Spinocerebellar ataxias (SCA) type 1, 2, 3, 6, and 7, associated with a (CAG)n repeat expansion in coding sequences, are the most prevalent autosomal dominant ataxias worldwide (approximately 60% of the cases). In addition, the phenotype of SCA2 expansions has been now extended to Parkinson's disease and amyotrophic lateral sclerosis. Their diagnosis is presently based on a PCR to identify small expanded alleles, followed by a second-level test whenever the suspect of false normal homozygous, or a CAT interruption in SCA1 needs to be verified. Next-generation sequencing still does not allow efficient detection of these repeats. Here, we show the efficacy of a novel, rapid, and cost-effective method to identify and size pathogenic expansions in SCA1-3, 6, and 7 and recognize large alleles or interruptions without a second-level test. Twenty-five healthy controls and 33 expansion carriers were analyzed: alleles migrated consistently in different PCRs/capillary runs, and homozygous subjects were always distinguishable from heterozygous carriers of both common and large (>100 repeats) pathogenic CAG expansions. Repeat number could be calculated counting the number of peaks, except for the largest SCA2 and SCA7 alleles. Interruptions in SCA1 were always visible. Overall, our method allows a simpler, cost-effective, and sensibly faster SCA diagnostic protocol compared to the standard technique and to the still unadapted next-generation sequencing.

SCA Tethering-PCR: A Rapid Genetic Test for the Diagnosis of SCA1-3, 6, and 7 by PCR and Capillary Electrophoresis

Cagnoli, Claudia;Brussino, Alessandro;Mancini, Cecilia;Ferrone, Marina;Giorgio, Elisa;Pozzi, Elisa;Cavalieri, Simona;Di Gregorio, Eleonora;Ferrero, Marta;Brusco, Alfredo
Last
2018

Abstract

Spinocerebellar ataxias (SCA) type 1, 2, 3, 6, and 7, associated with a (CAG)n repeat expansion in coding sequences, are the most prevalent autosomal dominant ataxias worldwide (approximately 60% of the cases). In addition, the phenotype of SCA2 expansions has been now extended to Parkinson's disease and amyotrophic lateral sclerosis. Their diagnosis is presently based on a PCR to identify small expanded alleles, followed by a second-level test whenever the suspect of false normal homozygous, or a CAT interruption in SCA1 needs to be verified. Next-generation sequencing still does not allow efficient detection of these repeats. Here, we show the efficacy of a novel, rapid, and cost-effective method to identify and size pathogenic expansions in SCA1-3, 6, and 7 and recognize large alleles or interruptions without a second-level test. Twenty-five healthy controls and 33 expansion carriers were analyzed: alleles migrated consistently in different PCRs/capillary runs, and homozygous subjects were always distinguishable from heterozygous carriers of both common and large (>100 repeats) pathogenic CAG expansions. Repeat number could be calculated counting the number of peaks, except for the largest SCA2 and SCA7 alleles. Interruptions in SCA1 were always visible. Overall, our method allows a simpler, cost-effective, and sensibly faster SCA diagnostic protocol compared to the standard technique and to the still unadapted next-generation sequencing.
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https://linkinghub.elsevier.com/retrieve/pii/S1525-1578(17)30389-6
ataxia; sca; method
Cagnoli, Claudia; Brussino, Alessandro; Mancini, Cecilia; Ferrone, Marina; Orsi, Laura; Salmin, Paola; Pappi, Patrizia; Giorgio, Elisa; Pozzi, Elisa; Cavalieri, Simona; Di Gregorio, Eleonora; Ferrero, Marta; Filla, Alessandro; De Michele, Giuseppe; Gellera, Cinzia; Mariotti, Caterina; Nethisinghe, Suran; Giunti, Paola; Stevanin, Giovanni; Brusco, Alfredo
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/1660638
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