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Polimorfismo nos loci ATXN2, CACNA1A e ATN1 : um estudo de potenciais genes modificadores da doença de Machado-Joseph em indivíduos açorianos saudáveis
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INTRODUÇÃO: a doença de Machado-Joseph (MJD) é uma ataxia espinocerebelosa (SCAs) hereditária autossómica dominante, causada por repetições CAG no gene ATXN3, que codificam um tracto de poliglutamina, e conduzem a um ganho de função neurotóxica da proteína correspondente, a ataxina-3, por mecanismos que não estão completamente conhecidos. As repetições CAG, porém, explicam apenas parte da variabilidade fenotípica observada nesta doença, sendo que a restante explicação poderá ser fornecida por variação em genes modificadores. As proteínas com um tracto de poliglutamina expandido têm tendência para formar agregados e, portanto, têm potencial para modular o fenótipo das doenças de expansão. De facto, estudos realizados em modelos animais e em doentes mostraram a existência de interacções entre as proteínas nativas codificadas pelos genes causais das diferentes SCAs e a ataxina-3 mutada. Com este trabalho pretendeu-se iniciar a investigação de um estudo caso-controlo da doença de Machado-Joseph, determinando as repetições CAG nos loci da SCA2, SCA6 e DRPLA. SUJEITOS e MÉTODOS: analisaram-se 78 indivíduos saudáveis açorianos não aparentados, selecionados por emparelhamento com uma série de doentes de MJD. Os loci ATXN3, ATXN2, CACNA1A e ATN1 foram amplificados por PCR e determinou-se o tamanho dos alelos por análise de fragmentos, num sequenciador automático. Para controlo de qualidade, a confirmação de alguns genótipos foi efectuada por sequenciação. Foram realizadas análises intra-populacionais (frequências alélicas e genotípicas, conformidade com o equilíbrio de Hardy-Weinberg, diversidade genética) e inter-populacionais (diferenciação populacional e distância genética). RESULTADOS: verificou-se, para todos os loci, uma diferença no tamanho dos alelos obtidos em ambas as técnicas utilizadas e efectuaram-se as correcções do respectivo tamanho do alelo. Na amostra estudada, todos os alelos observados estavam dentro da amplitude normal, tendo-se observado apenas um alelo intermediário no locus da SCA2. Todos os loci estavam em equilíbrio de Hardy-Weinberg. Os alelos 14 e 23 (29,5%), 22 (89,1%), 11 (35,9%) e 15 (23,7%) foram os mais frequentes nos loci da SCA3, SCA2, SCA6 e DRPLA, respectivamente. No locus ATXN2, os alelos 22 tinham 1-2 interrupções CAA, e os alelos 29 e 33 tinham apenas 1 CAA. Nos loci ATXN2 e ATN1 observou-se uma frequência relativa elevada de alelos normais grandes (9,6% e 10,5%, respectivamente). DISCUSSÃO: a confirmação, por sequenciação, do tamanho das repetições CAG veio reforçar a noção de que o uso de um marcador alélico na análise de fragmentos é essencial para uma genotipagem correcta. No locus ATXN2, o alelo intermediário observado poderá implicar uma susceptibilidade acrescida para desenvolver a SCA2; porém, a presença de uma interrupção poderá conferir-lhe uma maior estabilidade. Neste mesmo locus os alelos com 24-33 CAGs poderão estar associados com o de risco de desenvolvimento de outras doenças neurodegenerativas. No locus CACNA1A, a baixa frequência de alelos normais grandes parece ser coerente com a ausência de casos reportados de SCA6 nos Açores. No locus ATN1, a inexistência de correlação entre a elevada frequência de alelos normais grandes e a frequência da DRPLA poderá estar relacionada com o haplótipo associado a estes alelos. CONCLUSÃO: a genotipagem dos loci ATXN2, CACNA1A e ATN1 em indivíduos açorianos saudáveis permitirá a utilização desta amostra como grupo controlo num estudo de potenciais genes modificadores da MJD, usando doentes da população açoriana.
ABSTRACT: INTRODUCTION Machado-Joseph disease (MJD) is a spinocerebellar ataxia (SCAs) autosomal dominant inherited caused by CAG repeats in the ATXN3 gene, encoding a polyglutamine tract, and leading to a neurotoxic gain of function of the corresponding protein, ataxin-3, by mechanisms that are not fully known. The size of the CAG repeats, however, only partially explain the phenotypic variability observed in this disease, and the remaining explanation may be provided by variation in modifier genes. Proteins with expanded polyglutamine tracts tend to form aggregates and thus have the potential to modulate the phenotype of expansion diseases. In fact, studies both in animal models and in patients showed the existence of an interaction between the native proteins encoded by different SCAs loci and mutant ataxin-3. With this work we intended to begin the investigation of a case control study of Machado-Joseph disease, determining the CAG repeats at the SCA2, SCA6 and DRPLA loci. SUBJECTS and METHODS we analyzed 78 healthy, unrelated individuals born in the Azores selected by matching it with a series of patients with MJD. The ATXN3, ATXN2, CACNA1A and ATN1 loci were amplified by PCR and the fragment size was determined by fragments analysis in an automated sequencer. For quality control, a few genotypes were confirmed by sequencing. Intra-population (allele and genotype frequencies, conformity to Hardy-Weinberg expectations, genetic diversity) and inter-population (population differentiation and genetic distance) analyzes were performed. RESULTS for all loci, a difference in allele size was obtained with the two techniques used; the respective corrections in allele sizes were made. All alleles observed in this sample were in the normal range, and only one intermediate allele was observed at the SCA2 locus. All loci were in conformity with Hardy–Weinberg expectations. Alleles with 14 and 23 (29.5%), 22 (89.1%), 11 (35.9%) and 15 (23.7%) repeats were the most frequent in SCA3, SCA2, SCA6 and DRPLA loci, respectively. In the ATXN2 locus, alleles 22 had 1-2 CAA interruptions, and alleles 29 and 33 had only 1 CAA. A relatively high frequency of large normal alleles (9.6% and 10.5%, respectively) was observed at the ATXN2 and ATN1 loci. DISCUSSION the confirmation of the CAG repeats size by sequencing reinforces the idea that the use of an allelic ladder in fragment analysis is essential for accurate genotyping. The observed intermediate allele, at the ATXN2 locus, may increase the susceptibility for developing SCA2, but the interruption may confer in stability to this allele. In this locus the alleles with 24-33 CAGs may be associated with the risk for developing other neurodegenerative diseases. In the CACNA1A locus, the low frequency of large normal alleles seems to be consistent with the absence of reported cases of SCA6 in the Azores. In the ATN1 locus, the lack of correlation between DRPLA frequency and large-sized normal alleles may be related to the haplotype associated with these alleles. CONCLUSION the genotyping of the ATXN2, CACNA1A and ATN1 loci in healthy Azorean individuals will allow the use of this sample as a control group in a study of potential modifiers gene of MJD, using Azorean patients.
ABSTRACT: INTRODUCTION Machado-Joseph disease (MJD) is a spinocerebellar ataxia (SCAs) autosomal dominant inherited caused by CAG repeats in the ATXN3 gene, encoding a polyglutamine tract, and leading to a neurotoxic gain of function of the corresponding protein, ataxin-3, by mechanisms that are not fully known. The size of the CAG repeats, however, only partially explain the phenotypic variability observed in this disease, and the remaining explanation may be provided by variation in modifier genes. Proteins with expanded polyglutamine tracts tend to form aggregates and thus have the potential to modulate the phenotype of expansion diseases. In fact, studies both in animal models and in patients showed the existence of an interaction between the native proteins encoded by different SCAs loci and mutant ataxin-3. With this work we intended to begin the investigation of a case control study of Machado-Joseph disease, determining the CAG repeats at the SCA2, SCA6 and DRPLA loci. SUBJECTS and METHODS we analyzed 78 healthy, unrelated individuals born in the Azores selected by matching it with a series of patients with MJD. The ATXN3, ATXN2, CACNA1A and ATN1 loci were amplified by PCR and the fragment size was determined by fragments analysis in an automated sequencer. For quality control, a few genotypes were confirmed by sequencing. Intra-population (allele and genotype frequencies, conformity to Hardy-Weinberg expectations, genetic diversity) and inter-population (population differentiation and genetic distance) analyzes were performed. RESULTS for all loci, a difference in allele size was obtained with the two techniques used; the respective corrections in allele sizes were made. All alleles observed in this sample were in the normal range, and only one intermediate allele was observed at the SCA2 locus. All loci were in conformity with Hardy–Weinberg expectations. Alleles with 14 and 23 (29.5%), 22 (89.1%), 11 (35.9%) and 15 (23.7%) repeats were the most frequent in SCA3, SCA2, SCA6 and DRPLA loci, respectively. In the ATXN2 locus, alleles 22 had 1-2 CAA interruptions, and alleles 29 and 33 had only 1 CAA. A relatively high frequency of large normal alleles (9.6% and 10.5%, respectively) was observed at the ATXN2 and ATN1 loci. DISCUSSION the confirmation of the CAG repeats size by sequencing reinforces the idea that the use of an allelic ladder in fragment analysis is essential for accurate genotyping. The observed intermediate allele, at the ATXN2 locus, may increase the susceptibility for developing SCA2, but the interruption may confer in stability to this allele. In this locus the alleles with 24-33 CAGs may be associated with the risk for developing other neurodegenerative diseases. In the CACNA1A locus, the low frequency of large normal alleles seems to be consistent with the absence of reported cases of SCA6 in the Azores. In the ATN1 locus, the lack of correlation between DRPLA frequency and large-sized normal alleles may be related to the haplotype associated with these alleles. CONCLUSION the genotyping of the ATXN2, CACNA1A and ATN1 loci in healthy Azorean individuals will allow the use of this sample as a control group in a study of potential modifiers gene of MJD, using Azorean patients.
Description
Dissertação de Mestrado em Ciências Biomédicas.
Keywords
Doença de Machado-Joseph Doença Neurodegenerativa Epidemiologia Genética Genética Humana Machado-Joseph Disease (MJD)
Citation
Ferreira, Ana Filipa Bartolomeu – “Polimorfismo nos loci ATXN2, CACNA1A e ATN1 : um estudo de potenciais genes modificadores da doença de Machado-Joseph em indivíduos açorianos saudáveis”. Ponta Delgada : Universidade dos Açores. 2012. XIII, 68 p.. Dissertação de Mestrado.