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| Author(s): |
Marina Dorigatti Borges
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas |
| Defense date: | 2018-07-03 |
| Examining board members: |
Kleber Yotsumoto Fertrin;
Fernando Ferreira Costa;
Sara Teresinha Olalla Saad;
Sandra Fatima Menosi Gualandro
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| Advisor: | Kleber Yotsumoto Fertrin |
| Abstract | |
Aceruloplasminemia is an autosomal recessive hereditary disease caused by mutations in the CP gene, which encodes ceruloplasmin. This protein is involved in iron metabolism and abnormal function causes the intracellular accumulation of the metal in organs such as the brain, liver, and pancreas. The diagnosis is made upon the detection of absence of serum ceruloplasmin associated with neuroimaging tests suggestive of iron overload. In Brazil, there are no data on its prevalence or on which mutations are present in the population. This project aimed to characterize the molecular alterations that lead to aceruloplasminemia in Brazilian patients. Three patients and, when possible, first-degree relatives were included, as well as a healthy control and a negative control for glycosylphosphaditylinositol-anchored proteins (paroxysmal nocturnal hemoglobinuria - PNH). Peripheral blood samples were collected for the extraction of genomic DNA from leukocytes. Fragments of interest in the CP gene and in the CPP pseudogene, which shows possible association with the disease, were amplified through polymerase chain reaction (PCR) and sequenced. We also evaluated the expression of ferroportin and ceruloplasmin in peripheral blood monocytes and monocyte-derived primary macrophage. Data from first degree relatives of the patients were included in a systematic review of the clinical and molecular aspects of heterozygous aceruloplasminemia. Two new mutations were found in the CP gene: a replacement of a guanine by a thymidine at position 2879-1, a splice site mutation (patients 1 and 2), and a switch of a thymidine by a cytosine at position 2756 in exon 16, leading to a leucine substitution by a proline at codon 919, a missense mutation (patient 3). In silico analysis of the proteins showed that there is significant change in the predicted tertiary structure of the proteins due to these mutations. Immunophenotyping revealed that the protein expressions observed in macrophages were like those seen in monocytes. Both proteins were detected in peripheral blood monocytes and in B lymphocytes. The expressions found in B lymphocytes were similar across all individuals studied, but the expression in monocytes varied. The CP c.2879-1 G>T mutation presented with ceruloplasmin-GPI and ferroportin expressions like those found in a healthy control, whereas the CP c.2756 T>C mutation led to a considerable reduction of both proteins, a profile similar to what was found in PNH. These results support that ceruloplasmin-GPI stabilizes ferroportin in the cell membrane and shows that the different isoforms of the protein are expressed independently across different cell lineages. As for first-degree relatives, patient 1's sister and patient 3's son were confirmed to be heterozygotes for the mutation present in the family. They had low but still detectable levels of serum ceruloplasmin. We performed a systematic review of the scientific literature that showed that all described heterozygotes present reduced ceruloplasmin, but only part of them manifested neurological symptoms. This work pioneers in the characterization of aceruloplasminemia in the Brazilian population, its genetic variability, and heterogeneity of its clinical expression, as well as it contributes to expand the knowledge on the expression and importance of ceruloplasmin at the cellular level (AU) | |
| FAPESP's process: | 16/08072-9 - Characterization of molecular changes in patients with aceruloplasminemia |
| Grantee: | Marina Dorigatti Borges |
| Support Opportunities: | Scholarships in Brazil - Master |