The role of muscle biopsy in analysis of the dystrophin gene in Duchenne muscular dystrophy: experience of a national referral centre
Introduction
The dystrophin gene is of unparalleled size and complexity for a gene of such major clinical importance, consisting of 79 exons over 2.3 Megabases of genomic DNA and yielding a messenger RNA with an 11 kb coding region. However, following the description of the gene structure [1] mutational analysis for Duchenne and Becker muscular dystrophy was rapidly introduced because of the fortuitous finding that the majority of mutations consisted of large genomic deletions covering several exons [2], [3], [4].
The difference in phenotype between Duchenne muscular dystrophy (DMD) and the milder Becker muscular dystrophy (BMD) was explained in most cases by the ‘frame shift hypothesis’ [5]. If the deletion maintains an open reading frame a phenotype of Becker is observed. This corresponds to the finding of at least some (albeit mutant) dystrophin protein in a muscle biopsy. If the deletion shifts the translational reading frame of the transcript or create a stop codon Duchenne will be the outcome and no intact protein will be present [6].
Deletions account for ∼65% of cases and are currently detected using multiplex PCR primers which cover 18 exons [7], [8]. This has allowed widespread diagnostic applications which are particularly important in view of the devastating nature of the disease, the high new mutation rate [9] and the high frequency of somatic and germ line mosaicism in carrier mothers of isolated cases [10], [11].
The remaining approximately 35% of mutations provide a considerable technical challenge, not least because of the large number of exons, multiple promoters [12] and the low ratio of coding to intronic sequence. In the study of Tuffery et al. [13] point mutations arose preferentially on the grandpaternal chromosome during spermatogenesis, so most mothers are predicted to be carriers which makes molecular diagnosis particularly pressing.
Recent technical advances in automated sequencing have led to a protocol for rapid direct sequence analysis which relies on the amplification of a large number of exons at a single set of PCR temperatures. Although applicable to any multi-exon gene, a set of primers suitable for dystrophin analysis has been developed [14]. Undoubtedly this will be of use in laboratories with high throughput sequencing facilities. The primers have been designed to include the invariant sequences at intron/exon boundaries but will miss mutations deep in introns [15]. Although the method will detect sequence changes close to the splice points it will be difficult to interpret their functional outcome. For example, several cases have been published in which splice defects within the consensus sequence lead to the use of a nearby cryptic splice site rather than the anticipated exon-skipping [16]. In view of the importance of the structure and residual amount of the dystrophin mRNA in determining the phenotype, mRNA studies are an essential complementary step.
Efforts to use dystrophin mRNA as the template for mutational analysis were hampered by its low expression in peripheral blood. However, Chelly et al. [17] described so-called illegitimate transcription of the dystrophin gene and this was exploited by Roberts et al. [18] and Gardner et al. [19] to show that, using nested primers, dystrophin transcripts could be amplified from peripheral blood lymphocytes. Early studies showed that most dystrophin point mutations in DMD disrupted the translational reading frame [20] either through nonsense mutations or small frameshifting deletions or insertions [21], [22] and so the protein truncation test (PTT) developed by Roest et al. [23] which monitors an in vitro transcript for termination codons, is a suitable method for rapid screening. We now routinely use mRNA from muscle biopsies as the starting material for reverse transcription and mutational analysis. The muscle biopsies are a normal part of the diagnostic work up of the patients for dystrophin analysis.
We report here our experience over more than 10 years during which we have identified mutations in 79 out of 89 cases previously shown not to have gross rearrangements. No mutations have yet been found in the remaining 10 cases. We propose a general protocol for dystrophin point mutation analysis based on the spectrum of mutations found.
Section snippets
Referral of patients
Patients have been referred from the 10 of the 15 laboratories in France which perform standard dystrophin gene deletion analysis. If no deletion has been detected, a muscle biopsy and either DNA or a peripheral blood sample (for mutation confirmation on genomic DNA) are sent from the proband and his mother.
Protocol
The protocol is outlined in Fig. 1.
Dystrophin analysis
Muscle biopsies were analysed immunohistochemically for the presence of dystrophin using antibodies against the N-terminal (NCL-DYS3, aa 321–494),
Protein truncation assay
The PCR2 products are analysed in a PTT assay as previously described [16]. Protein products were produced using the TNT®-T7-Coupled Transcription/Translation System (Promega, Madison, WI, USA).
Results
The 79 mutations found in the 89 analysed samples (detection rate of 88.8%) are presented in Table 1. The most striking feature is the extreme heterogeneity both in position and type of mutation and there are only three examples of recurrent mutations (p.Arg1666X, p.Arg3190X, p.Arg3391X). Each of these occurs at a CpG site which is a known hot spot for mutations and indeed each of the three has been previously reported in the Leiden database (www.dmd.nl).
Forty-seven (71.2%) of the 66 small
Discussion
Initial mutational analysis of the dystrophin gene is particularly easy because of the high proportion of deletions which, due to the gene localisation on the X chromosome, can be readily detected by multiplex PCR. Analysis of the remaining nearly one third of mutations has been particularly challenging because of their heterogeneity and the size of the gene. The large series reported here shows that they divide into nonsense mutations (mutation of an amino acid to a stop codon) (39.3% of all
Acknowledgements
We also thank other clinicians who referred occasionally some patients or for making samples available to us: M. Blayau (Rennes), P. Boisseau (Nantes), C. Butori (Nice), H. Carrier (Lyon), M. Koenig (Strasbourg), I. Creveaux (Clermont-Ferrand), F. Fellman (Besançon), A. Joannard (Grenoble), J. Lespinasse (Chambery), B. de Martinville (Lille), CA. Maurage (Lille), L. Michel-Calemard (Lyon), V. Paquis (Nice), N. Philip (Marseille), M. Tardieu (Paris), and M. Smith (Australia).
The Association
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