Abstract
Introduction: The physiological body growth requires a plethora of hormonal, metabolic, and other growth factors involved in the hypothalamo-pituitary-somatotrope axis. Growth hormone (GH), is a polypeptide hormone secreted from the anterior lobe of the pituitary gland and plays a pivotal role in a number of physiological processes by promoting postnatal longitudinal body growth, lipid and carbohydrate metabolism, protein biosynthesis and activation of the immune system. GH deficiency (GHD) is diagnosed either by subnormal levels of serum GH during two hGH stimulation tests by pharmacological agents that physiologically stimulate GH secretion (classic form of GHD), or normal serum GH levels, but subnormal 24hr GH profile (Neurosecretory GH deficiency, GHND). Children with GHD and GHND have severe growth retardation and respond to exogenous human GH (hGH) therapy with significant catch-up growth. Short stature associated with isolated GH deficiency (GHD) is both sporadic and idiopathic, ...
Introduction: The physiological body growth requires a plethora of hormonal, metabolic, and other growth factors involved in the hypothalamo-pituitary-somatotrope axis. Growth hormone (GH), is a polypeptide hormone secreted from the anterior lobe of the pituitary gland and plays a pivotal role in a number of physiological processes by promoting postnatal longitudinal body growth, lipid and carbohydrate metabolism, protein biosynthesis and activation of the immune system. GH deficiency (GHD) is diagnosed either by subnormal levels of serum GH during two hGH stimulation tests by pharmacological agents that physiologically stimulate GH secretion (classic form of GHD), or normal serum GH levels, but subnormal 24hr GH profile (Neurosecretory GH deficiency, GHND). Children with GHD and GHND have severe growth retardation and respond to exogenous human GH (hGH) therapy with significant catch-up growth. Short stature associated with isolated GH deficiency (GHD) is both sporadic and idiopathic, but between 5 and 30% have an affected first degree relative consistent with a genetic etiology. Mutations identified on the GH gene (GH1) associate with the manifestation of familial isolated GH deficiency (IGHD). The GH1 gene, mapped to chromosome 17, is part of a GH gene cluster consisting of four other structural genes and is comprised by 5 exons and 5 introns. The proximal promoter region of GH1 exerts a highly polymorphic region, with at least 16 single nucleotide polymorphisms (SNPs) identified over a region of 535bp, manifested by a total of 40 haplotypes, some of which affect the GH1 expression. Aim: To identify possible changes in the sequence of the GH1 gene and its promoter region in patients with familial GH deficiency (GHD and GHND), that can possibly affect the transcription of GH1 and are involved in the pathogenesis of IGHD, together with the analysis of the inheritance pattern of such genetic changes. Patients and Methods: 33 IGHD patients (29 GHD και 4 GHND), their 1st degree relatives (22 families) and 31 controls were investigated. Genomic DNA was extracted from the lymphocytes of the subjects peripheral blood and the GH gene (GH1) was amplified by the Polymerase Chain Reaction (PCR). The samples were sequenced and the changes were identified according to the sequence M28466.1 of the NCBI blast database. In the patients full clinical history was recorded, along with other clinical characteristics such as bone age, growth velocity before and during hGH therapy, height and BMI SDS, whereas in the controls height and BMI SDS were recorded. Also in the patients, the levels of IGF-1 and other hormones of the pituitary were measured and the highest level of GH during the clonidine and L-Dopa hGH stimulation test were recorded, whereas in the 4 GHND patients with physiological levels of Hgh (>10ng/ml) during the stimulation tests a spontaneous 24hr GH profile was conducted. The sequencing results were analyzed for the frequencies of the genotypes of the identified SNPs and for any possible correlations with the clinical or biochemical characteristics of the patients and the controls, using the statistical program SPSS 20.0. Additionally, the possible functional role of the found mutations was assessed using specific programs. Results: The sequencing of GH1 and its promoter revealed 18 previously identified SNPs in the whole sample and 3 novel mutations in 3 of the 22 investigated families. Analysis with MatInspector of the 2 heterozygous nucleotide mutations located at the promoter regions -485GC and -400GA, respectively, revealed a binding sequence for the transcription factor E-twenty six-1 (ETS-1). These were identified in one GHD patient and its father and one GHND patient and its mother, respectively. The third heterozygous mutation (GA) at the +300 region of intron 1, was identified at the father but not the GHND patient of the family that had similar abnormal facial characteristics. Analysis of this mutation with ESEfinder3 and ASSP revealed a cryptic splicing position and disruption of the exon splicing enhancement (ESE) region by reducing the binding affinity of serine-arginine proteins (SRs). The frequency and correlation analysis showed that the GHD patients possible exert differential transcriptional activity at the GH1 gene via the SNPs at positions -278, -57 of the promoter, -6 of 5΄ UTR region, +1169 of intron 4, which is in accordance to previous studies, and also the newly reported SNP at -31 region. These SNPs associated also with decreased IGF-1 levels, especially SNP -57 that associated with decreased IGF-1 levels in the prepubertal GHD patients. Nevertheless, the -75 SNP did not seem to affect the expression of GH1. On the other hand, the SNPs identified in the GHND patients although similar to the GHD patients, they correlated with several growth parameters, like birth body length and weight, height and head circumference, irrespective to the IGF-1 levels. Conclusions: The 18 SNPs and 3 mutations identified in the sample seem to contribute partially and individually or in synergy to the transcriptional regulation of GH1 in the GHD and GHND patients. The SNPs contribution is differentially exerted in the GHD patients, when compared to the GHND patients and possibly reflects the different expression of the disease. The association of certain SNPs with the clinical and biochemical characteristics of the patients needs further investigation in large sample cohorts, whereas the changes identified in the study with a potential functional role, must be investigated in in vitro functional studies. The investigation of phenotypic variance in IGHD patients and their genetic predisposition can potentially improve our perception of the underlying mechanisms of growth and offer valuable information for the better therapeutic management of IGHD patients. Finally, while focusing on prediction models as well as ‘personalized’ growth-promoting therapy pharmacogenetics, pharmacogenomics and bioinformatics may play a major role in the future.
show more
