Loading...
Publication
Estudo da mutação W402X: frequência desta variante da α-L-iduronidase lisossomal na população portuguesa e análise da sua função
| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 2.49 MB | Adobe PDF |
Abstract(s)
As Doenças Lisossomais de Sobrecarga (DLSs) são um grupo de doenças metabólicas
hereditárias raras. Na população portuguesa, as DLSs têm uma prevalência de aproximadamente 1/4000
nados vivos. A Mucopolissacaridose tipo I (MPS I) é uma doença autossómica recessiva, que resulta da
actividade deficiente da enzima lisossomal α-L-iduronidase (IDUA), e está presente na população
portuguesa com uma prevalência de 1.33/100000 nados vivos. Apesar da existência de mais de 100
mutações causais, a mutação W402X é a causa genética mais frequente; a homozigotia para a mutação
W402X está associada ao fenótipo clínico mais grave (síndrome de Hurler, MPS IH). Assim, a relevância
do estudo desta mutação é evidenciada pelo facto de se tratar de uma mutação pan-europeia relacionada
com um fenótipo grave. Tem, inclusivamente, sido sugerido que esta mutação pode ser uma chave
essencial para a compreensão da patofisiologia da MPS IH.
Surgiram três questões principais que justificaram uma investigação mais aprofundada: qual
seria a frequência da mutação W402X na população portuguesa? Existiria um haplótipo comum nos
alelos mutados detectados? Seria possível melhorar o fenótipo e, talvez, recuperar parcialmente a função
deste mutante, utilizando mecanismos de supressão nonsense?
Neste trabalho determinou-se que a frequência alélica da mutação W402X na população
portuguesa em geral é de 0,20% (IC: 0,08-0,39), utilizando duas técnicas de genotipagem distintas, PCRRFLP
e PCR-dHPLC. Em termos de haplótipos foi encontrada variabilidade nos alelos mutados
detectados, o que parece indicar uma grande divergência entre os alelos W402X. Aparentemente o SNP
mais frequentemente associado à mutação W402X em portugueses e brasileiros é o Q33H, seria
interessante saber se na população irlandesa esta associação também se verifica.
Adicionalmente, ao caracterizar funcionalmente a mutação W402X do gene IDUA, comprovou-se
que, não só, esta mutação resulta em nonsense mediated decay, mas também que é susceptível a
mecanismos de supressão de nonsense com dois compostos distintos, a gentamicina e a cicloheximida
(CHX). Face aos resultados obtidos com os diferentes tratamentos, o efeito de supressão da CHX na
mutação nonsense W402X do gene IDUA provou ser o mais eficaz. Verificou-se que o aumento de
expressão do gene IDUA na linha celular de um doente MPS IH homozigótico para a mutação W402X
permitiu atingir níveis semelhantes aos da linha celular controlo não sujeita a tratamento.
Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic disorders. In the Portuguese population, LSDs have a prevalence of ≈1/4000 live births. Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder, which results from the defective activity of the lysosomal α-Liduronidase (IDUA), and is present in the Portuguese population at 1.33/100000 live births. Despite the existence of over 100 causal mutations, W402X is the most frequent genetic cause; homozygosity for W402X is associated to the most severe clinical phenotype (Hurler syndrome; MPS IH). Thus, the relevance of studying this mutation is evidenced by the fact that it is pan-european and related to a severe phenotype. It has, inclusively, been suggested that this mutation may hold an essential role for the comprehension of the MPS IH pathofisiology. Three major questions arose and were worth further investigation: what was the frequency of mutation W402X in the Portuguese population? Was there a common haplotype in the mutated alleles? Was it possible to ameliorate the phenotype and, perhaps, partially recover this mutant’s function using nonsense suppression mechanisms? In this work it was determined that the allelic frequency of mutation W402X in the Portuguese population in general is 0.20% (IC: 0.08-0.39) using two different techniques of genotyping, PCR-RFLP and PCR-dHPLC. In terms of haplotype, variability was found among the mutated alleles, which seems to indicate a wide divergence between alleles W402X. Apparently the SNP most frequently associated with mutation W402X in the Portuguese and Brazilian is the Q33H, it would be interesting to know if this association is also observed in the Irish population. In addition, by functionally characterizing mutation W402X of the IDUA gene, it turns out that not only does this mutation result in nonsense mediated decay, but it is also susceptible to suppression of nonsense with two different compounds, gentamicin and cycloheximide (CHX). Given the results obtained with different treatments, the effect of CHX on suppression of the nonsense mutation W402X of the IDUA gene proved to be the most efficient. It was found that the increase of the IDUA gene expression in a W402X homozygous cell line attained levels similar to those of the control cell line without treatment.
Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic disorders. In the Portuguese population, LSDs have a prevalence of ≈1/4000 live births. Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder, which results from the defective activity of the lysosomal α-Liduronidase (IDUA), and is present in the Portuguese population at 1.33/100000 live births. Despite the existence of over 100 causal mutations, W402X is the most frequent genetic cause; homozygosity for W402X is associated to the most severe clinical phenotype (Hurler syndrome; MPS IH). Thus, the relevance of studying this mutation is evidenced by the fact that it is pan-european and related to a severe phenotype. It has, inclusively, been suggested that this mutation may hold an essential role for the comprehension of the MPS IH pathofisiology. Three major questions arose and were worth further investigation: what was the frequency of mutation W402X in the Portuguese population? Was there a common haplotype in the mutated alleles? Was it possible to ameliorate the phenotype and, perhaps, partially recover this mutant’s function using nonsense suppression mechanisms? In this work it was determined that the allelic frequency of mutation W402X in the Portuguese population in general is 0.20% (IC: 0.08-0.39) using two different techniques of genotyping, PCR-RFLP and PCR-dHPLC. In terms of haplotype, variability was found among the mutated alleles, which seems to indicate a wide divergence between alleles W402X. Apparently the SNP most frequently associated with mutation W402X in the Portuguese and Brazilian is the Q33H, it would be interesting to know if this association is also observed in the Irish population. In addition, by functionally characterizing mutation W402X of the IDUA gene, it turns out that not only does this mutation result in nonsense mediated decay, but it is also susceptible to suppression of nonsense with two different compounds, gentamicin and cycloheximide (CHX). Given the results obtained with different treatments, the effect of CHX on suppression of the nonsense mutation W402X of the IDUA gene proved to be the most efficient. It was found that the increase of the IDUA gene expression in a W402X homozygous cell line attained levels similar to those of the control cell line without treatment.
Description
Dissertação de mestrado em Ciências da Saúde, apresentada à Escola de Ciências da Saúde da Universidade do Minho, 2012
Dissertação realizada sob a orientação científica da Doutora Olga Amaral, Assistente Principal na UID-P do Departamento de Genética do INSA e da Professora Doutora Patrícia Maciel, da Universidade do Minho. Aprovada em 18 de Abril de 2012.
Trabalho executado na UID-P do Departamento de Genética do INSA.
Dissertação realizada sob a orientação científica da Doutora Olga Amaral, Assistente Principal na UID-P do Departamento de Genética do INSA e da Professora Doutora Patrícia Maciel, da Universidade do Minho. Aprovada em 18 de Abril de 2012.
Trabalho executado na UID-P do Departamento de Genética do INSA.
Keywords
Doenças Lisossomais de Sobrecarga Doenças Genéticas Genética Humana IDUA MPS 1 Portugal Lysosomal Storage Diseases