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);
deletion / duplication screening
NOTE: 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.
Multiplex Ligation-dependent Probe Amplification (MLPA) the power of MLPA-analysis(Schwatz & Duno 2003) is that it
screens all 79 exons of the DMD-gene for deletion and
duplicationmutations. MLPA-analysis can also be performed using agarose gels (Lalic 2005) and arrays (Zeng 2008).
Recently, arrayCGH approaches have been published, using oligonucleotide tiling arrays
spanning the DMD-gene (Hegde 2008,
del Gaudio 2008, Saillour 2008). Compared to MLPA
these arrays precisely determine the deletion / duplication borders in the introns. Thusfar
this information seems not to add a lot to the diagnosis, while the cost of the assay is
significantly higher.
multiplex PCR (Beggs & Chamberlain kits)
multiplex PCR screens only 18 of the 79 exons of the DMD-gene and it will not detect
duplications present in 5-7% of the patients (den
Dunnen 1989, White & den
Dunnen 2006). Furthermore, additional analysis, e.g. Southern
blotting, will be required to determine the exact bordersof the
rearrangements detected, as well as to pick up duplications. Defining the deletion /
duplication borders is important to discriminate 'open reading frame' from 'reading
frame disrupting' changes (see NOTE).
other methods
many other quantitative methods have been used but none of them have found wide-spread
application. qPCR (quantitative-PCR - e.g. Ashton 2008) seems simple but is
technically demanding, especially when performed in mutliplex mode. Multiplex-Amplifiable Probe Hybridisation - (MAPH - White 2002) is a simple and
effective alternative for MLPA-screening, but it requires more input DNA and it is more
laborious. FISH, CA-repeat marker analysis and exon-specific qPCR are valuable tools to confirm
known rearrangments in carriers but they are not effective to screen
patients directly.
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)
high-resolution Melting Curve Analysis (hrMCA)
for DNA-based point mutation screening we currently prefer hrMCA (Al Momani, submitted
- details available on request). hrMCA is simple, cheap and very sensitive (>98%).
Applied as a pre-sequencing tool, resolving those fragments that contain variants, it is
very cost-effective.
Denaturing Gradient Gel-Electrophoresis (DGGE) DGGE (Hofstra
2004), having a close to 100% sensitivity, is once implemented a very effective
technique. However, DGGE is laborious, it uses several PCR and electrophoresis conditions
and it difficult to automate.
direct sequencing dirct sequencing (or SCAIP - single condition amplification/internal primer,
Flanigan
2003) is a straightforward and effective method but it is rather costly (>79
separate exon fragments to analyse) .
Single-Strand Conformation Analyis (SSCA) SSCA / DOVAM (detection of virtually all mutations, Mendell
2001 / Buzin
2005) is simple,cheap and effective but rather laborious (e.g. demanding
electrophoresis of all (>79) exon fragments each using several electrophoretic
conditions).
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).
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
DNA-analysis
large genetic rearrangements (deletions / duplications) For those interested we offer deletion / duplication screening of the
DMD-gene using both MLPA and MAPH
(for information please contact us; Johan den Dunnen - E-mail: ddunnen @ LUMC.nl).