A homozygous point mutation in the GH1 promoter (c.-223C > T) leads to reduced GH1 expression in siblings with isolated GH deficiency (IGHD)

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Madeira, Joao L. O. ; Jorge, Alexander A. L. ; Martin, Regina M. ; Montenegro, Luciana R. ; Franca, Marcela M. ; Costalonga, Everlayny F. ; Correa, Fernanda A. ; Otto, Aline P. ; Arnhold, Ivo J. P. ; Freitas, Helayne S. ; Machado, Ubiratan F. ; Mendonca, Berenice B. ; Carvalho, Luciani R.

• Document type: Journal article
• Source: EUROPEAN JOURNAL OF ENDOCRINOLOGY; v. 175, n. 2, p. K7-K15, AUG 2016.
• Web of Science Citations: 2

Abstract

Context: Mutations in the GH1 promoter are a rare cause of isolated growth hormone deficiency (IGHD). Objective: To identify the molecular aetiology of a family with IGHD. Design: DNA sequencing, electromobility shift (EMSA) and luciferase reporter assays. Setting: University Hospital. Patients: Three siblings (2M) born to consanguineous parents presented with IGHD with normal pituitary on MRI. Methods: The GH1 proximal promoter, locus control region, five exons and four introns as well as GHRHR gene were sequenced in genomic DNA by Sanger method. DNA- protein interaction was evaluated by EMSA in nuclear extracts of GH3 pituitary cells. Dual-luciferase reporter assays were performed in cells transiently transfected with plasmids containing four different combinations of GH1 allelic variants (AV). Results: The patients harboured two homozygous variants (c.-185T>C and c.-223C>T) in the GH1 promoter within a highly conserved region and predicted binding sites for POU1F1/SP1 and SP1 respectively. The parents and brother were carriers and these variants were absent in 100 controls. EMSA demonstrated absent binding of GH3 nuclear extract to the c.-223C>T variant and normal binding of both POU1F1 protein and GH3 nuclear extract to the c.-185T>C variant. In contrast to GH1 promoter with AV only at c.-185, the GH1 promoter containing the AV only at c.-223 and at both positions drove significantly less expression of luciferase compared with the promoter containing either positions wild type in luciferase reporter assays. Conclusion: To our knowledge, c.-223C>T is the first homozygous point mutation in the GH1 promoter that leads to short stature due to IGHD. (AU)

• FAPESP's process: 13/03236-5 - New approaches and methodologies in molecular-genetic studies of growth and pubertal development disorders
• Grantee: Alexander Augusto de Lima Jorge
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 10/05188-0 - Validation of a radioimmunoassay for 11-deoxycortisol measurement for the diagnosis of hyperandrogenic disorders
• Grantee: João Luiz de Oliveira Madeira
• Support Opportunities: Scholarships in Brazil - Scientific Initiation