open access

Vol 46, No 2 (2012)
ARTYKUŁ ORYGINALNY
Submitted: 2011-02-17
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Rapid detection of large expansions in progressive myoclonus epilepsy type 1, myotonic dystrophy type 2 and spinocerebellar ataxia type 8

Wioletta Krysa, Marta Rajkiewicz, Anna Sutek
DOI: 10.5114/ninp.2012.28253
·
Neurol Neurochir Pol 2012;46(2):113-120.

open access

Vol 46, No 2 (2012)
ARTYKUŁ ORYGINALNY
Submitted: 2011-02-17

Abstract

Background and purpose

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.

Material and 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.

Abstract

Background and purpose

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.

Material and 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.

Get Citation

Keywords

dynamic mutations, neurodegenerative disorders, repeat primed PCR (RP-PCR)

About this article
Title

Rapid detection of large expansions in progressive myoclonus epilepsy type 1, myotonic dystrophy type 2 and spinocerebellar ataxia type 8

Journal

Neurologia i Neurochirurgia Polska

Issue

Vol 46, No 2 (2012)

Pages

113-120

DOI

10.5114/ninp.2012.28253

Bibliographic record

Neurol Neurochir Pol 2012;46(2):113-120.

Keywords

dynamic mutations
neurodegenerative disorders
repeat primed PCR (RP-PCR)

Authors

Wioletta Krysa
Marta Rajkiewicz
Anna Sutek

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