Clinical diagnosis of heterozygous dystrophin gene deletions by fluorescence in situ hybridization
Introduction
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder which is lethal in early adulthood and affects one in ~3500 newborn males [1]. DMD is caused by mutations in the dystrophin gene, the largest gene found in nature to date, measuring 2.3 Mb and encoding a 14 kb mRNA 2, 3, 4. Becker muscular dystrophy (BMD) is an allelic but milder form, generally caused by mutations which do not disrupt the dystrophin gene's reading frame. In about two-thirds of DMD/BMD patients, large deletions or duplications are found in the dystrophin gene 3, 4. The deletions are not evenly distributed but are concentrated in a major and a minor hot spot around exons 45–47 and 7–9, respectively 3, 4, 5, 6.
Routine deletion detection in DMD/BMD males is performed using a simple and efficient multiplex polymerase chain reaction (PCR) assay amplifying 18 exons of the gene, focused on the most frequently deleted exons 5, 6, 7. The exact borders of the deletions are usually defined by hybridization of dystrophin cDNAs to Southern blots 3, 4. In females the detection of deletions, either using PCR or Southern blotting, is complicated by the presence of a second X-chromosome, carrying a normal gene. Discrimination between single and double dosage with both methods is feasible but technically demanding, requiring excellent technical performance and careful data interpretation 4, 8, 9.
In many diagnostic laboratories, fluorescence in situ hybridization (FISH) is part of the standard analytical tools available [10]. Since it allows the two X chromosomes to be examined individually, FISH to metaphase preparations constitutes an excellent alternative for diagnosing deletions in DMD/BMD heterozygotes 11, 12. Here we report the design of an optimal set of cosmids for deletion detection by FISH and its testing in a sample of 30 females, nine controls and 21 heterozygotes carrying deletions in the dystrophin gene. Based on the deletion frequency of individual dystrophin exons, as determined from 570 deletions (242 from São Paulo [13] and 328 from Leiden), six cosmid probes were selected from a contig spanning the 2.3 Mb dystrophin gene [14] in such a way that they would detect the highest possible number of deletions. To verify the robustness of the approach, the analysis was performed blindly and the results were compared with deletion data derived from molecular studies, i.e. multiplex PCR and Southern blot analysis. The efficiency and accuracy of the approach is discussed.
Section snippets
Samples
Patient phenotypes, either Duchenne muscular dystrophy (DMD), intermediate muscular dystrophy (IMD) or Becker muscular dystrophy (BMD), were classified as described by Jennekens [15]. The DMD heterozygotes belong to families ascertained through ABDIM (Brazilian Association for Muscular Dystrophies). Only heterozygotes that were known to carry deletions were chosen for the analysis. They were considered deletion carriers if they either constitute an obligatory link between two affected males,
Results
The probes used for the FISH experiments were selected on the basis of the distribution of deletions in affected patients. The data were collected from the São Paulo and Leiden diagnostic laboratories. The São Paulo set contains 231 deletions (174 DMD, 28 intermediate (IMD) and 29 BMD), the Leiden set 34 duplications and 278 deletions (202 DMD/IMD, 76 BMD). Fig. 1 shows the frequency of deletions per exon. Fig. 1 clearly shows a bipartite curve, with a major deletion hot spot centered around
Discussion
To our knowledge, this study describes the largest set of DMD/BMD deletions reported so far, including 570 deletions and 34 duplications. Comparison of the deletion profiles (Fig. 1) revealed two minor differences between the São Paulo and Leiden patient sets. First, the frequency of deletions in the minor hot spot is about 10% higher in Brazil. Second, the absolute peak lies at exon 46–47 in the Leiden and exon 50 in the São Paulo set. We believe that both differences can be explained by the
Acknowledgements
We thank Eric Meershoek for technical assistance, Lau Blonden for isolating the cosmids used in this study, and Dr. Rita de Cassia, M. Pavanello, Dr. Suely K. Marie, Marta Canovas, Antonia Cerqueira and Constancia Urbani for clinical evaluation of the patients. The studies were in part financed by grants from the `Muscular Dystrophy Group of Great Britain and Northern Ireland' (MDG), the Dutch `Prinses Beatrix Fonds', and the Brazilian institutions FAPESP, CNPq, PRONEX, FINEP, IAEA.
References (15)
- et al.
Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals
Cell
(1987) - et al.
FISH in genome research and molecular diagnostics
Curr Opin Genet Dev
(1995) - et al.
Diagnostic criteria for Duchenne and Becker muscular dystrophy and myotonic dystrophy
Neuromusc Disord
(1991) - et al.
Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus
Nature
(1989) - et al.
Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene
Nature
(1986) - et al.
Topography of the DMD gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications
Am J Hum Genet
(1989) - et al.
Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification
Nucl Acids Res
(1988)
Cited by (10)
Gene mutation spectrum analysis of 170 patients with duchenne/bay esian muscular dystrophy in southwest of China
2016, Journal of Sichuan University (Medical Science Edition)Gene diagnosis for nine Chinese patients with DMD/BMD by multiplex ligation-dependent probe amplification and prenatal diagnosis for one of them
2009, Journal of Clinical Laboratory AnalysisPGD for dystrophin gene deletions using fluorescence in situ hybridization
2006, Molecular Human ReproductionPrenatal diagnosis of de novo X;autosome translocations
2004, Clinical GeneticsTherapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients
2003, Human Molecular GeneticsLong-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation
2002, Journal of Clinical Investigation
- 1
Present address: Laboratory of Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.