Background: Isolated GH deficiency type II (IGHD II), the autosomal-dominant form of GH deficiency, is mainly caused by specific splicing mutations in the human GH (hGH) gene (GH-1). These mutations, occurring in and around exon 3, cause complete exon 3 skipping and produce a dominant-negative 17.5-kDa GH isoform that reduces the accumulation and secretion of wt-GH.
Objective and hypotheses: As the severity of IGHD II inversely correlates with the amount of 17.5-kDa produced and with the 17.5/22-kDa ratio, increasing the inclusion of exon 3 during splicing is expected to ameliorate disease symptoms. To test this hypothesis we modulated GH-1 splicing by overexpressing ASF/SF2, the alternative splicing factor 2, known to promote GH exon 3 inclusion in vitro and in vivo.
Method: Rat pituitary cell line stably expressing hGHRHR (GC-GHRHR cells) were transiently transfected with either wt-GH or with different GH-splice site mutants (IVS+2, IVS+6 and ISE+28), stimulated with GHRH (10 nM) and/or additionally transfected with ASF/SF2 for 24 h. The differences in the splicing pattern of wt-GH vs GH-splice site mutants were assessed by qRT-PCR and western blot. Extracellular GH-secretion was measured in aliquots of cultured medium by DSL-GH ELISA.
Results: At the mRNA level, overexpression of ASF/SF2, promoted exon 3 inclusion and increased the amount of full-length transcripts (P<0.05). At the protein level, we could observe an increased synthesis of the 22-kDa isoform (P<0.05) and therefore a decreased 17.5/22-kDa ratio (P<0.05). Overall, the switched balance between the two GH isoforms resulted in a statistically increase of GH secretion (P<0.01) in all GH-splice site mutants analysed. Moreover, the impact of ASF/SF2 overexpression was more pronounced in presence of GHRH (P<0.01).
Conclusion: While rhGH replacement therapy in IGHD II patients helps growth, it does not prevent the development of other pituitary hormone deficiencies in many of these patients. In this study we propose an alternative approach to IGHD II and we showed that, targeting GH-1 splicing, it is possible to significantly rescue the GH secretion and to reduce the 17.5/22-kDa ratio which has been established as a parameter of IGHD II clinical severity.
01 Oct 2015 - 03 Oct 2015