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DMD/BMD deletion detection using multiplex PCR

(last modified March 3, 2007)

Introduction
- history
Multiplex PCR: annotated images
- Chamberlain-set and Beggs-sets
Primer sequences for multiplex PCR
- described in detail in Current Protocols in Human Genetics
- additional sets
  - Abbs-5'-set
  - Abbs-3'-set
Other DMD-gene primers
- PCR per exon, sequence analysis, markers, Ca-repeats, mutation detectction, ...

Introduction: multiplex PCR

Multiplex PCR is a technique in which several PCR-products are amplified in one PCR-reaction. DMD is an X-linked disease, i.e. only one X-chromosome is present. Consequently, deletion of an exon can be determined by the failure to amplify that exon by PCR. Since deletion mutations are frequent in DMD/BMD (present in ~2/3 of cases) and since the deletions are non-randomly distributed across the gene (see "DMD gene deletion/duplication database"), a multiplex PCR co-amplifying the most frequently deleted exons provides a very simple, rapid and efficient diagnostic tool. DMD multiplex PCR was first described by Chamberlain et al. (1988) and became rapidly applied world-wide. The original 6-exon Chamberlain-set was later modified into a 9-exon (Chamberlain et al. 1990) and ultimately a 10-exon set (Beggs et al. 1991). A second set, the 9-exon Beggs-set (Beggs et al. 1990), was developed to increase the total number of deletions detected and to define the borders of the deletions in the deletion 'hotspot'. To cover some untested regions and to facilitate the determination of the borders of the deletions in a larger number of cases, Kunkel et al. 1991 developed a third 7-exon set. For specific purposes, several additional multiplex primer sets were published over the years (see below). An extensive discussion of the DMD/BMD multiplex PCR protocol and its characteristics can be found in Current Protocols in Human Genetics (by Alan Beggs and Johan den Dunnen).
NOTE: a 10-set quantitative multiplex PCR assay was descibed by Stockley et al. (2006).

Multiplex PCR and point mutation screening

When detection of point mutations in the DMD gene was still rather difficult, several studies used the the multiplex PCR products trying to identify such mutations. The techniques applied include single-strand conformation analysis (SSCA), hetero-duplex analysis (HD) and double-strand conformation analysis (DSCA). With these technologies, frequent polymorphic sequence variants can be detected in:

polymorphisms:
- coding region: exons 45 (45a, 45b)and 48
- intronic sequence: co-amplified with exon 3, 13 (13a, 13b), 17 (17a, 17b), 43 (43a, 43b), 45 and 48

Please note that since some of the primer sequences used in the multiplex sets are exonic, span or are to close to the exon/intron boundaries, this approach will fail to detect mutations which are at the primer annealing site and those outside the amplified region, in particular those affecting splicing. For efficient methods to scan the DMD gene for point mutations see "DNA-based diagnostic techniques for DMD / BMD".

Multiplex PCR images

Legend:
Size: size of PCR product (in base pairs). Exon: exon of DMD gene present in PCR fragment.