FIGURE LEGENDS
Figure 1. Molecular diagnostic studies. A . Familial pedigree and STRs segregation analysis. “del”: deletion; “-”: STR not analyzed; “dotted squares”: alleles implicated in STR49 retraction in III1. B. Upper figure, schematization of the DMD cxSV from III4. Bottom figure, MLPA result highlighting the deletion with a red dotted rectangle and the duplication with a blue full-line rectangle. C . AR-assay electropherograms and XIP for II11 and II3. “Mock”: Non-digested DNA; “HpaII”: HpaII-digested DNA; “n”:AR exon 1 STR allele; “AUC”: Area Under Curve.
Figure 2. Characterization of the cxSV in III4. A . SNP-array analysis. Deletion and duplication breakpoints are marked by rectangles and zoomed in to show the SNPs reference sequence (rs) from 5’ and 3’ interval breakpoints. “BAF”: B-allele frequency; “LRR”: Log R ratio (LRR); “+”: SNP present at normal dosage; “-”: SNP with null dosage; “++”: SNP with increased dosage; “black triangles”: LR-PCR primers (AF1, AR1 and AR2) location for deletion breakpoints delimitation.B. WGS IGV analysis. Upper figure, circles on the coverage track (blue histogram) indicate regions of interest encompassing the SVs breakpoints, where the deletion is characterized by null dosage (no bars) and the duplication by an increased dosage (higher bars). Relevant SNP-array SNPs are also depicted on the coverage track. Insets show a zoom in of the breakpoint area, inner circles highlight key chimeric reads presenting mismatches with the Reference Genome. Bottom figure, schematization of the key chimeric reads with their identification number (left, deletion chimeric reads; right, duplication chimeric reads), black spots delimit the portion of the read that maped on each side of the breakpoint junction. “+”: SNP present at normal dosage; “-”: SNP with null dosage; “++”: SNP with increased dosage; “black triangles”: PCR primers (DUP_F and DUP_R) location for duplication breakpoints delimitation. C . Sanger sequencing of the breakpoints junctions from deletion (upper figure) and duplication (bottom figure). Obtained DNA sequences are aligned with DMDReference Sequence (NG_012232.1, GRCh38.p13). The deletion present a 1 bp (“T”) of microhomology highlighted in boldface. The duplication shows a 18 bp insertion at the specific head-to-tail fusion marked in boldface.
Figure 3. Secondary structure analysis of SVs breakpoints. A 50 bp interval of the reference genome (GRCh38.p13) centered on each 5’ and 3’ breakpoints (blue box) of the deletion (A ) and the duplication (B ) were analyzed by Mfold. Also, the sites of directed or inverted insertions of the duplication were included in the analysis. “bkp”: breakpoint; “∆G”: Gibbs free energy [kcal/mol] associated with each secondary structure.
Figure 4. Schematic model of the putative recombination mechanism originating the deletion. A. Wide arrow indicates theDMD gene oriented towards the telomere (Xp tel, little circle), opposite to the centromere (X cen, line arrowhead). B. Paternal and Maternal Homologous X-chromosomes. “Black rectangles”: maternalDMD exons; “White rectangles”: paternal DMD exons; STR”n”: where number indicate their intronic location (introns 62, 49, 45 and 44); “STRs in bold”: Maternal alleles; “STRs underlined”: Paternal alleles; “Black zigzag lines”: eventual crossover breakpoints; “Chevrons”: Repetitive elements detected by Dfam algorithm (“Vertical lines”: LINEs, “Dotted”: LTRs, “Black”: MIRs, “White”: Alus, “Horizontal lines”: others); “Up-pointing black triangle”: Other relevant tandem repeats possibly involved in DNA ruptures as breakpoint stimulators [(TG)n and (T)n]. C.Unequal inter-chromosome recombination model. Schematic representation of a Non-allelic Non-homologous recombination event explaining both the origin of the deletion and the recombination between STR62 and STR44, observed from segregation analysis. D: Resulting III4´s chimeric X-chromosome structure, showing the deletion as the absence of STR45 and STR49.
Figure 5. Schematic representation of possible molecular mechanisms originating the SVs. A. Duplication FoSTeS mechanism. From top downwards. The 1st collapse of the replication fork may stop the DNA synthesis on the leading strand allowing the invasion of the lagging strand (on top), acting as a template to synthetize 7 bases representing an inverted insertion aided by the “biting” of the 3’ nucleotide “T”. A 2nd collapse stops the replication, permitting a 2nd template switching to the leading strand, near the 1st collapse´s sequence and not aided by microhomologies. This may proceed with the synthesis of an additional tract of 11 bases. Finally, a 3rd collapse may stop the synthesis at a dinucleotide “AG” on the 3’ end, which led to a 3rd template switching, invading the lagging strand several kb upstream defining the starting point of the duplicated region. “Black star”: Collapse sites of the replication fork. B. Deletion NHEJ model. DNA breaks located in both maternal homologous X-chromosomes were repaired by a classical route of NHEJ, where the gap structure may have been stabilized by a single bp “T” microhomology. “Black triangles”: 5’ and 3’ DNA breakpoints of the large deletion.
Supplementary Figure 1. Schematic representation of the chimeric reads involved in the SVs. Each figure shows the key WGS chimeric reads involving the deletion (A ) and the duplication (B ) breakpoints. Each read is shown with their respective ID number. The reads below represent sequences target of the deletion (A) and the duplication (B) breakpoints, relevant X-chromosome coordinates (NG_012232.1, GRCh38.p13) are depicted.