Changes of RAS Pathway Phosphorylation in Lymphoblastoid Cell Lines from Noonan Syndrome Patients Carrying Hypomorphic Variants in Two NS Genes

Noonan syndrome (NS) is an autosomal dominant multisystem disorder, characterized by variable expressivity and locus heterogeneity, being caused by mutations in one of a subset of RAS pathway genes. Nevertheless, for 20–30% of patients it is not possible to provide molecular diagnosis, suggesting that further unknown genes or mechanisms are involved in NS pathogenesis. Recently, we proposed a digenic inheritance of subclinical variants as an alternative NS pathogenic model in two NS patients negative for molecular diagnosis. They showed hypomorphic variants of RAS pathway genes co-inherited from both their healthy parents that we hypothesized to generate an additive effect. Here, we report on the phosphoproteome and proteome analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) performed on the immortalized peripheral blood mononuclear cells (PBMCs) from the two above trios. Our results indicate that the two unrelated patients show overlapped profiles in both protein abundances and their phosphorylation levels not reached by their parents. IPA software predicted RAS-related pathways as significantly activated in the two patients. Interestingly, they remained unchanged or only slightly activated in both patients’ parents. These findings suggest that the presence of one subclinical variant can activate the RAS pathway below the pathological threshold, which can instead be exceeded by the additive effect due to the co-presence of two subclinical variants causing NS, supporting our digenic inheritance hypothesis.

[1]  M. Zenker,et al.  Noonan syndrome: improving recognition and diagnosis , 2022, Archives of Disease in Childhood.

[2]  R. Elder,et al.  Noonan syndrome and pregnancy outcomes , 2022, Cardiology in the Young.

[3]  Jiang Zheng,et al.  Shenxiong glucose injection inhibits H2O2-induced H9c2 cell apoptosis by activating the ERK signaling pathway. , 2021, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[4]  F. He,et al.  A proteomic and phosphoproteomic landscape of KRAS mutant cancers identifies combination therapies. , 2021, Molecular cell.

[5]  J. Lacal,et al.  Integrated in silico MS-based phosphoproteomics and network enrichment analysis of RASopathy proteins , 2021, Orphanet Journal of Rare Diseases.

[6]  J. F. Longo,et al.  R-Ras subfamily proteins elicit distinct physiologic effects and phosphoproteome alterations in neurofibromin-null MPNST cells , 2020, Cell Communication and Signaling.

[7]  C. Battaglia,et al.  Digenic inheritance of subclinical variants in Noonan Syndrome patients: an alternative pathogenic model? , 2020, European Journal of Human Genetics.

[8]  B. Gelb,et al.  Congenital heart defects in Noonan syndrome: Diagnosis, management, and treatment , 2020, American journal of medical genetics. Part C, Seminars in medical genetics.

[9]  S. Baek,et al.  Neurodevelopmental Aspects of RASopathies , 2019, Molecules and cells.

[10]  H. Ohashi,et al.  Delineation of LZTR1 mutation-positive patients with Noonan syndrome and identification of LZTR1 binding to RAF1–PPP1CB complexes , 2018, Human Genetics.

[11]  D. Lacombe,et al.  RAS signalling in energy metabolism and rare human diseases. , 2018, Biochimica et biophysica acta. Bioenergetics.

[12]  E. I. Pierpont,et al.  Social skills in children with RASopathies: a comparison of Noonan syndrome and neurofibromatosis type 1 , 2018, Journal of Neurodevelopmental Disorders.

[13]  E. I. Pierpont,et al.  Social skills in children with RASopathies: a comparison of Noonan syndrome and neurofibromatosis type 1 , 2018, Journal of Neurodevelopmental Disorders.

[14]  J. Allanson,et al.  Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies , 2018, Scientific Reports.

[15]  J. Rosenfeld,et al.  Autosomal Recessive Noonan Syndrome Associated with Biallelic LZTR1Variants , 2017, Genetics in Medicine.

[16]  Frank McCormick,et al.  RAS Proteins and Their Regulators in Human Disease , 2017, Cell.

[17]  P. Sachdev,et al.  Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System , 2016, Scientific Reports.

[18]  L. Castagnoli,et al.  Alterations in the phosphoproteomic profile of cells expressing a non-functional form of the SHP2 phosphatase. , 2016, New biotechnology.

[19]  J. Allanson Objective studies of the face of Noonan, Cardio‐facio‐cutaneous, and Costello syndromes: A comparison of three disorders of the Ras/MAPK signaling pathway , 2016, American journal of medical genetics. Part A.

[20]  Yoichi Matsubara,et al.  Recent advances in RASopathies , 2015, Journal of Human Genetics.

[21]  J. F. Longo,et al.  Classic Ras Proteins Promote Proliferation and Survival via Distinct Phosphoproteome Alterations in Neurofibromin-Null Malignant Peripheral Nerve Sheath Tumor Cells , 2015, Journal of neuropathology and experimental neurology.

[22]  A. Heck,et al.  Phosphoproteomics-Mediated Identification of Fer Kinase as a Target of Mutant Shp2 in Noonan and LEOPARD Syndrome , 2014, PloS one.

[23]  A. Heck,et al.  PZR Coordinates Shp2 Noonan and LEOPARD Syndrome Signaling in Zebrafish and Mice , 2014, Molecular and Cellular Biology.

[24]  Jian Wang,et al.  Rare copy number variations containing genes involved in RASopathies: deletion of SHOC2 and duplication of PTPN11 , 2014, Molecular Cytogenetics.

[25]  Andreas Krämer,et al.  Causal analysis approaches in Ingenuity Pathway Analysis , 2013, Bioinform..

[26]  O. Gabrielli,et al.  SOS1 Mutations in Noonan Syndrome: Molecular Spectrum, Structural Insights on Pathogenic Effects, and Genotype–Phenotype Correlations , 2011, Human mutation.

[27]  J. Allanson,et al.  Noonan Syndrome: Clinical Features, Diagnosis, and Management Guidelines , 2010, Pediatrics.

[28]  M. Cisternino,et al.  Noonan syndrome associated with both a new Jnk‐activating familial SOS1 and a de novo RAF1 mutations , 2010, American journal of medical genetics. Part A.

[29]  M. Mann,et al.  Universal sample preparation method for proteome analysis , 2009, Nature Methods.

[30]  I. van der Burgt,et al.  Noonan Syndrome , 2019, Encyclopedia of Endocrine Diseases.

[31]  J. Wiśniewski Filter-Aided Sample Preparation for Proteome Analysis. , 2018, Methods in molecular biology.

[32]  C. Eyers Universal sample preparation method for proteome analysis , 2009 .