genetics/Medical genetics/Genetic counselling Health sciences/Medical research/Genetics research Noonan syndrome RAF1 hypertrophic cardiomyopathy clinical variability genotype-phenotype correlations functional domain Figures Figure 1 Figure 2 Figure 3 Introduction RASopathies are a group of clinically overlapping disorders caused by genetic variants in components or regulators of the RAS–mitogen-activated protein kinase (MAPK) signaling pathway, leading to upregulation of the signal transduction( 1 ). Pathogenic variants in most genes implicated in RASopathies follow an autosomal dominant inheritance pattern and cause disease through a gain-of-function mechanism, typically exhibiting complete penetrance with variable expressivity( 2 , 3 ). Less frequently, signal upregulation is caused by loss-of-function variants in genes encoding negative regulators of the RAS-MAPK cascade, as those encoding NF1 , LZTR1 , SPRED1 , SPRED2 and ERF ( 3 , 4 ). Among RASopathies, Noonan syndrome (NS) is the most common disorder, with an estimated prevalence of 1 in 1,000–2,500 individuals, and is also the more clinically heterogeneous ( 5 ). Although each clinical entity within the RASopathies presents with a distinct phenotype, they share common features including craniofacial features, cardiac anomalies, neurocognitive impairment, as well as cutaneous, musculoskeletal, ocular, and lymphatic abnormalities ( 6 – 10 ). Cardiac involvement is a hallmark of RASopathies, with congenital heart defects (CHDs) and hypertrophic cardiomyopathy (HCM) reported in 60–90% of patients ( 11 – 15 ). The prevalence, type, and severity of cardiovascular manifestations vary considerably depending on the underlying genotype ( 16 – 19 ). Numerous studies have investigated genotype–phenotype correlations, highlighting marked differences in the prevalence of clinical features across causative genes ( 20 – 27 ). Beyond gene–phenotype correlations, emerging evidence indicates variant- and domain-specific associations, documenting that distinct variants within the same gene can result in unique clinical manifestations ( 28 , 29 ). These observations underscore the importance of detailed variant/domain-level analysis to improve the clinical interpretation of genetic test results and refine prognostic assessment ( 5 , 30 – 33 ). Pathogenic variants in RAF1 accounts for approximately 5% of the NS ( 2 ). Previous studies identified these mutations as a major event associated with HCM ( 19 , 32 , 34 – 36 ). RAF1 is a member of the RAF serine/threonine-protein kinase family that function downstream of RAS in the MAPK signaling pathway ( 3 ). These proteins share structurally and functionally three conserved regions (CR): CR1 (amino acid residues 56–184 in RAF1 ), CR2 (residues 254–271), and CR3 (residues 349–609) (ref. 35). CR1 contains a Ras-binding domain along with a cysteine-rich domain, which have a regulatory role controlling the interaction of the kinase with activated Ras and plasma membrane translocation. Encompassing the phosphorylatable Ser259 residue, CR2 mediates the interaction with the 14-3-3 protein and functions as a key regulatory site keeping RAF1 in its catalytically inhibited conformation, basally. CR3 contains the kinase domain, where phosphorylation plays a key role in activation ( 37 ). In the absence of activating interactions with RAS, RAF proteins maintain a closed, autoinhibited conformation stabilized by a 14-3-3 protein dimer, which binds simultaneously to phosphorylated N- and C-terminal sites of the kinase. Upon binding to GTP-bound RAS proteins, RAF kinases undergo a conformational reorganization, favoring dimerization of the kinase and its catalytic activation, enabling downstream signaling through the phosphorylation of the MEK proteins, the second tiers of the MAPK cascade ( 36 , 38 ). The CR2 domain is a major mutational hotspot, as most germline pathogenic variants of RAF1 cluster at or near the regulatory Ser259 (ref. 35,36,38). Among these, substitutions of Ser257 represent the most common events (NSEuroNet database, https://nseuronet.com/php/ ). Studies have shown that affected individuals with variants within this region have a high lifetime risk of developing HCM, with nearly all presenting in early infancy and over 10% succumbing to cardiac failure within the first year of life ( 32 , 34 , 35 ). Although the phenotype associated with the c.770C > T variant has been thoroughly characterized ( 32 ), comprehensive genotype–phenotype studies of RAF1 variants remain limited, with available data from heterogeneous sources and lacking systematic collection and standardized phenotypic assessments. To address this gap, we conducted a systematic evaluation of the phenotypes associated with RAF1 variants by integrating newly collected, previously unpublished cases from a national Italian survey with a thorough literature review. Our goal was to generate robust data on RAF1 variant–phenotype correlations, specifically focusing on the clinical phenotype associated with p.Ser257Leu, other CR2-domain variants, and variants located outside the CR2 domain. Materials and Methods Retrospective Cohort - An observational retrospective study was conducted on patients with clinical diagnosis of NS and carrying heterozygous pathogenic or likely pathogenic variants in RAF1 (NM_002880.4) that had been followed at the participating institutions from 2010 to 2025 (retrospective cohort - RC). Local Ethical Committee approval (IRB 256/2022 protocol 68301 − 2022 Città della Salute e della Scienza di Torino) and signed informed consents were obtained for clinical and molecular data collection. Detailed information on each nuclear families was collected together with a shared clinical dataset including age at diagnosis, clinical diagnosis, deceased status and age at death, and occurrence of craniofacial features, electroencephalogram (EEG) anomalies and/or seizures, neurodevelopmental disorders (NDD), behavioral disorders (including autism spectrum disorder - ASD, and attention-deficit/hyperactivity disorder – ADHD), lymphatic disease, HCM, electrocardiogram (ECG) anomalies, and other congenital heart defects. During the years, molecular confirmation of the clinical diagnosis was attained by Sanger sequencing, parallel sequencing with custom-designed panels or clinical exome in the context of the diagnostic workflow on DNA derived from peripheral blood. Variants were classified according to American College of Medical Genetics and the Association for Molecular Pathology criteria ( 39 ). Literature search - Published cases were retrieved through a comprehensive literature review, focused on RAF1 related RASopathy clinical reports in PubMed (published in English). The search strategy targeted articles that specifically mentioned ‘RAF1’ and "Noonan" or "RASopathy" or "RASopathies" or "LEOPARD" or "lentiginosis" or "cardiomyopathy" or "RAS/MAPK"). Articles were filtered by three independent reviewers due to unavailability of the full text, preclinical studies and studies referring to somatic mutation. Articles were further filtered because of case duplication, referring to other diagnosis or other causative gene, poorly characterized or describing subjects with putative causative variants classified as a VUS (ACGM class 3). Subjects derived from the finally selected articles composed the literature cohort (LC) (Fig. 1 , Table S1 ). Clinical and molecular information was extracted as for the RC. Statistical analysis - Patients were divided into four groups: three according to genotype, based on the position of the variant within the three main protein domains (CR1, CR2, and CR3), which are the conserved regions implicated in the kinase structure (40–42). p.Ser257Leu, by far the most common variant encountered in RAF1 , was also considered as a fourth subgroup. For the few variants falling outside the putative coordinates of the domains, they were considered as part of one of the above-mentioned groups considering their proximity to one of the extremities. For statistical comparison, when not otherwise specified, patients with the c.Ser257Leu variant were considered within the group of CR2 domain (except for comparison with that specific variant). The χ2 test with Yates correction was used to assess differences between groups; for groups < 20, Fisher's exact test was used. Differences of continuous variables between groups was assessed by Mann–Whitney U test to account for non-normal distributions. A p-value < 0.05 was considered statistically significant. Results A total of 203 cases were gathered. Of these, forty cases were included through the multicenter collaboration (RC), while the LC comprised 163 affected individuals reported in 50 articles from the 236 analyzed. The distribution of RAF1 variants across the considered domains of the kinase was comparable between the LC and RC, with no statistically significant differences observed. Variants were predominantly clustered within CR2, with 83% ( n = 168) located in or near this region including one missense substitution involving a residue 17 amino acids upstream and one case 3 amino acids downstream. The p.Ser257Leu variant, also within the CR2 domain, was the most prevalent, accounting for 107 cases (53%). Less frequently, variants were identified within or near the CR3 domain ( n = 29, 14%), comprising 19 cases within CR3 and 10 cases located 3–41 residues downstream. Variants within or near CR1 domain accounted for 3% of cases ( n = 6), including 1 case 14 residues upstream (Fig. 2 ). No significant differences emerged from the comparison of clinical features reported in the RC and LC. The two cohorts were then merged and considered as a single group (Table 1). The comparative analysis revealed differences in the prevalence of major clinical features across the three considered regions. Significant differences also emerged by comparing p.Ser257Leu variants with other variants within the CR2 (Table 2, Fig. 3 ). Regarding the cardiological phenotype, HCM was markedly more frequent in subjects carrying substitutions involving residues within CR2 (89.4%) compared to residues outside this region (37.1%). HCM reached its highest prevalence (94.2%) in individuals carrying the p.Ser257Leu variant. On the other hand, no significant differences in HCM prevalence were observed between CR1 and CR3 domains. A similar trend was also observed for rhythm abnormalities, which were significantly more frequent in cases with variants within the CR2 domain (52.4% vs 23.5%). Conversely, the prevalence of other congenital heart defects, including septal defects, pulmonary valve dysplasia (VD), mitral VD, and aortic VD did not significantly differ across regions. A different prevalence pattern was observed for neurological involvement, which was more frequently observed in non-CR2 regions. NDDs were reported in 69.2% of patients with variants outside CR2, compared to 38.2% in the CR2 group, and 29.4% among individuals heterozygous for the p.Ser257Leu variant. No significant differences in NDD prevalence were observed between CR1 and CR3. Similarly, there were no significant inter-group differences regarding the prevalence of behavioral disorders and EEG anomalies/seizures. Musculoskeletal abnormalities were slightly but significantly less frequent in the individuals with CR2 variants (58.9%) and in the p.Ser257Leu group (55.0%) compared to those with CR1 or CR3 variants (80%). No significant differences were detected across groups in the prevalence of lymphatic dysplasia or dysmorphic facial features. Age at diagnosis varied significantly among groups: the median age was 0.25 years in the p.Ser257Leu group, 0.78 years ( n = 80) in the CR2 group, 4.6 years ( n = 5) in CR1, and 11.0 years ( n = 19) in CR3. At the time of reporting, 21 patients (10.3%) were deceased, all of whom carried CR2 variants. Age at death was remarkably early, with a mean of 1.59 ± 4.2 years; notably 18 of the 21 deaths occurred before the age of 3 years. Discussion This study constitutes a comprehensive effort to delineate genotype–phenotype correlations of constitutional RAF1 pathogenic variants and to clarify how variant location within the RAF1 protein shapes the clinical presentation. By comparing variants across the three major functional domains of the kinase, we identified both shared features and notable phenotypic differences, particularly in cardiac and neurodevelopmental outcomes, revealing clinically relevant patterns that may guide risk stratification, surveillance, and genetic counseling. The overall clinical profile of patients with RAF1 variants confirmed a high prevalence of HCM and other cardiac anomalies, often leading to early diagnosis and substantial mortality. In line with previous reports ( 34 ) this burden appears to be at least partially attributable to by the clustering of pathogenic variants within CR2, which emerges as a phenotypic hotspot for cardiac involvement. Variants in CR2 were associated with particularly severe cardiac phenotype. Among them, the p.Ser257Leu allele showed the strongest association, with nearly 95% of carriers affected by HCM. Importantly, this variant alone did not account for the increased prevalence of HCM associated with CR2, as other variants within the domain also contributed, underscoring CR2 as a critical regulatory region. This domain exerts an autoinhibitory function by mediating RAF1 interaction with the 14-3-3 protein dimers, and the disruption of this binding enhances signaling through the MAPK cascade by favoring stable RAF1 binding to GTP-bound Ras and kinase activation ( 37 ). These findings align with previous reports describing p.Ser257Leu as a driver of early-onset, rapidly progressive HCM with poor prognosis ( 32 , 34 , 35 ), while also expanding the evidence that additional CR2 variants can confer similarly severe cardiac phenotypes ( 43 – 45 ). Interestingly, despite the substantial burden of cardiac disease, patients carrying the p.Ser257Leu variant and other CR2-domain variants exhibited significantly lower frequencies of neurodevelopmental features compared to other genotypes. This observation raises the possibility that the contribution of CR2 variants to RAF1 functional dysregulation may be cell- or tissue-context dependent, predominantly affecting myocardial function while relatively sparing the central nervous system. This hypothesis warrants further investigation. Another noteworthy finding is the significantly younger age at diagnosis observed in patients with p.Ser257Leu and other CR2 variants, likely reflecting an earlier disease onset, along with a higher mortality rate, as indicated by the greater proportion of deceased individuals in this group. These results underscore the critical need for timely diagnosis, intensive cardiac monitoring, and proactive management in affected patients—potentially including early referral for advanced interventions such as cardiac transplantation or consideration of targeted therapeutic approaches ( 46 – 48 ). From a genotype–phenotype correlation perspective, our findings confirm that RAF1 variants are associated with distinct, non-uniform clinical manifestations across the gene. Instead, they exhibit clear variant- and domain-specific patterns of expressivity, reinforcing the importance of variant-level interpretation in both clinical management and genetic counseling. Despite the strengths of our approach — including both literature-derived and unpublished cases, and standardized data collection this study has limitations. The retrospective nature of data collection may introduce biases, particularly in the assessment of developmental and behavioral features. Moreover, the heterogeneity in clinical follow-up and cardiac assessment protocols across centers may affect the comparability of some findings. Lastly, rare variants were underrepresented, limiting our ability to draw conclusions on less common genotypes. Our findings reveal distinct domain- and variant-specific phenotypic profiles in RAF1 -related NS. Variants within CR2, especially p.Ser257Leu, are strongly associated with a high-risk cardiac phenotype and early mortality, whereas non-CR2 variants are more frequently linked to neurodevelopmental involvement. These findings underscore the clinical relevance of variant-level interpretation for risk stratification, management, and genetic counseling. They also highlight the growing need for domain- and variant-specific analyses across RASopathies to support more tailored clinical care. Declarations Data availability statement Full data are available from the corresponding author, upon reasonable request. Author Contribution Statement Conceptualization: AM, AG, MC, FR and EA; methodology: AM, AG, MC, and FR; validation: CL, MN, MCD, and SC; formal analysis: AG and FR; investigation: AG, MC, and FR; resources: AM; data curation: AG, MC, FR AM, CL, MN, MLD, EM, GL, SC, and MCD; writing-original draft preparation: AG, MC and FR; writing—review and editing: AM, AG, MC, FR, GR, CL, MN, MLD, MCD, FL, EM, IC, ET, IS, GM, EA, FS, FB, GZ, LT, RP, DC, AMV, EB, SM, SC, PD, EA, CR, GC, GBF, GL, ADL and MT; supervision: AM; project administration: AM; funding acquisition: AM. All authors have read and agreed to the published version of the manuscript. Funding This research was partially funded with the contribution of the Italian Association of patients affected by Noonan Syndrome and other RASopathies (Associazione Italiana Sindrome di Noonan e RASopatie ODV, www.sindromedinoonan.org), and Italian Ministry of Health (5 per 1000 2024 and RC 2022–2024). Ethical Approval The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of A.O.U. Città della Salute e della Scienza di Torino (approval number 256/2022 prot 68301 approved on 17/06/2022) for studies involving humans. Informed consent was obtained from all subjects involved in the study. Competing interests The authors declare no competing interests. References Zenker M. Clinical overview on RASopathies. American Journal of Medical Genetics Part C: Seminars in Medical Genetics. 2022;190(4):414–24. Tartaglia M, Gelb BD. NOONAN SYNDROME AND RELATED DISORDERS: Genetics and Pathogenesis. Annual Review of Genomics and Human Genetics. 22 settembre 2005;6(Volume 6, 2005):45–68. Tartaglia M, Aoki Y, Gelb BD. The molecular genetics of RASopathies: An update on novel disease genes and new disorders. Am J Med Genet C Semin Med Genet. dicembre 2022;190(4):425–39. Zenker M. Clinical manifestations of mutations in RAS and related intracellular signal transduction factors. Current Opinion in Pediatrics. agosto 2011;23(4):443. Roberts AE, Allanson JE, Tartaglia M, Gelb BD. Noonan syndrome. The Lancet. 26 gennaio 2013;381(9863):333–42. Davico C, D’Alessandro R, Borgogno M, Campagna F, Torta F, Ricci F, et al. Epilepsy in a cohort of children with Noonan syndrome and related disorders. Eur J Pediatr. agosto 2022;181(8):2919–26. Rauen KA. The RASopathies. Annu Rev Genomics Hum Genet. 2013;14:355–69. Libraro A, D’Ascanio V, Cappa M, Chiarito M, Digilio MC, Einaudi S, et al. Growth in Children With Noonan Syndrome and Effects of Growth Hormone Treatment on Adult Height. Front Endocrinol (Lausanne). 2021;12:761171. Davico C, Borgogno M, Campagna F, D’Alessandro R, Ricci F, Amianto F, et al. Psychopathology and Adaptive Functioning in Children, Adolescents, and Young Adults with Noonan Syndrome. J Dev Behav Pediatr. 1 marzo 2022;43(2):e87–93. Baldassarre G, Mussa A, Dotta A, Banaudi E, Forzano S, Marinosci A, et al. Prenatal features of Noonan syndrome: prevalence and prognostic value. Prenat Diagn. ottobre 2011;31(10):949–54. Karnik R, Geiger M. Cardiac Manifestations of Noonan Syndrome. Pediatr Endocrinol Rev. maggio 2019;16(Suppl 2):471–6. Prendiville TW, Gauvreau K, Tworog-Dube E, Patkin L, Kucherlapati RS, Roberts AE, et al. Cardiovascular disease in Noonan syndrome. Arch Dis Child. luglio 2014;99(7):629–34. Delogu AB, Limongelli G, Versacci P, Adorisio R, Kaski JP, Blandino R, et al. The heart in RASopathies. American Journal of Medical Genetics Part C: Seminars in Medical Genetics. 2022;190(4):440–51. Kim ST, Lee SY, Kim GB, Bae EJ, Ko JM, Song MK. Cardiovascular Characteristics and Progressions of Hypertrophic Cardiomyopathy and Pulmonary Stenosis in RASopathy Syndrome in the Genomic Era. The Journal of Pediatrics. novembre 2023;262:113351. Jhang WK, Choi JH, Lee BH, Kim GH, Yoo HW. Cardiac Manifestations and Associations with Gene Mutations in Patients Diagnosed with RASopathies. Pediatr Cardiol. dicembre 2016;37(8):1539–47. Tartaglia M, Gelb BD, Zenker M. Noonan syndrome and clinically related disorders. Best Practice & Research Clinical Endocrinology & Metabolism. 1 febbraio 2011;25(1):161–79. Monda E, Prosnitz A, Aiello R, Lioncino M, Norrish G, Caiazza M, et al. Natural History of Hypertrophic Cardiomyopathy in Noonan Syndrome With Multiple Lentigines. Circulation: Genomic and Precision Medicine. agosto 2023;16(4):350–8. Boleti O, Norrish G, Field E, Dady K, Summers K, Nepali G, et al. Natural history and outcomes in paediatric RASopathy-associated hypertrophic cardiomyopathy. ESC Heart Failure. 2024;11(2):923–36. Sun L, Xie YM, Wang SS, Zhang ZW. Cardiovascular Abnormalities and Gene Mutations in Children With Noonan Syndrome. Front Genet. 2022;13:915129. Swarts JW, Kleimeier LER, Leenders EKSM, Rinne T, Klein WM, Draaisma JMT. Lymphatic anomalies during lifetime in patients with Noonan syndrome: Retrospective cohort study. American Journal of Medical Genetics Part A. 2022;188(11):3242–61. Reynolds G, Gazzin A, Carli D, Massuras S, Cardaropoli S, Luca M, et al. Update on the Clinical and Molecular Characterization of Noonan Syndrome and Other RASopathies: A Retrospective Study and Systematic Review. International Journal of Molecular Sciences. gennaio 2025;26(8):3515. Komatsuzaki S, Aoki Y, Niihori T, Okamoto N, Hennekam RCM, Hopman S, et al. Mutation analysis of the SHOC2 gene in Noonan-like syndrome and in hematologic malignancies. J Hum Genet. dicembre 2010;55(12):801–9. Papadopoulou A, Bountouvi E. Skeletal defects and bone metabolism in Noonan, Costello and cardio-facio-cutaneous syndromes. Front Endocrinol [Internet]. 27 ottobre 2023 [citato 26 gennaio 2025];14. Disponibile su: https://www.frontiersin.org/journals/endocrinology/articles/ 10.3389/fendo.2023.1231828/full Gripp KW, Baker L, Robbins KM, Stabley DL, Bellus GA, Kolbe V, et al. The novel duplication HRAS c.186_206dup p.(Glu62_Arg68dup): clinical and functional aspects. Eur J Hum Genet. novembre 2020;28(11):1548–54. Cesarini L, Alfieri P, Pantaleoni F, Vasta I, Cerutti M, Petrangeli V, et al. Cognitive profile of disorders associated with dysregulation of the RAS/MAPK signaling cascade. American Journal of Medical Genetics Part A. 2009;149A(2):140–6. Riller Q, Rieux-Laucat F. RASopathies: From germline mutations to somatic and multigenic diseases. Biomed J. agosto 2021;44(4):422–32. Kouz K, Lissewski C, Spranger S, Mitter D, Riess A, Lopez-Gonzalez V, et al. Genotype and phenotype in patients with Noonan syndrome and a RIT1 mutation. Genetics in Medicine. 1 dicembre 2016;18(12):1226–34. Niihori T, Aoki Y, Ohashi H, Kurosawa K, Kondoh T, Ishikiriyama S, et al. Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia. J Hum Genet. 2005;50(4):192–202. Kontaridis MI, Swanson KD, David FS, Barford D, Neel BG. PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects. J Biol Chem. 10 marzo 2006;281(10):6785–92. Digilio MC, Sarkozy A, Pacileo G, Limongelli G, Marino B, Dallapiccola B. PTPN11 gene mutations: linking the Gln510Glu mutation to the «LEOPARD syndrome phenotype». Eur J Pediatr. novembre 2006;165(11):803–5. Uludağ Alkaya D, Lissewski C, Yeşil G, Zenker M, Tüysüz B. Expanding the clinical phenotype of RASopathies in 38 Turkish patients, including the rare LZTR1, RAF1, RIT1 variants, and large deletion in NF1. Am J Med Genet A. dicembre 2021;185(12):3623–33. Gazzin A, Fornari F, Niceta M, Leoni C, Dentici ML, Carli D, et al. Defining the variant-phenotype correlation in patients affected by Noonan syndrome with the RAF1:c.770C > T p.(Ser257Leu) variant. Eur J Hum Genet. agosto 2024;32(8):964–71. Ilic N, Krasic S, Maric N, Gasic V, Krstic J, Cvetkovic D, et al. Noonan Syndrome: Relation of Genotype to Cardiovascular Phenotype-A Multi-Center Retrospective Study. Genes (Basel). 13 novembre 2024;15(11):1463. Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, et al. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet. agosto 2007;39(8):1007–12. Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, Amo R, et al. Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet. agosto 2007;39(8):1013–7. Nakhaei-Rad S, Haghighi F, Bazgir F, Dahlmann J, Busley AV, Buchholzer M, et al. Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues. Commun Biol. 21 giugno 2023;6(1):657. Jeon H, Tkacik E, Eck MJ. Signaling from RAS to RAF: The Molecules and Their Mechanisms. Annu Rev Biochem. agosto 2024;93(1):289–316. García-Alonso S, Mesa P, Ovejero L de la P, Aizpurua G, Lechuga CG, Zarzuela E, et al. Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation. Molecular Cell. 15 settembre 2022;82(18):3438–3452.e8. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. maggio 2015;17(5):405–23. Xie Y, Li H, Luo X, Li H, Gao Q, Zhang L, et al. IBS 2.0: an upgraded illustrator for the visualization of biological sequences. Nucleic Acids Res. 5 luglio 2022;50(W1):W420–6. UniProt Consortium. UniProt: a hub for protein information. Nucleic Acids Res. gennaio 2015;43(Database issue):D204-212. Terrell EM, Morrison DK. Ras-Mediated Activation of the Raf Family Kinases. Cold Spring Harb Perspect Med. gennaio 2019;9(1):a033746. Sana ME, Spitaleri A, Spiliotopoulos D, Pezzoli L, Preda L, Musco G, et al. Identification of a novel de novo deletion in RAF1 associated with biventricular hypertrophy in Noonan syndrome. Am J Med Genet A. agosto 2014;164A(8):2069–73. Zheng J, Peng L, Cheng R, Li Z, Xie J, Huang E, et al. RAF1 mutation leading to hypertrophic cardiomyopathy in a Chinese family with a history of sudden cardiac death: A diagnostic insight into Noonan syndrome. Molecular Genetics & Genomic Medicine. 2024;12(1):e2290. Ratola A, Silva HM, Guedes A, Mota C, Braga AC, Oliveira D, et al. A Novel Noonan Syndrome RAF1 Mutation: Lethal Course in a Preterm Infant. Pediatr Rep. 25 maggio 2015;7(2):5955. Wolf CM, Zenker M, Boleti O, Norrish G, Russell M, Meisner JK, et al. Impact of MEK Inhibition on Childhood RASopathy-Associated Hypertrophic Cardiomyopathy. JACC: Basic to Translational Science. febbraio 2025;10(2):152–66. Gazzin A, Fornari F, Cardaropoli S, Carli D, Tartaglia M, Ferrero GB, et al. Exploring New Drug Repurposing Opportunities for MEK Inhibitors in RASopathies: A Comprehensive Review of Safety, Efficacy, and Future Perspectives of Trametinib and Selumetinib. Life (Basel). 6 giugno 2024;14(6):731. Mussa A, Carli D, Giorgio E, Villar AM, Cardaropoli S, Carbonara C, et al. MEK Inhibition in a Newborn with RAF1-Associated Noonan Syndrome Ameliorates Hypertrophic Cardiomyopathy but Is Insufficient to Revert Pulmonary Vascular Disease. Genes (Basel). 21 dicembre 2021;13(1):6. Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations There is no duality of interest Supplementary Files Table1.xlsx Table 1 - Clinical features of the total cohort including 203 subjects with a pathogenetic RAF1 variant. Denominator refers to the total number of subjects whose information was available. Abbreviations: NDD, neurodevelopmental delay; NS, Noonan syndrome; NSML, Noonan syndrome with multiple lentigines; EEG, electroencephalogram; ECG electrocardiogram; HCM, hypertrophic cardiomyopathy. Table2.xlsx Table 2 - clinical features of subjects with pathogenic variants involving different functional regions of RAF1 . * <0.05, **< 0.01, *** < 0.001. Denominator refers to the total number of subjects for whom the information was available. Supplementarytable1.xlsx Literature research summary Cite Share Download PDF Status: Published Journal Publication published 08 Jan, 2026 Read the published version in European Journal of Human Genetics → Version 1 posted Editorial decision: revise 13 Nov, 2025 Review # 2 received at journal 09 Nov, 2025 Review # 1 received at journal 02 Nov, 2025 Reviewer # 2 agreed at journal 15 Oct, 2025 Reviewer # 1 agreed at journal 12 Oct, 2025 Reviewers invited by journal 01 Oct, 2025 Submission checks completed at journal 25 Sep, 2025 First submitted to journal 23 Sep, 2025 Editor assigned by journal 23 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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VUS, variant of uncertain significance.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7696796/v1/1d9ccbb91c46c396b4835459.png"},{"id":93557063,"identity":"e1ce1df9-d4d5-44eb-90df-7ba469520d64","added_by":"auto","created_at":"2025-10-15 06:48:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1430481,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of variants along the RAF1 protein. Lollipops are scaled according to their relative frequency.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7696796/v1/0c896e0e3ca77686d21fedd0.png"},{"id":93557068,"identity":"b9911646-b976-4e12-9b7c-f78c9cb3e750","added_by":"auto","created_at":"2025-10-15 06:48:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86109,"visible":true,"origin":"","legend":"\u003cp\u003eHistograms showing the relative frequencies of major clinical features across subjects carrying pathogenic variants affecting different functional regions of \u003cem\u003eRAF1\u003c/em\u003e. CR, conserved region; NDD, neurodevelopmental disorder. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7696796/v1/177155ac31c7fc607bad7b42.png"},{"id":99865541,"identity":"db4ecdde-5be5-4e8a-b5fa-2936e7fab8d3","added_by":"auto","created_at":"2026-01-09 08:06:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4352759,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7696796/v1/0ebfa881-709d-4e44-adbe-1d819f300920.pdf"},{"id":93557048,"identity":"a77630ac-6f2d-4c7d-9b7d-418d0712b3d0","added_by":"auto","created_at":"2025-10-15 06:48:07","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1 - \u003c/strong\u003eClinical features of the total cohort including 203 subjects with a pathogenetic \u003cem\u003eRAF1\u003c/em\u003evariant. Denominator refers to the total number of subjects whose information was available. Abbreviations: NDD, neurodevelopmental delay; NS, Noonan syndrome; NSML, Noonan syndrome with multiple lentigines; EEG, electroencephalogram; ECG electrocardiogram; HCM, hypertrophic cardiomyopathy.\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7696796/v1/66a86db80840293b52c6204e.xlsx"},{"id":93557053,"identity":"847a0186-9dcc-49f7-adfe-e15267d030cd","added_by":"auto","created_at":"2025-10-15 06:48:07","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 2 -\u003c/strong\u003e clinical features of subjects with pathogenic variants involving different functional regions of \u003cem\u003eRAF1\u003c/em\u003e. \u003cem\u003e* \u0026lt;0.05, **\u0026lt; 0.01, *** \u0026lt; 0.001. \u003c/em\u003eDenominator refers to the total number of subjects for whom the information was available.\u003c/p\u003e","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7696796/v1/5fae7b328e610e0b9da4f753.xlsx"},{"id":93557069,"identity":"fca804bd-dc5b-4cab-9dba-69daec3bce79","added_by":"auto","created_at":"2025-10-15 06:48:07","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":26527,"visible":true,"origin":"","legend":"Literature research summary","description":"","filename":"Supplementarytable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7696796/v1/ea3a3c8341087f5e335e5d6a.xlsx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Domain-Specific Phenotypic Profiles in RAF1-Related Noonan Syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRASopathies are a group of clinically overlapping disorders caused by genetic variants in components or regulators of the RAS\u0026ndash;mitogen-activated protein kinase (MAPK) signaling pathway, leading to upregulation of the signal transduction(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePathogenic variants in most genes implicated in RASopathies follow an autosomal dominant inheritance pattern and cause disease through a gain-of-function mechanism, typically exhibiting complete penetrance with variable expressivity(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Less frequently, signal upregulation is caused by loss-of-function variants in genes encoding negative regulators of the RAS-MAPK cascade, as those encoding \u003cem\u003eNF1\u003c/em\u003e, \u003cem\u003eLZTR1\u003c/em\u003e, \u003cem\u003eSPRED1\u003c/em\u003e, \u003cem\u003eSPRED2\u003c/em\u003e and \u003cem\u003eERF\u003c/em\u003e (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Among RASopathies, Noonan syndrome (NS) is the most common disorder, with an estimated prevalence of 1 in 1,000\u0026ndash;2,500 individuals, and is also the more clinically heterogeneous (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough each clinical entity within the RASopathies presents with a distinct phenotype, they share common features including craniofacial features, cardiac anomalies, neurocognitive impairment, as well as cutaneous, musculoskeletal, ocular, and lymphatic abnormalities (\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Cardiac involvement is a hallmark of RASopathies, with congenital heart defects (CHDs) and hypertrophic cardiomyopathy (HCM) reported in 60\u0026ndash;90% of patients (\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The prevalence, type, and severity of cardiovascular manifestations vary considerably depending on the underlying genotype (\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Numerous studies have investigated genotype\u0026ndash;phenotype correlations, highlighting marked differences in the prevalence of clinical features across causative genes (\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Beyond gene\u0026ndash;phenotype correlations, emerging evidence indicates variant- and domain-specific associations, documenting that distinct variants within the same gene can result in unique clinical manifestations (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). These observations underscore the importance of detailed variant/domain-level analysis to improve the clinical interpretation of genetic test results and refine prognostic assessment (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePathogenic variants in \u003cem\u003eRAF1\u003c/em\u003e accounts for approximately 5% of the NS (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Previous studies identified these mutations as a major event associated with HCM (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). RAF1 is a member of the RAF serine/threonine-protein kinase family that function downstream of RAS in the MAPK signaling pathway (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). These proteins share structurally and functionally three conserved regions (CR): CR1 (amino acid residues 56\u0026ndash;184 in \u003cem\u003eRAF1\u003c/em\u003e), CR2 (residues 254\u0026ndash;271), and CR3 (residues 349\u0026ndash;609) (ref. 35). CR1 contains a Ras-binding domain along with a cysteine-rich domain, which have a regulatory role controlling the interaction of the kinase with activated Ras and plasma membrane translocation. Encompassing the phosphorylatable Ser259 residue, CR2 mediates the interaction with the 14-3-3 protein and functions as a key regulatory site keeping RAF1 in its catalytically inhibited conformation, basally. CR3 contains the kinase domain, where phosphorylation plays a key role in activation (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In the absence of activating interactions with RAS, RAF proteins maintain a closed, autoinhibited conformation stabilized by a 14-3-3 protein dimer, which binds simultaneously to phosphorylated N- and C-terminal sites of the kinase. Upon binding to GTP-bound RAS proteins, RAF kinases undergo a conformational reorganization, favoring dimerization of the kinase and its catalytic activation, enabling downstream signaling through the phosphorylation of the MEK proteins, the second tiers of the MAPK cascade (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe CR2 domain is a major mutational hotspot, as most germline pathogenic variants of \u003cem\u003eRAF1\u003c/em\u003e cluster at or near the regulatory Ser259 (ref. 35,36,38). Among these, substitutions of Ser257 represent the most common events (NSEuroNet database, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nseuronet.com/php/\u003c/span\u003e\u003cspan address=\"https://nseuronet.com/php/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Studies have shown that affected individuals with variants within this region have a high lifetime risk of developing HCM, with nearly all presenting in early infancy and over 10% succumbing to cardiac failure within the first year of life (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Although the phenotype associated with the c.770C\u0026thinsp;\u0026gt;\u0026thinsp;T variant has been thoroughly characterized (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), comprehensive genotype\u0026ndash;phenotype studies of \u003cem\u003eRAF1\u003c/em\u003e variants remain limited, with available data from heterogeneous sources and lacking systematic collection and standardized phenotypic assessments. To address this gap, we conducted a systematic evaluation of the phenotypes associated with \u003cem\u003eRAF1\u003c/em\u003e variants by integrating newly collected, previously unpublished cases from a national Italian survey with a thorough literature review. Our goal was to generate robust data on \u003cem\u003eRAF1\u003c/em\u003e variant\u0026ndash;phenotype correlations, specifically focusing on the clinical phenotype associated with p.Ser257Leu, other CR2-domain variants, and variants located outside the CR2 domain.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eRetrospective Cohort -\u003c/b\u003e An observational retrospective study was conducted on patients with clinical diagnosis of NS and carrying heterozygous pathogenic or likely pathogenic variants in \u003cem\u003eRAF1\u003c/em\u003e (NM_002880.4) that had been followed at the participating institutions from 2010 to 2025 (retrospective cohort - RC). Local Ethical Committee approval (IRB 256/2022 protocol 68301\u0026thinsp;\u0026minus;\u0026thinsp;2022 Citt\u0026agrave; della Salute e della Scienza di Torino) and signed informed consents were obtained for clinical and molecular data collection. Detailed information on each nuclear families was collected together with a shared clinical dataset including age at diagnosis, clinical diagnosis, deceased status and age at death, and occurrence of craniofacial features, electroencephalogram (EEG) anomalies and/or seizures, neurodevelopmental disorders (NDD), behavioral disorders (including autism spectrum disorder - ASD, and attention-deficit/hyperactivity disorder \u0026ndash; ADHD), lymphatic disease, HCM, electrocardiogram (ECG) anomalies, and other congenital heart defects. During the years, molecular confirmation of the clinical diagnosis was attained by Sanger sequencing, parallel sequencing with custom-designed panels or clinical exome in the context of the diagnostic workflow on DNA derived from peripheral blood. Variants were classified according to American College of Medical Genetics and the Association for Molecular Pathology criteria (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eLiterature search -\u003c/b\u003e Published cases were retrieved through a comprehensive literature review, focused on \u003cem\u003eRAF1\u003c/em\u003e related RASopathy clinical reports in PubMed (published in English). The search strategy targeted articles that specifically mentioned \u0026lsquo;RAF1\u0026rsquo; and \"Noonan\" or \"RASopathy\" or \"RASopathies\" or \"LEOPARD\" or \"lentiginosis\" or \"cardiomyopathy\" or \"RAS/MAPK\"). Articles were filtered by three independent reviewers due to unavailability of the full text, preclinical studies and studies referring to somatic mutation. Articles were further filtered because of case duplication, referring to other diagnosis or other causative gene, poorly characterized or describing subjects with putative causative variants classified as a VUS (ACGM class 3). Subjects derived from the finally selected articles composed the literature cohort (LC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cem\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/em\u003e). Clinical and molecular information was extracted as for the RC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis -\u003c/b\u003e Patients were divided into four groups: three according to genotype, based on the position of the variant within the three main protein domains (CR1, CR2, and CR3), which are the conserved regions implicated in the kinase structure (40\u0026ndash;42). p.Ser257Leu, by far the most common variant encountered in \u003cem\u003eRAF1\u003c/em\u003e, was also considered as a fourth subgroup. For the few variants falling outside the putative coordinates of the domains, they were considered as part of one of the above-mentioned groups considering their proximity to one of the extremities. For statistical comparison, when not otherwise specified, patients with the c.Ser257Leu variant were considered within the group of CR2 domain (except for comparison with that specific variant). The χ2 test with Yates correction was used to assess differences between groups; for groups\u0026thinsp;\u0026lt;\u0026thinsp;20, Fisher's exact test was used. Differences of continuous variables between groups was assessed by Mann\u0026ndash;Whitney U test to account for non-normal distributions. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 203 cases were gathered. Of these, forty cases were included through the multicenter collaboration (RC), while the LC comprised 163 affected individuals reported in 50 articles from the 236 analyzed. The distribution of \u003cem\u003eRAF1\u003c/em\u003e variants across the considered domains of the kinase was comparable between the LC and RC, with no statistically significant differences observed. Variants were predominantly clustered within CR2, with 83% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;168) located in or near this region including one missense substitution involving a residue 17 amino acids upstream and one case 3 amino acids downstream. The p.Ser257Leu variant, also within the CR2 domain, was the most prevalent, accounting for 107 cases (53%). Less frequently, variants were identified within or near the CR3 domain (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;29, 14%), comprising 19 cases within CR3 and 10 cases located 3\u0026ndash;41 residues downstream. Variants within or near CR1 domain accounted for 3% of cases (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6), including 1 case 14 residues upstream (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNo significant differences emerged from the comparison of clinical features reported in the RC and LC. The two cohorts were then merged and considered as a single group (Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eThe comparative analysis revealed differences in the prevalence of major clinical features across the three considered regions. Significant differences also emerged by comparing p.Ser257Leu variants with other variants within the CR2 (Table\u0026nbsp;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Regarding the cardiological phenotype, HCM was markedly more frequent in subjects carrying substitutions involving residues within CR2 (89.4%) compared to residues outside this region (37.1%). HCM reached its highest prevalence (94.2%) in individuals carrying the p.Ser257Leu variant. On the other hand, no significant differences in HCM prevalence were observed between CR1 and CR3 domains. A similar trend was also observed for rhythm abnormalities, which were significantly more frequent in cases with variants within the CR2 domain (52.4% vs 23.5%). Conversely, the prevalence of other congenital heart defects, including septal defects, pulmonary valve dysplasia (VD), mitral VD, and aortic VD did not significantly differ across regions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA different prevalence pattern was observed for neurological involvement, which was more frequently observed in non-CR2 regions. NDDs were reported in 69.2% of patients with variants outside CR2, compared to 38.2% in the CR2 group, and 29.4% among individuals heterozygous for the p.Ser257Leu variant. No significant differences in NDD prevalence were observed between CR1 and CR3. Similarly, there were no significant inter-group differences regarding the prevalence of behavioral disorders and EEG anomalies/seizures. Musculoskeletal abnormalities were slightly but significantly less frequent in the individuals with CR2 variants (58.9%) and in the p.Ser257Leu group (55.0%) compared to those with CR1 or CR3 variants (80%). No significant differences were detected across groups in the prevalence of lymphatic dysplasia or dysmorphic facial features. Age at diagnosis varied significantly among groups: the median age was 0.25 years in the p.Ser257Leu group, 0.78 years (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;80) in the CR2 group, 4.6 years (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) in CR1, and 11.0 years (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19) in CR3. At the time of reporting, 21 patients (10.3%) were deceased, all of whom carried CR2 variants. Age at death was remarkably early, with a mean of 1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2 years; notably 18 of the 21 deaths occurred before the age of 3 years.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study constitutes a comprehensive effort to delineate genotype\u0026ndash;phenotype correlations of constitutional \u003cem\u003eRAF1\u003c/em\u003e pathogenic variants and to clarify how variant location within the RAF1 protein shapes the clinical presentation. By comparing variants across the three major functional domains of the kinase, we identified both shared features and notable phenotypic differences, particularly in cardiac and neurodevelopmental outcomes, revealing clinically relevant patterns that may guide risk stratification, surveillance, and genetic counseling.\u003c/p\u003e\u003cp\u003eThe overall clinical profile of patients with \u003cem\u003eRAF1\u003c/em\u003e variants confirmed a high prevalence of HCM and other cardiac anomalies, often leading to early diagnosis and substantial mortality. In line with previous reports (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) this burden appears to be at least partially attributable to by the clustering of pathogenic variants within CR2, which emerges as a phenotypic hotspot for cardiac involvement. Variants in CR2 were associated with particularly severe cardiac phenotype. Among them, the p.Ser257Leu allele showed the strongest association, with nearly 95% of carriers affected by HCM. Importantly, this variant alone did not account for the increased prevalence of HCM associated with CR2, as other variants within the domain also contributed, underscoring CR2 as a critical regulatory region. This domain exerts an autoinhibitory function by mediating RAF1 interaction with the 14-3-3 protein dimers, and the disruption of this binding enhances signaling through the MAPK cascade by favoring stable RAF1 binding to GTP-bound Ras and kinase activation (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). These findings align with previous reports describing p.Ser257Leu as a driver of early-onset, rapidly progressive HCM with poor prognosis (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), while also expanding the evidence that additional CR2 variants can confer similarly severe cardiac phenotypes (\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, despite the substantial burden of cardiac disease, patients carrying the p.Ser257Leu variant and other CR2-domain variants exhibited significantly lower frequencies of neurodevelopmental features compared to other genotypes. This observation raises the possibility that the contribution of CR2 variants to RAF1 functional dysregulation may be cell- or tissue-context dependent, predominantly affecting myocardial function while relatively sparing the central nervous system. This hypothesis warrants further investigation.\u003c/p\u003e\u003cp\u003eAnother noteworthy finding is the significantly younger age at diagnosis observed in patients with p.Ser257Leu and other CR2 variants, likely reflecting an earlier disease onset, along with a higher mortality rate, as indicated by the greater proportion of deceased individuals in this group. These results underscore the critical need for timely diagnosis, intensive cardiac monitoring, and proactive management in affected patients\u0026mdash;potentially including early referral for advanced interventions such as cardiac transplantation or consideration of targeted therapeutic approaches (\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFrom a genotype\u0026ndash;phenotype correlation perspective, our findings confirm that \u003cem\u003eRAF1\u003c/em\u003e variants are associated with distinct, non-uniform clinical manifestations across the gene. Instead, they exhibit clear variant- and domain-specific patterns of expressivity, reinforcing the importance of variant-level interpretation in both clinical management and genetic counseling.\u003c/p\u003e\u003cp\u003eDespite the strengths of our approach \u0026mdash; including both literature-derived and unpublished cases, and standardized data collection this study has limitations. The retrospective nature of data collection may introduce biases, particularly in the assessment of developmental and behavioral features. Moreover, the heterogeneity in clinical follow-up and cardiac assessment protocols across centers may affect the comparability of some findings. Lastly, rare variants were underrepresented, limiting our ability to draw conclusions on less common genotypes.\u003c/p\u003e\u003cp\u003eOur findings reveal distinct domain- and variant-specific phenotypic profiles in \u003cem\u003eRAF1\u003c/em\u003e-related NS. Variants within CR2, especially p.Ser257Leu, are strongly associated with a high-risk cardiac phenotype and early mortality, whereas non-CR2 variants are more frequently linked to neurodevelopmental involvement. These findings underscore the clinical relevance of variant-level interpretation for risk stratification, management, and genetic counseling. They also highlight the growing need for domain- and variant-specific analyses across RASopathies to support more tailored clinical care.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFull data are available from the corresponding author, upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: AM, AG, MC, FR and EA; methodology: AM, AG, MC, and FR; validation: CL, MN, MCD, and SC; formal analysis: AG and FR; investigation: AG, MC, and FR; resources: AM; data curation: AG, MC, FR AM, CL, MN, MLD, EM, GL, SC, and MCD; writing-original draft preparation: AG, MC and FR; writing—review and editing: AM, AG, MC, FR, GR, CL, MN, MLD, MCD, FL, EM, IC, ET, IS, GM, EA, FS, FB, GZ, LT, RP, DC, AMV, EB, SM, SC, PD, EA, CR, GC, GBF, GL, ADL and MT; supervision: AM; project administration: AM; funding acquisition: AM. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially funded with the contribution of the Italian Association of patients affected by Noonan Syndrome and other RASopathies (Associazione Italiana Sindrome di Noonan e RASopatie ODV, www.sindromedinoonan.org), and Italian Ministry of Health (5 per 1000 2024 and RC 2022–2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of A.O.U. Città della Salute e della Scienza di Torino (approval number 256/2022 prot 68301 approved on 17/06/2022) for studies involving humans. Informed consent was obtained from all subjects involved in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZenker M. Clinical overview on RASopathies. American Journal of Medical Genetics Part C: Seminars in Medical Genetics. 2022;190(4):414\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTartaglia M, Gelb BD. NOONAN SYNDROME AND RELATED DISORDERS: Genetics and Pathogenesis. Annual Review of Genomics and Human Genetics. 22 settembre 2005;6(Volume 6, 2005):45\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTartaglia M, Aoki Y, Gelb BD. The molecular genetics of RASopathies: An update on novel disease genes and new disorders. Am J Med Genet C Semin Med Genet. dicembre 2022;190(4):425\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZenker M. Clinical manifestations of mutations in RAS and related intracellular signal transduction factors. Current Opinion in Pediatrics. agosto 2011;23(4):443.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoberts AE, Allanson JE, Tartaglia M, Gelb BD. Noonan syndrome. The Lancet. 26 gennaio 2013;381(9863):333\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavico C, D\u0026rsquo;Alessandro R, Borgogno M, Campagna F, Torta F, Ricci F, et al. Epilepsy in a cohort of children with Noonan syndrome and related disorders. Eur J Pediatr. agosto 2022;181(8):2919\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRauen KA. The RASopathies. Annu Rev Genomics Hum Genet. 2013;14:355\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLibraro A, D\u0026rsquo;Ascanio V, Cappa M, Chiarito M, Digilio MC, Einaudi S, et al. Growth in Children With Noonan Syndrome and Effects of Growth Hormone Treatment on Adult Height. Front Endocrinol (Lausanne). 2021;12:761171.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavico C, Borgogno M, Campagna F, D\u0026rsquo;Alessandro R, Ricci F, Amianto F, et al. Psychopathology and Adaptive Functioning in Children, Adolescents, and Young Adults with Noonan Syndrome. J Dev Behav Pediatr. 1 marzo 2022;43(2):e87\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaldassarre G, Mussa A, Dotta A, Banaudi E, Forzano S, Marinosci A, et al. Prenatal features of Noonan syndrome: prevalence and prognostic value. Prenat Diagn. ottobre 2011;31(10):949\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarnik R, Geiger M. Cardiac Manifestations of Noonan Syndrome. Pediatr Endocrinol Rev. maggio 2019;16(Suppl 2):471\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrendiville TW, Gauvreau K, Tworog-Dube E, Patkin L, Kucherlapati RS, Roberts AE, et al. Cardiovascular disease in Noonan syndrome. Arch Dis Child. luglio 2014;99(7):629\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDelogu AB, Limongelli G, Versacci P, Adorisio R, Kaski JP, Blandino R, et al. The heart in RASopathies. American Journal of Medical Genetics Part C: Seminars in Medical Genetics. 2022;190(4):440\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim ST, Lee SY, Kim GB, Bae EJ, Ko JM, Song MK. Cardiovascular Characteristics and Progressions of Hypertrophic Cardiomyopathy and Pulmonary Stenosis in RASopathy Syndrome in the Genomic Era. The Journal of Pediatrics. novembre 2023;262:113351.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJhang WK, Choi JH, Lee BH, Kim GH, Yoo HW. Cardiac Manifestations and Associations with Gene Mutations in Patients Diagnosed with RASopathies. Pediatr Cardiol. dicembre 2016;37(8):1539\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTartaglia M, Gelb BD, Zenker M. Noonan syndrome and clinically related disorders. Best Practice \u0026amp; Research Clinical Endocrinology \u0026amp; Metabolism. 1 febbraio 2011;25(1):161\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonda E, Prosnitz A, Aiello R, Lioncino M, Norrish G, Caiazza M, et al. Natural History of Hypertrophic Cardiomyopathy in Noonan Syndrome With Multiple Lentigines. Circulation: Genomic and Precision Medicine. agosto 2023;16(4):350\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoleti O, Norrish G, Field E, Dady K, Summers K, Nepali G, et al. Natural history and outcomes in paediatric RASopathy-associated hypertrophic cardiomyopathy. ESC Heart Failure. 2024;11(2):923\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun L, Xie YM, Wang SS, Zhang ZW. Cardiovascular Abnormalities and Gene Mutations in Children With Noonan Syndrome. Front Genet. 2022;13:915129.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSwarts JW, Kleimeier LER, Leenders EKSM, Rinne T, Klein WM, Draaisma JMT. Lymphatic anomalies during lifetime in patients with Noonan syndrome: Retrospective cohort study. American Journal of Medical Genetics Part A. 2022;188(11):3242\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReynolds G, Gazzin A, Carli D, Massuras S, Cardaropoli S, Luca M, et al. Update on the Clinical and Molecular Characterization of Noonan Syndrome and Other RASopathies: A Retrospective Study and Systematic Review. International Journal of Molecular Sciences. gennaio 2025;26(8):3515.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKomatsuzaki S, Aoki Y, Niihori T, Okamoto N, Hennekam RCM, Hopman S, et al. Mutation analysis of the SHOC2 gene in Noonan-like syndrome and in hematologic malignancies. J Hum Genet. dicembre 2010;55(12):801\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePapadopoulou A, Bountouvi E. Skeletal defects and bone metabolism in Noonan, Costello and cardio-facio-cutaneous syndromes. Front Endocrinol [Internet]. 27 ottobre 2023 [citato 26 gennaio 2025];14. Disponibile su: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.frontiersin.org/journals/endocrinology/articles/\u003c/span\u003e\u003cspan address=\"https://www.frontiersin.org/journals/endocrinology/articles/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2023.1231828/full\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2023.1231828/full\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGripp KW, Baker L, Robbins KM, Stabley DL, Bellus GA, Kolbe V, et al. The novel duplication HRAS c.186_206dup p.(Glu62_Arg68dup): clinical and functional aspects. Eur J Hum Genet. novembre 2020;28(11):1548\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCesarini L, Alfieri P, Pantaleoni F, Vasta I, Cerutti M, Petrangeli V, et al. Cognitive profile of disorders associated with dysregulation of the RAS/MAPK signaling cascade. American Journal of Medical Genetics Part A. 2009;149A(2):140\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRiller Q, Rieux-Laucat F. RASopathies: From germline mutations to somatic and multigenic diseases. Biomed J. agosto 2021;44(4):422\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKouz K, Lissewski C, Spranger S, Mitter D, Riess A, Lopez-Gonzalez V, et al. Genotype and phenotype in patients with Noonan syndrome and a RIT1 mutation. Genetics in Medicine. 1 dicembre 2016;18(12):1226\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNiihori T, Aoki Y, Ohashi H, Kurosawa K, Kondoh T, Ishikiriyama S, et al. Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia. J Hum Genet. 2005;50(4):192\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKontaridis MI, Swanson KD, David FS, Barford D, Neel BG. PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects. J Biol Chem. 10 marzo 2006;281(10):6785\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDigilio MC, Sarkozy A, Pacileo G, Limongelli G, Marino B, Dallapiccola B. PTPN11 gene mutations: linking the Gln510Glu mutation to the \u0026laquo;LEOPARD syndrome phenotype\u0026raquo;. Eur J Pediatr. novembre 2006;165(11):803\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUludağ Alkaya D, Lissewski C, Yeşil G, Zenker M, T\u0026uuml;ys\u0026uuml;z B. Expanding the clinical phenotype of RASopathies in 38 Turkish patients, including the rare LZTR1, RAF1, RIT1 variants, and large deletion in NF1. Am J Med Genet A. dicembre 2021;185(12):3623\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGazzin A, Fornari F, Niceta M, Leoni C, Dentici ML, Carli D, et al. Defining the variant-phenotype correlation in patients affected by Noonan syndrome with the RAF1:c.770C\u0026thinsp;\u0026gt;\u0026thinsp;T p.(Ser257Leu) variant. Eur J Hum Genet. agosto 2024;32(8):964\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIlic N, Krasic S, Maric N, Gasic V, Krstic J, Cvetkovic D, et al. Noonan Syndrome: Relation of Genotype to Cardiovascular Phenotype-A Multi-Center Retrospective Study. Genes (Basel). 13 novembre 2024;15(11):1463.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, et al. Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nat Genet. agosto 2007;39(8):1007\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRazzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, Amo R, et al. Germline gain-of-function mutations in RAF1 cause Noonan syndrome. Nat Genet. agosto 2007;39(8):1013\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNakhaei-Rad S, Haghighi F, Bazgir F, Dahlmann J, Busley AV, Buchholzer M, et al. Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues. Commun Biol. 21 giugno 2023;6(1):657.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJeon H, Tkacik E, Eck MJ. Signaling from RAS to RAF: The Molecules and Their Mechanisms. Annu Rev Biochem. agosto 2024;93(1):289\u0026ndash;316.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Alonso S, Mesa P, Ovejero L de la P, Aizpurua G, Lechuga CG, Zarzuela E, et al. Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation. Molecular Cell. 15 settembre 2022;82(18):3438\u0026ndash;3452.e8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. maggio 2015;17(5):405\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie Y, Li H, Luo X, Li H, Gao Q, Zhang L, et al. IBS 2.0: an upgraded illustrator for the visualization of biological sequences. Nucleic Acids Res. 5 luglio 2022;50(W1):W420\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUniProt Consortium. UniProt: a hub for protein information. Nucleic Acids Res. gennaio 2015;43(Database issue):D204-212.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTerrell EM, Morrison DK. Ras-Mediated Activation of the Raf Family Kinases. Cold Spring Harb Perspect Med. gennaio 2019;9(1):a033746.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSana ME, Spitaleri A, Spiliotopoulos D, Pezzoli L, Preda L, Musco G, et al. Identification of a novel de novo deletion in RAF1 associated with biventricular hypertrophy in Noonan syndrome. Am J Med Genet A. agosto 2014;164A(8):2069\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng J, Peng L, Cheng R, Li Z, Xie J, Huang E, et al. RAF1 mutation leading to hypertrophic cardiomyopathy in a Chinese family with a history of sudden cardiac death: A diagnostic insight into Noonan syndrome. Molecular Genetics \u0026amp; Genomic Medicine. 2024;12(1):e2290.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRatola A, Silva HM, Guedes A, Mota C, Braga AC, Oliveira D, et al. A Novel Noonan Syndrome RAF1 Mutation: Lethal Course in a Preterm Infant. Pediatr Rep. 25 maggio 2015;7(2):5955.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWolf CM, Zenker M, Boleti O, Norrish G, Russell M, Meisner JK, et al. Impact of MEK Inhibition on Childhood RASopathy-Associated Hypertrophic Cardiomyopathy. JACC: Basic to Translational Science. febbraio 2025;10(2):152\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGazzin A, Fornari F, Cardaropoli S, Carli D, Tartaglia M, Ferrero GB, et al. Exploring New Drug Repurposing Opportunities for MEK Inhibitors in RASopathies: A Comprehensive Review of Safety, Efficacy, and Future Perspectives of Trametinib and Selumetinib. Life (Basel). 6 giugno 2024;14(6):731.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMussa A, Carli D, Giorgio E, Villar AM, Cardaropoli S, Carbonara C, et al. MEK Inhibition in a Newborn with RAF1-Associated Noonan Syndrome Ameliorates Hypertrophic Cardiomyopathy but Is Insufficient to Revert Pulmonary Vascular Disease. Genes (Basel). 21 dicembre 2021;13(1):6.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Noonan syndrome, RAF1, hypertrophic cardiomyopathy, clinical variability, genotype-phenotype correlations, functional domain","lastPublishedDoi":"10.21203/rs.3.rs-7696796/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7696796/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePathogenic variants in \u003cem\u003eRAF1 \u003c/em\u003eare a common cause of Noonan syndrome (NS), accounting for approximately 5% of cases. Nonetheless, \u003cem\u003eRAF1\u003c/em\u003e-related NS is often associated with severe clinical features, particularly hypertrophic cardiomyopathy (HCM). Although initial studies highlighted the occurrence of genotype-phenotype correlations, a comprehensive analysis specifically focused on \u003cem\u003eRAF1 \u003c/em\u003evariants is still lacking.\u003cstrong\u003e \u003c/strong\u003eWe conducted a retrospective observational study combining newly collected cases of \u003cem\u003eRAF1\u003c/em\u003e-related NS from a national multicenter retrospective cohort (RC) with systematically reviewed cases from literature search (literature cohort, LC). Variants were classified by protein domain, while the most recurrent variant, p.Ser257Leu, was analyzed separately to assess variant- and domain-specific phenotype correlations.\u003c/p\u003e\n\u003cp\u003eA total of 203 cases were included. Variants in the CR2 domain accounted for 83% of cases, with p.Ser257Leu alone representing 53%. HCM was observed in 80.1% of affected individuals confirming its role as the predominant cardiac manifestation in \u003cem\u003eRAF1\u003c/em\u003e-related NS; neurodevelopmental features were reported in 44.5% of patients. The prevalence of clinical features varied significantly according to variant location. HCM was markedly more frequently associated with CR2 variants (89.4%) and in subjects heterozygous for the p.Ser257Leu change (94.2%) compared with non-CR2 variants (37.1%). Conversely, neurodevelopmental features were more common in patients with non-CR2 variants (69.2%) than in those with CR2 variants (38.2%) or p.Ser257Leu (29.4%). CR2 and p.Ser257Leu variants were associated with earlier age at diagnosis and increased mortality.\u003c/p\u003e\n\u003cp\u003eOur findings confirm and document more comprehensively domain- and variant-specific phenotypes in \u003cem\u003eRAF1\u003c/em\u003e-related NS, emphasizing the importance of variant-level interpretation in clinical management and genetic counseling.\u003c/p\u003e","manuscriptTitle":"Domain-Specific Phenotypic Profiles in RAF1-Related Noonan Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 06:48:02","doi":"10.21203/rs.3.rs-7696796/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-11-13T17:00:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-09T17:43:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-03T03:47:36+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-15T15:18:32+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-12T10:01:08+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-01T13:29:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-25T21:57:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Human Genetics","date":"2025-09-23T17:22:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-23T17:22:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c9559751-0f5d-4f0e-801a-36566ec2bab9","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55634077,"name":"Biological sciences/Genetics/Medical genetics/Genetic counselling"},{"id":55634078,"name":"Health sciences/Medical research/Genetics research"}],"tags":[],"updatedAt":"2026-01-09T08:06:38+00:00","versionOfRecord":{"articleIdentity":"rs-7696796","link":"https://doi.org/10.1038/s41431-025-02002-9","journal":{"identity":"european-journal-of-human-genetics","isVorOnly":false,"title":"European Journal of Human Genetics"},"publishedOn":"2026-01-08 05:00:00","publishedOnDateReadable":"January 8th, 2026"},"versionCreatedAt":"2025-10-15 06:48:02","video":"","vorDoi":"10.1038/s41431-025-02002-9","vorDoiUrl":"https://doi.org/10.1038/s41431-025-02002-9","workflowStages":[]},"version":"v1","identity":"rs-7696796","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7696796","identity":"rs-7696796","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}