Diagnosis of five spinocerebellar ataxia disorders by multiplex amplification and capillary electrophoresis.
The autosomal-dominant spinocerebellar ataxias (ADCA) are a heterogeneous group of neurodegenerative disorders with variable expression and phenotypic overlap. An accurate diagnosis relies on detection of a mutation in a specific causative gene, which is typically an abnormal number of CAG trinucleotide repeats. To streamline testing in a clinical setting, we converted our current panel of tests for the spinocerebellar ataxias (SCA) types SCA1, SCA2, SCA3, SCA6, and SCA7 from five independent amplification reactions analyzed by polyacrylamide gel electrophoresis (PAGE) to a single multiplex amplification reaction analyzed by capillary electrophoresis (CE). Multiplex amplification was facilitated by the use of chimeric primers; different lengths and fluorochromes distinguished the amplicons. During CE with commercially available molecular weight standards, the SCA amplicons migrated faster than predicted, thereby underestimating their length compared to that determined previously by PAGE. This was observed to varying degrees for each of the five loci, with the greatest size differential occurring in amplicons with greater (CAG)(n). To determine accurate amplicon length, and therefore an accurate number of CAG repeats, a size correction formula was calculated for each locus. This multiplex semi-automated assay has been reliable during 1 year of use in a clinical setting during which 57 samples were tested and five positive samples were detected.
Dorschner
Michael O
MO
Department of Medicine, Medical Genetics 357720, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195-7720, USA.
Barden
Deborah
D
Stephens
Karen
K
D000067696:Ataxin-1; D000067699:Ataxin-3; D000067719:Ataxin-7; D000067528:Ataxins; D015220:Calcium Channels / Q000235:genetics; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005784:Gene Amplification; D006801:Humans; D008297:Male; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics; D012097:Repressor Proteins; D020754:Spinocerebellar Ataxias / Q000175:diagnosis* / Q000235:genetics*; D018911:Trinucleotide Repeats; D020864:Calcium Channels, N-Type
D000067696:Ataxin-1; D000067699:Ataxin-3; D000067719:Ataxin-7; D000067528:Ataxins; D015220:Calcium Channels; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011506:Proteins; D012097:Repressor Proteins; C000593749:ATXN1 protein, human; C000593829:ATXN7 protein, human; C105656:CACNA1A protein, human; D020864:Calcium Channels, N-Type; C092341:ATXN3 protein, human