Leslie B. Gordon, MD, PHD
Hutchinson-Gilford progeria syndrome (Progeria) is a rare segmental premature aging syndrome that affects 200-250 children worldwide at any one time. It is caused by a mutation in the LMNA gene, whose normal lamin A protein product is central to nuclear structure and function in differentiated cells. The global expression of lamin A, and hence the aberrant protein produced in Progeria called progerin, results in a multisystem disease. Children with Progeria die of global, accelerated atherosclerosis at an average age of 13 years.
Christine Schramm,1 Deborah M. Fine,2 Michelle A. Edwards,1 Maike Krenz1 1Department of Medical Pharmacology & Physiology / Dalton Cardiovascular Research Center and 2Department of Veterinary Medicine and Surgery, University of Missouri-Columbia
Rationale: The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. We focused our studies on the Q510E mutation in Shp2, which is associated with a particularly severe form of hypertrophic cardiomyopathy in patients. It is still under debate whether patients with this particular mutation rather fall into the Noonan Syndrome or into the LEOPARD Syndrome category since clinical differentiation between these syndromes can be very challenging, in particular in infants. To unravel the underlying disease mechanism of this aggressive mutation, we tested the biochemical characteristics of the Q510E-Shp2 protein and investigated the downstream signaling events triggered by this mutation in a mouse model.
Yvonne E. Chiu, MD,1 Stefanie Dugan, MS,2 Donald G. Basel, MD, 2Dawn H. Siegel, MD1 1 Department of Dermatology-Division of Pediatric Dermatology, Medical College of Wisconsin and Children’s Hospital of Wisconsin 2 Department of Pediatrics-Section of Genetics, Medical College of Wisconsin and Children’s Hospital of Wisconsin
A 5 year old boy presented with a congenital depigmented patch of the forehead, as well as acquired white forelock, depigmentation of the medial eyebrows, and depigmented patches of the body. Additionally, he had started to develop multiple café-au-lait macules (CALMs) and freckling of the axillae and inguinal folds. His past history was significant for mild asthma, chronic sinusitis, surgical excision of a thyroglossal duct cyst, and umbilical hernia repair. There was no developmental delay or hearing loss. Given concerns by other providers for tuberous sclerosis, neurofibromatosis type 1 (NF1), and Waardenburg syndrome, the patient had a prior work-up consisting of a normal brain MRI, echocardiogram, audiogram, and ophthalmology exam. His family history was significant for similar skin findings in his father, paternal half-brother, paternal grandmother, and paternal great-grandfather.
1Mayes DA, 1Rizvi TA, 1,3Cancelas JA, 1Kolasinski N, 2Ciraolo CM, 4Stemmer-Rachamimov AO, and 1Ratner N Divisions of 1Experimental Hematology and Cancer Biology, and 2Pathology, Cincinnati Children’s Hospital Medical Center, 3Hoxworth Blood Center, University of Cincinnati, Department of Pathology4, Massachusetts General Hospital and Harvard Medical School.
OBJECTIVES Neurofibromas are tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. One idea within the field suggests that Nf1 loss must occur within progenitor cells present within a critical window during Schwann cell development in order for neurofibromas to form. To test this hypothesis and to examine whethermyelinating Schwann cells can serve as aneurofibroma cell of origin, Nf1 loss was induced at perinatal or adult timepoints using a tamoxifen-inducible Plp-CreERT driver.
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Martin Zenker, Hélène Cavé, Reza Ahmadian, Ype Elgersma, Patrick Raynal, Marco Tartaglia Institute of Human Genetics, University Hospital Magdeburg, Germany
The European network on Noonan syndrome and related disorders (NSEuroNet) is a EU-funded collaborative project that started its activities in 2010. The project is coordinated by Marco Tartaglia and aims at improving the knowledge about RASopathies at the levels of basic research as well as of practical clinical care. We provide a progress report on the RASopathy mutation and phenotype database that is a core part of the NSEuroNet project.
Martin Zenker Institute of Human Genetics, University Hospital Magdeburg, Germany
Noonan syndrome (MIM163950) is a clinically recognizable and genetically heterogeneous disorder. The common denominator of the various genetic alterations found in patients with Noonan syndrome and related disorders is that they cause dysregulation of RAS-MAPK signaling. Even if strict clinical criteria are applied, the molecular genetic lesion currently remains unidentified in about 10-20% of cases, thus suggesting additional genes for Noonan syndrome.
Noonan syndrome (NS) is one of several “RASopathies” caused by mutations in different components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase RAF1) account for ~3-5% of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM. Surprisingly, other NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of NS-associated RAF1 mutations, we generated “knock-in” mice that express kinase-activating (L613V) and impaired (D486N) RAF1 mutants, respectively.
Objective Neurofibromatosis (NF) encompasses a group of rare disorders affecting 100,000 Americans. The clinical course of NF is unpredictable: it can lead to deafness, blindness, amputation or disfigurement. The genes for the two major forms, NF1 and NF2, were identified in the early 1990’s, and the molecular signaling pathways are well understood. The primary funders of US NF research are the Congressionally Directed Medical Research Program for Neurofibromatosis Research ($10M-$25M/year) and NIH ($11-$15M/year). Major tools developed in the last 10 years include transgenic mouse models representing individual NF manifestations, and a Phase II Clinical Trials Consortium. In 2006, the Children’s Tumor Foundation (CTF) examined the NF research landscape looking for ‘funding gaps’ that if filled could rapidly advance NF discoveries to the clinic. The goal was to implement recommendations within 5 years.
The last few decades have seen rapid progress toward establishing neuropsychological profiles associated with Ras/MAPK pathway syndromes. Among affected individuals, cognitive functioning can vary from significant global intellectual disability to mild delays in specific domains. In a subset of individuals, no significant learning or behavior problems are seen. Research using animal models has begun to reveal neurobiological sources of differences in learning and memory processes in the RASopathies, as well as suggest potential methods to circumvent these differences. However, additional research is needed to identify the most useful neurocognitive targets of intervention in humans.
Cordeddu and co-workers (Nat. Genet. 2009, 41:1022-1026) reported that the invariant c.4A>G missense (p.Ser2Gly) change in the SHOC2 gene, which encodes a widely expressed non-membranous protein that positively modulates RAS-MAPK signal flow, underlies a distinctive RASopathy previously recognized as Noonan-like syndrome with loose anagen hair [OMIM 607721] by Mazzanti and colleagues (Am. J. Med. Genet. 2003, 118A:279-286). This mutation was demonstrated to promote N-myristoylation of the protein and its aberrant targeting to the plasma membrane. N-myristoylation is an irreversible cotranslational lipid modification, and is observed to occur in many signal transducers that require to be anchored to the cytoplasmic leaflet of cell membranes to carry out their function.
Costello Syndrome (CS) is a rare, complex, developmental disorder characterized by a number of features including— failure to thrive, characteristic facies, delay in intellectual development, hypertrophic cardiomyopathy, arrhythmia, and predisposition to both benign and malignant tumors. Past studies identifying gain-of-function mutations in the HRAS gene as the basis of human CS, and strong conservation with the mouse ortholog Hras1, have led to the development of a Gly12Val (G12V) mouse model of CS.
Lisa Ehrman1, Diana Nardini1, Tilat Rizvi1, Nancy Ratner1, Masato Nakafuku2, Jeffrey Robbins3, and Ronald R. Waclaw1 1Division of Experimental Hematology and Cancer Biology, 2Division of Developmental Biology, 3Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center Cincinnati, OH
Shp2 (PTPN11) is an intracellular protein tyrosine phosphatase that is critical for mediating cell signaling in response to growth factors. Germline mutations in PTPN11 have been identified Noonan and LEOPARD syndrome patients. Patients with either syndrome exhibit several congenital malformations including a variety of cardiovascular and skeletal defects. In addition to these defects, there is also an increased incidence of learning disabilities and cognitive impairment observed in both syndromes. The specific brain abnormalities that lead to these neurocognitive defects remain unknown. The goal of our study is to utilize mouse genetics to understand the role of Shp2 (PTPN11) in the telencephalon, which is the region of the brain most associated with higher neural functions like cognition and emotion.
Alcino J. Silva Departments of Neurobiology, Psychiatry and Biobehavioral Sciences, Psychology and Brain Research Institute, UCLA Los Angeles, California 90095 Our laboratory has been studying the molecular and cellular mechanisms responsible for learning deficits in Rasopathies, a class of genetic disorders that alter Ras signaling mechanisms, and that result in a broad range of somatic, neurologic and psychiatric phenotypes. Although our work started with studies of neurofibromatosis type I (NF1), we have also studied several other rasophaties. For example, recently we uncovered molecular and cellular mechanisms responsible for the learning deficits associated with Noonan syndrome (NS), a genetic disorder with an incidence of 1 in ~2,500. As with NF1 patients, a significant percentage of NS patients show cognitive deficits, such as learning disabilities and mental retardation. Mutations in the PTPN11 gene, which encodes the non-receptor protein tyrosine phosphatase SHP-2, account for ~50% of NS cases. Just as NF1, NS-associated SHP-2 mutants result in increases in Ras-ERK signaling, which is critically involved in learning and memory.
Maria T. Acosta, M.D. Children’s National Medical Center, Washington, DC, USA Background: Cognitive deficits are the most important long term co-morbidity in NF1 patients. In a mouse model of neurofibromatosis type 1(NF1), lovastatin improved cognitive deficits and executive functioning. Methods: Using a standard phase 1 study design, we examined the safety and biological impact of lovastatin as a treatment for the neurocognitive deficits in a group of 24 children 10-17 years old with diagnosis of NF1. A subset of patients underwent a 12-hour pharmacokinetics analyses.
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Rebecca Josowitz1, Sonia Mulero-Navarro1, Ilan Riess1, Sherly Pardo3 Sunita D’Souza2, Xonia Carvajal-Vergara2, Marco Tartaglia4, Ihor Lemischka2, Bruce D. Gelb1
1Child Health and Development and 2Black Family Stem Cell Institutes, Mount Sinai School of Medicine, New York, NY, USA, 10029, 3Recinto de Ciencias Medicas, Universidad de Puerto Rico, San Juan, Puerto Rico, 00936-5067, 4Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy, 00161.
Mutations in PTPN11 and BRAF, genes involved in the RAS/MAPK pathway, are implicated in a variety of clinical disorders, such as Noonan syndrome (NS), LEOPARD syndrome (LS), and cardio-facial-cutaneous syndrome (CFCS), members of a family of disorders termed the “RASopathies”. These disorders are characterized by skeletal and neurological defects, as well as a high prevalence of cardiovascular abnormalities. Hypertrophic cardiomyopathy (HCM) is observed in 90% of patients with PTPN11 mutations causing LS and 40% of patients with CFCS, yet PTPN11 mutations causing NS are negatively associated with HCM. In order to further elucidate the molecular mechanisms of RASopathy-associated HCM, we have generated human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes as representative models of these syndromes. We have previously demonstrated that hiPSCs derived from patients with LS exhibit altered RAS/MAPK signaling and their derived cardiomyocytes display a molecular phenotype consistent with the cardiac hypertrophy observed clinically.
Ami .V. Patel, Ph.D.,Nancy Ratner, Ph.D., Cincinnati Childrens Hospital Medical Center, Dept. of Experimental Hematology and Cancer Biology, Cincinnati, OH.
Malignant peripheral nerve sheath tumor (MPNST) is a soft tissue sarcoma that is highly invasive and eventually lethal. About 50% of MPNSTs have mutation and/or loss of the NF1 gene, while the rest are spontaneous. Many MPNSTs also show mutations in p53 and/or homozygous deletion of CDKN2. In an effort to identify novel genes that play key roles in MPNST tumorigenesis, we performed a genome wide micro-array analysis using human neurofibroma and MPNST (Miller et al., 2009). This analysis identified 130 genes that are up-regulated >3 fold in MPNSTs as compared to normal human Schwann cells. Over expression of these genes in tumors as compared to normal cells suggested that some might be critical for survival of MPNST cells. This hypothesis was directly tested by studying effects on survival of MPNST cells invitro post down regulation of the 130 genes using lenti-viral short hairpin (sh) RNA. For each shRNA experiment a shnon-targeting lenti-virus was used as a negative control and shSOX9, a construct previously shown to kill MPNST cells in vitro, was used as a positive control for an effect on cell survival. Screening was performed sequentially allowing for selection of genes with significant effects on survival. In screen I, T265 MPNST cells were exposed to each shRNA virus. In screen II, shRNAs were tested for effects on adenocarcinoma cells (A549) vs T265 cells. These two rounds of screens identified 8/130 genes specific for survival of T265 cells. Screen III wasperformed on four different MPNST cell lines, including T265 (NF1-/-), 8814 (NF1-/-), S462TY (NF1-/-) and SSTS26T (sporadic MPNST wild-type for NF1), to identify shRNA’s with similar or different effects on cell survival in the presence or absence of wild-type NF1gene. No significant difference in survival was observed for NF null MPNST cells vs sporadic MPNST cell line for any gene. This approach facilitates a streamlined pathway from genome wide gene expression analysis to identification of 8 novel genes critical to survival of cancer cells in vitro and potential therapeutic targets toward a cure for MPNSTs. Supported by NIH28840 (to NR). A.P. is the recipient of a DOD NF Postdoctoral Fellow Award.
Tetsuya Niihori, Yoko Aoki, Yoichi Matsubara Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan;
Costello syndrome (CS) is a congenital disease that is characterized by a distinctive facial appearance, failure to thrive, mental retardation and cardiomyopathy. In 2005, we identified that heterozygous germline mutations in HRAS caused CS. Several studies have shown that CS-associated HRAS mutations are clustered in codons 12 and 13, and mutations in other codons have also been identified. However, a comprehensive comparison of the substitutions identified in patients with CS has not been conducted. In the current study, we identified four mutations (p.G12S, p.G12A, p.G12C and p.G12D) in 21 patients and analyzed the associated clinical manifestations of CS in these individuals. <Read More>
Mayes D.A.,Rizvi T.A., Kolasinski N., Miller S.,& Ratner N. Department of Experimental Hematology & Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati OH
Neurofibromatosis type 1 patients are predisposed to central nervous system (CNS) phenotypes including enlarged brains, delayed acquisition of motor skills, brain tumors, and cognitive deficits. Imaging and pathologic analysis suggest that changes in white matter myelination may underlie both the enlargement of white matter tracts that contributes to megancephaly, and/or hyper-intense signals visualized on MRI. To study the role(s) of Nf1 and HRasin oligodendrocytes, we examined the optic nerve and corpus callosum,myelinated fiber tracts.
Perundurai S Dhandapany1, Susumu Hirabayashi2, Elizabeth Horvath1, Kimihiko Oishi1, Ross L Cagan2 and Bruce D. Gelb1
1Departments of Pediatrics and Genetics & Genomic Sciences and the Child Health and Development Institute, 2Department of Developmental and Regenerative Biology, Mount Sinai Medical School, One Gustave L. Levy Place, Box 1020, NY 10029, USA
BACKGROUND: Noonan syndrome (NS) is a pleiomorphic developmental disorder, for which increased RAS pathway signaling is the primary cause. Gain-of-function mutations in PTPN11, encoding SHP-2, are the major cause. While powerful Ras signaling inhibitors are in development for cancer, such drugs may not be useful for long-term use in children with NS. To find drugs that restore normal Ras signaling in NS, we are using a transgenic Drosophila model with the D61G mutation in corkscrew mutation (cswD61G), the fly orthologue of PTPN11.
Sonia Mulero-Navarro1, Ilan Riess1, Sherly Pardo3, Ana Sevilla2, Dung-Fang Lee2, Sunita D’Souza2, Helene Cave4, Marco Tartaglia5, Ihor Lemischka2, Bruce D. Gelb1
1Child Health and Development and 2Black Family Stem Cell Institutes, Mount Sinai School of Medicine, New York, NY, USA, 10029, 3Recinto de Ciencias Medicas, Universidad Puerto Rico, San Juan, Puerto Rico, 00936-5067, 4Departement Genetique Hopital Robert Debre AP-HP, Paris, France,5Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy, 00161
BACKGROUND: Noonan syndrome (NS) is a genetic developmental disorder caused by deregulation of the RAS/MAPK pathway. Germ-line mutations in PTPN11, which encodes SHP-2, a key component of the RAS/MAPK pathway, cause 50% of NS, while somatic mutations in this gene account for 35% of juvenile myelomonocytic leukemia (JMML). Children with NS and specific PTPN11 mutations are at increased risk for developing JMML, inferring that certain SHP-2 mutants result in abnormal differentiation and cell maturation in hematopoietic lineages. The molecular mechanisms resulting from SHP-2 dysregulation that lead to these abnormalities remain largely unexplored.
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Yuka Saito1, Yoko Aoki1, Tetsuya Niihori1, Akira Ohtake2, Atsushi Shibuya2, Kazuhito Sekiguchi,3, So-ichi Suenobu3, Taturo Izumi3, Hideki Muramatsu4, Seiji Kojima4, Shigeo Kure1,5, Shigeru Tsuchiya5, Yoichi Matsubara1
1) Dept Med Genet, Tohoku Univ Sch Med, Sendai, Japan; 2) Dept Pediatr, Saitama Med Univ, Moroyama, Saitama, Japan. 3) Dept Pediatr and Child Neurol, Oita Univ Facul Med, Oita, Japan 4) Dept. Pediatr, Nagoya Univ Graduate Sch Med, Nagoya, Japan 5) Dept. Pediatr. Tohoku Univ Sch Med
Cardio-facio-cutaneous (CFC) syndrome is a multiple congenital anomaly/mental retardation syndrome characterized by a distinctive facial appearance, ectodermal abnormalities and heart defects. Clinically, it overlaps with both Noonan syndrome and Costello syndrome. Mutations in KRAS, BRAF and MAP2K1/2 (MEK1/2) have been identified in patients with CFC syndrome. By age 20, the cumulative incidence of cancer was approximately 4% for Noonan syndrome and 15% for Costello syndrome (Christian P. Kratz. AJMD, 2011). For Costello syndrome, tumor-screening protocols have been proposed. In contrast, little attention has been paid to the development of tumors in patients with CFC syndrome.
Jessica Schwartz, Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI.
Growth Hormone (GH) is a major regulator of statural growth and metabolism. Changes in gene expression and in their regulatory transcription factors often underlie the physiological responses to GH. Recent insights in understanding GH signaling indicate that the interaction of GH with GH receptors on target cells initiates multiple signaling cascades that culminate in changes in transcription factors in the nucleus. Through analysis of profiles of GH-regulated genes, we find that in addition to well-recognized Stat5-mediated signaling, GH utilizes Ras/MAPK-mediated signaling to regulate the phosphorylation and function of C/EBP-CREB family transcription factors, which in turn modulate multiple genes in response to GH.
