Leiden Muscular Dystrophy pages

MLPA-analysis of the DMD (dystrophin) gene

(detecting deletions/duplications)

(modified last February 05, 2008)



Although ~95% of deletions can be detected in males using multiplex PCR, other methods must be used to determine duplications, as well as the carrier status of females. The most commonly applied methods are quantitative multiplex PCR and quantitative Southern blotting. The drawback of quantitative multiplex PCR is that often not all exons are examined, meaning that small and rare mutations are missed. Southern blotting is usually applied to determine the boundaries of the mutation, which is essential for distinguishing DMD from BMD, i.e. frame disrupting from open reading frame changes. Using high-quality Southern blots it is possible to perform a quantitative analysis and detect duplications. However, this technique is time consuming, it is difficult to exactly determine the duplication boundaries, it can be difficult to detect duplications in females and triplications will be missed.

We have developed a method to screen the entire DMD gene for deletions and duplications (White et al. [2002]). This method uses MAPH, Multiplex Amplifiable Probe Hybridisation, and analyses all 79 exons of the DMD gene in one assays (see DMD MAPH analysis). Recently, a similar method has been designed, based on MLPA (Multiplex Ligation-dependent Probe Amplification, Schouten et al. [2002]). The advantage of MLPA compared to MAPH is that a lower amount of input DNA is required and that MLPA is a one-tube assay.

A kit for MLPA analysis of the human DMD-gene can be obtained from MRC-Holland (SALSA P034 / P035 DMD test kit) as well as detailed MLPA-protocols. A paper describing our results using this kit has been published by Lalic et al. (2005). In addition we have published results using a modified kit that allows array-based MLPA-screening of the DMD-gene (Zeng et al. 2008). For those interested we offer deletion / duplication screening of the DMD-gene using both MLPA (for information please contact us; Johan den Dunnen - E-mail: ddunnen @ HumGen.nl).

MLPA on agarose gels

Usually, MLPA-products are fluorescently labelled and analysed using capillary gel-electrophoresis (e.g. using an automated DNA sequencer - see MLPA reference pictures). A nice feature of the MLPA-analysis of the DMD gene is that the amplification products can also be analysed using simple agarose gel-electrophoresis and ethidium-bromide staining. The only thing to do in this respect is to order the DMD MLPA-kit in a specific format. An example of the analysis of the 79-exons of the DMD-gene in this way is shown in Fig.1. An example of the analysis of a DMD-patient carrying a deletion is shown in Fig.2. When the number of PCR-cycles is optimised, agarose gel- electrophoresis even facilitates the detection of duplications in patients and deletions in carriers (White et al., in preparation).

Figure 1:   agarose gel-electrophoresis DMD MLPA-products

MLPA analysis of the human DMD-gene in a normal male. Content and length of the individual products is indicated in Table I. Picture kindly provided by Stefan White and Rolf Vossen.

Figure 2:   Agarose-gel analysis of DMD deletion patient

MLPA-analysis using agarose gel-electrophoresis of a DMD patient with a deletion of exons 4 to 13. Missing exons can be easily detected using the individual sets; P071 - exon 4, P073 - exon 13, P076 - exons 6, 8 and 10, P077 - exon 12. Not all sets are shown. Picture kindly provided by Stefan White and Rolf Vossen.

Tabular overview MLPA products

 Table I  -  DMD MLPA-products

exon 41146control130control142exon 11138Dp427m138exon 21154exon 51146exon 31154
exon 2170exon 42178exon 22186exon 61162exon 71162exon 52178exon 12170control184
exon 62194exon 3210exon 13210exon 32186control186control202exon 72194control211
exon 43218exon 63234exon 73234exon 53218exon 33226exon 23226control218exon 14242
exon 4242exon 74266exon 34258exon 44250exon 24258exon 54250control250exon 64266
control274exon 25298exon 45290exon 5282exon 55290exon 15282exon 75306exon 35298
exon 65306exon 36330exon 56322exon 16314exon 46322exon 6314exon 66338exon 26330
exon 76338exon 27370exon 47362exon 7354exon 57362exon 17354exon 77378exon 37370
exon 67378exon 38402exon 58394exon 18386exon 48394exon 8386exon 68410exon 28402
exon 78410exon 29442exon 49434exon 9426exon 59434exon 19426exon 79450exon 39442
exon 69450exon 40483exon 60475exon 10467exon 50475exon 10467exon 70491exon 30483

Individual DMD MLPA-probe sets P071 to P078 (obtained from MRC-Holland). Probe: probe for DMD-exon indicated or a control probe. bp: length of MLPA amplification product in base pairs (bp). An agarose-gel of these products can be seen in Fig.1.

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