Objective: GH insensitivity (GHI) is caused in the majority of cases by impaired function of the GH receptor (GHR). All but one knownGHRmutation are in the coding sequence or the exon/intron boundaries. We identified and characterised the first intronic defect occurring in the polypyrimidine tract of theGHRin a patient with severe GHI.Design: We investigated the effect of the novel defect on mRNA splicing using anin vitrosplicing assay and a cell transfection system.Methods: GHRwas analysed by direct sequencing. To assess the effect of the novel defect, two heterologous minigenes (wild-type and mutant L1-GHR8-L2) were generated by insertingGHRexon 8 and its flanking wild-type or mutant intronic sequences into a well-characterised splicing reporter (Adml-par L1–L2).³²P-labelled pre-mRNA was generated from the two constructs and incubated in HeLa nuclear extracts or HEK293 cells.Results: Sequencing of theGHRrevealed a novel homozygous defect in the polypyrimidine tract of intron 7 (IVS7-6T>A). This base change does not involve the highly conserved splice site sequences, and is not predictedin silicoto affect GHR mRNA splicing. Nevertheless, skipping of exon 8 from the mutant L1-GHR8-L2 mRNA was clearly demonstrated in thein vitrosplicing assay and in transfected HEK293 cells.Conclusion: Disruption of theGHRpolypyrimidine tract causes aberrant mRNA splicing leading to a mutant GHR protein. This is predicted to lack its transmembrane and intracellular domains and, thus, be incapable of transducing a GH signal.

Context: The GH receptor (GHR) pseudoexon 6Ψ defect is a frequent cause of GH insensitivity (GHI) resulting from a non-functioning GH receptor (GHR). It results in a broad range of phenotypes and may also be present in patients diagnosed as idiopathic short stature. Objective: Our objective was to correct aberrant GHR splicing and inclusion of 6Ψ using exon-skipping antisense oligonucleotides (ASOs). Design and Setting: Three ASOs binding the 5′ (ASO-5), 3′ (ASO-3), and branch site (ASO-Br) of 6Ψ were tested in an in vitro splicing assay and a cell transfection system. The wild-type (wt) and mutant (mt) DNA minigenes (wt- and mtL1-GHR6Ψ-L2, respectively) were created by inserting the GHR 6Ψ in a well-characterized splice reporter (Adml-par). For the in vitro splicing assay, the wt- and mtL1-GHR6Ψ-L2 were transcribed into pre-mRNA in the presence of [α³²P]GTP and incubated with ASOs in HeLa nuclear extracts. For the cell transfection studies, wt- and mtL1-GHR6Ψ-L2 cloned into pcDNA 3.1 were transfected with ASOs into HEK293 cells. After 48 h, RNA was extracted and radiolabeled RT-PCR products quantified. Results: ASO-3 induced an almost complete pseudoexon skipping in vitro and in HEK293 cells. This effect was dose dependent and maximal at 125–250 nm. ASO-5 produced modest pseudoexon skipping, whereas ASO-Br had no effect. Targeting of two splice elements simultaneously was less effective than targeting one. ASO-Br was tested on the wtL1-GHR6Ψ-L2 and did not act as an enhancer of 6Ψ inclusion. Conclusions: The exon-skipping ASO approach was effective in correcting aberrant GHR splicing and may be a promising therapeutic tool.

Background: Timing of puberty is associated with height, cardiovascular health and cancer risk, with a significant public health impact. Previous studies estimate that 60–80% of variation in the timing of pubertal onset is genetically determined. Self-limited delayed puberty (DP) segregates in an autosomal dominant pattern, but the underlying genetic background is unknown. Methods: We performed whole exome sequencing in 111 members of 18 families from our patient cohort with self-limited DP, with follow-up targeted re-sequencing of candidate genes in a further 42 families. For one candidate gene we defined tissue expression in human and mouse embryos. The effects of gene knockdown were investigated via neuronal migration assays, and in vivo using a transgenic zebrafish model with fluorescently-labelled GnRH neurons. N-terminal fragment mutations were interrogated via expression in mammalian cells. Results: We identified four rare heterozygous variants in IGSF10 in 29 members of ten unrelated families. All four variants were in evolutionarily conserved positions and were predicted by in silico analysis to have a deleterious effect on protein function. Statistical tests showed a significant difference in the prevalence of these mutations within DP cases compared to a general population (P=4.46×10⁻³), and a significant association between these mutations and the delayed puberty trait within our cohort (P=3.47×10⁻⁴). IGSF10 mRNA shows strong expression in the nasal mesenchyme in mouse and human embryos, during the time-period when GnRH neurons migrate from their nasal origin towards the hypothalamus. IGSF10 knockdown caused reduced migration of immature GnRH neurons in the in vitro analysis, and perturbed migration and extension of GnRH neurons in the transgenic zebrafish model. Reduced secretion of mutant protein was demonstrated by western blotting. Conclusions: We present our novel finding that IGSF10 mutations contribute to the phenotype of self-limited delayed puberty in humans, through impairment of migration of GnRH neurons during embryonic development.

Background: Abnormal pubertal timing affects over 4% of adolescents and is associated with adverse health and psychosocial outcomes. Previous studies estimate that 60–80% of variation in the timing of pubertal onset is genetically determined. However, despite this strong heritability, little is known about the genetic control of human puberty. Self-limited delayed puberty (DP) segregates in an autosomal dominant pattern, but in the majority of patients the neuroendocrine pathophysiology and its genetic regulation remain unclear. Methods: We performed whole exome sequencing in 52 members of seven families from our patient cohort with DP, with follow-up targeted re-sequencing of candidate genes in a further 42 families. The functional consequences of the identified mutations in one candidate gene were interrogated via expression of WT and mutant proteins in mammalian cells. For this gene we defined tissue expression in human and mouse embryos by in situ hybridization and immunohistochemistry. The effects of gene knockdown were investigated via in vitro neuronal migration assays, and in vivo using a transgenic zebrafish model with fluorescently labeled GnRH neurons. Results: We identified four rare mutations in IGSF10 in ten unrelated families, which are tightly associated with the DP trait within our cohort (P=3.47×10⁻⁴). The identified mutations are in evolutionarily conserved positions, and two mutations result in intracellular retention with failure in secretion of the N-terminal fragment of the protein. IGSF10 mRNA is strongly expressed in the nasal mesenchyme in mouse and human embryos, during the time-period when GnRH neurons migrate from their nasal origin towards the hypothalamus. IGSF10 knockdown caused reduced migration of immature GnRH neurons in the in vitro analysis, and perturbed migration and extension of GnRH neurons in the zebrafish model. Conclusions: We present our novel finding that mutations in IGSF10 cause delayed puberty in humans, through misregulation of GnRH neuronal migration during embryonic development.