Clinical diagnosis of heterozygous dystrophin gene deletions by fluorescence in situ hybridization

Abstract

Two-thirds of patients affected by Duchenne or Becker muscular dystrophy (DMD/BMD) carry large intra-genic deletions in the dystrophin gene. In males, the deletions can be efficiently detected using multiplex polymerase chain reaction (PCR) and Southern blotting. In contrast, deletion detection in carrier females is complicated by the presence of a normal gene copy on the second X-chromosome. We have analyzed the boundaries of 570 deletions and 34 duplications in the dystrophin gene identified in the São Paulo and Leiden diagnostic laboratories. The data were used to select an optimal set of cosmid probes for the detection of the most frequently deleted areas of the dystrophin gene. Six cosmids were evaluated in fluorescence in situ hybridization (FISH) experiments to assess deletions in 21 heterozygous deletion-carriers and nine controls. No discrepancy was found between the FISH analysis and the molecular data, demonstrating the accuracy of the technique for carrier detection in Duchenne and Becker muscular dystrophy.

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.