Gene Diagnostics
Genetic analysis requires a blood sample containing leucocytes. These white blood cells have cell nuclei (unlike the red blood cells) which contain the hereditary material, DNA.
About 60% of Duchenne patients have large deletions spanning one or more exons of the dystrophin gene. A further 5% have large duplications of genetic material. Because these deletions and duplications are more numerous in certain regions of the gene, by analysing just 19 of the 79 exons that make up the dystrophin gene, 98% of these mutations can be detected. In about two thirds of these cases, the nature of the genetic error, i.e. whether the reading frame is disrupted or not, can be deduced, and thus it can be predicted whether the mutation will lead to a Duchenne or Becker dystrophy. For the remaining third, further exons must be amplified and analysed.
The large deletions or duplications are detected using multiplexed polymerase chain reaction (PCR) primers to amplify the exons of interest. These primers are short synthetic DNA sequences which attach themselves to the beginning and end of an exon sequence. The gene fragments obtained in this way are separated by electrophoresis, during which fragments migrate different distances on a gel layer depending on their size. When made visible these are seen as bands like unevenly spaced rungs on a ladder. A �missing� band represents a deletion.
If a large deletion or duplication is not found by this method, the mutation is presumed to be a point mutation (either a deletion, a substitution or an insertion of one or several nucleotides). To characterise such an abnormality unequivocally, the base sequences of most of the exons would have to be determined. Such sequence determination is difficult and is not yet routinely offered. In these cases, a final diagnosis can only be made by investigation of muscle tissue obtained by a biopsy. The dystrophin protein is analyzed here in a western blot.
In this method, after electrophoretic separation, the protein pattern is transferred to another carrier material by blotting, where it is made visible with antibodies. Or fluorescent antibodies are attached to the dystrophin, which can then be detected under the microscope as a bright line around the healthy muscle cells. In Duchenne dystrophy, it is not seen there or only in traces, and in Becker dystrophy, these lines are often frayed, interrupted and mostly much weaker than normal.
If a gene analysis gives an unequivocal result, a muscle biopsy is often no longer necessary. Small children could thus avoid this surgical procedure. However, if a muscle biopsy is necessary, then it can also be performed as a needle biopsy under local anesthesia.