Loading...
Publication
Hemocromatose Hereditária Não-Clássica - pesquisa de mutações em genes relacionados com o metabolismo do ferro e estudos funcionais de uma nova variante da proteína hemojuvelina
| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 2.18 MB | Adobe PDF |
Authors
Advisor(s)
Abstract(s)
[PT] O ferro é um dos micronutrientes mais importantes a nível biológico, participando
em diversos processos biológicos. Por outro lado, o seu excesso ou défice no
organismo estão associados a consequências negativas para a saúde. A
Hemocromatose Hereditária (HH) é um distúrbio da regulação da homeostase do ferro
caracterizado por uma elevada absorção intestinal de ferro levando à sua acumulação
em diversos órgãos. As consequências mais frequentes desta doença são o
desenvolvimento de cirrose hepática, carcinoma hepatocelular, cardiomiopatias,
artralgias, hiperpigmentação da pele e problemas endócrinos. O gene HFE é o
principal gene associado a esta patologia (HH-clássica ou do tipo I). Os genótipos
mais frequentes são a homozigotia para a alteração p.C282Y e a heterozigotia
composta p.C282Y/p.H63D. No entanto, existem outros genes envolvidos no
desenvolvimento de hemocromatose hereditária não clássica. Entre eles, encontra-se
o gene TfR2 que codifica o recetor 2 da transferrina, estando associado à
hemocromatose hereditária do tipo III e o gene SLC40A1 codificante da ferroportina e
associado à hemocromatose hereditária do tipo IV. A forma mais grave de HH é, no
entanto, a hemocromatose juvenil (HJ) ou tipo II associada a alterações nos genes que
codificam a hemojuvelina (HJV) ou a hepcidina (HAMP).
Um dos objetivos do trabalho subjacente a esta dissertação consistiu na
implementação no laboratório de uma metodologia de pesquisa rápida, pouco
dispendiosa e em larga escala, de alterações em genes relacionados com o
metabolismo do ferro. Deste modo, foi possível realizar a identificação e caraterização
de mutações em 6 genes relacionados com o metabolismo do ferro (HFE, HJV, HAMP,
TfR2, SLC40A1 e FTL) em 88 doentes com sobrecarga em ferro (ferritina sérica
superior a 200 ng/mL e saturação de transferrina superior a 60%), e diagnóstico
negativo para Hemocromatose Hereditária do tipo I. Os DNAs foram preparados
utilizando a metodologia Treu Seq Custum Amplicon e sequenciados recorrendo à
tecnologia de Next Generation Sequencing, illumina.
Através desta metodologia foram detetadas 1242 alterações correspondentes a 55
variantes genéticas diferentes, 16 das quais não estão descritas nas bases de dados
disponíveis. Dessas 5 são potencialmente patogénicas (3 são missense HFE,
p.Y230C, TfR2, p.L750V e TfR2, p.A777V; 1 localiza-se numa região de splicing no
gene TfR2 (c.967-1 G>C) e 1 localiza-se na região 5’-UTR do gene HAMP (-25 G>A).
iii
Foram realizados estudos in silico, recorrendo à ferramenta bioinformática
PolyPhen-2, de modo a identificar possíveis efeitos que estas mutações poderão ter
na estrutura das proteínas resultantes. Para além disso, também se procedeu à
análise do impacto da alteração nucleotídica a nível do splicing utilizando o software
Human Splicing Finder.
Dado o tipo de transmissão autossómica recessiva da hemocromatose hereditária,
foi possível, através da metodologia utilizada, justificar a sobrecarga em ferro
(estabelecer uma associação genótipo/fenótipo) em 5 dos indivíduos em estudo.
O outro objetivo deste trabalho foi dar início ao estudo funcional in vitro da
variante p.P124L da proteína HJV em células eucarióticas. Para tal procedeu-se à
clonagem do cDNA wild-type HJV no vetor de expressão pEGFP-N1, originando uma
proteína de fusão constituída por HJV e GFP (Green Fluorescent Protein).
Posteriormente, e recorrendo a microscopia confocal e western-blot será analisada a
sua distribuição intracelular na linha celular HeLa transfetada e o seu efeito no
processamento da HJV.
[ENG] Iron is one of the most important as it, participates in many relevant biological processes. On the other hand, its surplus or deficits in the body are associated with negative consequences for health. The Hereditary Hemochromatosis (HH) is a disorder of iron homeostasis regulation characterized by high intestinal absorption of iron leading to its accumulation in various organs. The main consequences of this disease are the development of liver cirrhosis, hepatocellular carcinoma, cardiomyopathies, arthralgia, skin hyperpigmentation and endocrine problems. The HFE gene is the main gene associated with this disease (HH-classical or type I). The most common genotypes are homozygous for p.C282Y change and compound heterozygosity for p.C282Y / p.H63D. Nevertheless, there are other genes involved in the development of non-classical hereditary hemochromatosis. Among them is the TfR2 gene encoding the transferrin receptor 2, is associated with hereditary hemochromatosis type III and SLC40A1 gene encoding ferroportin and associated with hereditary hemochromatosis type IV. The most severe form of HH is, however, juvenile hemochromatosis (HJ) or type II associated with changes in the genes encoding the hemojuvelin (HJV) and hepcidin (HAMP). One goal of the work underlying this thesis was the implementation in the laboratory of a methodology for quick search, less expensive and large-scale changes in genes related to iron metabolism. Thus, it was possible to realize the identification and characterization of mutations in six genes related to iron metabolism (HFE, HJV, HAMP, TfR2, SLC40A1 and FTL) in 88 patients presenting iron overload (serum ferritin higher than 200 ng/ml and transferrin saturation above 60%), and a negative diagnosis of type I, Hereditary Hemochromatosis. DNAs were prepared using the methodology Treu Custum Sequence Amplicon and sequenced using the Next Generation Sequencing technology, illumina. Through this methodology 1242 changes were detected corresponding to 55 different genetic variants, 16 of which are not yet described in the available databases. Out of the 16 observed variants 5 are potentially pathogenic (3 are missense HFE, p.Y230C, TFR2, p.L750V and TFR2, p.A777V; one is located in a splicing region of TFR2 gene (c.967-1 G> C) and one located in the 5'-UTR region of the gene HAMP (- 25 G> A). In silico studies were performed using the bioinformatics tool PolyPhen-2, in order to identify possible effects that these variations can have upon the protein v structure. In addition, we also examined the impact of nucleotide change the splicing level using the Human Splicing Finder software. Due to autosomal recessive inheritance of hereditary hemochromatosis, it was possible, through the methodology used, justify the iron overload (establishing an association genotype / phenotype) in 5 of the study subjects. The other goal of this work was to start the in vitro characterization of the HJV variant p.P124Lin eukaryotic cells. Accordingly, we have cloned the wild-type HJV cDNA into the pEGFP-N1 expression vector, thus originating a recombinant protein formed by HJV and GFP (Green Fluorescent Protein). Later on, and using confocal microscopy and western blot analyses, its intracellular distribution and effect upon HJV processing will be evaluated.
[ENG] Iron is one of the most important as it, participates in many relevant biological processes. On the other hand, its surplus or deficits in the body are associated with negative consequences for health. The Hereditary Hemochromatosis (HH) is a disorder of iron homeostasis regulation characterized by high intestinal absorption of iron leading to its accumulation in various organs. The main consequences of this disease are the development of liver cirrhosis, hepatocellular carcinoma, cardiomyopathies, arthralgia, skin hyperpigmentation and endocrine problems. The HFE gene is the main gene associated with this disease (HH-classical or type I). The most common genotypes are homozygous for p.C282Y change and compound heterozygosity for p.C282Y / p.H63D. Nevertheless, there are other genes involved in the development of non-classical hereditary hemochromatosis. Among them is the TfR2 gene encoding the transferrin receptor 2, is associated with hereditary hemochromatosis type III and SLC40A1 gene encoding ferroportin and associated with hereditary hemochromatosis type IV. The most severe form of HH is, however, juvenile hemochromatosis (HJ) or type II associated with changes in the genes encoding the hemojuvelin (HJV) and hepcidin (HAMP). One goal of the work underlying this thesis was the implementation in the laboratory of a methodology for quick search, less expensive and large-scale changes in genes related to iron metabolism. Thus, it was possible to realize the identification and characterization of mutations in six genes related to iron metabolism (HFE, HJV, HAMP, TfR2, SLC40A1 and FTL) in 88 patients presenting iron overload (serum ferritin higher than 200 ng/ml and transferrin saturation above 60%), and a negative diagnosis of type I, Hereditary Hemochromatosis. DNAs were prepared using the methodology Treu Custum Sequence Amplicon and sequenced using the Next Generation Sequencing technology, illumina. Through this methodology 1242 changes were detected corresponding to 55 different genetic variants, 16 of which are not yet described in the available databases. Out of the 16 observed variants 5 are potentially pathogenic (3 are missense HFE, p.Y230C, TFR2, p.L750V and TFR2, p.A777V; one is located in a splicing region of TFR2 gene (c.967-1 G> C) and one located in the 5'-UTR region of the gene HAMP (- 25 G> A). In silico studies were performed using the bioinformatics tool PolyPhen-2, in order to identify possible effects that these variations can have upon the protein v structure. In addition, we also examined the impact of nucleotide change the splicing level using the Human Splicing Finder software. Due to autosomal recessive inheritance of hereditary hemochromatosis, it was possible, through the methodology used, justify the iron overload (establishing an association genotype / phenotype) in 5 of the study subjects. The other goal of this work was to start the in vitro characterization of the HJV variant p.P124Lin eukaryotic cells. Accordingly, we have cloned the wild-type HJV cDNA into the pEGFP-N1 expression vector, thus originating a recombinant protein formed by HJV and GFP (Green Fluorescent Protein). Later on, and using confocal microscopy and western blot analyses, its intracellular distribution and effect upon HJV processing will be evaluated.
Description
Paula Faustino: Grupo de Investigação em Hemoglobinopatias, Metabolismo do Ferro e
Patologias Associadas (Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge)
Keywords
Hemocromatose Hereditária Ferro Sobrecarga em Ferro Metabolismo do Ferro Hemojuvelina HFE HJV HAMP TfR2 SLC40A1 FTL Next Generation Sequencing p.P124L Doenças Genéticas Iron Hereditary Hemochromatosis Iron Overload