Background: Familial isolated growth hormone deficiency (IGHD) is a disorder with about 5–30% of patients having affected relatives. Among those familial types, IGHD type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32–71-GH, a GH peptide which cannot fold properly. The mechanism by which this mutant GH may alter the controlled secretory pathway and therefore suppress the secretion of the normal 22-kDa GH product of the normal allele is not known in detail. Previous studies have shown variance depending on cell type, transfection technique used, as well as on the method of analysis performed. Aim: The aim of our study was to analyse and compare the subcellular distribution/localization of del32–71-GH or wild-type (wt)-GH (22-kDa GH), each stably transfected into AtT-20, a mouse pituitary cell line endogenously producing ACTH, employed as the internal control for secretion assessment. Methods: Co-localizationof wt- and del32–71 mutant GH form was studied by quantitative confocal microscopy analysis. Using the immunofluorescent technique, cells were double stained for GH plus one of the following organelles: endoplasmic reticulum (ER anti-Grp94), Golgi (anti-βCOP) or secretory granules (anti-Rab3a). In addition, GH secretion and cell viability were analysed in detail. Results/Conclusions: Our results show that in AtT-20 neuroendocrine cells, in comparison to the wt- GH, the del32–71-GH has a major impact on the secretory pathway not only affecting GH but also other peptides such as ACTH. The del32–71-GH is still present at the secretory vesicles’ level, albeit in reduced quantity when compared to wt-GH but, importantly, was secretion-deficient. Furthermore, while focusing on cell viability an additional finding presented that the various splice site mutations, even though leading eventually to the same end product, namely del32–71-GH, have different and specific consequences on cell viability and proliferation rate.

Keywords:
Growth hormone, Isolated growth hormone deficiency, Confocal microscopy, GH secretion, ACTH secretion, Cell viability
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