Whole-Genome Sequencing Identified New Structural Variations in the DMD Gene That Cause Duchenne Muscular Dystrophy in Two Girls

Figure 1. Histological analysis including immunohistochemistry of the skeletal muscle tissue of patient 1. (a) Atrophic and round fibers alternating with hypertrophic fibers in a Gömöri trichrome stain (original magnification ×200). (b) Loss of dystrophin 1 on the sarcolemma of many fibres as well as reduced staining intensity on many of them in a Dys1 (rod domain) stain (original magnification ×200). (c) Loss of dystrophin 2 on the sarcolemma of many fibres as well as reduced and irregular/incomplete staining intensity on many of them in a Dys2 (C-terminus) stain (original magnification ×200). (d) Severe loss of dystrophin 3 on the sarcolemma of many fibres approaching complete loss of staining in a Dys3 (N-terminus) stain (original magnification ×200). (e) Loss of nNOS on the sarcolemma of myofibres in an nNOS stain (original magnification ×200). (f) Compensatory upregulation of utrophin with different intensity on the sarcolemma of the myofibres in an utrophin stain (original magnification ×200).

Figure 2. Immunofluorescence staining with antibodies against Dys1 (a), Dys2 (b), and Dys3 (c) showing the reduced dystrophin in most muscle fibers. Compensatory upregulation of utrophin (d).

Figure 3. (a) Sequence surrounding the breakpoint (g.31628083) in intron 54 of DMD on chromosome X. Black lines show the sequence of the intron that matches the reference sequence; pink lines show parts of the sequence of chromosome 5 not matching the reference sequence of DMD. (b) Sequence surrounding the breakpoint (g.40072329) on chromosome 5. Black lines show the sequence of chromosome 5 that matches the reference sequence; pink lines show the sequence of intron 54 of the DMD gene not matching the reference sequence on chromosome 5. (c) PCR fragments containing the breakpoints on chromosomes 5 and X in patient 1 (P1) and a healthy control (ctr) beside a 100 bp ladder (M). The clean blank values were removed from the gel image for better clarity. (d) Electropherogram of the fragment amplified with primers Chr5-F and DMD-Int54-R compared to the reference sequence (Ref Seq) of DMD and chromosome 5. The red line indicates the breakpoint. The guanine in DMD outlined in red is deleted due to the translocation. On the left, patient 1 sequence matches the reference of the DMD gene, and on the right, patient 1 sequence matches the reference on chromosome 5.

Figure 4. (a) Sequence surrounding the breakpoint area (g.32798883–g.32798891) in intron 7 of DMD on chromosome X. Black lines show the sequence of the intron that matches the reference sequence; pink lines show parts of the sequence of chromosome 19 not matching the reference sequence of DMD. (b) Sequence surrounding the breakpoint area (g.39820677–g.39820693) on chromosome 19. Black lines show the sequence of chromosome 19 that matches the reference sequence; pink lines show the sequence of intron 7 of the DMD gene not matching the reference sequence on chromosome 19. (c) PCR fragments containing the breakpoints on chromosomes 19 and X in patient 2 (P2) and a healthy control (ctr) beside a 100 bp ladder (M) Two additional controls and the clean blank value were removed from the gel image for better clarity. (d) Electropherogram of the fragment amplified with primers DMD-Int7-F and Chr19-R compared to the reference sequence (Ref Seq) of DMD and Chr19. The sequence outlined in red is gained due to translocation. The breakpoint lies in this area. On the left, patient 2 sequence matches the reference of the DMD gene, and on the right, patient 2 sequence matches the reference on chromosome 19.

Figure 5. (a) GTG banding of chromosome 5 (Chr 5) and X (Chr X) of index patient 1. The derivative chromosomes are marked with arrows. (b) FISH with two different probes labeling SHOX on the terminal site of the Xp of the normal chromosome X in red and the centromere of chromosome X with DXZ1 in green. The white arrow points to the derivative chromosome 5 displaying SHOX signal at the terminal region of 5p. (c) FISH with probe ASP (arm-specific paint)-Xp which marks the complete short arm of the normal X chromosome in red (green arrow). The white arrow marks the derivative X chromosome showing partial labelling close to the centromere. Note near-complete labelling of the short arm of the derivative chromosome 5.

Figure 6. GTG banding of chromosome 19 (Chr 19) and X (Chr X) of index patient 2. The possible breakpoints involving the translocation between the long arm of chromosome 19 and the short arm of chromosome X are marked with the red arrows.

Figure 7. Fragment analysis of the CAG repeat in the AR gene of patient 1, (a) undigested and (b) digested with HhaI. The peak with a size of 279 bp is almost completely lost through digestion (red arrow). (c) Fragment lengths of the undigested AR repeats of the patient’s mother and (d) her father. The repeat lengths of 276 bp and 279 bp in the mother represent the two different alleles of the mother. The father has only one allele; the smaller stutter pre-peaks are artificially created by this technique.

Figure 8. Fragment analysis of the CAG repeat in the AR gene of patient 2, (a) undigested and (b) digested with HhaI. The peak with a size of 279 bp is almost completely lost through digestion (red arrow). The peak previous to the 282 bp peak is an artifact (stutter peak of the 282 bp repeat fragment) resulting from the method used. (c) Fragment lengths of the undigested AR repeats of the patient’s mother and (d) her father. The repeat lengths of 282 bp and 285 bp in the mother represent the two different alleles of the mother. The father has only one allele; the smaller stutter pre-peaks are artificially created by this technique.