De novo germline disorders of the Ras-MAPK pathway:clinical delineation, molecular diagnosis and pathogenesis

  • Emma Burkitt Wright

    Student thesis: Phd


    This work sought to investigate the clinical phenotypes and molecular basis ofcardio-facio-cutaneous syndrome (CFC), a germline disorder of the Ras-MAPKpathway, like Noonan syndrome (NS) and neurofibromatosis type I, caused bymutations in genes encoding proteins that act within this signal transduction pathway.CFC is most commonly due to mutation in BRAF, and less commonly MAP2K1,MAP2K2 or KRAS. A proportion of patients currently have no mutation identified.Mutations and clinical features of patients with a molecular diagnosis of CFC wereinvestigated, which demonstrated a wide range of causative mutations, and someunclassified variants. Both known and novel clinical features of CFC were identified. Astrong association between severe contractures and the p.(Tyr130Cys) mutation inMAP2K1 was found, which has not previously been reported.In contrast to the large number of patients with a confirmed molecular diagnosis,several with a highly suggestive clinical phenotype have been found to have no mutationin any of the known CFC genes. The molecular basis of these presentations wasinvestigated by conventional Sanger sequencing of candidate genes. Fourteen patientswith the p.(Ser2Gly) mutation in SHOC2 were identified, with clinical presentationsconsistent with CFC, NS or CS. Target enrichment and massively parallel sequencing ofselected genes was undertaken in ten patients. Mutations in known genes were identifiedin four patients (including the positive control). Candidate causative variants in novelgenes were suggested in two further patients, one of which was confirmed on Sangersequencing. Whole exome sequencing of patient-parent trios was also undertaken toidentify de novo variants. Three trios were analysed, and in one patient with a clinicaldiagnosis of CFC, a frameshift mutation in NF1 was identified, which was confirmed bySanger sequencing to be present and de novo.The molecular effects of CFC-associated mutations in BRAF on Ras-MAPKpathway signalling were studied in cell culture systems, using Western blotting forERK1/2 phosphorylation, in vitro kinase assays and luciferase assays, to assess activity ofdownstream targets of the Ras-MAPK pathway. Altered pathway activity wasdemonstrated for novel variants that had not previously been characterised at themolecular level, which was in keeping with the findings of the effects of previouslystudied mutations.The cardiac phenotype in animal models of CFC, CS and NS/CFC was exploredusing expression microarrays to identify potentially important genes and pathways in thepathogenesis of hypertrophic cardiomyopathy (a progressive but potentially treatabledisease feature) in these conditions. A signature of increased expression of Myh7, theembryonic form of myosin, was identified in the heart of the mouse model of CFC dueto a B-Raf mutation at four weeks postnatal age, but comparative analysis suggestedsignificant differences in either the mechanisms causing cardiac phenotypes, or thetimescales over which these may exert their effects, in the three models.In summary, the most significant findings of this work were that SHOC2 mutationis a frequent cause of a severe NCFC presentation, and massively parallel sequencingcan be an effective means of molecular investigation of this group of disorders. Novelfeatures of CFC syndrome that were identified include severe contractures inassociation with p.(Tyr130Cys) mutations in MAP2K1. The analysis of mouse models ofthe NCFCs was hampered by heterogeneity within the expression microarray results,and low levels of expression of the H-Ras mutant allele in the mouse model of Costellosyndrome.
    Date of Award1 Aug 2014
    Original languageEnglish
    Awarding Institution
    • The University of Manchester
    SupervisorGraeme Black (Supervisor), Alan Whitmarsh (Supervisor) & Bronwyn Kerr (Supervisor)


    • Ras-MAPK pathway signal transduction
    • germline disorders
    • cardio-facio-cutaneous syndrome

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