title: A homozygous point mutation in the GH1 promoter (c.-223C>T) leads to reduced GH1 expression in siblings with isolated GH deficiency (IGHD).
abstract: BACKGROUND
  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.
  
  
  METHODS
  DNA sequencing, electromobility shift (EMSA) and luciferase reporter assays.
  
  
  METHODS
  University Hospital.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  To our knowledge, c.-223C>T is the first homozygous point mutation in the GH1 promoter that leads to short stature due to IGHD.
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: João L O
authors.initials: JL
authors.lastname: Madeira
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42 Unidade de Endocrinologia-Genética - LIM/25Disciplina de Endocrinologia da Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil.
authors.forename: Alexander A L
authors.initials: AA
authors.lastname: Jorge
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: Regina M
authors.initials: RM
authors.lastname: Martin
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: Luciana R
authors.initials: LR
authors.lastname: Montenegro
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: Marcela M
authors.initials: MM
authors.lastname: Franca
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: Everlayny F
authors.initials: EF
authors.lastname: Costalonga
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: Fernanda A
authors.initials: FA
authors.lastname: Correa
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: Aline P
authors.initials: AP
authors.lastname: Otto
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: Ivo J P
authors.initials: IJ
authors.lastname: Arnhold
authors.affiliation: Laboratório de Metabolismo e Endocrinologia do Departamento de Fisiologia e Biofísica do Instituto de Ciências Biomédicas da Universidade de São Paulo (ICB-USP)São Paulo, Brazil.
authors.forename: Helayne S
authors.initials: HS
authors.lastname: Freitas
authors.affiliation: Laboratório de Metabolismo e Endocrinologia do Departamento de Fisiologia e Biofísica do Instituto de Ciências Biomédicas da Universidade de São Paulo (ICB-USP)São Paulo, Brazil.
authors.forename: Ubiratan F
authors.initials: UF
authors.lastname: Machado
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42.
authors.forename: Berenice B
authors.initials: BB
authors.lastname: Mendonca
authors.affiliation: Unidade de Endocrinologia do DesenvolvimentoLaboratório de Hormônios e Genética Molecular LIM/42 lucianic@gmail.com.
authors.forename: Luciani R
authors.initials: LR
authors.lastname: Carvalho
mesh_terms: D000328:Adult; D000483:Alleles; D004393:Dwarfism, Pituitary / Q000235:genetics*; D005260:Female; D005838:Genotype; D006720:Homozygote; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D008297:Male; D010375:Pedigree; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D035781:Siblings
chemical_list: D019382:Human Growth Hormone