1- Departamento de Biologia, Universidade dos Açores, Ponta Delgada, Açores, Portugal.
2- Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Porto, Portugal.
3- Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
4- Hospital de Santo Espírito da Ilha Terceira, SEEBMO, Angra do Heroísmo, Açores, Portugal.
5- Especialidade de Genética Médica; Área da Mulher, Criança e Adolescente; Hospital de Dona Estefânia, Centro Hospitalar de Lisboa Central, EPE, Lisboa;
6- Departamento de Neurologia, Hospital do Divino Espírito Santo, Ponta Delgada, Açores, Portugal.
7- Departamento de Biologia, Universidade dos Açores, Rua da Mãe de Deus, Ponta Delgada, 9501-801, Açores, Portugal.
8- Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Porto, Portugal.
9 - Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
- Molecular Diagnosis & Therapy, December 2016; 20(6):617-622
Resumo:
INTRODUCTION AND OBJECTIVE: Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder for which the routine molecular testing is based on PCR and automated capillary electrophoresis. When only a normal allele is detected by standard PCR, the hypothesis of a failed amplification of the expanded allele must be raised. In such cases, complementary techniques such as Southern Blot or triplet repeat primed PCR (TP-PCR) have to be applied. For SCA3, TP-PCR is implemented in some diagnostic laboratories, but a tested protocol has yet to be published. The purpose of this study was to develop and test a TP-PCR protocol for SCA3.
METHODS: Sixty-five blood samples previously genotyped by standard PCR were used in the TP-PCR assay. Fourteen buccal swab samples were also analyzed to confirm the robustness of the technique. The reproducibility of the TP-PCR was evaluated by analyzing all samples in a second laboratory.
RESULTS: The results obtained by TP-PCR confirmed the previous PCR results for 64 blood samples; in one sample an expanded allele, previously undetected by PCR, was identified. The results obtained for the buccal swab samples were totally concordant with those obtained for blood. Furthermore, the results obtained in the alternative laboratory were in full agreement with the results obtained in our study.
CONCLUSION: The present TP-PCR protocol developed for SCA3 should constitute a reliable complementary technique to overcome the limitations of standard PCR.
Palavras Chave: SCA3; Machado-Joseph Disease; TP-PCR