A quantitative polymerase chain reaction (PCR) assay completely discriminates between Duchenne and Becker muscular dystrophy deletion carriers and normal females.
Duchenne/Becker muscular dystrophy (DMD/BMD) is a severe X-linked myopathy. In 65% of the patients, the mutations responsible for the disease are macrodeletions in the dystrophin-encoding gene that can be identified with multiplex polymerase chain reaction (PCR) technology. We developed a method for quantitative PCR analysis of deletion carriers involving the use of phosphorimager-based scanning of radioactive-labelled PCR products. We calculated the ratios between the areas of two peaks, one corresponding to the deleted segments to be analysed and the other taken as a reference. In carriers, these ratios (R value) were always about half those obtained in normal females. The final diagnostic result, the diagnostic index (DI), is the ratio of the R values between the propositus and a normal subject. We also assessed the variability of each step of the procedure and the overall variability of the DI value, thus obtaining cut-off values that completely discriminated BMD/DMD deletion carriers from normal females. We were also able to classify, as either 'carrier' or 'normal', several females whose status was not identified with linkage analysis.
Pastore
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L
Dipartimento di Biochimica e Biotecnologie Mediche, Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli Federico II, Italy.
Caporaso
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MG
Frisso
G
G
Orsini
A
A
Santoro
L
L
Sacchetti
L
L
Salvatore
F
F
D017931:DNA Primers; D016189:Dystrophin / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006580:Genetic Carrier Screening; D006801:Humans; D009136:Muscular Dystrophies / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012016:Reference Values; D012044:Regression Analysis; D015203:Reproducibility of Results; D017384:Sequence Deletion; D014960:X Chromosome
D017931:DNA Primers; D016189:Dystrophin