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A repeat-primed PCR assay for pentanucleotide repeat alleles in spinocerebellar ataxia type 37

Abstract

Spinocerebellar ataxia 37 (SCA37) is caused by an (ATTTC)n insertion in a polymorphic ATTTT repeat in the non-coding region of DAB1. The non-pathogenic alleles have a configuration [(ATTTT)7–400], whereas pathogenic alleles have a complex structure of [(ATTTT)60–79(ATTTC)31–75(ATTTT)58–90]. Molecular diagnosis of SCA37 is laborious because about 7% of the pentanucleotide repeat alleles in DAB1 are larger than 30 units and, thus, fail to amplify with standard PCR conditions, resulting in apparently homoallelism or in complete lack of PCR amplification in several cases. The molecular test currently available requires long-range PCR and sequencing analysis for the detection and characterization of these large alleles. We developed a simple assay capable of rapidly detecting the presence or absence of large pentanucleotide repeat sizes. This assay is based on repeat-primed PCR followed by high-throughput capillary electrophoresis. Combining the standard PCR with RP-PCR allows completion of the diagnosis in more than 80% of individuals, minimizing the number of samples that require long-range PCR followed by Sanger sequencing analysis. This assay meets many of the requirements for pre-screening of large cohorts of affected individuals.

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Acknowledgements

This work was funded by Fundo Europeu de Desenvolvimento Regional-FEDER funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by funding from FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação, Portugal, in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274); by Grant PTDC/SAU-GMG/098305/2008, from FCT, to I.S. J.R.L. was supported by scholarships from Grant PTDC/GMG-SAU/098305/2008, FCT, PEst-C/SAU/LA0002/2013 and EMBO (ASTF494-2015). C.L.O. was supported by a scholarship from PEst-C/SAU/LA0002/2013. This work was also funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), Portugal, that supports the Norte-01-0145-FEDER-000008—Porto Neurosciences and Neurologic Disease Research Initiative at I3S.

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Correspondence to Isabel Silveira.

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Loureiro, J.R., Oliveira, C.L., Sequeiros, J. et al. A repeat-primed PCR assay for pentanucleotide repeat alleles in spinocerebellar ataxia type 37. J Hum Genet 63, 981–987 (2018). https://doi.org/10.1038/s10038-018-0474-3

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