Leiden Muscular Dystrophy pages

DNA-based diagnostic techniques for DMD / BMD

(last modified October, 2008)

My opinion

Based on the latest results regarding the frequency of DMD-mutations identified causing Duchenne / Becker muscular dystrophy [see Table, White & den Dunnen 2006, Aartsma-Rus 2006] the most powerful DNA-based techniques currently available to reveal molecular changes in patients are (to be performed in this order);

  1. deletion / duplication screening
    to reliably predict the consequences of any rearrangmenent (incl. deletions / duplications) in the DMD gene on the dystrophin reading frame (i.e. in-frame or out-of-frame) it is essential to analyse DMD mRNA. Predictions based on DNA findings are predcitions only.
  2. point mutation screening
    we consider RNA-based point mutation screening as the most powerful technique to screen for deleterious, non-exon-deletion / duplication changes in the DMD-gene. By amplifying the entire DMD coding region from an RNA template, all deleterious truncating mutations will be resolved, including those affecting RNA-splicing. The Protein Truncation Test (PTT), an RNA-based screening mehtod, has been proven to be very effective. However, PTT is not the simplest method to implement and an RNA sample, preferably from a muscle biopsy,  is not always available. PTT on lymphocyte RNA is possible, but more difficult to perform (Tuffery-Giraud 2004). An alternative is to use RNA obtained after MyoD-induced in vitro muscle differentiation. The cDNA fragments obtained after RT-PCR can also be used for sequencing to determine the mutations present (Hamed 2006, see Primers for DMD RNA RT-PCR

    Compared to DGGE we consider SSCA and DHPLC as good but more laborious alternatives. Direct sequencing is very powerful, but also more costly.

    With few exceptions, mostly only the protein coding regions of the DMD gene are analysed. Studies analysing other regions (promoters, 5'UTR and 3'UTR) have so far not revealed many changes (e.g. Tubiello 1995, Flanigan 2003).

  3. haplotyping
    when no change can be detected using the above mentioned techniques, haplotype analysis (i.e. identifying the risk chromosome) is the only available technique to perform a DNA-based analysis. In rare cases, a cytogenetic analysis may reveal translocations or large inversions. For primers see PCR of sequences in/around the DMD-gene.

Next to DNA-based analysis, dystrophin staining on a muscle biopsy and PTT-analysis of an RNA sample (blood-derived or from a muscle biopsy), provide the most powerful techniques to perform a molecular diagnosis for DMD/BMD. In exceptional cases, a myoD-induced myo-differentiation can be performed.

JT den Dunnen


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