FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.
ABSTRACT The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.
- SourceAvailable from: Albino Bacolla[Show abstract] [Hide abstract]
ABSTRACT: Different types of human gene mutation may vary in size, from structural variants (SVs) to single base-pair substitutions, but what they all have in common is that their nature, size and location are often determined either by specific characteristics of the local DNA sequence environment or by higher order features of the genomic architecture. The human genome is now recognized to contain "pervasive architectural flaws" in that certain DNA sequences are inherently mutation prone by virtue of their base composition, sequence repetitivity and/or epigenetic modification. Here, we explore how the nature, location and frequency of different types of mutation causing inherited disease are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment. The mutability of a given gene or genomic region may also be influenced indirectly by a variety of noncanonical (non-B) secondary structures whose formation is facilitated by the underlying DNA sequence. Since these non-B DNA structures can interfere with subsequent DNA replication and repair and may serve to increase mutation frequencies in generalized fashion (i.e., both in the context of subtle mutations and SVs), they have the potential to serve as a unifying concept in studies of mutational mechanisms underlying human inherited disease.Human Mutation 08/2011; 32(10):1075-99. · 5.21 Impact Factor
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ABSTRACT: As prostate cancer progresses to the lethal castration resistant and metastatic form, genetic and epigenetic adaptation, clonal selection, and evolution of the tumor microenvironment contribute to the emergence of unique biologic characteristics under the selective pressure of external stresses. These stresses include the therapies applied in the clinic or laboratory and the exposures of cancers to hormonal, paracrine, or autocrine stimuli in the context of the tumor micro- and macro-environment. The androgen receptor (AR) is a key gene involved in prostate cancer etiology and oncogenesis, including disease development, progression, response to initial hormonal therapies, and subsequent resistance to hormonal therapies. Alterations in the AR signaling pathway have been observed in certain selection contexts and contribute to the resistance to agents that target hormonal regulation of the AR, including standard androgen deprivation therapy (ADT), anti-androgens such as enzalutamide, and androgen synthesis inhibition with abiraterone acetate. One such resistance mechanism is the synthesis of constitutively active AR variants lacking the canonical ligand binding domain. This review focuses on the etiology, characterization, biologic properties, and emerging data around the clinical characteristics of AR variants, and suggests approaches to full-length AR and AR variant biomarker validation, assessment, and systemic targeting in the clinic.Endocrine Related Cancer 05/2014; · 5.26 Impact Factor
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ABSTRACT: Identification of the isochromosome 12p (i(12p)) associated with Pallister-Killian syndrome is complicated by the low frequency of this supernumerary chromosome in PHA stimulated peripheral blood lymphocytes, and frequently requires cytogenetic analysis of fibroblast cells. Recently, it has been shown that array CGH techniques are able to detect tetrasomy 12p in peripheral blood, even when not identified by traditional cytogenetic techniques. We studied 15 patients with a previous cytogenetic and clinical diagnosis of Pallister-Killian syndrome using genome-wide SNP arrays to investigate the ability of this platform to identify the i(12p) in blood and tissue. Array analysis verified tetrasomy 12p in all samples from fibroblasts, but was only able to detect it in 46% of blood samples. The genotyping information available from the SNP arrays allowed for the detection of as low as 5% mosaicism, as well as suggesting a Meiosis II origin for the isochromosome in the majority of patients. Analysis of the percentage of abnormal cells with patient age at time of study suggests that the frequency of the i(12p) decreased with age in blood, but not in fibroblasts. These highlight the power of SNP arrays in detecting and characterizing the isochromosome 12p in Pallister-Killian syndrome as well as underscoring the important utility of traditional cytogenetic techniques. © 2012 Wiley Periodicals, Inc.American Journal of Medical Genetics Part A 11/2012; · 2.30 Impact Factor
FoSTeS, MMBIR and NAHR at the human proximal
Xp region and the mechanisms of human Xq
George Koumbaris1, Hariklia Hatzisevastou-Loukidou2, Angelos Alexandrou1,
Marios Ioannides1, Christodoulos Christodoulou1, Tomas Fitzgerald3, Diana Rajan3,
Stephen Clayton3, Sophia Kitsiou-Tzeli4, Joris R. Vermeesch5, Nicos Skordis6,
Pavlos Antoniou1, Ants Kurg7, Ioannis Georgiou8, Nigel P. Carter3and Philippos C. Patsalis1,∗
1Department of Cytogenetics and Genomics, The Cyprus Institute of Neurology and Genetics, Nicosia 2370, Cyprus,
2Laboratory of Cytogenetics, First Pediatric Department, Hippocration General Hospital, Thessaloniki 54642, Greece,
3The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK,
4Department of Medical Genetics, University of Athens, St Sophia Children’s Hospital, Athens 11527, Greece,5Centre
for Human Genetics, University Hospital, Catholic University of Leuven, 3000 Leuven, Belgium,6Pediatric Endocrine
Unit, Makarios III Hospital, Nicosia 1474, Cyprus,7Institute of Molecular and Cell Biology, University of Tartu, Tartu
51010, Estonia and8Medical School, University of Ioannina, Ioannina 45110, Greece
Received January 5, 2011; Revised and Accepted February 18, 2011
The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS)
and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze com-
plex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the
long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization
(aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and
MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion
and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based
mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mech-
anisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-alle-
lic homologous recombination between palindromic low copy repeats and highly homologous palindromic
LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-
associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent
recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region
as a chromosomal rearrangement hotspot.
Mammalian autosomal chromosomes undergo homologous
recombination during meiosis, ensuring proper chromosomal
segregation and providing the means for increasing genetic
diversity and for repairing chromosomal damage. While for
autosomes, a homologous chromosome is always available
for pairing during meiosis, human sex chromosomes face par-
ticular challenges. The lack of a homologous Y chromosome
in male meiosis has led to the progressive decay of the Y
chromosome and the evolution of massive palindromic low
copy repeats (LCRs or segmental duplications) in the male-
specific region of the Y chromosome (MSY), which harbor
critical genes for spermatogenesis (1). Even though these
palindromes predate the human-chimpanzee divergence, they
exhibit sequence identity .99% suggesting that they are
undergoing extensive sequence homogenization by gene con-
version, thus providing a mechanism for maintaining the
∗To whom correspondence should be addressed at: Department of Cytogenetics and Genomics, The Cyprus Institute of Neurology and Genetics, 6,
International Airport Avenue, Nicosia 2370, Cyprus. Tel: +357 22392600; Fax: +357 22358237; Email: firstname.lastname@example.org
# The Author 2011. Published by Oxford University Press. All rights reserved.
For Permissions, please email: email@example.com
Human Molecular Genetics, 2011
HMG Advance Access published March 8, 2011
at Library on March 9, 2011
integrity of critical spermatogenesis genes in the absence of
homologous recombination (1). The MSY palindromes also
predispose to chromosomal rearrangements since they can
act as substrates for non-allelic homologous recombination
(NAHR), resulting in non-reciprocal exchanges and the for-
mation of isodicentric Y chromosomes which are responsible
for a significant proportion of cases with spermatogenic
failure, Turner syndrome and sex reversal (2).
In the case of the X chromosome, while a homologue is
available for pairing and recombination during female
meiosis, the same challenges to those faced by the Y chromo-
some are present in male meiosis where only the pseudoauto-
somal regions of the mammalian sex chromosomes can pair
and recombine. Also, while the pericentromeric region of
the human X chromosome is not enriched for segmental dupli-
cations (3,4), in analogy to the MSY, the proximal Xp region
is highly enriched for large and highly homologous palindro-
mic segmental duplications which harbor genes that are
expressed mainly or exclusively in testes (5). The proximal
Xp palindromes, like the MSY palindromes, predate the
human-chimpanzee divergence and yet exhibit very high
arm-to-arm sequence identity (5). The structural similarities
between the MSY and the proximal Xp palindromes and the
occurrence of analogous non-pathogenic recombination pro-
cesses in both the MSY and proximal Xp palindromes, as
evidenced by the extensive arm-to-arm sequence homogeniz-
ation, prompted us to hypothesize that analogous processes
of pathogenic copy number change are likely to operate in
proximal Xp. In order to test this hypothesis and investigate
whether the proximal Xp palindromes catalyze the formation
of isochromosomes of the long arm of chromosome X
(i(Xq)), we analyzed a large number of Turner syndrome
patients carrying i(Xq) chromosomes. Most i(Xq) chromo-
somes have breakpoints in proximal Xp, consist of mirror-
image Xq arms and lack most or all of the Xp arm. The
i(Xq) is the most common human isochromosome and the
most frequent structural abnormality in Turner syndrome
(6,7). Previous studies have established that i(Xq) chromo-
somes can be of either paternal or maternal origin (8,9) with
approximately equal frequencies and that i(Xq) formation is
not associated with increased parental age (8). It has also
been shown that the majority of i(Xq) are dicentric isochromo-
somes and consist of identical long arms, suggesting that they
originate from a single X chromosome both in the male and
female germline, and that most of them do not arise by centro-
mere misdivision (8–10). The mechanism of i(Xq) formation
The detailed analysis of 34 i(Xq) chromosomes allowed us
to gain insights into the mechanisms of i(Xq) formation and
identify specific genomic architectural elements which cata-
lyze the formation of i(Xq) chromosomes via recombination
and replication-based mechanisms.
Characterization of i(Xq) chromosomes by STR analysis
We first investigated whether the i(Xq) chromosomes are
formed by an interchromosomal or intrachromosomal mechan-
ism, by undertaking the amplification of a series of highly
polymorphic short tandem repeat markers (STRs) spanning
the X chromosome (Supplementary Material, Table S1).
Homozygosity was observed at all tested Xp STRs. This is
consistent with the deletion of almost the entire Xp arm on
the isochromosome. In all but two cases, one or two different
alleles were observed for the Xq markers. These results are
consistent with an intrachromosomal mechanism involving
sister chromatids. Interchromosomal recombination between
homologous X chromosomes would lead to the presence of
three alleles for most Xq markers, one derived from the long
arm of the normal X chromosome and two different ones
from the two different Xq arms of the i(Xq). In two cases,
only one allele was detected at all tested loci due to very
high mosaicism for a 45,X cell line. In another case, three
alleles were observed at one Xq marker and in an additional
case three alleles were detected at two markers. These findings
suggest that the i(Xq) in this study consist of two identical
arms, which are derived from sister chromatids. In the two
cases where triallelic patterns were observed for one and two
STRs, respectively, the possibility that the isochromosome
originates from two homologous chromosomes cannot be
excluded. However, since in these cases one or two alleles
were observed at the majority of Xq STRs, the observed
triallelic pattern of these markers is more likely caused by
rare variants and/or allelic recombination at these loci.
High-resolution characterization of i(Xq) chromosomes by
whole-genome tiling path array comparative genomic
Conventional cytogenetic methods such as karyotyping and
fluorescence in situ hybridization (FISH) cannot efficiently
distinguish monocentric i(Xq) from dicentric i(Xq) which
harbor two centromeres in very close proximity. In order to
unambiguously distinguish monocentric i(Xq) from dicentric
i(Xq), we characterized the breakpoints of all 34 i(Xq) by
using a whole-genome tiling path array (WGTPA) (11,12).
We were able to identify the breakpoints of all 34 cases
within two to four consecutive array clones (breakpoint resol-
ution ?200–500 Kb) (Fig. 1D).
In 15 out of the 34 i(Xq) cases, the array clones correspond-
ing to the short arm of chromosome X revealed a deletion of
the entire euchromatic portion of the short arm, and all the
long arm clones revealed a duplication of the entire long
arm. Representative X-chromosome WGTPA data from five
cases are shown in Supplementary Material, Figure S1A.
Figure 1. Microarray-determined breakpoints of idic(Xq) in relation to regional genomic architecture. (A) Expanded view of the 7 Mb proximal Xp region in
which all the idic(Xq) breakpoints were localized. (B) Breakpoint annotation in relation to the underlying genomic architecture. The arms of the LCR palin-
dromes i(Xq)-P1 to i(Xq)-P6 are denoted as paired green triangles (P1–P6). The LINE, L1 palindrome i(Xq)-LINEP is shown as a red triangle pair
(LINEP). Breakpoint sequences in regions of no extended homology are denoted as blue rectangles. (C) High-resolution custom oligo aCGH breakpoints of
idic(Xq). Red bars denote proximal Xp sequences that are duplicated on the idic(Xq). Lighter red bars denote breakpoint intervals. Color gradients in four
cases indicate breakpoint uncertainty (see text). (D) WGTPA breakpoints of idic(Xq). Blue bars denote proximal Xp sequences that are duplicated on the
idic(Xq). Lighter blue bars denote breakpoint intervals. (E) Segmental duplications in proximal Xp. The large, complex LCR cluster which includes
i(Xq)-P1 to i(Xq)-P5 can be seen between ?52 and 53 Mb.
2 Human Molecular Genetics, 2011
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Human Molecular Genetics, 2011 3
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These 15 i(Xq) cases have breakpoints within centromeric het-
erochromatin and are thus defined as cytogenetically mono-
The remaining 19 of the 34 i(Xq) cases had WGTPA-
determined breakpoints in euchromatic sequences within a
7 Mb region of proximal Xp and are thus defined as cytogen-
etically dicentric (idic(Xq)). The breakpoints of 8 of these 19
cases were localized within a complex cluster of large, highly
homologous, direct and inverted LCRs (ChrX: ?52–53 Mb).
The breakpoints of the remaining 11 cases were localized
more proximally in an LCR-poor part of this 7 Mb region
where only one large and highly homologous LCR palindrome
is present (Fig. 1A, B, D, E).
Refinement of cytogenetically monocentric i(Xq)
breakpoints by real-time polymerase chain reaction
In order to refine the WGTPA-determined breakpoints of the
cytogenetically monocentric i(Xq) and determine whether
they are structurally monocentric or structurally dicentric,
primers were designed and quantitative real-time polymerase
chain reaction (RT-PCR) was undertaken in the last unique
sequence on proximal Xp, which lies deep into the centromeric
heterochromatin, 606 Kb from ZXDA, the most proximal gene
on Xp. This sequence lies within monomeric DXZ1 sequences
close to the DXZ1 Array Junction which separates monomeric
DXZ1 DNA from the higher order, DXZ1 array (13,14) (Sup-
plementary Material, Fig. S1B). The functional centromere of
chromosome X was previously delimited within the DXZ1
array (14). In most cases, we were able to determine whether
meric alpha satellite DNA or more proximally, potentially
function. In four cases, the breakpoints were localized within
monomeric alpha satellite sequences, thusthese i(Xq)are struc-
the breakpoints lie within monomeric alpha satellite sequences,
or DXZ1 sequences due to very low mosaicism; however, these
i(Xq) are apparently structurally dicentric, as evidenced by the
high mosaicism for a 45,X cell line. Finally, in eight cases, the
breakpoints were localized more proximally, and lie either
within the most proximal part of the monomeric DXZ1
sequences, or within the DXZ1 array. Four of these eight
cases are mosaic for a 45,X cell line, while the other four have
a 46,X,i(Xq) karyotype. Considering that only structurally
dicentric isochromosomes can behave as functionally dicentric
can only be functionally monocentric) and give rise to a second
four of these eight i(Xq) can be structurally dicentric.
Ultra-high-resolution characterization of idic(Xq)
breakpoints by custom oligonucleotide array comparative
The WGTPA comparative genomic hybridization (CGH)
identification of a common 7 Mb breakpoint interval and the
considerable overlap of many idic(Xq) breakpoints with
large and highly homologous LCR palindromes prompted us
to investigate the potential involvement of specific palin-
dromes in idic(Xq) formation and determine the fine-scale
structure of the idic(Xq) breakpoint junctions. Two custom
high-resolution (mean resolution:
ultra-high-resolution (mean resolution: ?20 bp) microarrays
targeting the 7 Mb breakpoint region and individual WGTPA-
determined breakpoints were designed. While typical microar-
ray designs do not include probes in repetitive genomic
regions, in this study we followed an alternative approach
carefully designing array probes which provide comprehen-
sive coverage of both unique sequences in proximal Xp and
also of palindrome arms and spacers.
the junctions of highly homologous large LCR palindromes
defined as i(Xq)-P1 to i(Xq)-P6 (Fig. 1B and C, Supplementary
region was associated with at least one idic(Xq) breakpoint and
targeted more than once. The breakpoints of two idic(Xq) were
localized to i(Xq)-P2. This is the biggest palindrome in the
regionandconsistsof142 Kbarmsthathave .99.9%sequence
the idic(Xq) from the twin patients along with the breakpoint of
another unrelated case were localized to i(Xq)-P4. This is the
second biggest palindrome in the region. It consists of 60 Kb
arms that share .99.5% sequence identity. The two arms are
separated by a very small 0.2 Kb spacer. Besides their very
ized by additional complexity. The proximal junction of the
massive i(Xq)-P2 palindrome is flanked by one of the few
remaining gaps in the human reference genome, and the arms
and spacers of both palindromes are partially duplicated and
organized in complex duplicon clusters in this region and in
other parts of the X chromosome. The breakpoint of another
case was localized in i(Xq)-P3. This palindrome consists of
29 Kb arms that have .99.8% sequence identity and are separ-
ated by a 22 Kb spacer. Additional complexity is also present at
this palindrome as both arms and the spacer are duplicated in
multiple positions in this region, and the palindrome in its
entirety comprises the central section of i(Xq)-P2. The break-
points of cases GK28 and GK17 were localized to the junctions
of i(Xq)-P1 and i(Xq)-P5, respectively. These two palindromes
flank the complex LCR cluster. The i(Xq)-P1 flanks the LCR
cluster distally and consists of 36 Kb arms that have sequence
identity .99.8% and are separated by a 100 Kb spacer. This
palindrome does not exhibit additional complexity and has an
breakpoint-associated palindromes. The i(Xq)-P5 flanks the
LCR cluster proximally and consists of 39 Kb arms that have
97.9% sequence identity and are separated by a 15 Kb spacer.
Finally, the breakpoint of the last palindrome-associated
idic(Xq) was localized more proximally at i(Xq)-P6. This is
the most proximal large and highly homologous palindrome
and consists of 27 Kb arms that have sequence identity
.99.8% and are separated by a 13 Kb spacer. Overall, a prefer-
ence for high sequence identity, large arm size and small spacer
length was observed, as the palindromes that exhibited these
characteristics were targeted more than once. The presence of
unique sequences at the spacers of i(Xq)-P1, i(Xq)-P5 and
4 Human Molecular Genetics, 2011
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i(Xq)-P6, and the absence of additional complexity at the arm
sequences of these palindromes allowed the characterization
of the palindrome-specific breakpoint sequences (Fig. 2, Sup-
plementary Material, Figs S2 and S3). Sequences immediately
distal to the breakpoint palindromes are deleted and sequences
immediately proximal to the palindromes are duplicated on
the isochromosomes. More importantly, as can be seen in
Figure 2, Supplementary Material, Figures S2 and S3, the
Figure 2. The fine-scale structure of palindrome-associated idic(Xq) breakpoints. (A) idic(Xq) breakpoint characterized by WGTPA CGH. Highlighted break-
point clones exhibit intermediate ratios in relation to duplicated and deleted clones. (B) Expanded view of highlighted breakpoint region showing
non-repeat-filtered aCGH data from the high-resolution oligo array (GK28-HRO) and repeat-filtered aCGH data from the ultra-high-resolution oligo array
(GK28-UHRO_RF) in relation to i(Xq)-P1. Light-red highlighted probes represent sequences immediately distal to the left palindrome arm and are deleted.
Light-blue highlighted probes represent sequences immediately proximal to the right palindrome arm and are duplicated. Light-yellow highlighted probes rep-
resent sequences corresponding to the palindrome arms and unique-sequence spacer and are present in one copy on the isochromosome. For clarity, chromosome
X is not shown to scale.
Human Molecular Genetics, 20115
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palindrome arms and spacer are intact on the isochromosome
(present in one copy). These results suggest that crossovers
(COs) between palindrome arms are likely to be the initiating
event giving rise to the idic(Xq) that have palindrome-
associated breakpoints. The extensive complexity of i(Xq)-P2,
i(Xq)-P3 and i(Xq)-P4, which are characterized by complex
duplication patterns and a reference sequence gap, and also
the high mosaicism for a 45,X cell line made such determi-
nations not possible in cases GK14, GK37 and GK63.
In 4 of the 19 idic(Xq) cases, the breakpoints were localized
within a LINE, L1 rich region. The high-resolution aCGH
revealed the same breakpoint in all four cases. The breakpoint
was localized to the junctions of a LINE, L1 palindrome
defined as i(Xq)-LINEP (Fig. 1, Supplementary Material,
Fig. S4). This palindrome consists of two almost identical
?5.5 Kb LINE, L1PA3 repeats arranged in an inverted orien-
tation. The two repeats have .99% sequence identity and are
separated by a very short spacer which consists of 30 bp of
overlap between the two inverted L1s. This is the most recom-
binogenic structure we identified in this study and catalyzes
the formation of four idic(Xq).
The breakpoints of the remaining 6 of the 19 idic(Xq) cases
were localized in regions of no extended homology (Fig. 1,
Supplementary Material, Fig. S5). The breakpoint of one of
these cases could not be refined extensively due to very high
mosaicism for a 45,X cell line. Another case had a breakpoint
in a direct intrachromosomal segmental duplication. The
breakpoints of the remaining four cases were localized in
interspersed repeat-rich sequences.
Characterization of non-LCR associated idic(Xq)
breakpoints by sequencing
Unique primers were designed and RT-PCR was undertaken in
order to refine and further characterize the idic(Xq) chromo-
somes whose breakpoints were localized in regions of no
extended homology. Based on the custom oligo aCGH and
RT-PCR results, primers spanning the breakpoints were
designed and breakpoint junctions were obtained by PCR in
three cases and were cycle sequenced.
Microhomologies of 2–5 bp were identified at the break-
points of all three cases (Fig. 3). The microhomologies are
present in inverted orientation on opposite strands at the prox-
imal and distal breakpoint junctions. A microhomology of
2 bp (GC) is present at the breakpoint of case GK16. The prox-
imal junction is located in an SVA repeat and the distal junc-
tion is located in unique sequences (Supplementary Material,
Fig. S5). The distal and proximal breakpoint junctions of
this idic(Xq) are 3.69 Kb apart. Three base pairs of microho-
mology (GAG) are present at the breakpoint of case GK27.
The proximal breakpoint junction of this case is located in a
SINE, Alu repeat and the distal breakpoint junction is
located in an LTR, ERVL repeat. The breakpoint junctions
are 1.97 Kb apart. Finally, five bases of microhomology
(TTTAT) are present at the breakpoint of case GK32. The
two junctions are 6.82 Kb apart and both reside in LINE, L1
repeats. In all three cases, the chromosomal region between
the proximal and distal junctions (defined as template switch
region) is neither duplicated nor deleted, but present in one
copy on the isochromosome. No additional complexity was
detected at the breakpoints. Even though the breakpoints of
the remaining two cases were thoroughly refined, we were
unable to isolate them by PCR, which suggests that further
breakpoint complexity might be present, thus rendering them
refractory to PCR analysis.
In order to characterize the molecular basis of i(Xq) formation
and investigate the role of the proximal Xp LCR palindromes
in the formation of X-chromosome rearrangements, we ana-
lyzed i(Xq) chromosomes from a large number of Turner syn-
drome cases using high-resolution molecular methodologies
including high- and ultra-high-resolution aCGH, and sequen-
cing. We were able to map the breakpoints of 34 i(Xq)
chromosomes, identify the molecular mechanisms which
may be responsible for the formation of the idic(Xq) and
show that most if not all dicentric i(Xq) are catalyzed by the
human genome’s underlying genomic architecture.
Intrachromosomal recombination between inverted alpha
satellite repeats may be the prominent mechanism for the
formation of cytogenetically monocentric i(Xq)
The breakpoints of 15 out of 34 cases were localized in centro-
meric heterochromatin. Even though crossing over is sup-
pressed near and within centromeres (16) in order to prevent
non-disjunction and chromosome breakage (17,18), unequal
thought to underlie the concerted evolution of centromeric
sequences (13,19,20).Topoisomerase IIa has been shown to
cleave hairpins formed in human centromeric DNA (21).
Such hairpin structures can theoretically form by inverted
sequences, and the recent discovery of a small polymorphic
inversion in chromosome X centromeric sequences (22)
suggests that inversions within centromeric DNA may not be
a rare phenomenon. Also, substantial double-strand break
(DSB) formation was documented near centromeres in
budding yeast (23), and a recent study has found evidence
of widespread gene conversion in maize centromeres where
crossing over is also suppressed (24). It appears that DSB for-
mation is frequent at centromeres and that recombination in
centromeric sequences is regulated at the level of DNA repair.
The i(Xq) chromosomes which have breakpoints in centro-
meric sequences can theoretically result from CO resolution of
NAHR events between inverted centromeric sequences on
sister chromatids. When recombination occurs more proxi-
mally within the DXZ1 array, the resulting isochromosome
would carry a truncated DXZ1 array and would be function-
ally monocentric. A more distal NAHR event within mono-
meric alpha satellite DNA would give rise to a structurally
dicentric i(Xq), carrying two DXZ1 arrays. Depending on
the degree of coordination between the two DXZ1 arrays
and the effect/extent of the poorly understood phenomenon
of centromere inactivation, such a structurally dicentric iso-
chromosome could be either functionally dicentric, or func-
tionally monocentric. Our data are in agreement with
previous reports (8,10) and suggest that the vast majority of
gene conversion are
6 Human Molecular Genetics, 2011
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i(Xq) chromosomes are structurally dicentric. Alternatively,
isochromosome formation by the classical model of centro-
mere misdivision (25) or via a replication-based mechanism
cannot be excluded.
NAHR between palindromic sequences catalyzes the
formation of recurrent-breakpoint idic(Xq) chromosomes
The breakpoints of nine idic(Xq) chromosomes were localized
to the junctions of large and highly homologous palindromes
which in analogy to the MSY palindromes on the Y chromo-
some are enriched for genes that are predominantly or exclu-
sively expressed in testes. Also, the breakpoints of four
idic(Xq) were localized to a LINE, L1 palindrome.
on these findings, and the fact that long palindromic sequences
are recombinogenic structures which can extrude into hairpins
or cruciforms and induce the formation of DSBs via the action
of structure-specific nucleases, we hypothesize that recombi-
nation between the highly homologous arms of proximal Xp
palindrome-associated idic(Xq) and propose a model which
offers a mechanistic interpretation of how these rearranged
for theformation ofthe
chromosomes arise (Supplementary Material, Fig. S6). We
propose that a DSB within one arm of a palindrome can
initiate a NAHR event in the same manner that DSBs initiate
homologous recombination during meiosis. DSB formation
followed by 5′to 3′resection and strand invasion of homolo-
gous sequences on the opposing palindrome arm, on the sister
chromatid and subsequent DNA repair synthesis, ligation and
CO resolution of the resulting double Holliday junctions can
lead to the formation of an isodicentric chromosome and an
acentric fragment. The acentric fragment is eventually lost
due to the lack of a centromere, and the isodicentric chromo-
some is stabilized through inactivation of one of the two cen-
tromeres. Incomplete centromere inactivation is a frequent
phenomenon, and results in mosaic 45,X/46,X,i(Xq) karyo-
A number of testable predictions stem from the proposed
model. First, the resulting isochromosome should carry a
duplication of the entire Xq arm. Second, if the NAHR
event involves two sister chromatids, the two Xq arms
should be identical. Third, the Xp sequences proximal to the
palindrome should be duplicated, and the Xp sequences
distal to the palindrome should be deleted. Finally, the arms
Figure 3. Microhomology-associated breakpoints. (A) Replication of the entire Xq arm and a small part of proximal Xp, represented as a solid horizontal line
above the chromosome X illustration, followed by a template switch (dotted line) more distally and replication in the reverse direction to the end of the chromo-
some can catalyze the formation of idic(Xq) that have non-recurrent breakpoints. Proximal and distal junctions (P.J. and D.J.) represent the sites of replication
disruption and resumption, respectively. The template switch region (T.S.R.) is intact on the idic(Xq) (present in one copy). Microhomologies are illustrated as
vertical red lines. Alternatively, the proximal-distal template switch could occur in the reverse order. (B) Three sequenced breakpoint junctions. Plus strand
proximal reference sequence is shown at the top. Minus strand distal reference sequence is shown at the bottom. Breakpoint sequence is shown in the
middle. Microhomology bases (green letters) are boxed. All sequences are shown 5′–3′.
Human Molecular Genetics, 20117
at Library on March 9, 2011
and the spacer of the breakpoint-associated palindrome should
be intact on the isochromosome (no deletions or duplications
of palindrome sequences). As shown above, all four predic-
tions were experimentally tested and the results support the
Previous studies have localized the breakpoints of isochro-
mosome 17q within large palindromic LCRs on 17p11 (26)
and the breakpoints of the isodicentric Y chromosome
within MSY palindromes (2), suggesting that pericentromeric
LCR palindromes play an important role in isochromosome
formation. A resent study (27) identified breakpoints of eight
idic(Xq) cases within proximal Xp and the breakpoints of
seven of these cases were localized within regions encompass-
ing four of the six i(Xq) palindromes identified here. Even
though palindrome probe coverage was not provided in this
study and thus, the copy number state of sequences within
the palindrome arms and spacers could not be determined,
the emerging breakpoint landscape for the NAHR-catalyzed
i(Xq) chromosomes appears to be characterized by highly
recurrent breakpoints involving most, if not all, of the highly
homologous palindromic LCRs in proximal Xp and a single
highly homologous LINE, L1 palindrome.
recombination, or non-homologous end joining catalyzes
the formation of non-recurrent-breakpoint idic(Xq)
The lack of extended homology at the breakpoints of six
idic(Xq) precludes the possibility of being generated by a
homologous recombination repair mechanism, since extensive
homology of up to 300 bp is needed for homologous recombi-
nation in humans (28). Even though non-homologous end
joining (NHEJ) cannot be excluded as a potential mechanism
of formation, the microhomologies at the breakpoints of three
cases, along with perfect sequence preservation of both the
proximal and distal breakpoint-junction sequences, suggest
that the involvement of a replication-based mechanism is
more likely. The absence of the common ‘information scar’
(29) left at the breakpoints of many NHEJ-repaired sequences
implies that no end processing took place. For these break-
points to have been generated by NHEJ, DSB formation at
different positions in close proximity, at sites of perfect micro-
homology, on the two sister chromatids, would be needed in
all three cases. Such an event would then need to be repaired
distally and not locally by joining together opposite strand
sequences from the two DSB sites. These findings can be
microhomology-mediated replication-dependent recombina-
tion mechanism (30) such as serial replication slippage
(SRS) (31–33), break-induced SRS (34), fork stalling and
template switching (FoSTeS) or microhomology-mediated
break-induced replication (MMBIR) (35,36). The presence
of breakpoint microhomology, the relatively close proximity
of the breakpoint junctions and the nearby presence of
complex LCRs are the hallmarks of the recently described
FoSTeS (35), and MMBIR mechanisms (36). FoSTeS pro-
vides the conceptual and mechanistic framework to explain
the formation of non-recurrent and complex rearrangements
which cannot be explained by NAHR or NHEJ. FoSTeS
events tend to occur in regions where complex genomic archi-
tecture is thought to impede replication fork progression. Dis-
engagement of the lagging strand and annealing at a site of
inverted microhomology at a nearby replication fork moving
in the opposite direction
Fig. S7A), or at any nearby single-stranded sequence having
inverted microhomology with the free 3′-end, followed by
resumption of replication to the end of the chromosome
(FoSTeS X 1) is consistent with our findings in all three
cases whose breakpoints were sequenced.
MMBIR repair is also consistent with our findings. MMBIR
incorporates further molecular mechanistic details based on
the break-induced replication (BIR) repair mechanism and
mediates the repair of single-ended DSBs arising from replica-
tion fork collapse in cells under stress (36,37). In such an
event, if a replication fork encounters a DNA nick, a single-
ended DSB could be generated (Supplementary Material,
Fig. S7Bb, Bc, Bd). Subsequent 5′to 3′resection could
allow the 3′-end to anneal to a nearby single-stranded
sequence which shares inverted microhomology with the
3′-end, and prime DNA synthesis in the opposite direction
(Supplementary Material, Fig. S7Be and Bf). Establishment
of a replication fork with both leading and lagging strand syn-
thesis (Supplementary Material, Fig. S7Bg) would result in the
formation of anidic(Xq)
Five of the six breakpoint junctions in the three sequenced
cases reside within interspersed repeats. Repetitive sequences
have a higher propensity to form non-B DNA structures and
can promote the formation of DNA nicks, DSBs or stall repli-
cation forks inducing chromosomal rearrangements (38,39).
Also, at both the proximal and distal breakpoint junctions of
the three sequenced idic(Xq) breakpoints, sequences which
can form secondary structures when in single stranded form
were identified (Supplementary Material, Figs S8 and S9).
Such sequences can potentially form hairpins or fold into
more complex secondary structures on the lagging strand tem-
plate during replication, where ?200 bp of single-stranded
DNA is needed for Okazaki fragment priming (40). Whether
these structures form in vivo and whether they can cause repli-
cation forks to stall or collapse remains unknown. However, it
is interesting to note that in all six breakpoint junctions where
alternative DNA structures can potentially form, the microho-
mology junctions are located within terminal or internal
hairpin loops/bulges near single-/double-strand transition
regions (Supplementary Material, Figs S8 and S9). Such struc-
tures can be cleaved by structure-specific nucleases such as the
Artemis:DNA-PKcs complex (38,41). Also, since the micro-
homology junctions reside within single-stranded regions,
they can potentially be accessed by collapsed replication
forks for annealing via MMBIR. Nevertheless, since predicted
secondary structures are prevalent throughout the genome, the
actual significance of these observations cannot be currently
Further support for the involvement of a replication-based
mechanism in the formation of the non-recurrent breakpoint
idic(Xq) comes from two recent studies showing that dicentric
and acentric chromosomes in yeast are readily formed by a
replication-based mechanism that involves template switching
and may not require DSBs (42,43). Also, recently, post-zygotically
8 Human Molecular Genetics, 2011
at Library on March 9, 2011
formed human isochromosomes were detected in cleavage-stage
The identification of these non-homologous recombination-
catalyzed isochromosomes suggests that this region is suscep-
tible to both recombination-based and replication-based
rearrangements. While replication-based mechanisms such as
FoSTeS and MMBIR have been shown to catalyze the for-
mation of other chromosomal rearrangements (35,45–47),
this is, to our knowledge, the first report of the involvement
of replication-based mechanisms in the formation of human
isochromosomes. Such replication-based mechanisms can
also provide a potential explanation for the genesis of the
complex segmental duplications found in proximal Xp. Theor-
etically, initial direct or inverted duplications could readily
form via replication-based mechanisms which do not require
extended homology. These initial ‘seeder’ LCRs could then
serially propagate and disperse through repeated NAHR-
catalyzed duplication events giving rise to complex segmental
duplication clusters such as the ones seen in proximal Xp.
Human proximal Xp: a chromosomal rearrangement
A large number of diverse chromosomal rearrangements have
been previously localized in proximal Xp. Approximately 30%
of the non-syndromic X-linked mental retardation genes are
located in Xp11 (48,49) and the recently described recurrent
dup(X)(p11.22-p11.23) which is associated with speech
delay and electroencephalographic anomalies (50) exhibits
breakpoints that overlap with i(Xq)-P4. This palindrome cata-
lyzes the formation of idic(Xq) chromosomes in three cases in
the present study. Also, breakpoints of X-chromosome translo-
cations and more complex rearrangements were previously
mapped within Xp11.2 (51,52). This region is also associated
with cancer. A variant associated with prostate cancer risk was
recently identified in proximal Xp (53), and the reciprocal
translocations t(X;18) have X-chromosome breakpoints in
i(Xq)-P4 and other large LCRs in proximal Xp which harbor
‘cancer antigen’ genes (54). Polymorphic inversions involving
four of the six i(Xq) palindromes (Supplementary Material,
Fig. S10) and overall a significant enrichment for polymorphic
inversions in proximal Xp were previously identified (55).
These data, along with the data presented in this study,
suggest that both interchromatid and intrachromatid recombi-
nation involving the proximal Xp LCR palindromes is a fre-
Classical meiotic homologous recombination can be alter-
natively resolved via non-crossover (NCO) or CO pathways.
NCO resolution of Holliday junctions results in gene conver-
sion, while CO resolution results in crossing over (56).
Considering the mechanistic analogies between meiotic hom-
ologous recombination and NAHR, we postulate that NCO
resolution of recombination events between opposing palin-
drome arms on the same chromatid (Supplementary Material,
Fig. S11Aa) or on sister chromatids (Supplementary Material,
Fig. S11Ba) would lead to gene conversion. Alternatively, CO
resolution of intrachromatid recombination between opposing
palindrome arms (Supplementary Material, Fig. S11Ab) would
lead to inversions, while CO resolution of interchromatid
recombination between opposing palindrome arms would
(Supplementary Material, Fig. S11Bb). We propose that the
extensive arm-to-arm sequence homogenization, the identified
polymorphic inversions and the isochromosome formation
involving the palindromic sequences in proximal Xp constitute
alternative outcomes of recombination events between palin-
The unique biology of the X chromosome and the diverse
genomic architecture of human proximal Xp, which is charac-
terized by a high concentration of recombinogenic structures,
render this region a hotspot for both recombination-based and
evidently replication-based rearrangements which provide sig-
nificant insights into the genesis of not only LCRs, but also of
complex constitutional and somatic rearrangements, such as
the ones seen in genomic disorders and many cancers. The
human proximal Xp region offers an insightful look into
the evolutionary and mutational processes which shaped the
to theformation of dicentricisochromosomes
MATERIALS AND METHODS
The i(Xq) cases in this study consisted of 32 unrelated Turner
syndrome patients that had either a 46,X,i(Xq) karyotype or a
45,X/46,X,i(Xq) karyotype exhibiting various degrees of
mosaicism. The study also included two identical twins with
a 45,X/46,X,i(Xq) karyotype.
Informed consent was obtained from all subjects. The
screening protocols and study design were approved by the
review boards of all institutes involved in the study and by
the Cyprus National Bioethics Committee. DNA was extracted
from peripheral blood using the Gentra Puregene genomic
DNA purification kit (Qiagen, Valencia, CA, USA) and,
where material was available, cultures were setup and karyo-
typing was repeated using standard techniques in order to
confirm the presence of the abnormal chromosome.
Quantitative fluorescent polymerase chain reaction amplifica-
tion was performed as previously described (57). Three
markers were derived from the short arm and five markers
from the long arm of chromosome X. The identity and cytoge-
netic position of these markers are shown in Supplementary
Material, Table S1.
The initial screening of all cases was carried out using a
WGTPA consisting of 26 574 BAC clones covering 93.7%
of the euchromatic sequence of the human genome (11,12).
In summary, 150 ng of test and reference DNA (male or
female) was differentially labeled using the Bioprime Labeling
Kit (Invitrogen, Carlsbad, CA, USA) and hybridized on the
arrays using an automated slide processor (HS.4800, Tecan
Inc., Mannedorf, Switzerland). Two hybridizations were run
for each sample in a dye-swap mode. The arrays were
scanned using the Agilent DNA microarray scanner (Agilent
Technologies Inc., Santa Clara, CA, USA). Fluorescent
Human Molecular Genetics, 20119
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intensities were extracted, dye-swap experiments were fused
and ratios were calculated using the BlueFuse software (Blue-
Gnome Ltd, Cambridge, UK). Aberration calling was carried
out using the BlueFuse software and custom Perl scripts (11).
FISH validation of the WGTPA results was carried out in 11
i(Xq) cases in order to empirically assess the accuracy of the
array results before proceeding with the design of targeted
high-resolution oligonucleotide arrays. In summary, the
WGTPA-determined breakpoint BAC clones were isolated
from the X-chromosome tiling path array library. Probes
were labeled using the Abbott Nick Translation Kit and
Abbott Orange dUTP (Abbott Laboratories, Abbott Park, IL,
USA). Hybridizations were carried out according to the man-
ufacturer’s recommended protocols.
High-resolution and ultra-high-resolution targeted aCGH
Once the breakpoints of the isochromosomes were determined
ultra-high-resolution oligonucleotide arrays (Agilent Technol-
ogies Inc.; NimbleGen Systems Inc., Madison, WI, USA) were
designed in order to refine the breakpoint-junction determi-
nation. The NimbleGen array (custom 385K) was a non-
design covering the proximal region of Xp in which all the iso-
chromosome breakpoints were localized by the WGTPA. The
array consisted of 345 782 probes tiling the region ChrX:
51,000,131–58,597,616 in an unbiased manner (mean resol-
ution: ?20 bp). The Agilent array (custom 44 K) consisted
of 3723 autosomal probes, 75 chromosome Y probes, 39 089
probes covering the region ChrX: 51,373,761–58,598,658
(mean resolution: ?180 bp) and 215 backbone probes cover-
ing the rest of the X chromosome in low resolution. The auto-
somal probes were used for normalization. The chromosome X
backbone probes and chromosome Y probes were used as con-
trols to assess the hybridization efficiency. The custom 44 K
arraywas alsoa non-segmental
non-repeat-masked design and was carefully enriched for
probes in subregions of proximal Xp. These subregions were
identified as isochromosome breakpoint junctions by the
WGTPA. The two microarray designs were not subjected to
repeat masking or segmental-duplication masking in order to
investigate the potential involvement of both segmental dupli-
cations and interspersed repeats in the formation of the iso-
chromosomes. LCR and repeat masking of the data was
applied during data analysis where necessary. The probes on
the custom 44 K array were assigned confidence scores
based on GC content, Tm, sequence complexity, hairpin DG
and homology with the rest of the genome (Agilent Technol-
ogies Inc.). Probe scores were used during data analysis to
filter out poor-performing probes. Hybridizations and analysis
using the custom 385 K array were performed by NimbleGen.
The cases were also screened in-house using the custom 44 K
array following the Agilent CGH protocol with the following
modifications. Test and reference DNA (500 ng each) were
differentially labeled overnight using the Bioprime Labeling
extraction were carried out according to Agilent’s rec-
ommended protocols. Self–self hybridizations were carried
out to empirically assess probe performance. Normalization
and data analysis were carried out using custom scripts. Satu-
rated probes and low-quality probes were removed from the
data based on the results of the self–self control hybridiz-
ations. Repeat masking was carried out using the RepMask
3.2.7 track (58) from the UCSC Genome Browser March
2006 assembly and custom perl scripts. Probe filtering was
performed using probe confidence scores.
In those cases where the array-determined breakpoints were
mapped in alpha satellite repeats, or in clusters of interspersed
repeats, array-based confirmation was not possible. In these
cases, unique primers were designed and RT-PCR was per-
formed to confirm and refine the array-determined breakpoint
junctions. RT-PCR primers were designed using Primer3 (59)
and were BLASTed (60) against the reference genome.
RT-PCR was carried out using the SYBR Green PCR
Master mix (Applied Biosystems, Foster City, CA, USA)
and reactions were run on the ABI 7900 Real-Time PCR
System (Applied Biosystems).
Sequencing and sequence analysis
In three cases, we were able to PCR amplify the breakpoint
junctions by using outward-facing primers. PCR reactions
were carried out using the illustra rTaq DNA Polymerase
(GE Healthcare UK Limited, Little Chalfont, UK). Sequencing
was carried out using the BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems). Sequencing reactions
were run on a 3130xl Genetic Analyzer (Applied Biosystems).
DNA sequences were analyzed by comparing them to the
reference genome using BLAT (61). Secondary structure pre-
dictions were carried out using mfold (62). Two hundred base
pairs flanking the breakpoint microhomology junctions were
analyzed for cases GK16 and GK27 and 1Kb flanking the
breakpoint junctions of the idic(Xq) from case GK32.
Sequences were analyzed using default mfold parameters.
Untangle modes were used in order to avoid overlaps.
Supplementary Material is available at HMG online.
We thank the patients and their families for their participation
and Dr Heike Fiegler for assisting with experiments.
Conflict of Interest statement. None declared.
This work was supported by the Cyprus Research Promotion
Foundation (EPYEJ 0406). AK received support from the
Estonian Government (SF0180027s10), (ETF 7617).
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