Background: 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.
Method: Genomic DNA from patients with familial GHD was enriched for the coding exons using hybrid capture technology and GH1 was sequenced using Next Generation Sequencing technology. Plasmids containing genes for WT and A34T GH1 were transfected into NCI HEK295 cells. RNA was analysed by RT PCR. The p.A34T mutant protein was expressed in bacteria and studied by chemical denaturation and proteolysis. Denaturation was quantified using fluorescence spectroscopy. Proteolysis was determined by SDS-PAGE and western blot.
Results: GHD was identified in three female siblings aged 3.25-6.33 years (Ht SDS −3.21 to −1.13, peak GH 2.96.6 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 heterozygous variant (c.178G>A; p.Ala34Thr) that had not been previously described, and was not found in the Broad ExAc dataset representing >60,000 children without severe childhood-onset disease. Functional studies of the mutant GH protein showed reduced stability to denaturation and proteolysis compared to native GH. This mutation leads to alternate splicing resulting in increased expression of the smaller isoform of GH missing exon 3. PCR analysis of RNA revealed loss of signal for the A34T mutant of GH1.
Conclusion: The 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. In addition, functional studies of the mutant GH protein show reduced stability to denaturation and proteolysis. The mutant RNA is unstable. Binding studies of the mutant protein to the GHR are underway to determine the mechanism causing the dominant negative phenotype.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology