Autosomal Dominant Growth Hormone Deficiency Due to a Novel c.178G>A Mutation in the GH1 Gene is Caused by Alternative Splicing to Produce a Small GH Isoform

Abstract

IntroductionGrowth hormone (GH) is a member of the somatotropin/prolactin family of hormones and plays a vital role in human physiology. Mutations in GH1 cause isolated growth hormone deficiency. Several disease‐causing mutations have been reported in patients with IGHD. The most frequent cause of familial growth hormone deficiency (GHD) is Type II autosomal dominant GHD (isolated GHD type II) due to several heterozygous GH1 mutations. These mutations have been shown to (a) produce shorter isoforms of GH that do not bind to growth hormone receptors, (b) cause diminished secretion of GH, or (c) result in misfolded GH protein.MethodsGenomic DNA from patients with familial GHD was enriched for the coding exons using hybrid capture technology, and GH1 was sequenced. The p.A34T mutant protein was expressed in bacteria, and binding to GHR was studied by Surface Plasmon Resonance technology. Computational prediction of transcription indicated that alternative splicing is likely to produce a shorter GH variant with skipping of exon 3 in GH1. Mammalian cell‐based studies incorporating transfections of the whole GH1 gene containing all exons and introns, were used to study transcriptional effects. RNA was isolated from cells transfected with WT and mutant GH1 gene and analyzed by RT PCR using primers in the second and fifth exons of GH1 that could identify all possible isoforms of GH1 mRNA.ResultsGHD was identified in three female siblings aged 3.25–6.33 years (Ht SDS −3.21 to −1.13, peak GH 2.9–6.6 ng/mL); their mother had previously been diagnosed with GHD at age 12.33 years (Ht SDS −3.44, GH peak < 2 ng/mL). Sequencing of GH1 identified a novel heterozygous variant (c.178G>A; p.Ala34Thr) not found in the Broad ExAc dataset representing >60,000 children without the severe childhood‐onset disease. Functional studies using whole gene transfection showed that the c.178G>A mutation leads to alternate splicing resulting in increased production of the shorter 17.5kD isoform of GH due to exon 3 skipping. Results were confirmed by quantitative RT‐PCR as well as GH secretion assays, which showed a lower level of GH production from cells transfected with the GH1 gene containing the c.178G>A mutation. The SPR based receptor binding assay and cell proliferation assay using bacterially expressed proteins showed that once produced, the GH protein with the A34T mutation behaves similar to WT GH protein. All these results confirm that the cause of GHD due to the c.178G>A mutation in GH1 is due to altered transcription leading to the production of the shorter 17.5 kD isoform of GH protein and not due to the amino acid change A34T that is caused by the mutation.ConclusionThe presence of a heterozygous GH1 variant (c.178G>A, p.Ala34Thr) in four individuals with GHD suggests that this is a novel cause of IGHD type II. Production of the smaller 17.5 kD GH isoform results in reduced overall GH secretion and loss of binding to GHR due to competition with the normal GH protein, explaining the dominant‐negative phenotype.Support or Funding InformationUnrestricted research grants from Novo Nordisk AG, & Sandoz Pharmaceuticals AG, Switzerland.