Identification of genetic mutations in patients with familial central diabetes insipidus

• Autor(a) principal: Francisco, Ângela Sofia Fernandes Alves
• Data de Publicação: 2012
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/3266
• topic: Diabetes insípida - Mutação AVP (Arginine vasopressin); Diabetes insípida - Gene AVP (Arginine vasopressin)

Diabetes insipidus (DI) is associated with defects that involve the secretion and the action of hormone arginine vasopressin (AVP) resulting in the excretion of abnormally large volumes of diluted urine. The most common defect that results in disease development is the deficient secretion of the hormone AVP and the disease is referred to as central or neurohypophyseal DI. The AVP hormone is synthesized in magnocellular neurons, that originate in the supraoptic and paraventricular nuclei of the hypothalamus and are projected to neurohypophysis, and the destruction of these neurons leads to a deficiency of AVP hormone, resulting in neurohypophyseal DI. The familial form of disease represents 1% of all causes of neurohypophyseal DI and the main points of the disease are: it is associated with mutations in one allele of the AVP gene, and it is caused by postnatal development of deficient AVP secretion, proposed to result from selective degeneration of the magnocellular neurons. The aims of this thesis are: to review AVP mutations described in the scientific literature, to expand the spectrum of mutations through the analysis of additional patients with DI and to characterize the functional consequences of identified novel AVP mutations. To achieve these aims a bibliographic research was developed; genetic studies were performed to amplify and to sequence the three exons of the AVP gene in 9 patients; an expression vector containing the desired mutations was constructed by subcloning, site-directed mutagenesis and enzymatic digestion; and the functional studies were initialized by optimization of transfection and immunocytochemistry assays for WT AVP cDNA expression vector. Three mutations were identified: c.154T>C, c.289C>G and c.343G>T. The first two mutations are novel and the last mutation is already described in the scientific literature. The AVP cDNA from the expression vector was subcloned in the pVAX/lacZ vector and the mutations were inserted in the AVP cDNA by site-directed mutagenesis and enzymatic digestion. The mutated AVP cDNAs were sequenced and have been prepared to be inserted in the expression vector. The transfection and immunocytochemistry protocols have been optimized for WT AVP cDNA expression vector. This study allowed the increase in the number of mutations from 70 to 72 different mutations, although further work is necessary in order to understand the molecular mechanisms responsible for the development of the disease and to give help and information to patients affected with this disease.