ESPE Abstracts (2014) 82 P-D-1-1-136

A Homozygous Point Mutation in the GH1 Promoter ([mdash]161T[gt]C) Leads to Reduced GH Expression in Siblings with Isolated GH Deficiency

Joao Madeiraa, Regina Martina, Luciana Montenegroa, Marcela Francaa, Everlayny Costalongaa, Fernanda Correaa, Aline Ottoa, Ivo Arnholda, Helayne Freitasb, Ubiratan Machadob, Berenice Mendoncaa, Alexander Jorgea & Luciani Carvalhoa

aDivision of Endocrinology, University of Sao Paulo Medical School, Sao Paulo, Sao Paulo, Brazil; bDepartment of Physiology and Biophysics, Institute of Biomedical Science, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil

Introduction: Mutations in the GH1 promoter are a rare cause of isolated GH deficiency (IGHD). In order to find the molecular cause of short stature due to IGHD, three siblings (2 M) born to consanguineous parents without mutations in the GHRHR and GH1 coding regions were screened for mutations in the GH1 promoter and locus control region. All patients harbored two variants (c.−123T>C and −161C>T) in homozygous state in the GH1 promoter, not found in 100 controls. The parents and a brother with normal stature were carriers. Patients presented proportionate short stature (height SDS from −4.1 to −5.8) and normal pituitary at MRI. At first evaluation, low IGF1 and IGFBP3 levels, in addition to decreased GH peak to hypoglycemia test (4.8 ng/ml by RIA), were found in all siblings. At adulthood IGF1 and IGFBP3 were low as well as GH peak at hypoglycemia tests (2.5–2.8 ng/ml – IFMA). Nucleotides −123T and −161C are within a highly conserved region among species and predicted binding sites for POU1F1/SP1 and NF1 respectively. Functional study was performed aiming to check the effect of these variants on the phenotype.

Methods: DNA–protein interaction was evaluated by EMSA. In order to perform transient transfection and dual luciferase reporter assay, three plasmids were constructed containing both positions WT (WTWT) or mutated (MUTMUT) or only mutated for −161 position (−161MUT).

Results: EMSA demonstrated less affinity of GH3 nuclear extract to −161C>T variant and normal affinity of POU1F1 protein and GH3 nuclear extract for −123T>C variant. The transfected WTWT mean values were significantly higher compared to MUTMUT (20.2±2.24 vs 11.1±2.7, P<0.01), and to −161MUT (11.3±2.1 vs 5.2±0.8, P<0.01).

Conclusion: To our knowledge, c.−161C>T is the first point mutation in the GH1 promoter that leads to short stature due to IGHD.