Microarray-based mutation detection in the dystrophin gene

Abstract

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans>2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6 to 10% of males with either DMD or BMD. The remaining 30 to 35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, multiplex ligation-dependent probe amplification (MLPA), detection of virtually all mutations-SSCP (DOVAM-S), and single condition amplification/internal primer sequencing (SCAIP); however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution comparative genomic hybridization (CGH) microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin.

Figure 1

Representative example of dystrophin CGH array design. A sample 240-bp region, including the entire exon 44 and 92 bp of flanking intronic sequence, of the dystrophin gene shows array CGH probe distribution. Each thin blue line represents one probe. Exon 44 is represented by the thick blue line at the bottom of the figure.

Figure 2

Validation of targeted CGH dystrophin array for males and females. The dystrophin coordinates are represented at the top, with exon 1 to 79 from right to left. The representative array results shown here for males and females are displayed in the scatter plot. Each pair shows results of CGH analysis using NimbleGen DNACopy (A) and GLAD analysis (B). 1) 15 Mv - male with duplication of exons 2–4; 2) 5 Mv - male with deletion of exon 44; 3) 2 Mv - male with deletion of exons 17–44; 4) 8 Mv - male with deletion of exons 48–52; 5) 16 Fv - female with deletion of exons 46–55; 6) 20 Fv - female with deletion of exons 49–50; and 7) 23 Fv - female with duplication of exons 18–38.

Figure 2

Validation of targeted CGH dystrophin array for males and females. The dystrophin coordinates are represented at the top, with exon 1 to 79 from right to left. The representative array results shown here for males and females are displayed in the scatter plot. Each pair shows results of CGH analysis using NimbleGen DNACopy (A) and GLAD analysis (B). 1) 15 Mv - male with duplication of exons 2–4; 2) 5 Mv - male with deletion of exon 44; 3) 2 Mv - male with deletion of exons 17–44; 4) 8 Mv - male with deletion of exons 48–52; 5) 16 Fv - female with deletion of exons 46–55; 6) 20 Fv - female with deletion of exons 49–50; and 7) 23 Fv - female with duplication of exons 18–38.

Figure 3

Targeted CGH dystrophin array for clinical samples. The dystrophin gene coordinates are represented at the top, with exon 1 to 79 from right to left. The representative array results shown here for males and females are displayed in the scatter plot. Each pair shows results of CGH analysis using NimbleGen SegMNT. 1) 10 Mc - male with deletion of exon 18 (c.2169−?_2292+?del); 2) 4 Mc - male with deletion of exons 45–54 (c.6439−?_8027+?del); 3) 2 Mc -male with duplication of exon 2–4 (c.32−?_264+?dup); 4) 23 Mc - male with duplication of exons 35–44 (c.4846−?_6438+?dup); 5) 31 Mc - male with a 33-kb deletion in intron 1(c.31+?_32−?del) and an 11-kb deletion in intron 2 (c.93+?_94−?del); 6) 35 Fc - female with deletion of exons 49–50 (c.6615−?_7309+?del); and 7) 33 Fc - female with duplication of exon 44 (c.6291−?_6438+?dup); and 8) 34 Fc - female with duplication of exons 8–11 (c.650−?_1331+?dup).

Figure 4

CGH and MLPA analysis of exon 8–13 (c.650−?_1602+?del) familial deletion mutation. Panel A - Exons 1 to 79 are represented on the CGH scatter plot from right to left. Scatter plots shown are for CGH analysis on the male proband with deletion of exons 8–13 (above) and carrier testing for the proband’s mother for exons 8–13 showing no deletion of exons 8–13 (below). Panel B -Confirmatory analysis of the negative findings for the mother using MLPA showed no deletion of exons 8–12 and a deletion of exon 13. Exon peaks are marked with black arrows, and exon 13 is marked with a red arrow. Other peaks on the panel are internal controls for the MLPA reaction. Panel C - Subsequent sequencing of exon 13 on the maternal sample detected a SNP (c.1554T> A: p.D518E) where the MLPA probe binds. Presence of the SNP interfered with hybridization of the probe, giving the appearance of an exon 13 deletion.

Figure 4

CGH and MLPA analysis of exon 8–13 (c.650−?_1602+?del) familial deletion mutation. Panel A - Exons 1 to 79 are represented on the CGH scatter plot from right to left. Scatter plots shown are for CGH analysis on the male proband with deletion of exons 8–13 (above) and carrier testing for the proband’s mother for exons 8–13 showing no deletion of exons 8–13 (below). Panel B -Confirmatory analysis of the negative findings for the mother using MLPA showed no deletion of exons 8–12 and a deletion of exon 13. Exon peaks are marked with black arrows, and exon 13 is marked with a red arrow. Other peaks on the panel are internal controls for the MLPA reaction. Panel C - Subsequent sequencing of exon 13 on the maternal sample detected a SNP (c.1554T> A: p.D518E) where the MLPA probe binds. Presence of the SNP interfered with hybridization of the probe, giving the appearance of an exon 13 deletion.

Figure 5

Dystrophin testing algorithm.