RESUMEN
Isolated growth hormone deficiency type II (IGHD II) is characterized by short stature due to dominant-negative mutations of the human growth hormone gene (GH1). Most of the known mutations responsible for IGHD II cause aberrant splicing of GH1 transcripts. We have recently shown that mutations that cause exon 3 skipping and produce a dominant-negative 17.5-kDa isoform in humans also cause a dose-dependent disruption of GH secretory vesicles when expressed in GC cells and transgenic mice. We show here that overexpression of the dominant-negative 17.5-kDa isoform also destroys the majority of somatotrophs, leading to anterior pituitary hypoplasia in transgenic mice. It is, therefore, important to understand the regulation of GH1 splicing and why its perturbation causes IGHD II. We demonstrate that dual splicing enhancers are required to ensure exon 3 definition to produce full-length 22-kDa hormone. We also show that splicing enhancer mutations that weaken exon 3 recognition produce variable amounts of the 17.5-kDa isoform, a result which could potentially explain the clinical variability observed in IGHD II. Non-canonical splicing mutations that disrupt splicing enhancers, such as those illustrated here, demonstrate the importance of enhancer elements in regulating alternative splicing to prevent human disease.
Asunto(s)
Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/genética , Errores Innatos del Metabolismo/genética , Mutación , Empalme del ARN , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Exones , Humanos , Intrones , Ratones , Ratones Transgénicos , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genéticaAsunto(s)
Enanismo Hipofisario/genética , Eliminación de Gen , Hormona de Crecimiento Humana/genética , Enanismo Hipofisario/diagnóstico , Enanismo Hipofisario/epidemiología , Enanismo Hipofisario/fisiopatología , Mutación del Sistema de Lectura , Frecuencia de los Genes , Hormona de Crecimiento Humana/inmunología , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Familial isolated GH deficiency type II (IGHD II) is caused, in some cases, by heterogeneous IVS3 mutations that affect GH mRNA splicing. We report here our finding an A-->G transition of the fifth base of exon 3 (E3+ 5 A-->G) in affected individuals from an IGHD II family. This mutation disrupts a (GAA)(n) exon splice enhancer (ESE) motif immediately following the weak IVS2 3' splice site. The mutation also destroys an MboII site used to demonstrate heterozygosity in all affected family members. To determine the effect of ESE mutations on GH mRNA processing, GH(3) cells were transfected with expression constructs containing the normal ESE, +5 A-->G, or other ESE mutations, and cDNAs derived from the resulting GH mRNAs were sequenced. All ESE mutations studied reduced activation of the IVS2 3' splice site and caused either partial E3 skipping, due to activation of an E3+ 45 cryptic 3' splice site, or complete E3 skipping. Partial or complete E3 skipping led to loss of the codons for amino acids 32-46 or 32-71, respectively, of the mature GH protein. Our data indicate that the E3+ 5 A-->G mutation causes IGHD II because it perturbs an ESE required for GH splicing.