Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Growth Hormone Receptor (GHR) 6Ω Pseudoexon Activation: A Novel Cause of Severe Growth Hormone Insensitivity

doi: 10.1210/clinem/dgab550

Figure Lengend Snippet: Expression of wild-type (WT) and mutant transcripts in affected family members with the heterozygous c.618+836 T > G growth hormone receptor ( GHR ) 6Ω variant. The GHR 6Ω pseudoexon also diminishes GH-dependent STAT5B activation and accumulates extracellularly. A, Complementary DNA (cDNA) was prepared from dermal fibroblasts derived from a healthy control (HC) and patients 2 (P2) and P3 and both parents. Schematic showing the locations of the primers ( GHR cDNA exon 4F [forward] and GHR cDNA exon 8R [reverse] [blue arrows]) used to amplify the region encompassing WT GHR , the GHR 6Ω pseudoexon insertion. A “normal” 705–base pair (bp) polymerase chain reaction (PCR) product was seen in all samples. An additional larger (856-bp) PCR product was seen in P2, P3, and their mother, who were heterozygous for the c.618+836 T > G GHR 6Ω variant, indicating the additional 151-bp 6Ω pseudoexon insertion. B, Schematic showing the locations of the primers ( GHR cDNA pseudo F1 [forward] and GHR cDNA exon 8R [reverse] [blue arrows]). The forward primer at the junction of the 6Ω pseudoexon insertion means only sequences containing the GHR 6Ω pseudoexon insertion are amplified. The expected 387-bp PCR product is seen in patient 2, patient 3, and their mother, all of whom are heterozygous for the c.618+836 T > G GHR variant. C, Whole-cell lysates from untreated or GH-stimulated (20 minutes) HEK293 cells transfected with pcDNA3.1 empty vector, WT GHR or 6Ω GHR mutant constructs. Representative immunoblots of 3 experiments are shown. D, Immunoblot analysis of conditioned media with anti-GH binding protein (BP) antibody from HEK293 cells transfected with the 6Ω GHR mutant construct showing extracellular accumulation of the truncated mutant 6Ω GHR protein. B actin, β actin; K2M, kindred 2 mother; K2F kindred 2 father.

Article Snippet: Primary dermal fibroblasts of normal human neonatal origin, (ATCC PCS201010) were used as controls.

Techniques: Expressing, Mutagenesis, Variant Assay, Activation Assay, Derivative Assay, Control, Polymerase Chain Reaction, Amplification, Transfection, Plasmid Preparation, Construct, Western Blot, Binding Assay