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.