ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2019) 66 OC4.7 | DOI: 10.1530/endoabs.66.OC4.7

Novel variants in the Leucine-zipper-like transcription regulator 1 (LZTR1) gene cause Noonan syndrome phenotype by upregulation of the RAS-MAPKinase pathway

Sumana Chatterjee1, Debora Bertola2, Chizo Agwu3, Maria Karantza4, Emily Cottrell1, Lucy Shapiro1, Avinaash V Maharaj1, Jack Williams1, Martin O Savage1, Carles Gaston-Massuet1, Louise A Metherell1 & Helen L Storr1

1Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary University London, London, UK; 2Department of Paediatrics, University of Sao Paulo, Sao Paolo, Brazil; 3Department of paediatrics, Sandwell and West Birmingham Hospitals, Birmingham, UK; 4Mitera Children’s Hospital, Athens, Greece

Objectives: Noonan Syndrome (NS) is an autosomal dominant multi-system disorder characterised by short stature (SS), distinctive facial features and cardiovascular abnormalities. Mutations in multiple genes regulating the RAS-MAPK pathway have been identified in NS including 5 recently described novel LZTR1 variants. We identified 2 novel LZTR1 variants in patients with features of growth hormone insensitivity and NS. The molecular function of LZTR1 is unknown and we aimed to assess the impact of the LZTR1 variants on (1) LZTR1 protein expression and (2) RAS-MAPK pathway function.

Methods: Targeted and whole exome sequencing data were analysed by Ingenuity Variant Analysis using established bioinformatic pipelines. We identified 2 novel heterozygous missense LZTR1 gene variants [c.466A>G; p.K156E and c.23G>C; p.G8A] in 2 subjects and 5 previously published 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]. Site-Directed Mutagenesis generated the 7 LZTR1 variants in WT-LZTR1 vector (pcDNA-Myc-Hist-LZTR1) and constructs were verified by Sanger sequencing. HEK293 cells were transiently transfected with variant and WT LZTR1 in 3 technical replicates. Western blot (WB) analysis was performed, using anti-c-Myc, anti-ERK and anti-pERK antibodies (anti-beta-actin/GAPDH as controls).

Results: The 7 patients had characteristic facial features of NS (downslanting palpebral fissures, hypertelorism, short nose), 6/7 had cardiac defects and 5/7 had short stature (height SDS −2.3 to −1.8). WB confirmed significantly reduced mean LZTR1 protein expression in the 7 mutants (0.064±0.01, 0.046±0.01, 0.053±0.01, 0.042±0.003, 0.046±0.01, 0.039±0.005 and 0.058±0.003, respectively) vs WT(0.277± 0.02); P≤0.001 for all variants. There was significant increase in mean p-ERK:total ERK ratios in mutant (0.0067±0.0008, P=0.0281; 0.0049±0.0003, P=0.0139; 0.0099±0.0006, P=0.0049; 0.0058±0.0003, P=0.0081; 0.0057±0.0003, P=0.0079; 0.006±0.0003, P=0.006; 0.0056±0.0002, P=0.0047, respectively) vs WT(0.0026±0.0002) suggesting enhanced ERK phosphorylation and up-regulation of RAS-MAPK pathway.

Conclusions: Novel LZTR1 variants reduce LZTR1 protein expression. Enhanced RAS-MAPK signalling would be consistent with other NS-causing gene mutations e.g. PTPN11. LZTR1 may negatively regulate this critical cellular pathway and further functional work is underway.