Agwu, C

Andrews, A

Bertola, DR

Chatterjee, S

Cottrell, E

Gaston-Massuet, C

Maharaj, A

Metherell, LA

Savage, MO

Shapiro, L

Storr, HL

Williams, J

Hormone Research in Paediatrics

Queen Mary Research Online

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 Chatterjee1 Debora Romeo Bertola2, Chizo Agwu3, Avinaash Maharaj1, Jack Williams1, Emily Cottrell1, Lucy Shapiro1, Afiya Andrews1, Martin O. Savage1, Carles Gaston-Massuet1, Louise A. Metherell1, Helen L. Storr1 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 RASMAPK pathway could contribute to growth retardation. Reference 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. 

English

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

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

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