ESPE Abstracts (2023) 97 P1-109

ESPE2023 Poster Category 1 Growth and Syndromes (75 abstracts)

Unique proteomic signatures of Noonan Syndrome-associated LZTR1 variants detected by phosphopeptide analysis

Sumana Chatterjee 1 , D&eactue;bora Bertola 2 , Chizo Agwu 3 , Lucy Shapiro 1 , Carles Gaston-Massuet 1 , Louise Metherell 1 , Avinaash Maharaj 1 & Helen Storr 1

1Centre for Endocrinology, William Harvey Research Institute, Queen Mary University London, London, United Kingdom. 2Department of Paediatrics, University of São Paulo, São Paulo, Brazil. 3Institute of Clinical Sciences, College of Medicine and Dental Sciences, University of Birmingham, Birmingham, United Kingdom

Background: Noonan syndrome (NS) is caused by variants in multiple genes regulating the RAS/MAPK signalling cascade. NS can present with growth failure associated with growth hormone insensitivity (GHI; low IGF-I and normal/elevated GH levels). Variants in LZTR1 lead to NS, although the interaction of LZTR1 with the RAS/MAPK and the GH-IGF-1 pathways remain to be elucidated.

Objectives: To gain insights into the functional role of LZTR1 genetic variants by phosphoproteomic analysis.

Methods: A novel heterozygous missense LZTR1 variant (p.K156E) was identified in a GHI subject with clinical features of NS by our whole genome short stature panel. We also examined a published (but not characterised) LZTR1 variant causing NS and GHI (p.G248R). LZTR1 WT, LZTR1 variants (generated by site directed mutagenesis) and empty vector (EV) constructs were transiently transfected into HEK 293T cells using Lipofectamine™ 3000. Cell lysates were extracted, phosphopeptides enriched and pellets reconstituted. Differences in phosphorylation patterns between WT, variant and EV constructs were reported as fold over WT and statistical significance assessed using unpaired two-tailed t-tests. Analysis of transcriptomic data was conducted using Ingenuity Pathway Analysis.

Results: Kinase activity estimation (KSEA) demonstrated significant enrichment of MAPK1 (mitogen-activated protein kinase 1) suggesting increased ERK activation in both variants. This corroborates our previously generated in vitro data where through Western Blot analysis of transfected HEK293 cell lysates, we observed increase in p-ERK/total ERK ratios in several LZTR1 variants compared to WT. Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1), major effectors of the DNA damage response (DDR), were both preferentially activated in LZTR1 variants. Ingenuity pathway analysis and filtering of molecules based on P-values revealed a target histone acetyltransferase inhibitor, NOC2L (NOC2 Like Nucleolar Associated Transcriptional Repressor), upregulated in both variants.

Conclusions: Phosphoproteomic analysis of two LZTR1 variants, corroborated enhanced MAPK1 signalling and revealed specific phosphopeptide signatures indicative of upregulation of the DNA damage response (DDR) consistent with previous data demonstrating activation of the DDR and chromosomal instability in germline LZTR1 variants. Upregulation of a novel target, NOC2L, was concordant for both LZTR1 variants. Its involvement in cell cycle regulation makes it a potential regulator of growth either downstream of or independent of LZTR1, although further work is needed to characterise this target. The phosphoproteomic analysis thus reveals unique molecular signatures characteristic of LZTR1 variants causing NS and growth failure.

Volume 97

61st Annual ESPE (ESPE 2023)

The Hague, Netherlands
21 Sep 2023 - 23 Sep 2023

European Society for Paediatric Endocrinology 

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