Article Figures & Data
Figures
- Figure 1 Pedigree of Families A–G
Index patient for each family denoted with black arrow. Affected members colored in red, and carriers part colored in red.
- Figure 2 Muscle RNA Studies of DMD in Patients
(A) RNA-seq read coverage of DMD exons in muscle RNA from AII:1, BIV:1, DII:1, EII:1, and GII:1 and 2 GTEx controls. Red arrows indicate the reduction in read depth, which corresponds with the location of DMD structural variants for BIV:1, DII:1, EII:1, and GII:1. (B–G) RT-PCR studies of muscle-derived RNA of patients with splicing abnormalities and 3 male controls (C1, quadriceps, 6.5 years; C2, vastus lateralis, 17 years; C3, unknown, 20 years). Primers used are listed at the bottom right of each gel image and are labeled according to their location (exon; Ex, intron; In, pseudoexon; P) and orientation (forward; F, reverse; R). Bridging primers span a splice junction and are denoted by X/Y, where X and Y are exons the primer spans. All results were confirmed by Sanger sequencing. (B) RT-PCR showing reduced levels of correctly spliced DMD transcript (exons 43 and 44) in AII:1 and BIV:1 compared with controls. AII:1 shows the inclusion of a 128-bp pseudoexon. (C) Primers specific to the 128 bp pseudoexon revealed that the inclusion is specific to AII:1 (Sanger sequencing showed that the faint bands in C1 were non-DMD sequences). Sequencing reveals that faint bands in AII:1 correspond to multiple pseudoexons in DMD incorporated into a minority of DMD transcripts. (D) Various chr8 pseudoexons and LINC00251 exons are included in DMD transcripts as a result of the chr8 insertion in BIV:1. The lowest band detected in all samples in the top gel corresponds to non-DMD sequences. (E) RT-PCR confirms the inclusion of a 84-bp pseudoexon in CII:2 in the majority of DMD transcripts. Normal splicing can only be detected in very low levels in CII:2 by bridging primers. The 92 bp pseudoexon is absent in control samples. (F) RT-PCR of DII:1 confirms that exon 51 is absent from all DMD transcripts. A bridging primer indicates that skipping of both exons 50 and 51 is a low frequency event observed in both controls and DII:1. (G) GAPDH loading controls to indicate that similar concentrations of complementary DNA were used for both control and patient samples. DMD = Duchenne muscular dystrophy; DMD = DMD gene or transcript; RT-PCR = reverse transcription PCR.
- Figure 3 Schematics of Variants Identified in Families A–G
(A) Family A: intronic c.6290+30954C>T (black arrow) creates a cryptic donor splice site, leading to inclusion of a 128-bp pseudoexon (red, within DMD intron 43) into the DMD mRNA, causing a frameshift and stop codon (red arrow) encoded by exon 44 (ex44). Gene direction is demonstrated by gray arrows. Reading frame between exons is shown by shape complementarity. (B) Family B: insertion of 116,284 bp of chr8 (red sequence) into DMD intron 43. The insertion includes LINC00251 exons 1–3 (black outlined exons). A 124-bp sequence of intron 43 of DMD (chrX:32,276,895-32,277,018) is duplicated as part of the structural rearrangement and now flanks the chr8 insertion. In addition, there is an insertion of 13 bp (insGCCTTTGCCCACA, shown in green) adjacent to 1 copy of the 124-bp duplication. mRNA studies show evidence for numerous, different abnormal splicing events from DMD exon 43 to various pseudoexons (red exons) and LINC00251 exons (red exons with black outlines) within the chr8 insertion. Low levels of normal DMD splicing (from exons 43 and 44; blue exons) are also observed. Frame of splicing in pseudoexons and LINC00251 exons not shown. (C) Family C: intronic c.3603+820G>T (black arrow) increases the strength of the polypyrimidine tract leading to use of a cryptic acceptor splice site (3/5 algorithms within Alamut Visual biosoftware predictions; MaxEntScan, NNSPLICE, and GeneSplicer) leading to inclusion of a 84-bp pseudoexon (red, within DMD intron 26) into the DMD mRNA, encoding a stop codon (red arrow) 39 nucleotides into the pseudoexon. Gene direction is demonstrated by gray arrows. Reading frame between exons is shown by shape complementarity. (D) Family D: inversion of DMD exon 51 and flanking adjacent intronic sequence. Flanking the structural rearrangement are 2 intronic deletions (orange 3.5 kb and purple 44 bp) and an insertion of CCAATA (green). mRNA studies show exon 51 skipping, causing a frameshift and a premature stop codon (TAG, encoded by exon 52; red arrow). (E) Family E: A 2.6-Mb inversion on the X chromosome between 2 breakpoints; A in intron 45 of CFAP47, 1.9 Mb upstream of exon 1 of DMD (GRCh37:chrX:35,180,364) and B in intron 18 of DMD (GRCh37:chrX:32,521,892, NM_004006.2). This reverses the orientation of exons 1–18 of DMD, which are now joined to CFAP47 sequences upstream of exon. The DMD gene is in blue, exons dark blue, and introns light blue. Intergenic sequence (non-DMD ChrX in green). (F) Family F: A 4.1-Mb inversion on the X chromosome between 2 breakpoints; A is 3.8 Mb upstream of exon 1 of DMD (GRCh37:chrX:36236087) and B in intron 44 of DMD (GRCh37:chrX:32122714). This reverses the orientation of exons 1–44 of DMD, which are now joined to intergenic sequence upstream of exon 1. This is accompanied by duplication of exons 31–37 (orange) and exons 43 and 44 (purple) around the breakpoint. (G) Family G: A 119.8-Mb inversion on the X chromosome between 2 breakpoints; A in an intergenic region on the q arm of the X chromosome, 118 Mb upstream of exon 1 of DMD (GRCh37:chrX:151,194,962), and B in intron 60 of DMD (GRCh37:chrX:31,379,010, NM_004006.2). This reverses the orientation of exons 1–60 of DMD, which are now joined to intergenic sequence upstream of exon 1. In addition, 2 deletions were identified at these breakpoints; an intronic 63 bp deletion (orange, GRCh37:chrX:31,378,947-31,379,009) and an intergenic 18 bp deletion (purple, GRCh37:chrX:151,194,963-151,194,980). X chromosome displayed in unusual orientation with q arm to the left, so the DMD gene is presented with exons in order. DMD = DMD gene or transcript. mRNA = messenger RNA.
- Figure 4 Western Blot Panel for All Patients
(A.a) Western blot was performed on skeletal muscle from index patients from families A and B (AII:1 and BIV:1) against DYS1 (rod domain epitope) and Rb-DMD (C-terminal epitope) with serial dilutions (1/2, 3/4, 5/6, and 9/10) human control skeletal muscle. Muscle lysate derived from an individual with Duchenne muscular dystrophy and undetectable levels of dystrophin by Western blot (DMD control; deltoid, 14-year-old boy, GRCh37:chrX:32364116G>A, NM_004006.2:c.5530C>T, p.Arg1844*) were added to diluted controls to normalize total protein loading in each lane of the gel. Loading controls: a-actinin-2 and myosin (coomassie). (A.b) Image J23 was used to measure the densities of the patient and serially diluted controls bands to create a standard curve. Quantification of relative dystrophin levels was performed by comparing patient sample densities to the control standard curves across the 3 gels shown. AII:1 demonstrates 15.5% ± 1.9% levels of dystrophin protein relative to controls. BIV:1 demonstrates 9.6% ± 1.7% levels of dystrophin protein relative to control. (B) Western blot analysis on skeletal muscle from patient CII:2 against DYS1 shows undetectable levels of dystrophin compared with controls. Loading controls: myosin (coomassie). (C) Western blot analysis on skeletal muscle from patients DII:1, EII:1, FII:6, and GII:1 against DYS1 compared with human control skeletal muscle. DII:1 shows very low levels of dystrophin. EII:1 FII:6, and GII:1 show undetectable levels of dystrophin. Loading controls: a-actinin-2 and sarcomeric actin. Male controls used: C1, tibialis anterior, 16 years; C2, unknown, 5.5 years; C3, unknown, 14 years; C4, quadriceps, 4.5 years. DMD = Duchenne muscular dystrophy.
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