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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Loureiro JR, Oliveira CL, Silveira I. Unstable repeat expansions in neurodegenerative diseases: nucleocytoplasmic transport emerges on the scene. Neurobiol Aging. 2016;39:174–83.
Sequeiros J, Martins S, Silveira I. In Handbook of Clinical Neurology Vol. 103, pp. 227–51, (Netherlands 2012).
Huang M, Verbeek DS. Why do so many genetic insults lead to Purkinje Cell degeneration and spinocerebellar ataxia? Neurosci Lett. (2018) https://doi.org/10.1016/j.neulet.2018.02.004.
Paulson HL, Shakkottai VG, Clark HB, Orr HT. Polyglutamine spinocerebellar ataxias—from genes to potential treatments. Nat Rev Neurosci. 2017;18:613–26.
Coutinho P, Ruano L, Loureiro J, Cruz V, Barros J, Tuna A, et al. Hereditary ataxia and spastic paraplegia in Portugal: a population-based prevalence study. JAMA Neurol. 2013;70:746–55.
Nelson DL, Orr HT, Warren ST. The unstable repeats–three evolving faces of neurological disease. Neuron. 2013;77:825–43.
Garcia-Murias M, Quintans B, Arias M, Seixas AI, Cacheiro P, Tarrio R, et al. ‘Costa da Morte’ ataxia is spinocerebellar ataxia 36: Clinical and genetic characterization. Brain. 2012;135:1423–35.
Alonso I, Jardim L, Artigalas O, Saraiva-Pereira M, Matsuura T, Ashizawa T, et al. Reduced penetrance of intermediate size alleles in spinocerebellar ataxia type 10. Neurology. 2006;66:1602–4.
Silveira I, Alonso I, Guimarães L, Mendonça P, Santos C, Maciel P, et al. High germinal instability of the (CTG)n at the SCA8 locus of both expanded and normal alleles. Am J Hum Genet. 2000;66:830–40.
Silveira I, Miranda C, Guimaraes L, Moreira M, Alonso I, Mendonca P, et al. Trinucleotide repeats in 202 families with ataxia - A small expanded (CAG)(n) allele at the SCA17 locus. Arch Neurol. 2002;59:623–9.
Seixas AI, Vale J, Jorge P, Marques I, Santos R, Alonso I, et al. FXTAS is rare among Portuguese patients with movement disorders: FMR1 premutations may be associated with a wider spectrum of phenotypes. Behav Brain Funct. 2011;7:19.
Sato N, Amino T, Kobayashi K, Asakawa S, Ishiguro T, Tsunemi T, et al. Spinocerebellar ataxia type 31 is associated with “inserted” penta-nucleotide repeats containing (TGGAA)n. Am J Hum Genet. 2009;85:544–57.
Seixas AI, Loureiro JR, Costa C, Ordonez-Ugalde A, Marcelino H, Oliveira CL, et al. A pentanucleotide ATTTC repeat insertion in the non-coding region of DAB1, mapping to SCA37, causes Spinocerebellar Ataxia. Am J Hum Genet. 2017;101:87–103.
Ishikawa K, Dürr A, Klopstock T, Müller S, De Toffol B, Vidailhet M, et al. Pentanucleotide repeats at the spinocerebellar ataxia type 31 (SCA31) locus in Caucasians. Neurology. 2011;77:1853–5.
Cagnoli C, Michielotto C, Matsuura T, Ashizawa T, Margolis RL, Holmes SE, et al. Detection of large pathogenic expansions in FRDA1, SCA10, and SCA12 genes using a simple fluorescent repeat-primed PCR assay. J Mol Diagn. 2004;6:96–100.
Chen L, Hadd A, Sah S, Filipovic-Sadic S, Krosting J, Sekinger E, et al. An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis. J Mol Diagn. 2010;12:589–600.
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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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