Rare CNVs provide novel insights into the molecular basis of GH and IGF-1 insensitivity.

OBJECTIVE: Copy number variation (CNV) has been associated with idiopathic short stature, small for gestational age and Silver-Russell syndrome (SRS). It has not been extensively investigated in growth hormone insensitivity (GHI; short stature, IGF-1 deficiency and normal/high GH) or previously in IGF-1 insensitivity (short stature, high/normal GH and IGF-1). DESIGN AND METHODS: Array comparative genomic hybridisation was performed with ~60 000 probe oligonucleotide array in GHI (n = 53) and IGF-1 insensitivity (n = 10) subjects. Published literature, mouse models, DECIPHER CNV tracks, growth associated GWAS loci and pathway enrichment analyses were used to identify key biological pathways/novel candidate growth genes within the CNV regions. RESULTS: Both cohorts were enriched for class 3-5 CNVs (7/53 (13%) GHI and 3/10 (30%) IGF-1 insensitivity patients). Interestingly, 6/10 (60%) CNV subjects had diagnostic/associated clinical features of SRS. 5/10 subjects (50%) had CNVs previously reported in suspected SRS: 1q21 (n = 2), 12q14 (n = 1) deletions and Xp22 (n = 1), Xq26 (n = 1) duplications. A novel 15q11 deletion, previously associated with growth failure but not SRS/GHI was identified. Bioinformatic analysis identified 45 novel candidate growth genes, 15 being associated with growth in GWAS. The WNT canonical pathway was enriched in the GHI cohort and CLOCK was identified as an upstream regulator in the IGF-1 insensitivity cohorts. CONCLUSIONS: Our cohort was enriched for low frequency CNVs. Our study emphasises the importance of CNV testing in GHI and IGF-1 insensitivity patients, particularly GHI subjects with SRS features. Functional experimental evidence is now required to validate the novel candidate growth genes, interactions and biological pathways identified.

GHR gene transcript heterogeneity may explain phenotypic variability in GHR pseudoexon (6Ψ) patients.

OBJECTIVES: The homozygous GH receptor (GHR) pseudoexon (6Ψ) mutation leads to growth hormone insensitivity (GHI) with clinical and biochemical heterogeneity. We investigated whether transcript heterogeneity (6Ψ-GHR to WT-GHR transcript ratio) and/or concurrent defects in other short stature (SS) genes contribute to this. METHODS: 6Ψ-GHR and WT-GHR mRNA transcripts of 4 6Ψ patient (height SDS -4.2 to -3.1) and 1 control fibroblasts were investigated by RT-PCR. Transcripts were quantified by qRT-PCR and delta delta CT analysis and compared using ANOVA with Bonferroni correction. In eleven 6Ψ patients, 40 genes known to cause GHI/SS were analysed by targeted next generation sequencing. RESULTS: RT-PCR confirmed 6Ψ-GHR transcript in the 6Ψ patients but not control. 6Ψ-GHR transcript levels were comparable in patients 1 and 3 but significantly different among all other patients. The mean 6Ψ:WT transcript ratios ranged from 29-71:1 for patients 1-4 and correlated negatively with height SDS (R=-0.85; p<0.001). Eight deleterious variants in 6 genes were detected but the number of gene hits did not correlate with the degree of SS in individual 6Ψ patients. CONCLUSION: Variable amounts of 6Ψ- and WT-GHR transcripts were identified in 6Ψ patients but no 6Ψ transcript was present in the control. Higher 6Ψ:WT GHR transcript ratio correlated with SS severity and may explain the phenotypic variability. Analysis of known SS genes suggested that phenotypic variation is independent of the genetic background. This is the first report of transcript heterogeneity producing a spectrum of clinical phenotypes in different individuals harbouring an identical homozygous genetic mutation.

Phenotypic spectrum and responses to recombinant human IGF1 (rhIGF1) therapy in patients with homozygous intronic pseudoexon growth hormone receptor mutation.

BACKGROUND: Patients with homozygous intronic pseudoexon GH receptor (GHR) mutations (6Ψ) have growth hormone insensitivity (GHI) (growth failure, IGF1 deficiency and normal/elevated serum GH). We report 9 patients in addition to previously described 11 GHR 6Ψ patients and their responses to rhIGF1 therapy. METHODS: 20 patients (12 males, 11 families, mean age 4.0 ± 2.2 years) were diagnosed genetically in our centre. Phenotypic data and responses to rhIGF1 treatment were provided by referring clinicians. Continuous parametric variables were compared using Student t-test or ANOVA. RESULTS: 10/20 (50%) had typical facial features of GHI, 19/20 (95%) from consanguineous families and 18/20 (90%) of Pakistani origin. At diagnosis, mean height SDS: -4.1 ± 0.95, IGF1 SDS: -2.8 ± 1.4; IGFBP3 SDS: -3.0 ± 2.1 and mean basal and peak GH levels: 11.9 µg/L and 32.9 µg/L, respectively. 1/12 who had IGF1 generation test, responded (IGF1: 132-255 ng/mL). 15/20 (75%; 11M) received rhIGF1 (mean dose: 114 µg/kg twice daily, mean duration: 5.3 ± 2.5 years). Mean baseline height velocity of 4.7 ± 1.1 cm/year increased to 7.4 ± 1.8 cm/year (P = 0.001) during year 1 of therapy. Year 3 mean height SDS (-3.2 ± 1.0) was higher than pre-treatment height SDS (-4.3 ± 0.8) (P = 0.03). Mean cumulative increase in height SDS after year 5 was 1.4 ± 0.9. Difference between target height (TH) SDS and adult or latest height SDS was less than that of TH SDS and pre-treatment height SDS (2.1 ± 1.2 vs 3.0 ± 0.8; P = 0.02). CONCLUSION: In addition to phenotypic heterogeneity in the cohort, there was mismatch between clinical and biochemical features in individual patients with 6Ψ GHR mutations. rhIGF1 treatment improved height outcomes.