Endocrine Abstracts

JOINT100

Human growth hormone (hGH), or somatotropin, a 217-amino acid glycoprotein produced by the somatotropic cells in the anterior pituitary, is encoded by the GH1 gene, located on chromosome 17 (17q22-24). Mutations in GH1 are associated with Isolated Growth Hormone Deficiency (IGHD), characterized by a spectrum of pituitary hormone deficiencies and growth failure in children, and it can result from mutations in GH1 or related genes. IGHD is classified into four types based on inheritance patterns: Type IA and IB (autosomal recessive) Type II (Autosomal dominant), and Type III (X-linked), all of which result in short stature. We have investigated five functionally uncharacterized but clinically reported GH1 missense variants (A39T, R42L, C79G, Q110E, and R160W), identified in patients with IGHD of various types and genetic background s. Pathogenicity analysis tools such as PANTHER, PhD-SNP, SIFT, Meta-SNP, and E-SNPs & GO were utilized to assess the pathogenicity scores for each mutation, highlighting C79G and R42L as the most deleterious mutations. Among the mutations, Q110E is predicted to have the most significant stabilizing effect. Protein free energy ΔΔG calculations (1. 882 kcal/mol) suggested that it enhances protein stability, reducing the likelihood of denaturation or unfolding. Conversely, A39T appears to significantly decrease protein flexibility compared to the other five mutations, as indicated by ΔΔSVib (-4. 925 kcal/mol*K) predictions, which suggests that A39T induces a more rigid and ordered protein structure. Additionally, multiple sequence alignment and phylogenetic analysis with Clustal Omega, phylogenetic analysis with MEGA, domain prediction with SMART, 3D structures modeling, and motif analysis with MEME, confirmed high conservation these residues among primates, underscoring their importance. To complement computational insights, the variants were introduced into the wild-type GH1 gene via site-directed mutagenesis and expressed in E. coli. Recombinant proteins were purified using immobilized metal affinity chromatography (IMAC), yielding high-quality preparations suitable for downstream functional studies. These findings provide a detailed computational and experimental framework for understanding GH1 variant effects, offering a foundation for future In vitro studies aimed at advancing personalized approaches to IGHD diagnosis and treatment.