RESUMO
CONTEXT: Mutations within the PROP1 gene represent one of the main causes of familial combined pituitary hormone deficiency (CPHD). However, most of the cases are sporadic with an unknown genetic cause. OBJECTIVE: The aim of this study was the search for low penetrance variations within and around a conserved regulatory element in the intron 1 of PROP1, contributing to a multifactorial form of the disease in sporadic patients. METHODS AND PATIENTS: A fragment of 570 bp encompassing the conserved region was sequenced in 107 CPHD patients and 294 controls, and an association study was performed with the four identified variants, namely c.109+435G>A (rs73346254), c.109+463C>T (rs4498267), c.109+768C>G (rs4431364), and c.109+915_917ins/delTAG (rs148607624). The functional role of the associated polymorphisms was evaluated by luciferase reporter gene expression analyses and EMSA. RESULTS: A statistically significant increased frequency was observed in the patients for rs73346254A (P = 5 × 10(-4)) and rs148607624delTAG (P = 0.01) alleles. Among all the possible allele combinations, only the haplotype bearing both risk alleles showed a significantly higher frequency in the patients vs. controls (P = 4.7 × 10(-4)) and conferred a carrier risk of 4.19 (P = 1.2 × 10(-4)). This haplotype determined a significant decrease of the luciferase activity in comparison with a basal promoter and the other allelic combinations in GH4C and MCF7 cells (P = 4.6 × 10(-6); P = 5.5 × 10(-4), respectively). The EMSA showed a differential affinity for nuclear proteins for the alternative alleles of the two associated variations. CONCLUSIONS: Variations with a functional significance conferring susceptibility to CPHD have been identified in the PROP1 gene, indicating a multifactorial origin of this disorder in sporadic cases.
Assuntos
Proteínas de Homeodomínio/genética , Hormônio do Crescimento Humano/deficiência , Adolescente , Idade de Início , Células Cultivadas , Criança , Pré-Escolar , Sequência Conservada , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Variação Genética , Vetores Genéticos , Hormônios/sangue , Humanos , Hipotálamo/patologia , Lactente , Fator de Crescimento Insulin-Like I/deficiência , Íntrons/genética , Luciferases/genética , Imageamento por Ressonância Magnética , Masculino , Mutação/genética , Mutação/fisiologia , Penetrância , Hipófise/patologia , Hormônios Hipofisários/sangue , Polimorfismo de Nucleotídeo Único/genética , Transfecção , Adulto JovemRESUMO
Matrix metalloproteinase 8 (MMP-8) has been reported to have a key role in several pathologic conditions, like heart diseases, osteoarthritis, multiple sclerosis, and various other inflammatory conditions. Therefore, there is a great interest regarding the development of MMP-8 selective inhibitors. In the recent years, immobilized enzyme reactors (IMERs) proved to be an efficient alternative to solution-based assays. Besides the recycling of the enzyme, IMER approach allows a simple way to determine affinity data and thus the ranking of inhibiting potency of the compounds under study, especially when coupled to MS. In this study, the immobilization of MMP-8 was investigated in terms of type of support, kinetic parameters, storage and pH stability. Epoxy activated silica resulted the best matrix for the preparation of an immobilized enzyme reactor (IMER) containing human MMP-8. The IMER was successfully used for the online screening of known MMP-8 inhibitors in zonal chromatography and inhibition experiments.