title: Isolated autosomal dominant growth hormone deficiency: stimulating mutant GH-1 gene expression drives GH-1 splice-site selection, cell proliferation, and apoptosis.
abstract: The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.
authors.affiliation: Paediatric Endocrinology, University Children's Hospital, Inselspital, University of Bern, CH-3010 Bern, Switzerland.
authors.forename: Souzan
authors.initials: S
authors.lastname: Salemi
authors.forename: Shida
authors.initials: S
authors.lastname: Yousefi
authors.forename: Didier
authors.initials: D
authors.lastname: Lochmatter
authors.forename: Andrée
authors.initials: A
authors.lastname: Eblé
authors.forename: Johnny
authors.initials: J
authors.lastname: Deladoëy
authors.forename: Iain C A F
authors.initials: IC
authors.lastname: Robinson
authors.forename: Hans-Uwe
authors.initials: HU
authors.lastname: Simon
authors.forename: Primus E
authors.initials: PE
authors.lastname: Mullis
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D053938:DNA Fragmentation; D003907:Dexamethasone / Q000494:pharmacology; D005091:Exons / Q000235:genetics; D005799:Genes, Dominant; D005938:Glucocorticoids / Q000494:pharmacology; D006130:Growth Disorders / Q000235:genetics* / Q000503:physiopathology; D019382:Human Growth Hormone / Q000172:deficiency / Q000235:genetics*; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D010641:Phenotype; D010902:Pituitary Gland / Q000166:cytology*; D022821:RNA Splice Sites / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D014162:Transfection
chemical_list: D005938:Glucocorticoids; D022821:RNA Splice Sites; D019382:Human Growth Hormone; D003907:Dexamethasone