ARRAY-COMPARATIVE GENOMIC HYBRIDIZATION
RESULTS IN CLINICALLY AFFECTED CASES
WITH APPARENTLY BALANCED CHROMOSOMAL
REARRANGEMENTS
Satkin NB, Karaman B, Ergin S, Kayserili H, Kalelioglu IH, Has R, Yuksel A, Basaran S
*Corresponding Author: Nihan B. Satkin, Ph.D., Department of Medical Genetics, Istanbul University
Faculty of Medicine, Millete Street, 34093, Istanbul, Turkey. Tel.: +90-536-561-0313.
Fax: +90-212-414-2000. E-mail: bilgenihan@gmail.com
page: 25
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DISCUSSION
Molecular karyotyping allowed us to detect genomewide
chromosomal imbalances even in size of kbs using
DNA copy number and/or SNP variation probes [10].
Recent reports have suggested that ABCRs in patients
having abnormal phenotypes can be more complex at the
molecular level than suspected by karyotyping. Therefore,
ABCRs either de novo or familial, should be investigated at
the molecular level, if the phenotype is affected [5,11-15].
Molecular karyotyping reveals the breakpoints of the
rearrangements, especially for inversions and insertions,
more precisely than the karyotyping seen in our cases 10,
14, and 21. As CMA is a genome-wide technique, it allowed
us to detect the imbalances elsewhere in the genome such as in our cases 15, 16, 20, and 21. Eight CNVs at
different locations, apart from the suspected breakpoints,
indicates the complexity of the CCRs and effectiveness
of CMA [16].
No molecular imbalances were observed in our familial
ABCRs (five prenatal, seven postnatal). However,
Gijsbers et al. [16] reported a deletion at the unrelated
chromosome in one case of four familial translocation
cases (25.0%). Sismani et al. [5] determined two CNVs at
the translocation breakpoints in one out of the six familial
translocations (16.6%). In another study from Schluth-
Bolard et al. [14] containing 14 familial cases (seven translocations,
five inversions, two CCRs), imbalance rates
were 14.3% for translocations, 40.0% for inversions and
50.0% for CCR and one inversion case had two different
CNVs, one at the breakpoint and the other one at an independent
region. Tabet et al. [15] reported four familial
translocation and three inversion cases, of which one had
a deletion at the related region of the inversion (14.3%).
Sezin et al. [17] reported four familial cases with no cryptic
imbalance. To sum up, the CNV rate in familial cases was
14.9% in a total of 47 cases. Six of eight imbalanced cases
were at the breakpoints (75.0%), whereas the remaining
two were at unrelated locations (25.0%). These results
show that the risk for imbalances is higher for inversions
and CCRs than translocations, and familial ABCRs can
also have imbalances at the breakpoints or somewhere
in the genome, coincidentally or not. If aCGH reveals no
imbalances in cases presenting with phenotypical findings,
they should be further investigated for monogenic
disorders [18]. In our postnatal series, six of the seven
familial ABCR carriers had a consanguineous marriage,
which enhances the possibility of monogenic disorders.
The rate of de novo imbalances detected by CMA was
higher in postnatal than prenatal cases (50.0 vs. 25.0%) in
this study, because postnatal cases were preselected due
to their distinct abnormal phenotype. In our center, CMA
was offered in the presence of pathological fetal ultrasound
findings after normal fetal karyotypes, but also in cases
with de novo ABCRs, even if ultrasound findings were normal.
A prenatal case presented with polyhydramnios and
abdominal cysts had a 1.2 Mb deletion containing GRID1,
Mir_544, AK097624, LOC100507470, AX746544, 7SK
genes at the 10q23.1 band. Van Bon et al. [19] reported 12
patients having a deletion on 10q22.3-q23.3. One patient
had an atrioventricular septal defect (AVSD), dysmorphic
features, diaphragmatic eventration and undescended testes,
and CMA revealed a small interstitial GRID1 deletion
comprising exons 5 to 8, 0.2 Mb in size. The GRID1 gene
has been also reported to be associated with schizophrenia [20,21]. Because the ultrasound findings of our case was
not compatible with the cases of Van Bon et al. [19], and
deletions of this region have not yet been associated with
a particular phenotype, this deletion was interpreted as
variations of unknown significance (VUS). In another
case, presenting with only choroid plexus cysts, a 5.5 Mb
duplication was inherited from the healthy mother. Surprisingly,
prenatal cases presenting with major structural
anomalies (cases 31, 32, 33 and 34) had no imbalances by
CMA, and further studies to search for monogenic disorders
were planned. De Gregori et al. [11] also reported 14
de novo prenatal translocations including one presenting
with abnormal USG findings, one had normal USG but
delayed psychomotor development at 6 months of age and
none of them had an imbalance by CMA. Evangelidou et
al. [22] reported one familial and four de novo translocation
cases with abnormal USG findings; all cases were also
found to be normal by CMA. Due to the limited number
of published cases, experiences with prenatal cases are not
sufficient to estimate a risk figure for prenatal ABCRs with
abnormal USG findings. When de novo ABCRs detected
by fetal karyotyping, independent from the phenotypical
findings, molecular karyotyping should be applied, because
clinical findings are limited with only ultrasound
examination.
Variations of unknown significance detected in prenatal
CMA studies are confusing and genetic counseling
is difficult. Therefore, to decrease the VUS possibility, the
standards and guidelines of 2013 [9] recommends reporting
the variations that are >400 kb for both deletions and
duplications in prenatal and postnatal studies by whole
genome array platforms. Different countries have their approach
for prenatal cases, such as including genes number
[>18 genes (Belgium), size of imbalances (>500 kb for deletions
and >1.0 Mb for duplications (Canada)], choosing
array platforms reducing densities of the probes [23-25].
In this study, we initially used higher CMA resolution
(1.4 Mb) for both postnatal and prenatal cases. Detected
imbalances in prenatal cases were over 1.0 Mb, which
could be confirmed by 180 K resolution. In some cases, a
lower resolution can cause difficulties to search the whole
genome or the size of the CNV may differ from the actual
size due to the limited number of probes [26].
Parental array studies showed that the detected duplication
at 16p11 in case 26 was inherited maternally, which,
coincidentally, was on one of the ABCR-involved chromosomes.
Imbalances (size range 579 kb to 4.6 Mb) including
deletions and duplications at 16p11 showing incomplete
penetrance/variable expressivity, can be associated with
global development delay, behavioral problems, epilepsy,
autism [27]. The critical region comprising breakpoints
four and five (BP4-BP5) (600 kb, chr16; 29.6-30.2 mb-
HG19) called 16p11.2 microdeletion/microduplication
syndrome is reported in about three in 10,000 [28]. Most
of the 16p11.2 microduplications (70.0%) are familial, and
the clinical findings are variable from severe to mild [29].
The size of microduplication in our case was larger than the
critical region, the healthy carrier mother decided to continue
the pregnancy. Clinical evaluations of the newborn
revealed normal results, clinical follow-up controls of the
baby were planned. In general, the detection of duplications
is troublesome, either cytogenetically or clinically
due to the nonspecific and variable phenotypes. Based on
these experiences, it was expected that the frequency of
the duplications is higher by CMA than in karyotyping
in patients with behavioral problems or autism without
distinct dysmorphic features [30]. Therefore, genetic counseling
for de novo duplications detected prenatally is still
challenging.
When the data of published postnatal de novo series
are combined, 35.9% of the apparently balanced de novo
translocations were, in fact, unbalanced. This rate was
20.0% in our series (Table 3). Imbalance rate in inversions
was as much as in translocations (37.0%). There was only
one inversion patient in our series and she had a deletion.
As expected, the highest submicroscopic imbalance rate
(74.4%) was observed in CCRs. In our series, it was 62.5%.
The difference (~12.0%) could be explained by the presence
of two Xp21-autosome translocation cases in our
series. Both cases manifested female carriers of Duchenne
muscular dystrophy (DMD) had also been investigated by
multiplex ligation-dependent probe amplification (MLPA)
(MRC-Holland, Amsterdam, The Netherlands) and by next
generation sequencing (NGS) techniques, and no mutations
were detected. The causing factor was not imbalances
at the molecular level, but the possibly skewed X
inactivation, where the X chromosome carrying the normal
dystrophin gene is preferentially inactivated to save
the translocated autosomal segment on the derivative X
chromosome [31]. If these two cases were excluded, the
imbalance rate would be 83.3% in our series.
The imbalances at unrelated regions of the breakpoints/
chromosomes observed in five CCR cases (cases 10,
15, 16, 20, 21) demonstrate the advantage of genome-wide
array studies. Multiple breaks and consecutive micro-deletions
in these cases show the complexity of the CCRs, and
support that de novo CCRs occur due to the multiple breaks
in the genome and increased genomic instability. The term
‘chromothripsis’ is used to describe ‘chromosome shattering,’
which means chromosomes are first fragmented into
many pieces and then the fragments stick back together
randomly due to the DNA repair processes [32]. Analysis
of CCRs in patients with congenital disorders showed that
chromothripsis is not applicable for all complex germline rearrangements. The term ‘chromosoanasynthesis,’ which
means repeated chromosome synthesis was suggested by
Liu et al. [33] for describing multiple template switch
events that may occur during the germline CCRs formation
process. Many more CCRs should be investigated
by whole-genome analysis to increase the knowledge and
understanding the underlying occurrence mechanism of
the CCRs [34-36].
Altogether, the imbalance rate of de novo ABCRs is
24.6% at the breakpoints and 19.2% at different regions
apart from the breakpoints. These rates can be used in
genetic counseling related to de novo ABCRs.
In conclusion: 1) the current study supports previous
studies, that the cryptic genomic imbalances are high
(43.8%) in patients of de novo ABCRs with abnormal
phenotype. 2) Patients presenting with multiple congenital
anomalies, intellectual disability, and carrying de novo
or familial ABCRs, whatever the type of rearrangement
(translocation, inversion, or CCR), should be studied systematically
by CMA. 3) Chromosomal microarray investigation
is more important in cases with de novo ABCRs
detected prenatally, as the results are essential for genetic
counseling and decision-making process of the parents.
Studies focused on prenatal ABCR carriers with abnormal
ultrasound findings are limited and no submicroscopic imbalance
was reported. Abnormality rate detected by CMA
was 25.0% in our de novo prenatal cohort. 4) Chromosomal
microarray promises to detect genome-wide imbalances at
the kb level. However, further studies are needed for other
mechanisms such as gene fusions or gene disruptions that
might explain the phenotype of affected ABCR carriers.
All unexplained patients should be examined for single
gene disorders.
Declaration of Interest. The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of this article.
Funding. This study was supported by the Istanbul
University Scientific Research Project Unit [Project Nos:
34325, 8563 and 25099].
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