OR20-3 Decreased growth hormone (GH) expression due to double homozygous mutations in the GH1 promoter region is associated to isolated growth hormone deficiency (IGHD) in 3 siblings

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: OR20-Genetics of Growth
Bench to Bedside
Sunday, June 16, 2013: 11:15 AM-12:45 PM
Presentation Start Time: 11:45 AM
Room 122 (Moscone Center)
João Luiz de Oliveira Madeira*1, Regina Matsunaga Martin2, Luciana Ribeiro Montenegro3, Marcela Moura Franca4, Everlayny Fiorot Costalonga5, Fernanda de Azevedo Correa6, Aline Pedrosa Otto4, Ivo J Arnhold7, Helayne Soares de Freitas8, Ubiratan Fabres Machado8, Berenice Bilharinho Mendonca7, Alexander Augusto Lima Jorge9 and Luciani Renata Silveira Carvalho10
1Hospital das Clínicas, University of São Paulo, Medical School, São Paulo, Brazil, 2Hosp das Clinicas de Fac de Med, Sao Paulo SP, Brazil, 3HC-FMUSP, Sao Paulo, Brazil, 4Univ of Sao Paulo, Sao Paulo, Brazil, 5University of Sao Paulo, Vit?ria - ES, Brazil, 6Hospital das Clinical - FMUSP, Sao Paulo- SP, Brazil, 7Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 8University of São Paulo, São Paulo, Brazil, 9University of Sao Paulo, Sao Paulo, Brazil, 10Hosp das Clinicas, Sao Paulo, Brazil
Background and aims: The incidence of short stature due to growth hormone (GH) deficiency is estimated between 1:4,000-10,000 live births. Horan et al. described several polymorphisms in the proximal GH1 promoter region that influence its transcriptional activity. Millar et al. first described short stature patients harboring heterozygous microdeletions and gene conversions in the GH1 promoter region, which is a good candidate region for mutations in IGHD patient. We studied 3 siblings (2 M, 1 F) with short stature born to consanguineous parents (first cousins). The 3 siblings presented proportionate severe postnatal short stature (height SDS from -4.1 to -5.8 for a target height of -2.4 SDS). When first evaluated, they presented low IGF-1 and IGFBP-3 levels and decreased GH peak level to hypoglycemia test (4.8 ng/mL by RIA). The pituitary was normal at MRI. At adulthood without GH treatment, IGF-1 and IGFBP-3 levels were extremely low in the 3 siblings and in 2 patients GH peak levels were 2.5 to 2.8 ng/mL after induced hypoglycemia. The DNA analysis of GHRHR and GH1 coding region were normal, and GH1 proximal and distal promoter (locus control region - LCR) were sequenced. The 3 patients harbored, in addition to haplotype 8 described by Horan et al, 2 single nucleotide changes (c.-123 T>C and -161 C>T) in homozygous state, that were previously described separately as polymorphisms. The parents and a normal brother are heterozygous for these allelic variations. Nucleotide -123T is a predicted binding site for the transcription factors PIT1 (activator) and SP1 (repressor), while -161C is a predicted binding site for the activator NF1. To analyze the effect of the double homozygous allelic variants c.-161 C>T and c.-123 T>C on the phenotype, transient transfection was performed in triplicate at least 3 times in the GH3 cell line. The DNA-protein interaction was tested by EMSA using mutated and wild type promoter region and GH3 nuclear extract beside PIT1 protein. Results: In the transfected cells the mean values of GH1 wild type promoter expression were 4 times higher in comparison to the mutant (p<0.001). EMSA demonstrated less affinity of GH3 nuclear extract to -161 mutant region due to the lost of activator NF1 binfind site, but higher affinity to -123 mutant region probably due to enhanced binding to the repressor SP1. Conclusion: The double homozygous mutations in the proximal GH1 promoter region are the disease causing in the siblings with IGHD.


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Sources of Research Support: Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP