Severe growth failure associated with a novel heterozygous nonsense mutation in the GHR transmembrane domain leading to elevated growth hormone binding protein

Abstract

Objective: To report a novel mutation in GHR and to characterize a novel mechanism of nonclassical growth hormone insensitivity.

Context: Laron syndrome (LS) is a well-described disorder of growth hormone insensitivity due to mutations in the growth hormone receptor (GHR) that leads to short stature. Biochemically, LS patients classically have elevated levels of growth hormone (GH), but low levels of insulin-like growth factor (IGF)-1, IGF binding protein (IGFBP)-3 and GH binding protein (GHBP).

Design: Case presentation with in vitro functional studies.

Patients: A young male Caucasian child with short stature was found to have growth hormone insensitivity manifested by elevated levels of GH and GHBP.

Measurements: Growth hormone stimulation tests revealed baseline GH level of 20.9 µg/L and maximum stimulated GH level of 52.7 µg/L and GHBP level of 4868 pmol/L. GHR gene sequencing revealed a novel heterozygous nonsense mutation (c.800G > A, p.Trp267*) in the transmembrane domain of the receptor. Immunoblot analysis of transfected GHR p.Trp267* in HEK293 revealed inhibition of GH-induced STAT5 signalling that was overcome with increasing doses of recombinant human GH.

Results: Using an in vitro model, we show that elevated levels of GHBP inhibit the action of GH. Furthermore, our studies demonstrate that this inhibition by GHBP can be overcome by increasing doses of recombinant human GH.

Conclusions: To our knowledge, this is the first study to demonstrate in vitro that elevated levels of GHBP attenuate the effect of GH and inhibit GH-induced signalling, thereby leading to short stature. Though this inhibition was overcome in vitro with supraphysiologic doses of GH, significantly above endogenously available GH, it remains to be seen whether such an effect can be replicated in vivo.

Keywords: GHR; growth hormone; growth hormone insensitivity; growth hormone receptor; hormone replacement; paediatric endocrinology; pituitary; short stature.

FIGURE 1

The CDC growth chart (male) of the patient carrying the heterozygous GHR mutation. The time frame of rhIGF-1 treatment is indicated on the growth curve. CDC, Centers for Disease Control and Prevention

FIGURE 2

Identification of heterozygous GHR c.800G > A (p.Trp267*) in exon 8. Schematic of the GHR gene, exons 2–10, encoding for GHR domains as indicated. TM, transmembrane. Electropherograms indicating heterozygous ic.800G > A identified in the patient and his mother

FIGURE 3

Transient transfected GHR p.Trp267* in HEK293 is secreted and inhibits GH-induced STAT5 activation. A, Schematic of transfection and post-transfection procedures, In some experiments, the 48 h post-transfection serum-free media (SFM) was removed and replaced with fresh SFM (indicated as ‘Fresh SFM’) for 0 or 48 h, prior to GH treatment (100 ng/mL). B, Immunoblot analysis of GH-treated transfected HEK293 cell lysates. GHBP in conditioned media (CM) were also analysed. Proteins immuno-detected are as indicated. C, Effect on GH-induced STAT5 signalling in the presence of increasing proportion of CM carrying GHR p.Trp267* mixed with Fresh SFM. D, Effects on GH-induced STAT5 signalling with increasing dose of GH