Full text
| |
| Author(s): |
Simone Caixeta de Andrade
Total Authors: 1
|
| Document type: | Master's Dissertation |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas |
| Defense date: | 2006-02-21 |
| Examining board members: |
Carmen Sílvia Bertuzzo;
Omar Magid Hauache
|
| Advisor: | Lilia F. R. de Souza Li |
| Abstract | |
The CASR belongs to family C of the G protein coupled receptors and it is activated by the interaction with extracellular calcium, which is responsible for adjusting extracellular calcium set point adjusting PTH and calcium excretion. Calcium Sensing Receptor mutations are related to Familial Hypocalciuric Hypercalcemia (FHH) and Neonatal Severe Hyperparathyroidism (NSHPT) when inactivating and to Autosomal Dominant Hypocalcemia (ADH) when activating. Neonatal Severe Hyperparathyroidism (NSHPT) is a rare disease characterized by hypercalcemia, calcium levels close to those incompatible with life, markedly elevated PTH levels, severe bone demineralization and neonatal symptoms as hypotonia and poor weight gain. Familial Hypocalciuric Hypercalcemia is a familial disease with Autosomal dominant inheritance, in which parents are usually affected, generally asymptomatic, mild ¿ to ¿ moderate hypercalcemia and normal PTH levels (but not suppressed) and hypocalciuria. In ADH, affected individuals¿ present hypocalcemia, PTH at the lower limit or normal range, hyperphosphatemia and hypercalciuria. The objective of this work was study of two families (S and J) with Neonatal Severe Hyperparathyroidism and Familial Hypocalciuric Hypercalcemia, search for new mutations and analyze the expression pattern of mutated receptors. Three new missense mutations were found: c.1913.G>T, c.2.T>G and c.2244.C>G. The mutation c.1913.G>T was identified at family S. and resulted in Arginine to Leucine change at codon 638. The silent mutation c.2244.C>G didn¿t change the amino acid Proline at codon 748. A novel mutation in exon 2, T to G transition at nucleotide 2, changing Metionine to Arginine was identified at family M. The mutation disrupts the original Kozak sequence (AXXATGG), altering the protein start site. Computational analysis using a program that predicts start sites showed that the putative new translation start site was in the exon 3 in frame. A portion of the gene containing the mutation and five cryptic Kozak sequences (-226 to 66) was used to analyze the expression of the mutant receptor (p.M1?). To analyze the expression pattern of Family S, the mutated cDNAs was inserted in a vector, using site direct mutagenesis. Western blot was performed to analyze the expression analysis of the mutated receptors. The p.R638L receptor showed similar expression pattern compared with the wild type receptor, presenting the monomeric forms of 140 (immature, partial glycosylated) and 160kDa (mature, glycosylated) and other forms higher than 200kDa. The mutation c.2244.C>G showed similar expression pattern compared with the wild type receptor. In contrast, Western blot expression levels of the mutant receptor p.M1? was dramatically reduced. Immunocytochemistry experiments showed strong staining at the cell surface of nonpermeabilized and permeabilized HEK293 cells expressing the wild type receptor. The same pattern was observed for the mutant receptor p.R638L, suggesting correct maturation and trafficking. While the mutant receptor p.M1? was not expressed on the cell surface and the staining was only identified inside permeabilized cells, suggesting that the mutant receptor was trapped within the endoplasmatic reticulum and was not expressed at the plasmatic membrane (AU) | |