Triplet Repeat Primed PCR (TP-PCR) in Molecular Diagnostic Testing for Spinocerebellar Ataxia Type 3 (SCA3)
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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.
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.
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.
The present TP-PCR protocol developed for SCA3 should constitute a reliable complementary technique to overcome the limitations of standard PCR.
KeywordsMyotonic Dystrophy Spinocerebellar Ataxia Type Buccal Swab Friedreich Ataxia Expanded Allele
Compliance with Ethical Standards
Conflict of interest
ARVM, AR, NK, MR, BFB, ARR, TK, JV, JB-A and ML declare no conflicts of interests.
Ethical approval and informed consent
The present work was conducted as part of the ESMI project (European Spinocerebellar Ataxia Type 3/Machado-Joseph Disease Initiative) which was approved by the Ethical Board of the Hospital do Divino Espírito Santo (Ponta Delgada, Azores, Portugal). Informed consent was provided by all participants.
PhD fellowship M3.1.2/F/006/2011 (Mafalda Raposo) and postdoctoral fellowships M3.1.7/F/031/2011 (Amanda Ramos) and M3.1.3/F/004/2009 (Nadiya Kazachkova) were supported by Fundo Regional para a Ciência (FRC), Government of the Azores.
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