Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations

Oishi, Kimihiko; Gaengel, Konstantin; Krishnamoorthy, Srinivasan; Kamiya, Kazutaka; Kim, In Kyong; Ying, Huiwen; Weber, Ursula; Perkins, Lizabeth A.; Tartaglia, Marco; Mlodzik, Marek; Pick, Leslie; Gelb, Bruce D.

doi:10.1093/hmg/ddi471

Cited by 67 publications

(66 citation statements)

References 55 publications

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“…However, eye development, which also depends on Ras/MAPK signaling, is not affected in these mutants (SI Appendix, Fig. S7), which is consistent with a previous report on GOF Csw (the Drosophila ortholog of Shp2) mutations (31). Furthermore, because ectopic wing vein formation was sensitive to the weak mutation D44G and the other assays were not, this suggests that some cellular contexts are more sensitive to small perturbations in the Ras/MAPK pathway.…”

Section: Discussionsupporting

confidence: 90%

“…Ras/MAPK signaling is involved in wing formation, and we quantified the defects induced by GOF mutations, which include formation of stereotypic ectopic veins (31,42). Surprisingly, D44G, which does not show any significant deviation from WT in zebrafish and fly embryonic assays, displayed ectopic wing veins, suggesting either that this mutation displays more activity in this context or that wing development is more sensitive to pathway perturbations (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Although previous studies have indirectly observed mutationbased severity of effects using cultured cells (16,27,28) and model organisms (29)(30)(31) by comparing a few mutations, in no case were the studies comprehensive. To address this more systematically, we develop a framework for the quantitative comparison of MEK1 mutants found in human diseases.…”

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confidence: 99%

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In vivo severity ranking of Ras pathway mutations associated with developmental disorders

Jindal

Goyal

Yamaya

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Germ-line mutations in components of the Ras/MAPK pathway result in developmental disorders called RASopathies, affecting about 1/1,000 human births. Rapid advances in genome sequencing make it possible to identify multiple disease-related mutations, but there is currently no systematic framework for translating this information into patient-specific predictions of disease progression. As a first step toward addressing this issue, we developed a quantitative, inexpensive, and rapid framework that relies on the early zebrafish embryo to assess mutational effects on a common scale. Using this assay, we assessed 16 mutations reported in MEK1, a MAPK kinase, and provide a robust ranking of these mutations. We find that mutations found in cancer are more severe than those found in both RASopathies and cancer, which, in turn, are generally more severe than those found only in RASopathies. Moreover, this rank is conserved in other zebrafish embryonic assays and Drosophila-specific embryonic and adult assays, suggesting that our ranking reflects the intrinsic property of the mutant molecule. Furthermore, this rank is predictive of the drug dose needed to correct the defects. This assay can be readily used to test the strengths of existing and newly found mutations in MEK1 and other pathway components, providing the first step in the development of rational guidelines for patient-specific diagnostics and treatment of RASopathies.T he Ras/MAPK (rat sarcoma/mitogen-activated protein kinase) signaling pathway is involved in essentially all aspects of organismal development, from the first cell divisions in the early embryo to postnatal development and growth (1-3). Given its critical function, it is not surprising that deregulated Ras/ MAPK signaling, resulting from either genetic or environmental perturbations, can lead to developmental abnormalities. A large class of such abnormalities, known as RASopathies, is associated with activating germ-line mutations in many components of the Ras pathway (4). Estimated to affect 1/1,000 human births, these abnormalities are characterized by a broad spectrum of phenotypes, including cardiac defects, craniofacial dysmorphisms, and neurocognitive delays (4). Although the origins of these phenotypes are still poorly understood, studies of model organisms show that many of the observed structural and functional defects can indeed be mimicked by targeted introduction of mutations found in RASopathies (5). Hundreds of such mutations have already been identified, and many more are likely to be discovered by the sequencing of affected individuals (6).Importantly, these studies identify new mutations in the very same components that are mutated in cancer and have been extensively studied both in vitro and in vivo (7-9). In particular, multiple new mutations were identified in MEK1, a MAPK kinase (10) and an important target for anticancer therapeutics (11)(12)(13)(14)(15)(16)(17)(18)(19). Because it is commonly believed that cancer mutations would be embryonic lethal when inherited through t...

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Section: Discussionsupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

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In vivo severity ranking of Ras pathway mutations associated with developmental disorders

Jindal

Goyal

Yamaya

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Mutations identified were checked for de novo occurrence whenever parental DNAs were available. For the 140 individuals with NS or a clinically related phenotype selected from the ISS and UCSC, Rome, Italy, exons 15,16,19,24,25,26,29, 31 and 32 and flanking intronic sequences were considered, based on the identification of possible pathogenic mutations in the first cohort. For all variants detected, an in silico-based method was used to assess the effect of the mutation (Alamut software, version 2.1; http://www.interactivebiosoftware.com/) in addition to an assessment of variant pathogenicity according to guidelines by the CMGS and VKGL, for the British and Dutch Molecular Genetic Societies, respectively.…”

Section: Mutation Analysismentioning

confidence: 99%

Heterozygous germline mutations in A2ML1 are associated with a disorder clinically related to Noonan syndrome

et al. 2014

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Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphisms and congenital heart defects. To date, all mutations known to cause NS are dominant, activating mutations in signal transducers of the RAS/ mitogen-activated protein kinase (MAPK) pathway. In 25% of cases, however, the genetic cause of NS remains elusive, suggesting that factors other than those involved in the canonical RAS/MAPK pathway may also have a role. Here, we used family-based whole exome sequencing of a case-parent trio and identified a de novo mutation, p.(Arg802His), in A2ML1, which encodes the secreted protease inhibitor a-2-macroglobulin (A2M)-like-1. Subsequent resequencing of A2ML1 in 155 cases with a clinical diagnosis of NS led to the identification of additional mutations in two families, p.(Arg802Leu) and p.(Arg592Leu). Functional characterization of these human A2ML1 mutations in zebrafish showed NS-like developmental defects, including a broad head, blunted face and cardiac malformations. Using the crystal structure of A2M, which is highly homologous to A2ML1, we identified the intramolecular interaction partner of p.Arg802. Mutation of this residue, p.Glu906, induced similar developmental defects in zebrafish, strengthening our conclusion that mutations in A2ML1 cause a disorder clinically related to NS. This is the first report of the involvement of an extracellular factor in a disorder clinically related to RASopathies, providing potential new leads for better understanding of the molecular basis of this family of developmental diseases.

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“…Whereas several studies show that LS mutations downregulate the level of phosphorylated ERK (Kontaridis et al, 2006;Stewart et al, 2010), the role of LS-Shp2 mutation on the MAPK pathway is still controversial (Oishi et al, 2006(Oishi et al, , 2009Edouard et al, 2010). Increased RAS/MAPK signaling is implicated in the gain-of-function phenotypes that are caused by expression of the LS Drosophila ortholog of PTPN11, corkscrew (csw) (Oishi et al, 2009), suggesting a mechanism by which NS and LS variants may result in similar phenotypes.…”

Section: Introductionmentioning

confidence: 99%

Noonan and LEOPARD syndrome Shp2 variants induce heart displacement defects in zebrafish

et al. 2014

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Germline mutations in PTPN11, encoding Shp2, cause Noonan syndrome (NS) and LEOPARD syndrome (LS), two developmental disorders that are characterized by multiple overlapping symptoms. Interestingly, Shp2 catalytic activity is enhanced by NS mutations and reduced by LS mutations. Defective cardiac development is a prominent symptom of both NS and LS, but how the Shp2 variants affect cardiac development is unclear. Here, we have expressed the most common NS and LS Shp2-variants in zebrafish embryos to investigate their role in cardiac development in vivo. Heart function was impaired in embryos expressing NS and LS variants of Shp2. The cardiac anomalies first occurred during elongation of the heart tube and consisted of reduced cardiomyocyte migration, coupled with impaired leftward heart displacement. Expression of specific laterality markers was randomized in embryos expressing NS and LS variants of Shp2. Ciliogenesis and cilia function in Kupffer's vesicle was impaired, likely accounting for the left/right asymmetry defects. Mitogen-activated protein kinase (MAPK) signaling was activated to a similar extent in embryos expressing NS and LS Shp2 variants. Interestingly, inhibition of MAPK signaling prior to gastrulation rescued cilia length and heart laterality defects. These results suggest that NS and LS Shp2 variant-mediated hyperactivation of MAPK signaling leads to impaired cilia function in Kupffer's vesicle, causing left-right asymmetry defects and defective early cardiac development.

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Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations

Cited by 67 publications

References 55 publications

In vivo severity ranking of Ras pathway mutations associated with developmental disorders

In vivo severity ranking of Ras pathway mutations associated with developmental disorders

Heterozygous germline mutations in A2ML1 are associated with a disorder clinically related to Noonan syndrome

Noonan and LEOPARD syndrome Shp2 variants induce heart displacement defects in zebrafish

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