ESPE Abstracts (2019) 92 RFC14.4

GHR Transcript Heterogeneity May Explain the Phenotypic Variability in Patients with Homozygous GHR Pseudoexon (6ψ) Mutation

Sumana Chatterjee1, Steven J Rose2, Talat Mushtaq3, Emily Cottrell1, Avinaash V Maharaj1, Jack Williams1, Martin O Savage1, Loiuse A Metherell1, Helen L Storr1

1Centre for Endocrinology, William Harvey Research Institute, Queen Mary University London, London, United Kingdom. 2Birmingham Heartland Hospital, Heart of England NHS Foundation Trust, Birmingham, United Kingdom. 3The Leeds Teaching Hospital NHS Trust, Leeds, United Kingdom

Objectives: The homozygous GHR pseudoexon (6ψ) mutation leads to aberrant splicing of the GHR gene with clinical and biochemical heterogeneity. We investigated whether the phenotypic variability could be explained by transcript heterogeneity i.e. ratio of abnormal (6ψ GHR) to normal (WT GHR) transcripts and/or the presence of concurrent defects in other short stature (SS) genes.

Methods: 6ψ GHR and WT GHR mRNA transcripts from 4 6ψ patients' fibroblasts (height SDS -3.6, -3.4, -4.2 and -3.8) and 1 control subject were investigated by reverse-transcriptase PCR (RT-PCR) using intron skipping primers. Transcripts (mean±SD) were quantified by qRT-PCR and double delta CT analysis (5 experimental repeats) and compared using ANOVA with Bonferroni correction. In eleven 6ψ patients, 63 genes known to cause SS were analysed by targeted sequencing.

Results: RT-PCR confirmed the presence of WT transcript (193 bp) in 6ψ patients and control. 6ψ transcript (217 bp) was seen in all 4 6ψ patients but not control. Direct sequencing verified predicted mRNA sequences. 6ψ transcript expression was significantly different amongst patients (1±0, 0.334±0.032, 0.549±0.005, 0.960±0.071) p values<0.001, except between patients 1 & 4. The mean 6ψ:WT transcript ratios (40.33, 29.69, 72.74, 47.39) correlated negatively with height SDS (R=-0.96, p value <0.01) in all 4 6ψ patients.

Genetic analysis of 11 6ψ patients revealed 9 deleterious variants in 6 genes. However, there was no correlation between the number of gene hits and degree of short stature in individual 6ψ patients.

Conclusion: Varying amounts of 6ψ and WT GHR transcripts were identified in 6ψ patients, with no 6ψ transcript identified in the control. A higher 6ψ:WT GHR transcript ratio correlates with the severity of short stature and thus may explain the phenotypic variability. Genetic changes in other known SS genes do not appear to account for the phenotypic variation.