The DMD gene was the first gene identified by reverse genetics. Mutations in the gene cause Duchenne (DMD) and Becker (BMD) muscular dystrophies. Both the frequency and devastating nature of these conditions make DMD one of the most extensively studied genes among the rare genetic disorders [1–3].
This intense research has provided molecular tools for the identification of the causative mutation in about 98% of patients, combining MLPA to detect exonic deletions/duplications (75–80% of mutations) and direct sequencing to identify small mutations (up to 20% of mutations). Nevertheless, some mutations remain unidentified. Furthermore it is well known that the large size (2.2 Mb) of the gene makes it prone to complex rearrangements which are impossible to define precisely using routine molecular diagnostic techniques.
As a consequence, there are a considerable number of DMD/BMD patients in whom no causative mutation has been identified. This impacts on genetic diagnosis, genetic prognosis, clinical confirmation, carrier detection, prenatal diagnosis and genetic counselling for the families involved.
Furthermore, the recent opportunities in terms of innovative therapeutic approaches [4, 5] highlight the relevance for patients and families of obtaining a correct molecular diagnosis, which is required in order to be included in innovative trials. Indeed the increased availability of experimental but highly mutation specific therapies, summarised in the concept of "personalised medicine" [6, 7], makes the identification of private mutations in the DMD gene necessary to be eligible for these trials.
In the last few years genome scanning technologies have enabled the detection of previously unrecognised large (>1 kb) copy-number variations (CNVs) in human DNA. While many of these variants do exist as polymorphisms, some of them can change the copy number of critical genes or genomic regions, or alter gene regulation and underlie monogenic disorders, developmental abnormalities and a variety of complex genetic disorders [8–11].
Therefore there is a wide consensus on the potential of array-CGH to determine CNVs for research and clinical purposes, in terms of providing robust and precise measurement of CNVs, scalability and very high resolution .
Although CGH was initially considered as a strategy for improving cytogenetic resolution by detecting fine chromosome imbalances [13, 14], recently other applications have been envisaged such as cancer studies , complex syndromes, mental retardation, Mendelian disorders and polygenic traits .
The flexibility of CGH arrays is also due to the availability of both commercial and custom arrays, which are designed on demand, therefore it is possible to investigate any region of interest with the appropriate resolution.
Dhami et al.  designed a single strand PCR-based CGH array in order to detect exon deletions/duplications in a few genes, including DMD.
This strategy demonstrated the ability to identify CNVs, however, in the same way as MLPA and other techniques, it only investigated coding regions.
We have applied the CGH technique in a novel full-gene approach which investigates the presence of CNVs in the entire genomic region of the DMD gene. Our custom designed high density-comparative genomic hybridisation array (DMD-CGH) based on in situ synthesis of 60 mer probes with intervals of 260 bp, allowed us to obtain a full map of CNVs in the gene, including the non coding regions which have not been investigated previously.
Our studies allowed us to validate our array for accurately detecting previously identified rearrangements, to define intronic breakpoints precisely and to identify three pathogenic purely intronic CNVs. We corroborated the CGH studies by RNA analysis, therefore validating the significance of the gene imbalances identified. Transcription analysis of the full DMD transcript furthermore disclosed three rare splicing mutations due to small intronic changes, missed by the CGH analysis.