Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization

Abstract

Duplications and deletions are known to cause a number of genetic disorders, yet technical difficulties and financial considerations mean that screening for these mutations, especially duplications, is often not performed. We have adapted multiplex amplifiable probe hybridization (MAPH) for the screening of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. We have engineered probes for each of the 79 exons of the DMD gene, and we analyzed them by using a 96-capillary sequencer. We screened 24 control individuals, 102 patients, and 23 potential carriers and detected a large number of novel rearrangements, especially small, one- and two-exon duplications. A duplication of exon 2 alone was the most frequently occurring mutation identified. Our analysis indicates that duplications occur in 6% of patients with DMD. The MAPH technique as modified here is simple, quick, and accurate; furthermore, it is based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should not require new equipment. Together, these features should allow easy implementation in routine diagnostic laboratories. Furthermore, the methodology should be applicable to any genetic disease, it should be easily expandable to cover >200 probes, and its characteristics should facilitate high-throughput screening.

Figure 1

Outline of the MAPH technique. Probes are prepared such that all can be amplified with one primer pair. After overnight hybridization to immobilized genomic DNA, unbound probes are removed by stringent washing. Bound probes are then released and amplified in a quantitative manner. By fluorescent labeling and capillary electrophoresis, it is possible to both discriminate and quantify each probe. Changes in peak heights correspond to copy-number changes (i.e., deletions and duplications).

Figure 2

Example of trace patterns obtained from an unaffected male individual. The numbers refer to DMD exon numbers: “1.x” and “2.x” (where x is the exon number) refer to BRCA1 and BRCA2, respectively, and “NF2” denotes a probe homologous to the first exon of the NF2 gene. Asterisks (*) indicate control peaks, and unlabeled peaks indicate noise. Probes range in size from 151 bp (DMD exon 34) to 602 bp (DMD exon 2).

Figure 3

Patterns obtained from analysis of patients by use of probe set A. A, Male patient's duplicated exon 2, male patient's duplicated exons 14–21, and male control individual. B, Female carrier's duplicated exons 52–54 and female control individual.

Figure 4

Analysis of different patient samples. A, Male patient L1's duplicated exons 8–13, with SD 0.05. B, Female carrier D36's deleted exons 10–46, with SD 0.05 C, Female carrier D70's duplicated exons 3–7, with SD 0.06.

Figure 5

Independent mutations detected during the present study. Vertical bars represent the 18 exons tested using the Chamberlain et al. (1988) and Beggs et al. (1990) kits. A, Mutations detected in samples in which point mutations and deletions had been excluded, mainly by multiplex PCR. B, All other mutations detected.