The seventh international RASopathies symposium: Pathways to a cure—expanding knowledge, enhancing research, and therapeutic discovery

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed

PECAM-1 engagement counteracts ICAM-1-induced signaling in brain vascular endothelial cells

Interactions between leukocytes and vascular endothelial cells are mediated by a complex set of membrane adhesion molecules which transduce bi-directional signals in both cell types. Endothelium of the cerebral blood vessels, which constitute the blood–brain barrier, strictly controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells, we previously documented the role of ICAM-1 in activation of the tyrosine phosphorylation of several actin-binding proteins and subsequent rearrangements of the actin cytoskeleton. In the present study, we show that, whereas PECAM-1 is known to control positively the trans-endothelial migration of leukocytes via homophilic interactions between leukocytes and endothelial cells, PECAM-1 engagement on brain endothelial surface unexpectedly counteracts the ICAM-1-induced tyrosine phosphorylation of cortactin and rearrangements of the actin cytoskeleton. We present evidence that the PECAM-1-associated tyrosine phosphatase SHP-2 is required for ICAM-1 signaling, suggesting that its activity might crucially contribute to the regulation of ICAM-1 signaling by PECAM-1. Our findings reveal a novel activity for PECAM-1 which, by counteracting ICAM-1-induced activation, could directly contribute to limit activation and maintain integrity of brain vascular endothelium

Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133

The expression of three microRNAs, miR-1, miR-206 and miR-133 is restricted to skeletal myoblasts and cardiac tissue during embryo development and muscle cell differentiation, which suggests a regulation by muscle regulatory factors (MRFs). Here we show that inhibition of C2C12 muscle cell differentiation by FGFs, which interferes with the activity of MRFs, suppressed the expression of miR-1, miR-206 and miR-133. To further investigate the role of myogenic regulators (MRFs), Myf5, MyoD, Myogenin and MRF4 in the regulation of muscle specific microRNAs we performed gain and loss-of-function experiments in vivo, in chicken and mouse embryos. We found that directed expression of MRFs in the neural tube of chicken embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in a loss of miR-1 and miR-206 expression. Taken together our results demonstrate differential requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis

Inhibition of the tyrosine phosphatase SHP-2 suppresses angiogenesis in vitro and in vivo

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitro as well as new vessel growth ex vivo (p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction ( p ! 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis ( annexin V staining, p ! 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n=3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth. Copyright (C) 2007 S. Karger AG, Basel

The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Toward a Therapeutic Approach

The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion

Rapidly progressive hypertrophic cardiomyopathy in an infant with Noonan syndrome with multiple lentigines: Palliative treatment with a rapamycin analog

Noonan syndrome with multiple lentigines (NSML) frequently manifests with hypertrophic cardiomyopathy (HCM). Recently, it was demonstrated that mTOR inhibition reverses HCM in NSML mice. We report for the first time on the effects of treatment with a rapamycin analog in an infant with LS and malignant HCM. In the boy, progressive HCM was diagnosed during the first week of life and a diagnosis of NSML was established at age 20 weeks by showing a heterozygous Q510E mutation in PTPN11. Immunoblotting with antibodies against pERK, pAkt, and pS6RP in fibroblasts demonstrated enhanced Akt/mTOR pathway activity. Because of the patient's critical condition, everolimus therapy was started at age 24 weeks and continued until heart transplantation at age 36 weeks. Prior to surgery, heart failure improved from NYHA stage IV to II and brain natriuretic peptide values decreased from 9,600 to <1,000 pg/ml, but no reversal of cardiac hypertrophy was observed. Examination of the explanted heart revealed severe hypertrophy and myofiber disarray with extensive perivascular fibrosis. These findings provide evidence that Akt/mTOR activity is enhanced in NSML with HCM and suggest that rapamycin treatment could principally be feasible for infantile NSML. The preliminary experiences made in this single patient indicate that therapy should start early to prevent irreversible cardiac remodelling

RhoA signaling in cardiomyocytes protects against stress-induced heart failure but facilitates cardiac fibrosis

The Ras-related guanosine triphosphatase RhoA mediates pathological cardiac hypertrophy, but also promotes cell survival and is cardioprotective after ischemia/reperfusion injury. To understand how RhoA mediates these opposing roles in the myocardium, we generated mice with a cardiomyocyte-specific deletion of RhoA. Under normal conditions, the hearts from these mice showed functional, structural, and growth parameters similar to control mice. Additionally, the hearts of the cardiomyocyte-specific, RhoA-deficient mice subjected to transverse aortic constriction (TAC)-a procedure that induces pressure overload and, if prolonged, heart failure-exhibited a similar amount of hypertrophy as those of the wild-type mice subjected to TAC. Thus, neither normal cardiac homeostasis nor the initiation of compensatory hypertrophy required RhoA in cardiomyocytes. However, in response to chronic TAC, hearts from mice with cardiomyocyte-specific deletion of RhoA showed greater dilation, with thinner ventricular walls and larger chamber dimensions, and more impaired contractile function than those from control mice subjected to chronic TAC. These effects were associated with aberrant calcium signaling, as well as decreased activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT. In addition, hearts from mice with cardiomyocyte-specific RhoA deficiency also showed less fibrosis in response to chronic TAC, with decreased transcriptional activation of genes involved in fibrosis, including myocardin response transcription factor (MRTF) and serum response factor (SRF), suggesting that the fibrotic response to stress in the heart depends on cardiomyocyte-specific RhoA signaling. Our data indicated that RhoA regulates multiple pathways in cardiomyocytes, mediating both cardioprotective (hypertrophy without dilation) and cardio-deleterious effects (fibrosis). The Ras-related guanosine triphosphatase RhoA mediates pathological cardiac hypertrophy, but also promotes cell survival and is cardioprotective after ischemia/reperfusion injury. To understand how RhoA mediates these opposing roles in the myocardium, we generated mice with a cardiomyocyte-specific deletion of RhoA. Under normal conditions, the hearts from these mice showed functional, structural, and growth parameters similar to control mice. Additionally, the hearts of the cardiomyocyte-specific, RhoA-deficient mice subjected to transverse aortic constriction (TAC)-a procedure that induces pressure overload and, if prolonged, heart failure-exhibited a similar amount of hypertrophy as those of the wild-type mice subjected to TAC. Thus, neither normal cardiac homeostasis nor the initiation of compensatory hypertrophy required RhoA in cardiomyocytes. However, in response to chronic TAC, hearts from mice with cardiomyocyte-specific deletion of RhoA showed greater dilation, with thinner ventricular walls and larger chamber dimensions, and more impaired contractile function than those from control mice subjected to chronic TAC. These effects were associated with aberrant calcium signaling, as well as decreased activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT. In addition, hearts from mice with cardiomyocyte-specific RhoA deficiency also showed less fibrosis in response to chronic TAC, with decreased transcriptional activation of genes involved in fibrosis, including myocardin response transcription factor (MRTF) and serum response factor (SRF), suggesting that the fibrotic response to stress in the heart depends on cardiomyocyte-specific RhoA signaling. Our data indicated that RhoA regulates multiple pathways in cardiomyocytes, mediating both cardioprotective (hypertrophy without dilation) and cardio-deleterious effects (fibrosis)

The sixth international RASopathies symposium: Precision medicine—From promise to practice

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS‐mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life‐limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion

Leopard syndrome

LEOPARD syndrome (LS, OMIM 151100) is a rare multiple congenital anomalies condition, mainly characterized by skin, facial and cardiac anomalies. LEOPARD is an acronym for the major features of this disorder, including multiple Lentigines, ECG conduction abnormalities, Ocular hypertelorism, Pulmonic stenosis, Abnormal genitalia, Retardation of growth, and sensorineural Deafness. About 200 patients have been reported worldwide but the real incidence of LS has not been assessed. Facial dysmorphism includes ocular hypertelorism, palpebral ptosis and low-set ears. Stature is usually below the 25th centile. Cardiac defects, in particular hypertrophic cardiomyopathy mostly involving the left ventricle, and ECG anomalies are common. The lentigines may be congenital, although more frequently manifest by the age of 4–5 years and increase throughout puberty. Additional common features are café-au-lait spots (CLS), chest anomalies, cryptorchidism, delayed puberty, hypotonia, mild developmental delay, sensorineural deafness and learning difficulties. In about 85% of the cases, a heterozygous missense mutation is detected in exons 7, 12 or 13 of the PTPN11 gene. Recently, missense mutations in the RAF1 gene have been found in two out of six PTPN11-negative LS patients. Mutation analysis can be carried out on blood, chorionic villi and amniotic fluid samples. LS is largely overlapping Noonan syndrome and, during childhood, Neurofibromatosis type 1-Noonan syndrome. Diagnostic clues of LS are multiple lentigines and CLS, hypertrophic cardiomyopathy and deafness. Mutation-based differential diagnosis in patients with borderline clinical manifestations is warranted. LS is an autosomal dominant condition, with full penetrance and variable expressivity. If one parent is affected, a 50% recurrence risk is appropriate. LS should be suspected in foetuses with severe cardiac hypertrophy and prenatal DNA test may be performed. Clinical management should address growth and motor development and congenital anomalies, in particular cardiac defects that should be monitored annually. Hypertrophic cardiomyopathy needs careful risk assessment and prophylaxis against sudden death in patients at risk. Hearing should be evaluated annually until adulthood. With the only exception of ventricular hypertrophy, adults with LS do not require special medical care and long-term prognosis is favourable

Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a “second hit,” that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome