ESPE Abstracts (2021) 94 P2-287

ESPE2021 ePoster Category 2 Growth and syndromes (to include Turner syndrome) (56 abstracts)

Novel LZTR1 mutations in subjects with features of Noonan Syndrome and GH insensitivity negatively regulate GH-induced IGF-I production and hyperactivate GH-induced ERK1/2 activation in response to GH in vitro

Sumana Chatterjee 1 , Debora Romeo Bertola 2 , Chizo Agwu 3 , Avinaash Maharaj 1 , Jack Williams 1 , Emily Cottrell 1 , Lucy Shapiro 1 , Afiya Andrews 1 , Martin O. Savage 1 , Carles Gaston-Massuet 1 , Louise A. Metherell 1 & Helen L. Storr 1

1Centre for Endocrinology, William Harvey Research Institute, Queen Mary University London, London, United Kingdom; 2Department of Genetics, Children’s Institute, Faculty of Medicine, University of Sao Paulo, São Paulo, Brazil; 3Department of Paediatric Diabetes and Endocrinology, Sandwell and West Birmingham NHS Trust, Birmingham, United Kingdom

Background: Noonan Syndrome (NS) can overlap clinically and biochemically with growth hormone insensitivity [GHI; short stature (SS), low IGF-I and normal/elevated GH levels]. Mutations in multiple genes regulating RAS/MAPK pathway have been identified in NS including LZTR1 variants. Function of LZTR1 is poorly understood and its role in growth retardation is unknown.

Objectives: To functionally characterise 6 novel LZTR1 variants (5 previously published1 and 1 identified in our GHI patient cohort) and determine their impact on the GH-IGF-I axis.

Methods: A novel heterozygous missense LZTR1 variant (c.466A>G; p.K156E) was identified in a GHI subject (V1) by our SS whole genome panel. 5 previously published NS-associated heterozygous inactivating missense LZTR1 variants [c.742G>A;p.G248R, c.850C>T;p.R284C, c.740G>A;p.S247N, c.356A>G;p.Y119C and c.859C>T;p.H287Y (V2-6, respectively)] were also studied. V1-6 LZTR1 vectors were generated by site-directed mutagenesis and verified by Sanger sequencing. Western blot (WB) analysis of transfected HEK293 cell lysates was performed using anti-c-Myc & anti-ERK/anti-pERK antibodies (anti-beta-actin/GAPDH antibodies as controls). Supernatant from transfected & GH-stimulated (24 hours) HepG2 cells were assessed by ELISA. Cell lysates from transfected (with V1, 2 & 5) & GH-stimulated (20 minutes) HepG2 cells were subjected to WB analysis (anti-ERK/anti-pERK antibodies & anti-STAT5/anti-pSTAT5 antibodies).

Results: All 6 subjects had characteristic facial features of NS and cardiac defects. 2 subjects (V1 & 2) had features of SS & GHI (height/IGF-I SDS of -2.3/-2.3 and -2.1/-2.2 respectively). All variants showed significantly reduced LZTR1 protein expression and increase in p-ERK/total ERK ratios compared to WT, latter suggesting up-regulation of RAS-MAPK pathway. Compared to WT (0.54±0.12), GH-induced mean IGF-I levels were significantly lower in V1 & 2 (0.28±0.03 & 0.29±0.07, respectively; both P <0.05), but not in V3-6. IGF-I rise following GH stimulation in all 6 subjects correlated negatively with the subject’s height SDS (P < 0.001). Following GH stimulation, as compared to WT, a significant increase in p-ERK/total ERK ratios but no difference in p-STAT5/total STAT5 ratios were observed in V1 & 2. This suggests GH-induced ERK1/2 hyperactivation and upregulation of RAS-MAPK pathway in variants causing SS.

Conclusion: Novel LZTR1 variants in NS cause reduced LZTR1 protein expression and enhanced RAS-MAPK signalling, similar to that observed in PTPN11 and SOS1 mutations. GHI-causing LZTR1 mutants negatively regulate GH-induced IGF-I production and hyperactivate ERK1/2 activation in response to GH in vitro. This suggests that dysregulation of GH-induced RAS-MAPK pathway could contribute to growth retardation.

1 Yamamoto GL, Aguena M, et al. Rare variants in SOS2 and LZTR1 are associated with Noonan syndrome. J. Med. Genet. 2015.52: 413-421.

Volume 94

59th Annual ESPE (ESPE 2021 Online)

22 Sep 2021 - 26 Sep 2021

European Society for Paediatric Endocrinology 

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