William E. Tidyman

Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder involving characteristic craniofacial features, cardiac defects, ectodermal abnormalities, and developmental delay. We demonstrate that heterogeneous de novo missense mutations in three genes within the mitogen-activated protein kinase (MAPK) pathway cause CFC syndrome. The majority of cases (18 out of 23) are caused by mutations in BRAF, a gene frequently mutated in cancer. Of the 11 mutations identified, two result in amino acid substitutions that occur in tumors, but most are unique and suggest previously unknown mechanisms of B-Raf activation. Furthermore, three of five individuals without BRAF mutations had missense mutations in either MEK1 or MEK2, downstream effectors of B-Raf. Our findings highlight the involvement of the MAPK pathway in human development and will provide a molecular diagnosis of CFC syndrome.

HRAS mutations in Costello syndrome: detection of constitutional activating mutations in codon 12 and 13 and loss of wild-type allele in malignancy.

Costello syndrome (CS) is a complex developmental disorder involving characteristic craniofacial features, failure to thrive, developmental delay, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Based on similarities with other cancer syndromes, we previously hypothesized that CS is likely due to activation of signal transduction through the Ras/MAPK pathway [Tartaglia et al., 2003 ]. In this study, the HRAS coding region was sequenced for mutations in a large, well‐characterized cohort of 36 CS patients. Heterogeneous missense point mutations predicting an amino acid substitution were identified in 33/36 (92%) patients. The majority (91%) had a 34G → A transition in codon 12. Less frequent mutations included 35G → C (codon 12) and 37G → T (codon 13). Parental samples did not have an HRAS mutation supporting the hypothesis of de novo heterogeneous mutations. There is phenotypic variability among patients with a 34G → A transition. The most consistent features included characteristic facies and skin, failure to thrive, developmental delay, musculoskeletal abnormalities, visual impairment, cardiac abnormalities, and generalized hyperpigmentation. The two patients with 35G → C had cardiac arrhythmias whereas one patient with a 37G → T transversion had an enlarged aortic root. Of the patients with a clinical diagnosis of CS, neoplasia was the most consistent phenotypic feature for predicating an HRAS mutation. To gain an understanding of the relationship between constitutional HRAS mutations and malignancy, HRAS was sequenced in an advanced biphasic rhabdomyosarcoma/fibrosarcoma from an individual with a 34G → A mutation. Loss of the wild‐type HRAS allele was observed, suggesting tumorigenesis in CS patients is accompanied by additional somatic changes affecting HRAS. Finally, due to phenotypic overlap between CS and cardio‐facio‐cutaneuos (CFC) syndromes, the HRAS coding region was sequenced in a well‐characterized CFC cohort. No mutations were found which support a distinct genetic etiology between CS and CFC syndromes.

Noonan, Costello and cardio-facio-cutaneous syndromes: dysregulation of the Ras-MAPK pathway.

A class of developmental disorders caused by dysregulation of the Ras-induced mitogen-activated protein kinase (MAPK) cascade (the Ras-MAPK pathway) has emerged. Three of these disorders - Noonan, Costello and cardio-facio-cutaneous syndromes - have overlapping phenotypic features characterised by distinctive facial dysmorphia, cardiac defects, musculoskeletal and cutaneous abnormalities, and neurocognitive delay. The germline mutations associated with these disorders are in genes that encode proteins of the Ras-MAPK pathway. In vitro studies have determined that the overwhelming majority of these mutations result in increased signal transduction down the pathway, but usually to a lesser degree than somatic mutations in the same genes that are associated with cancer. The Ras-MAPK pathway is essential in the regulation of the cell cycle, differentiation, growth and senescence, so it is not surprising that germline mutations that affect its function have profound effects on development. Here we review the clinical consequences of the known molecular lesions associated with Noonan syndrome, Costello syndrome and cardio-facio-cutaneous syndrome, and explore possible therapeutic modalities for treatment.

Prader-Willi syndrome resulting from an unbalanced translocation: characterization by array comparative genomic hybridization

Prader–Willi syndrome (PWS) is caused by lack of expression of paternally inherited genes on chromosome 15q11→15q13. Most cases result from microdeletions in proximal chromosome 15q. The remainder results from maternal uniparental disomy of chromosome 15, imprinting center defects, and rarely from balanced or unbalanced chromosome rearrangements involving chromosome 15. We report a patient with multiple congenital anomalies, including craniofacial dysmorphology, microcephaly, bilateral cryptorchidism, and developmental delay. Cytogenetic analysis showed a de novo 45,XY,der(5)t(5;15)(p15.2;q13), ‐15 karyotype. In effect, the proband had monosomies of 5p15.2→pter and 15pter→15q13. Methylation polymerase chain reaction analysis of the promoter region of the SNRPN gene showed only the maternal allele, consistent with the PWS phenotype. The probands expanded phenotype was similar to other patients who have PWS as a result of unbalanced translocations and likely reflects the contribution of the associated monosomy. Array comparative genomic hybridization (array CGH) confirmed deletions of both distal 5p and proximal 15q and provided more accurate information as to the size of the deletions and the molecular breakpoints. This case illustrates the utility of array CGH in characterizing complex constitutional structural chromosome abnormalities at the molecular level.

Precocious terminal differentiation of premigratory limb muscle precursor cells requires positive signalling

The timing of myogenic differentiation of hypaxial muscle precursor cells in the somite lags behind that of epaxial precursors. Two hypotheses have been proposed to explain this delay. One attributes the delay to the presence of negative‐acting signals from the lateral plate mesoderm adjacent to the hypaxial muscle precursor cells located in the ventrolateral lip of the somitic dermomyotome (Pourquié et al. [ 1995 ] Proc. Natl. Acad. Sci. USA 92:3219–3223). The second attributes the delay to an absence of positive‐acting inductive signals, similar to those from the axial structures that induce epaxial myotome development (Pownall et al. [ 1996 ] Development 122:1475–1488). Because both studies relied principally upon changes in the expression pattern of mRNAs specific to early muscle precursor cell markers, we revisited these experiments using two methods to assess muscle terminal differentiation. First, injection of fluorescent dyes before surgery was used to determine whether ventrolateral lip cells transform from epithelial cells to elongated myocytes. Second, an antibody to a terminal differentiation marker and a new monoclonal antibody that recognises avian and mammalian Pax3 were used for immunohistochemistry to assess the transition from precursor cell to myocyte. The results support both hypotheses and show further that placing axial structures adjacent to the somite ventrolateral lip induces an axial pattern of myocyte terminal differentiation and elongation. Developmental Dynamics 229:591–599, 2004.

Abnormal Ras Signaling in Costello Syndrome (CS) Negatively Regulates Enamel Formation

RASopathies are syndromes caused by gain-of-function mutations in the Ras signaling pathway. One of these conditions, Costello syndrome (CS), is typically caused by an activating de novo germline mutation in HRAS and is characterized by a wide range of cardiac, musculoskeletal, dermatological and developmental abnormalities. We report that a majority of individuals with CS have hypo-mineralization of enamel, the outer covering of teeth, and that similar defects are present in a CS mouse model. Comprehensive analysis of the mouse model revealed that ameloblasts, the cells that generate enamel, lacked polarity, and the ameloblast progenitor cells were hyperproliferative. Ras signals through two main effector cascades, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. To determine through which pathway Ras affects enamel formation, inhibitors targeting either PI3K or MEK 1 and 2 (MEK 1/2), kinases in the MAPK pathway, were utilized. MEK1/2 inhibition rescued the hypo-mineralized enamel, normalized the ameloblast polarity defect and restored normal progenitor cell proliferation. In contrast, PI3K inhibition only corrected the progenitor cell proliferation phenotype. We demonstrate for the first time the central role of Ras signaling in enamel formation in CS individuals and present the mouse incisor as a model system to dissect the roles of the Ras effector pathways in vivo.

Molecular and Functional Analysis of a Novel MEK2 Mutation in Cardio-Facio-Cutaneous Syndrome: Transmission Through Four Generations

Cardio‐facio‐cutaneous (CFC) syndrome is one of the RASopathies and is caused by alteration of activity through the Ras/mitogen‐activated protein kinase (MAPK) pathway due to heterozygous de novo mutations in protein kinases BRAF, MEK1, or MEK2. CFC is a rare multiple congenital anomaly disorder in which individuals have characteristic dysmorphic features, cardiac defects, ectodermal anomalies and developmental delay. We report a

Peripheral muscle weakness in RASopathies

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Craniofacial and dental development in cardio-facio-cutaneous syndrome: the importance of Ras signaling homeostasis

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Skeletal muscle pathology in Costello and cardio-facio-cutaneous syndromes: developmental consequences of germline Ras/MAPK activation on myogenesis.

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