title: Rapid detection of large expansions in progressive myoclonus epilepsy type 1, myotonic dystrophy type 2 and spinocerebellar ataxia type 8.
abstract: OBJECTIVE
  Human genetic disorders associated with multiple unstable repeats resulting in long DNA expansions are difficult to identify by conventional polymerase chain reaction (PCR) in routine molecular testing, and therefore require time-consuming hybridisation. To improve and expedite the diagnostic methods for progressive myoclonus epilepsy (EPM1), myotonic dystrophy 2 (DM2) and spinocerebellar ataxia 8 (SCA8) caused by dynamic mutations, we adapted a repeat primed PCR (RP-PCR) assay which was previously developed for testing of other triplet repeat disorders.
  
  
  METHODS
  The new algorithm for molecular analysis was to run a standard PCR to yield alleles in an amplifiable range and then run a RP-PCR to detect larger expansions. Electrophoresis and visualisation of PCR products on an automatic sequencer were applied to determine normal and pathogenic alleles comprising (C4GC4GCG)n in EPM1 in 44 subjects, (CCTG)n in DM2 in 76 individuals and (CTG)n in SCA8 in 378 patients.
  
  
  RESULTS
  The protocol combining conventional PCR and RP-PCR proved to be a rapid and reliable test to diagnose the above named disorders. Among 44 individuals tested for EPM1, two expanded alleles were identified in 7 patients. Out of 76 apparently homozygous subjects, RP-PCR allowed us to detect 56 expansions specific to DM2, and out of 378 ataxia patients, a large allele of the ATXN8OS gene (SCA8) was found in 25 subjects.
  
  
  CONCLUSIONS
  Here, for the first time, we report detection of large expansions in EPM1 and SCA8 patients. This RP-PCR assay is high throughput, reproducible and sensitive enough to be successfully used for diagnostic purposes.
authors.affiliation: Zakład Genetyki, Instytut Psychiatrii i Neurologii w Warszawie, ul. Sobieskiego 9, 02-957 Warszawa. krysa@ipin.edu.pl
authors.forename: Wioletta
authors.initials: W
authors.lastname: Krysa
authors.forename: Marta
authors.initials: M
authors.lastname: Rajkiewicz
authors.forename: Anna
authors.initials: A
authors.lastname: Sułek
mesh_terms: D000465:Algorithms; D001483:Base Sequence; D006580:Genetic Carrier Screening / Q000379:methods; D006801:Humans; D009223:Myotonic Dystrophy / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013132:Spinocerebellar Degenerations / Q000175:diagnosis* / Q000235:genetics*; D020194:Unverricht-Lundborg Syndrome / Q000175:diagnosis* / Q000235:genetics*