DISCUSSION
DMD is one of the most prevalent among the rare diseases
(Mendell et al., 2012). It
is caused by sequence variants in DMD , the largest gene of the
human genome, which is located in a genomic region with high rates of
recombination. These characteristics make DMD highly susceptible
to mutation (Nguyen &
Yokota, 2019). The current work presents a familial case of DMD with a
cxSV, a multi-technique and bioinformatic approach was implemented in
pursuit of 2 major aims. The first one focus on the molecular diagnosis
and genetic assessment of the family, including manifesting carrier and
prenatal studies. While the second one, centered on finding possible
explanations of the origin of SVs in DMD .
The design of the prenatal diagnosis molecular strategy was based on the
previously detected DMD deletion of exons 45-54 in the affected
alive male (III4). On the one hand, we performed a CVS maternal cell
contamination study to determine the usefulness of the fetal material.
On the other hand, we decided to evaluate the presence of the deletion
by 2 different means, MLPA and the co-segregation of the deletion by
STRs. Therefore, 2 STRs mapping within the deletion and 2 flanking it
were selected for the haplotype study.
A priori, we were expecting that II1 and II3 shared III4´s
deletion, so it was a surprise when studies revealed the existence of 2
SVs in the family. A duplication of exons 38-43, shared by all family
members, probing to be the ancestral familial causing mutation. Also,
the well-known deletion, found only in III4, suggesting being the second
mutational event i.e. a de novo alteration. Therefore, this case
works as an example of the importance of retesting historic patients
diagnosed by multiplex-PCR, where deletion´s boundaries might not have
been precisely determined or other SVs could have been missed out,
affecting eligibility and effectiveness of mutation-dependent therapies.
We would like to hallmark the usefulness of the old-fashioned STRs
segregation studies as a tool to identify homologous recombination and
STRs retraction/expansion events, determine parental origin of the
haplotypes, corroborate sample´s gender and discard CVS maternal cell
contamination. In this case, STRs revealed a recombination between STR44
and STR62 in III4, a retraction of STR49 in III1, the existence of 2
different paternal X-chromosomes in II1 and II3, an estimation of female
gender in the fetus and the exclusion of CVS contamination.
Regarding the AR-Assay, the skewed XIP observed in II1 suggests that the
fully active X-chromosome should be the maternal one, carrying the
duplication, giving the presence of DMD symptomatology. On the contrary,
as expected, her asymptomatic sister (II3) had a random inactivation.
Because of the STRs and MLPA results, II1 and II3 were already expected
to present different paternal X-chromosomes but to share the maternal
one, as both carried the duplication. Remarkably, they did not share anyAR allele, corroborating the double paternity and suggesting
another recombination event that separated the DMD causative
mutation from the AR allele.
Although, AR-assay has been recently validated on amniotic fluid for DMD
symptomatology prediction, we could not test the XIP in the fetus, as
their parents did not agree on performing a second puncture
(He et al., 2019). There
are conflicting reports in the literature about the heritability of the
XIP (Renault et al.,
2007; Viggiano et al., 2017). Therefore, future XIP studies may be
needed in case any clinical symptoms arise in III1.
Several authors reported the occurrence of non-contiguous
rearrangements, within the same DMD allele, with frequencies up
to 2% (Kerr et al., 2013).
In our studied cohort, we estimated a cxSV rate of 1.4% (6/437 DMD
patients), encompassing deletions-duplications, non-contiguous
duplications and large deletion plus a 20pb insertion. The present case
offered an extra advantage, the possibility of establishing a mutational
timeline thanks to familial segregation study of the detected cxSV.
Molecular diagnosis encompasses the study of index cases, usually
lacking a complete familial analysis. The obtained molecular alterations
have a “photo effect”, like a picture of the resulting rearrangement
at a specific moment careless of timeline determination. This lack of
information is more obvious when dealing with cxSVs, where is impossible
to differentiate between 2 concurrent alterations and 2 different
mutational events that take place in different times/generations.
Studying chronologically these cxSVs could be useful to unravel if the
first alteration acts as a predisposing factor to the latter.
Focusing on our second aim, centered on finding the molecular mechanisms
underlying the origin of the observed SVs, we decided to precisely
determine their breakpoints. For this purpose, we used several practical
approaches, including SNP-array, WGS and customized PCRs. Followed by a
bioinformatic approach, screening the surroundings of the SVs’
breakpoints for the identification of DNA DSB stimulator motifs, which
have been found statistically significantly more frequent at breakpoint
junctions than expected by chance according to Abelleyro et al(2020) (Abelleyro et al.,
2020).
While deletion breakpoints were neatly and accurately identified by
SNP-array and LR-PCR, the characterization of duplication breakpoints
was particularly challenging, as the SNP-array could not certainly
delimitate the single/double-doses transitions. It was only after WGS
analysis that the identification of the reads mapping on the tandem
duplication breakpoint junction permitted its precise delimitation.
Therefore, these results remark the importance of revalidating the SV´s
boundaries detected by SNP-array.
Regarding the tandem segmental duplication, this event may be adequately
explained either by MMBIR or FoSTeS models, both involving de
novo DNA synthesis. However, considering the principle of maximum
parsimony and the complexity of the event, characterized by at least 3
strand invasions preceded by replication fork collapses and facilitated
by microhomologies, supports a FoSTeS mechanism over MMBIR (Figure 5A).
In addition, the necessary DNA strand break collapses may have been
stimulated by the presence of Jurka hexa-nucleotides, secondary
structures as well as repetitive elements at relevant locations
(Vissers et al., 2009).
Concerning the deletion, it seems to occur de novo concurrently
with a meiotic recombination event switching STRs alleles between
maternal homologous, given the coincident X-chromosome location of both
molecular events. The most likely scenario suggests that these two
concomitant molecular events may actually represent a single
inter-chromosomal recombination event, which could have taken place
either in II3’s oocyte meiosis or in II3´s oogonia mitosis, making the
deletion inheritable. The above mentioned evidence indicates that II3
carries the DMD duplication, but does not have the deletion in
peripheral blood, suggesting that the complex series of events took
place in her germline resulting in a chimeric X-chromosome with STRs
alleles from both progenitors (I1 and I2) and both SVs in phase.
Furthermore, our recombination hypothesis relies on the presence of
repetitive elements relatively near, both, 5’ and 3’ deletion
breakpoints, supporting a putative inter-chromosome non-allelic pairing
structure or ectopic synapsis suitable for a localized recombination
(Figure 4) (Liu et al.,
2012).
At the molecular level, the breakpoint junctions of the deletion point
out the hypothesis of a classical NHEJ, given that the event does not
present any molecular characteristic that could imply an association
with a DNA replication dependent mechanism. We detected just a
single-thymine of microhomology, which does not provide sufficient
evidence to sub-classify the event as MMEJ (Figure 5B). Moreover,
supporting the classical NHEJ model DNA DSB stimulating motifs were
found at both deletion breakpoints, such as Jurka hexa-nucleotides and
SAR on the 5’ breakpoint and Ig heavy chain switch region on the 3’
deletion breakpoint
(Gale et al.,
1992; Jurka, 1997; Rabbitts et al., 1981). Other possible stimulators
for DBS, possibly exerting its action, could be LTRs, STRs and highly
stable secondary structures, which have also been detected at both
deletion breakpoints. Furthermore, 2 of these motifs might be related,
as long STRs have the ability to form non-B-DNA structures.
Resuming the chronology of the molecular events that gave rise to the
cxSV, the duplication could have favored the approach of the regions
involved in the deletion and stabilize an unequal pairing between the
duplicated X-chromosome and its unduplicated counterpart. This theory
supports both previously mentioned hypothesis for the origin of the
deletion. On the one hand, the duplication could have helped the
previously mentioned repetitive elements in the formation of the ectopic
synapsis during female gametogenesis. While, it could have also been
implicated in the unequal pairing that resulted in NHEJ mechanism.
On the other hand, we want to highlight the important role that
secondary structures must be exerting in the origin of SVs, given that
we found them in 7/8 breakpoints. Its implication for DSB formation is
suggested by the fact that if we randomly select 50pb throughout the
genome it is very likely that they form secondary structures, but if we
simulate random breakpoints and analyze 25bp at each side of it, the
chances of finding secondary structures involving them are greatly
reduced (Abelleyro et al.,
2020).
In conclusion, the strategy implemented in the present work allowed us
to provide an accurate diagnosis and genetic assessment, enabling early
diagnosis and the selection of the appropriate standard of care and
mutation-specific treatments. Finally, the thorough characterization of
the SV´s breakpoints and the analysis of their molecular scars not only
widen the understanding of the molecular mechanisms involved in their
generation, but also may help the development of new therapeutic
approaches.