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MLPA-analysis of the DMD (dystrophin) gene

(detecting deletions/duplications)

(modified last February 05, 2008)

Introduction
MLPA analysis on agarose gels
- agarose-gel images for DMD-kits P071 to P078
- DMD deletion patient analysed on an agarose gel
reference pictures DMD MLPA
tabular overview MLPA products
kits P071 to P078 (exons / length in bp)

Introduction

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

Legend:
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

Legend:
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

P071		P072		P073		P074		P075		P076		P077		P078
probe	bp	probe	bp	probe	bp	probe	bp	probe	bp	probe	bp	probe	bp	probe	bp
exon 41	146	control	130	control	142	exon 11	138	Dp427m	138	exon 21	154	exon 51	146	exon 31	154
exon 2	170	exon 42	178	exon 22	186	exon 61	162	exon 71	162	exon 52	178	exon 12	170	control	184
exon 62	194	exon 3	210	exon 13	210	exon 32	186	control	186	control	202	exon 72	194	control	211
exon 43	218	exon 63	234	exon 73	234	exon 53	218	exon 33	226	exon 23	226	control	218	exon 14	242
exon 4	242	exon 74	266	exon 34	258	exon 44	250	exon 24	258	exon 54	250	control	250	exon 64	266
control	274	exon 25	298	exon 45	290	exon 5	282	exon 55	290	exon 15	282	exon 75	306	exon 35	298
exon 65	306	exon 36	330	exon 56	322	exon 16	314	exon 46	322	exon 6	314	exon 66	338	exon 26	330
exon 76	338	exon 27	370	exon 47	362	exon 7	354	exon 57	362	exon 17	354	exon 77	378	exon 37	370
exon 67	378	exon 38	402	exon 58	394	exon 18	386	exon 48	394	exon 8	386	exon 68	410	exon 28	402
exon 78	410	exon 29	442	exon 49	434	exon 9	426	exon 59	434	exon 19	426	exon 79	450	exon 39	442
exon 69	450	exon 40	483	exon 60	475	exon 10	467	exon 50	475	exon 10	467	exon 70	491	exon 30	483
Dp427c	491

Legend
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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