BackgroundCostello syndrome (CS) and cardio-facio-cutaneous syndrome (CFCS) belong to the RASopathies, a group of neurodevelopmental disorders with skeletal anomalies. Due to their rarity, the characterization of the musculo-skeletal phenotype in both disorders has been poorly characterized.Patients and methodsHerein we reported data on orthopedic findings and functional status of a large sample of CS and CFCS patients. Thirty-four patients (CS=17 and CFCS=17) were recruited. Functional and disability evaluations were performed by assessing the 6-minute walking test (6MWT) and Pediatric Outcomes Data Collection Instrument (PODCI). Genotype/phenotype correlation was also provided.ResultsOrthopedic manifestations are highly prevalent in CS and CFCS and overlap in the two disorders. Overall, patients with CS harboring the recurrent HRAS Gly12Ser substitution show a more severe skeletal phenotype compared to patients carrying the Gly12Ala and Gly13Cys variants. Among CFCS patients, those with the MAP2K1/2 variant show different skeletal characteristics compared to BRAF variants, with a higher prevalence of orthopedic abnormalities. Functional assessment showed that patients with CS and CFCS reached lower values compared to the general population, with CFCS patients displaying the lowest scores.ConclusionsOrthopedic manifestations appear universal features of CS and CFCS and they can evolve across patients’ life. Longitudinal assessment of disability status by using 6MWT and PODCI could be useful to evaluate the functional impact of orthopedic manifestations on patients’ outcome and help planning a tailored treatment of these comorbidities.

We developed a new class of inhibitors of protein–protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2–20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein–protein interactions in the function of SHP2.

Costello syndrome (CS) is a neurodevelopmental disorder with a distinctive musculoskeletal phenotype and reduced bone mineral density (BMD) caused by activating de novo mutations in the HRAS gene. Herein, we report the results of a prospective study evaluating the efficacy of a 4-year vitamin D supplementation on BMD and bone health. A cohort of 16 individuals ranging from pediatric to adult age with molecularly confirmed CS underwent dosages of bone metabolism biomarkers (serum/urine) and dual-energy X-ray absorptiometry (DXA) scans to assess bone and body composition parameters. Results were compared to age-matched control groups. At baseline evaluation, BMD was significantly reduced (p ≤ 0.05) compared to controls, as were the 25(OH)vitD levels. Following the 4-year time interval, despite vitamin D supplementation therapy at adequate dosages, no significant improvement in BMD was observed. The present data confirm that 25(OH)vitD and BMD parameters are reduced in CS, and vitamin D supplementation is not sufficient to restore proper BMD values. Based on this evidence, routine monitoring of bone homeostasis to prevent bone deterioration and possible fractures in adult patients with CS is highly recommended. K E Y W O R D S bone metabolism biomarkers, bone mineral density, Costello syndrome, patient-centered care, personalized medicine, RASopathies 1 | INTRODUCTION Costello syndrome (CS, OMIM #218040) is a rare multisystem disorder belonging to a family of syndromes affecting development and growth collectively known as the RASopathies. These rare diseases share the upregulation of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway as a common pathogenetic mechanism (Aoki et al., 2016;Tartaglia & Gelb, 2010). This signaling cascade controls a wide array of cellular processes (e.g., proliferation, migration, Abbreviations: 25(OH)vitD, 25 hydroxy vitamin D; B-ALP, bone alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; CFCS, cardio-facio-cutaneous syndrome; CS, Costello syndrome; DXA, dual-energy X-ray absorptiometry; F-BMD, femur bone mineral density; FFM, fat free mass; FM, fat mass; FN-BMD, femoral neck bone mineral density; L-BMD, lumbar bone mineral density; MAPK, mitogen-activated protein kinase; NF1, neurofibromatosis type 1; NS, Noonan syndrome; PTH, parathyroid hormone; S-BMC, subtotal bone mineral content; S-BMD, subtotal bone mineral density; WBLH, whole body less head.

Cardio‐facio‐cutaneous syndrome (CFCS) is a rare disorder characterized by distinctive craniofacial appearance, cardiac, neurologic, cutaneous, and musculoskeletal abnormalities. It is due to heterozygous mutations in BRAF, MAP2K1, MAP2K2, and KRAS genes, belonging to the RAS/MAPK pathway. The role of RAS signaling in bone homeostasis is highly recognized, but data on bone mineral density (BMD) in CFCS are lacking. In the present study we evaluated bone parameters, serum and urinary bone metabolites in 14 individuals with a molecularly confirmed diagnosis of CFCS. Bone assessment was performed through dual X‐ray absorptiometry (DXA); height‐adjusted results were compared to age‐ and sex‐matched controls. Blood and urinary bone metabolites were also analyzed and compared to the reference range. Despite vitamin D supplementation and almost normal bone metabolism biomarkers, CFCS patients showed significantly decreased absolute values of DXA‐assessed subtotal and lumbar BMD (p ≤ 0.05), compared to controls. BMD z‐scores and t‐scores (respectively collected for children and adults) were below the reference range in CFCS, while normal in healthy controls. These findings confirmed a reduction in BMD in CFCS and highlighted the importance of monitoring bone health in these affected individuals.

Kinesins are motor proteins involved in microtubule-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as ‘ciliopathies’. Among kinesins, kinesin-1 is the most abundant microtubule motor for transport of cargoes towards the plus end of microtubules. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.

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Mutations of PTPN11, the gene coding for the Src homology 2 domain-containing phosphatase 2 (SHP2), cause childhood leukemias and developmental disorders. SHP2 inhibitors targeting the catalytic site or an allosteric pocket lack specificity or are ineffective on pathogenic variants. In addition, several data indicate that increased association with cognate proteins, through its SH2 domains, rather than enhanced catalytic activity, is the main effect of mutations causing hyperactivation of SHP2-mediated signaling. We developed peptide-based molecules with low nM affinity to the N-SH2 domain and high specificity. These molecules bind to pathogenic variants of SHP2 with an affinity up to 20 times higher than to the wild-type protein, in contrast to allosteric inhibitors, and were able to revert the effects of a pathogenic SHP2 mutation in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool to investigate the role of protein-protein interactions in the function of SHP2.TABLE OF CONTENTS GRAPHICS

Germline activating mutations in HRAS cause Costello Syndrome (CS), a cancer prone multisystem disorder characterized by reduced postnatal growth. In CS, poor weight gain and growth are not caused by low caloric intake. Here we show that constitutive plasma membrane translocation and activation of the GLUT4 glucose transporter, via ROS-dependent AMPKα and p38 hyperactivation, occurs in CS, resulting in accelerated glycolysis, and increased fatty acid synthesis and storage as lipid droplets in primary fibroblasts. An accelerated autophagic flux was also identified as contributing to the increased energetic expenditure in CS. Concomitant inhibition of p38 and PI3K signaling by wortmannin was able to rescue both the dysregulated glucose intake and accelerated autophagic flux. Our findings provide a mechanistic link between upregulated HRAS function, defective growth and increased resting energetic expenditure in CS, and document that targeting p38 and PI3K signaling is able to revert this metabolic dysfunction.