Observation and prediction of recurrent
human translocations mediated by NAHR
between nonhomologous chromosomes
Zhishuo Ou,1,9Pawe1 Stankiewicz,1,9Zhilian Xia,1Amy M. Breman,1Brian Dawson,1
Joanna Wiszniewska,1Przemyslaw Szafranski,1M. Lance Cooper,1Mitchell Rao,1
Lina Shao,1Sarah T. South,2Karlene Coleman,3Paul M. Fernhoff,3Marcel J. Deray,4
Sally Rosengren,5Elizabeth R. Roeder,6Victoria B. Enciso,6A. Craig Chinault,1
Ankita Patel,1Sung-Hae L. Kang,1Chad A. Shaw,1James R. Lupski,1,7,8
and Sau W. Cheung1,10
1Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA;2Departments of Pediatrics
and Pathology, University of Utah, Salt Lake City, Utah 84112, USA;3Children’s Healthcare of Atlanta, Atlanta, Georgia 30033, USA;
4Department of Neurology, Miami Children’s Hospital, Miami, Florida 33155, USA;5Hartford Hospital, Hartford, Connecticut 06102,
USA;6Department of Pediatrics, UTHSCSA, San Antonio, Texas 78229, USA;7Department of Pediatrics, Baylor College of Medicine,
Houston, Texas 77030, USA;8Texas Children’s Hospital, Houston, Texas 77030, USA
Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were analyzed. Both of the
breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ~359-kb and ~215-kb low-copy repeat (LCR) clusters,
respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation
to 24 bp within interchromosomal paralogous LCRs of ~130 kb in length and 94.7% DNA sequence identity located in
olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for trans-
location formation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal
translocation formation, we performed computational genome-wide analyses and identified 1143 interchromosomal LCR
substrate pairs, >5 kb in size and sharing >94% sequence identity that can potentially mediate chromosomal trans-
locations. Additional evidence for interchromosomal NAHR mediated translocation formation was provided by se-
quencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped
within 55 bp in ~7.8-kb paralogous subunits of 95.3% sequence identity located in the ~579-kb (chr 8) and ~287-kb
(chr 12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations t(4;11) and t(8;12) and
potentially many other interchromosomal translocations throughout the human genome. Furthermore, we provide
a computationally determined genome-wide ‘‘recurrent translocation map.’’
[Supplemental material is available online at http:/ /www.genome.org. The sequence data from this study have been
submitted to GenBank (http:/ /www.ncbi.nlm.nih.gov/genbank/) under accession nos. HM989976 and HM989977 and to
the NCBI Gene Expression Omnibus (http:/ /www.ncbi.nlm.nih.gov/geo/) under accession nos. GSM578535–GSM578540,
GSM578936–GSM578940, GSM578965, and GSM579028.]
Reciprocal (non-Robertsonian) translocations are one of the most
frequently occurring human chromosomal aberrations. Balanced
reciprocal translocations are found in one in approximately 600
individuals (Van Dyke et al. 1983); thus, one in approximately 300
couples are at risk for having chromosomally unbalanced off-
spring. In most cases, carriers of balanced reciprocal translocations
do not have an abnormal phenotype but may experience repro-
ductive issues such as infertility or multiple miscarriages. Inter-
comparative genomic hybridization) that up to 40% of the appar-
ently balanced reciprocal chromosome translocations in patients
with an abnormal phenotype are accompanied by a chromosome
imbalance, either at the translocation breakpoints or elsewhere in
2008). Littleis known, however, about the mechanisms or genomic
sequences involved in the formation of non-neoplastic reciprocal
translocations (Abeysinghe et al. 2003; Higgins et al. 2008).
translocations have been described in humans. The most frequent
translocation, t(11;22)(q23;q11), is the result of a rearrangement
between palindromic AT-rich cruciform structures in 11q23 and in
low-copy repeat (LCR) LCR22-3a in 22q11.2 (Zackai and Emanuel
1980; Kurahashi et al. 2000; Edelmann et al. 2001; Kurahashi and
Emanuel 2001; Ashley et al. 2006; Kato et al. 2006). Carriers of the
9These authors contributed equally to this work.
E-mail email@example.com; fax (713) 798-8937.
Article published online before print. Article and publication date are at
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21:33–46 ? 2011 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/11; www.genome.org
at risk of having progeny with the supernumerary der(22) syn-
drome (Emanuel syndrome; MIM 609029), resulting from asym-
metric 3:1 meiotic segregation (Zackai and Emanuel 1980; Shaikh
et al. 1999; McDermid and Morrow 2002). Recently, Sheridan et al.
(2010) reported a second recurrent AT-rich palindrome-mediated
translocation t(8;22)(q24.13;q11.21). The third recurrent trans-
location examined, t(4;8)(p16;p23), has been shown to resultfrom
a crossover between the olfactory receptor-gene cluster LCRs
(Giglio et al. 2002; Maas et al. 2007). However, for this latter t(4;8),
the precise breakpoint location or crossover was not determined at
nucleotide sequence resolution.
We report the molecular and clinical data on four unrelated
families with the same recurrent unbalanced chromosomal trans-
location der(4)t(4;11)(p16.2;p15.4), leadingto monosomy4p16.2-
pter and trisomy 11p15.4-pter. When isolated, each genomic im-
balance results in distinct, well-characterized syndromes. Deletion
of 4p16.3 includes two proposed critical regions WHSCR1 and
WHSCR2 (Zollino et al. 2008), and manifests clinically as Wolf-
Hirschhorn syndrome (WHS; MIM 194190). Duplication of the
imprinted 11p15.5 region results in Beckwith–Wiedemann syn-
drome (BWS; MIM 130650) when paternally inherited, or Russell–
Silver syndrome (RSS; MIM 180860) when maternally inherited.
However, when the imbalances are present together, the clinical
manifestation reported in the literature represent a unique phe-
notype with overlapping featuresof WHS,BWS,or RSS, depending
on the parental origin of the duplicated chromosome.
We provide evidence that this recurrent translocation be-
tween chromosomes 4 and 11 [t(4;11)] arises by nonallelic ho-
mologous recombination (NAHR) mediated by interchromosomal
paralogous LCRs. To investigate the genomic potential for recur-
rent translocations to occur by NAHR, we analyzed the genome-
wide distribution of interchromosomal LCRs with >94% sequence
identity and between 5–10 kb, 10–20 kb, 20–30 kb, 30–40 kb, 40–
105, 45, and 162 pairs of interchromosomal LCRs, respectively,
that may potentially act as NAHR substrate pairs. To demonstrate
utility of this computationally generated ‘‘potential recurrent trans-
location map’’ we sequenced the NAHR crossover sites within
der(8)t(8;12)(p23.1;p13.31). We demonstrate that NAHR between
interchromosomal LCRs on nonhomologous chromosomes me-
diate recurrent constitutional translocations potentially through-
out the human genome and provide a computationally deter-
mined genome-wide ‘‘recurrent translocation map.’’
Genomic rearrangements identified by chromosomal
microarray analysis and chromosomal studies
Chromosomal microarray analysis (CMAV5 BAC, V6 BAC, and V6
OLIGO) (Cheung et al. 2005), initially performed on patients 1, 2,
and 3 (see Supplemental Notes) identified similar genomic im-
balances: terminal deletion of 4p16.2-pter and duplication of
11p15.4-pter. Subsequent fluorescence in situ hybridization (FISH)
analysis confirmed the CMA results and revealed an unbalanced
translocation der(4)t(4;11)(p16.2;p15.4) in each patient. Two
other related patients (patients 4 and 4U; referred to as patient 2
and patient 3 in South et al. 2008) with identical cytogenetic
breakpoints were obtained for further molecular analyses on five
subjects in total.
Translocation breakpoints map to LCR
Using a high-resolution SNP array, we fine-mapped the translo-
cationbreakpointsinpatients1,2,3,4,and4Utolarge LCRs, ;359
4U share the same breakpoints in both 4p16.2 and 11p15.4 (South
et al. 2008). This high-resolution genome analysis also confirmed
that the WHS critical regions WHSCR1 and WHSCR2 at 4p16.3
were deleted and the BWS and RSS genomic regions containing
imprinted domains at 11p15.4 were duplicated (Table 1).
Parental origin of rearranged genomic sequences
FISH analysis using the same probes on the parental samples from
patients 1 and 3 showed a balanced t(4;11) in their fathers. No
parental samples were available for patient 2.
MS-MLPA analysis of DNA samples from patients 2, 4, and 4U
showed increased peak intensities of IGF2 and KCNQ1, consistent
with a duplication of the 11p15 region. The methylation studies
revealed a difference in the methylation pattern in the 11p15
imprinted region in patients 2, 4, and 4U. Gain of differentially
patients 2 and 4, whereas gain of DMR1 methylation was detected
in patient 4U (Fig. 1). These patterns are indicative of a maternal
origin for the duplicated 11p15 region in patients 2 and 4, and
paternal origin of the duplicated 11p15 region in patient 4U,
consistent with the respective clinical findings.
Genomic architecture and sequence analyses
Bioinformatic analysis was performed comparing the breakpoint
regions within the LCR blocks in 4p16.2 with that of the 11p15.4
region (genomebuild GRCh37/hg19). This analysis revealedthat a
359-kb LCR cluster in 4p16.2 (genomic position 3.88–4.24 Mb)
shares 204 kb of significant homology (DNA sequence identity
>94%) with the 215-kb genomic segment on 11p15.4 (genomic
position 3.41–3.62 Mb). Both paralogous regions harbor all the
breakpoints of the five t(4;11) cases we studied (Fig. 2).
To further delimit the exact crossover and the nature of the
surrounding sequences in proximity to the strand exchange, we
the long-range PCR products amplified from the patient’s DNA
enabled narrowing of the NAHR site. PCR amplification of the
genomic DNA from patient 3 with forward primer GCCTAAACT
ATTTCTCAGCAAGGAGGAAGG and reverse primer CCCGAGTG
product (Fig. 2C). Subsequent DNA sequencing analysis allowed
us to narrow the NAHR sites to the 24-bp regions between
chr4:3,940,888–3,940,911 and chr11:3,426,699–3,426,722 (Fig.
directly oriented (centromere to telomere direction) LCRs of ;130
(5 kb on each side of the breakpoints) revealed the recently pro-
posed homologous recombination ‘‘hotspot’’ associated sequence
motif CCNCCNTNNCCNC 1287 bp and 3209 bp telomeric and
636 bp centromeric to the chromosome 11p15.4 breakpoint and
642 bp centromeric to the chromosome 4p16.2 breakpoint. This
13-bp homologous recombination hotspot associated motif is
H3 lysine 4 trimethylation (Baudat et al. 2010; Myers et al. 2010;
Parvanov et al. 2010).
34 Genome Research
Ou et al.
Clinical manifestations of chromosome imbalances
Table 2. Our patients 1 and 3 with the der(4)t(4;11) of paternal ori-
gin demonstrate a unique phenotype. Their growth parameters are
within the normal range, resembling neither WHS nor BWS. The
facial features of patient 1 include arched eyebrows, short philtrum,
Features of patient 3 include micrognathia, seizures, and feeding
difficulties consistent with WHS, whereas renal and cardiac anom-
alies occur in WHS and BWS. Macrosomia, macroglossia, and ab-
dominal wall defect, which are defined as major features for BWS,
were not detected in patients 1 and 3. Our data are consistent with
WHS features over the BWSfeatures(Russoetal. 2006;Mikhail et al.
2007; South et al. 2008; Thomas et al. 2009).
Patient 2, with a methylation pattern consistent with a
maternal origin of t(4;11), has partial features of WHS and RSS,
including prenatal onset of growth deficiency seen in both WHS
and RSS. Hypertelorism, high arched eyebrows, cleft palate, and
downturned mouth corners are seen in WHS, and the low nasal
Interestingly, most of these features were also observed in patient
4, in whom the derivative chromosome was also found to be ma-
ternally inherited (Table 2; Supplemental Notes).
A genome-wide recurrent translocation map
NAHR is a major mechanism for recurrent interstitial (i.e., within
and between homologous chromosomes) rearrangements using
eitherdirectlyoriented (for deletions andduplications) orinversely
oriented (for inversions) LCRs as homologous recombination sub-
strates (Stankiewicz and Lupski 2002). We now sought to identify
genome-wide interchromosomal LCRs that could potentially me-
diate rearrangements of nonhomologous chromosomes via NAHR.
The formation of a stable reciprocal translocation mediated
by interchromosomal NAHR between LCRs is dependent upon the
orientation of the LCRs and the chromosome arms involved. In-
terchromosomal NAHR between LCRs mapping in the same ori-
entation and on the same chromosome arms (i.e., p-arm of one
chromosome versus the p-arm of the other) or those in inverted
orientation on opposite chromosome arms (i.e., p-arm from one
chromosome versus q-arm of the other) are predicted to result in
NAHR-mediated stable, monocentric reciprocal translocation chro-
mosomes. In contrast, LCRs in opposite orientation on the same
chromosome arms or those in the same orientation on opposite
chromosome arms are predicted to result in either unstable di-
centric or acentric chromosomes (Fig. 3).
We analyzed the human haploid genomic reference DNA se-
quence (NCBI36/hg18) for interchromosomal LCRs of >5 kb in
length and >94% DNA sequence identity, and identified 1902 se-
quences that correspond to our inclusion criteria. We further seg-
mented our genomic sequence analysis to include LCRs with 5–10
kb, 10–20 kb, 20–30 kb, 30–40 kb, 40–50 kb, and >50 kb in length,
resulting in the identification of 295, 352, 184, 105, 45, and 162
pairs of interchromosomal LCRs, respectively. We also constructed
a global view of potential translocations mediated by the inter-
chromosomal NAHR between LCRs with >94% identity and >5 kb
in size; parameters empirically shown to support recurrent trans-
location (Fig. 4). The global view was divided into 25% each, based
on the size of the LCRs for easy visualization (Fig. 4A–D). Some of
the potential interchromosomal NAHR pairs represent olfactory
receptor gene repeats (Fig. 4, green lines). Importantly, both known
recurrent translocations, t(4;8) (Giglio et al. 2002) and t(4;11),
reportedherein,are predicted bythistranslocationmap(Fig. 4,red
Predicted recurrent translocations
To test the hypothesis that the identified 1902 candidate in-
terchromosomal LCRs or 1143 LCR NAHR substrate pairs can po-
tentially mediate different recurrent chromosomal translocations,
we queried our patient database and found 105 patients with un-
balanced translocations detected by array CGH analysis using
CMA V6 OLIGO (44K), V7 OLIGO(105K), and V8 OLIGO (180K)
Summary of the parental origin, genomic rearrangement, and breakpoints of patients with recurrent t(4;11)
Patient 1der(4)t(4;11) Paternal4 p16.2-pter
3.39 - 4.85
2.87 - 3.57
? - 4.39
3.37 - 3.57
3.81 - 3.84
3.36 - 3.58
3.81 - 3.84
3.36 - 3.58
Patient 2 der(4)t(4;11)Maternal
Patient 3 der(4)t(4;11)Paternal
South et al. (2008) patient 2a
South et al. (2008) patient 3b
South et al. (2008) patient 1der(4)t(4;11)Maternal
Russo et al. (2006) patient 3der(4)t(4;11)Paternal
Thomas et al. (2009) family 5der(4)t(4;11) Paternal
Thomas et al. (2009) family 6der(4)t(4;11)Paternal
Mikhail et al. (2007)der(4)t(4;11)Paternal
aDesignated as patient 4 in our study.
bDesignated as patient 4U in our study.
Recurrent translocations by NAHR
arrays; custom whole genome arrays with
varying densities of backbone interrogat-
ing oligonucleotides. In addition to the
three cases with t(4;11), we found seven
cases with t(4;8)(p16.2;p23.1), five with
the derivative chromosome 4, and two
with the derivative chromosome 8, and
(patients 5 and 6).
Bioinformatic analysis of the t(8;12)
breakpoint regions revealed an ;579-kb
LCR cluster (genomic position 7.52-8.10
Mb) on chromosome 8p23.1 and an
;287-kb LCR cluster on chromosome
12p13.31 (genomic position 8.31–8.60
Mb) that share 285 kb of significant ho-
mology (DNA sequence identity >94%).
Both paralogous regions harbor all four
breakpoints of the t(8;12) cases we stud-
ied (Fig. 5; Supplemental Table 1).
DNA sequencing of the t(8;12) pa-
tient 5–specific ;12-kb LR-PCR product
amplified with forward primer TTCTTAAT
ATCACTTTTCCCCACTCTAGTTC and re-
verse primer GTGTAAGACGTCGATACG
ATACGGCACTTC, enabled narrowing the
NAHR sites to the 55-bp regions between
chr8:7,884,979–7,885,033 and chr12:8,
374,239–8,374,293 flanked by two paralo-
gous sequence variants (Fig. 5). Sequence
analyses of 10-kb flanking regions revealed
a homologous recombination ‘‘hotspot’’
Locations of the LCR and breakpoints
from empiric data
The chromosomal distribution of the
identified 1902 LCR sequences is detailed
in Figure 6 and Supplemental Table 4. We
calculated that 33.64% of all 1902 in-
terchromosomal LCRs map to the sub-
telomeric most distal 5 Mb regions of all
chromosomes. From our clinical aCGH pa-
unbalanced constitutional chromosomal
translocations; of these, 85 translocations
had breakpoints that were resolved using
custom whole genome clinical microarrays
(105K or 180K). The line graph shows the
density estimate for the distribution of
LCRs on each chromosome. The red hash
marks below the curve show the location
of the LCR midpoints (Fig. 6). The 170
breakpoint coordinates from the aCGH-
detected 85 translocations are depicted in
green hash marks below the line plot.
To determine whether the observed
translocation breakpoints are more closely
t(4;11). (A) Pedigrees illustrating the parental origin of the duplicated 11p material if the balanced
translocation is present in the father (left), or the mother (right). (Blue) Paternal inheritance; (pink)
maternal inheritance. (B) Schematic representation of the imprinted region at 11p15. The imprinted
11p15 region consists of two independent domains that are regulated by differentially methylated
and IGF2 genes. The centromeric DMR2 is maternally methylated and regulates expression of imprinted
genes in this region including CDKN1C, KCNQ1OT1, and KCNQ1. CH3 represents the methylated allele.
(C) Partial profile of MS-MLPA HhaI digestion/ligation products for patients 2, 4, and 4U (blue) com-
pared to normal control (red). Increased peak intensities of IGF2 and KCNQ1 in patients 2, 4, and 4U
indicate duplication of the 11p15 region. Gain of methylation in the HhaI sensitive DMR2 was detected
in patients 2 and 4, whereas gain of methylation in the HhaI sensitive DMR1 was detected in patient 4U.
The results demonstrated that maternal inheritance (M) of the duplicated 11p15 region in patients 2
and 4 and paternal inheritance (P) of the duplicated 11p15 region in patient 4U.
Methylation pattern and copy number analysis by MS-MLPA of patients with recurrent
Ou et al.
36 Genome Research
by chance, we computed the minimum distance between the
distal endpoint (furthest from telomere) of each translocation
event and the LCRs located on that chromosome. We used the
absolute value of the difference between the coordinate of the
translocation distal breakpoint and the midpoints of the LCRs. In
order to create a reference distribution for these values, we per-
formed a Monte Carlo simulation, drawing 10,000 random co-
ordinates along each chromosome and computing the minimum
LCR absolute distance statistic for each draw. We standardized our
observed translocation-LCR distance values by subtracting the
simulation-derived mean and dividing by the simulation-derived
standard deviations determined by the random draws for each
chromosome. We then performed an analysis of the standardized
distances to the LCRs using a Wilcoxon signed rank test with
continuity correction, and this analysis determines a P-value of
1.410 3 10?7for the observed translocation breakpoints against
the null hypothesis that the breakpoints are located a random
distance from the predicted LCRs against the alternative hypoth-
esis that the breakpoints are closer to the LCRs than would be
expected by chance. The median standardized distance between
the observed and expected distance from a breakpoint to an LCR
is ?0.6285 standardized units, indicating together with the
Wilcoxon P-value that the observed breakpoints are signifi-
cantly closer to the LCRs than would be expected by chance.
(Legend on next page)
Recurrent translocations by NAHR
Balanced reciprocal translocations are one of the most commonly
observed chromosomal abnormalities in humans. However, the
molecular mechanisms of formation of these rearrangements re-
main elusive with the exception of the recurrent translocations,
t(11;22)(q23;q11), t(8;22)(q24.13;q11.21), and t(4;8)(p16;p23)
(Kurahashi et al. 2000; Edelmann et al. 2001; Kurahashi and
Emanuel 2001; Giglio et al. 2002; Sheridan et al. 2010). NAHR be-
tween interchromosomal LCRs on different (i.e.,nonhomologous)
chromosomes has been suggested to result in chromosomal trans-
locations (Lupski 1998; Stankiewicz and Lupski 2002). We now
provide molecular evidence to support NAHR between inter-
chromosomal LCRs as a potential major mechanism for recurrent
For NAHR to occur, it has been proposed that 300–500 bp of
perfect DNA sequence identity is the minimal efficient process-
ing segment required to mediate meiotic NAHR between intra-
chromosomal LCRs (Reiter et al. 1998). LCRs of >10 kb in size
and with >95%–97% DNA sequence identity have been shown
interstitial or intrachromosomal genomic rearrangements (Lupski
1998; Stankiewicz and Lupski 2002, 2006; Shaw and Lupski 2004;
Lupski and Stankiewicz 2005; Sharp et al. 2005). The distance be-
tween LCRs is another factor apparently influencing NAHR, since
apart often correlate with large LCRs (Lupski 1998; Stankiewicz
and Lupski 2002).
The ‘‘rules’’ for NAHR mediated interstitial chromosomal
rearrangements have enabled predictions of genomic instability
regions proneto deletions/duplicationscausinggenomicdisorders
(Sharp et al. 2005). Five novel genomic disorders have been elu-
cidated by this predictive ‘‘interstitial rearrangement map’’ and
informed design of human genomic microarrays for array CGH
analysis; these include: 1q21.1 microdeletion/microduplication
(Brunetti-Pierri et al. 2008; Mefford et al. 2008), 15q13.3 micro-
deletion (Sharp et al. 2008; Ben-Shachar et al. 2009; Miller et al.
microduplication associated with renal disease, diabetes, and epi-
lepsy (Mefford et al. 2007; Moreno-De-Luca et al. 2010; Nagamani
et al. 2010), and 17q21.3 microdeletion/microduplication (Koolen
et al. 2006; Sharp et al. 2006; Shaw-Smith et al. 2006).
The understanding of the NAHR mechanism combined with
the availability of the human genome sequence (International
bioinformatics as a tool to predict hotspots for genomic instability
that may be prone to recurrent translocations. Genome-wide bio-
informatic analyses revealed 1902 interchromosomal LCR sub-
strates or 1143 pairs, >5 kb in size and sharing >94% sequence
identity that can potentially mediate recurrent chromosomal
translocations via NAHR.
From our clinical aCGH patient databases, there were 105 pa-
tients with unbalanced constitutional chromosomal translocations
with 85 of these translocations identified using custom whole ge-
nome clinical microarrays (105K or 180K). We found threerecurrent
translocations matching our predictions; seven t(4;8)(p16.2;p23.1),
three t(4;11)(p16.2;p15.4), and two t(8;12)(p23.1;p13.31) with a
detection rate of 12/105 = ;11%. Although NAHR-mediated re-
ciprocal translocations appear to be rare events, we contend that
the frequency of NAHR in reciprocal translocations at the level of
the genomic sequence has not been systematically assessed. Prior
to our analysis, only two translocations, t(11;22)(q23;q11) and
t(8;22)(q24.13;q11.21), have had their breakpoints sequenced.
Thet(4;11) reportedin the currentstudy is onlythe thirdrecurrent
translocation, in which the breakpoint region is established at the
nucleotide sequence level. This was accomplished using arrays to
identify unbalanced translocations, which narrowed the trans-
location breakpoint regions to a genome resolution level high
enough for PCR amplification of the junction fragments.
Although the frequency of reciprocal translocation is rela-
tively common, most are detected by GTG banded chromosome
analysis. The resolution of banding is usually between 5–10 Mb
DNA from patient 1 (left). The mean normalized log2(Cy3/Cy5)ratio of each BAC clone is plotted on the x-axis as dots with error bars, and arranged along
the vertical axis from chromosome 1 at the top to chromosomes X and Y at the bottom. All 11 clones on the 4p subtelomeric region showed displacement
to the left, indicating a deletion of 4p16.2-p16.3 material, whereas five clones on the 11p subtelomeric region are shifted to the right, indicating a du-
plication of 11p15.5 material in the patient versus the reference DNA. The results of FISH analysis of metaphase chromosomes prepared from the patient’s
peripheral blood lymphocytes with probe RP11-371C18 specific for chromosome region 11p15.5 (red) show the presence of 11p15.5 material on the
derivativechromosome4[der(4)] (arrow), whereasthe resultsofFISHanalysiswith probeRP11-478C1 specific forchromosome region4p16.3 (red)show
the deletion of4p16.3 material (arrow). The CMAand FISH analysesrevealed an unbalanced translocation between 4p16 and11p15.CMAprofile ofDNA
from patient 2 tested on V5 BAC array (middle). As in patient 1, the 4p deletion and 11p duplication were detected by displacement of 11 clones and five
clones on the corresponding region, respectively. The results of FISH analysis with the PAC probe RP5-998N23 (red) specific for 11p15.5 indicate the
presence of 11p15.5 material on der(4), whereas the results of FISH analysis in patient 2 with the 4p subtelomeric probe D4S3359 (green) show the
deletion of 4pter material. CMA profile of DNA from patient 3 tested on BAC emulated Version 6 OLIGO array (right) revealed the same genomic
aberrations as patients 1 and 2. The results of FISH analysis with RP13-870H17 (red) specific for 11p15.5 indicate the presence of 11p15.5 material on
Five t(4;11) cases mapped this to NAHR substrate pair. Summary of the sequence similarity BLAST2 analysis of the 350-kb sequence surrounding the
4p16.2 (top) and 11p15.4 (bottom) breakpoint regions. The different color horizontal arrows depict the homologous LCR subunits. The numbers above
and below the lines represent genomic distance (megabases) from 4p and 11p telomeres, according to NCBI human genome build 37 (GRCh37/hg19;
Feb. 2009). The regions between 4p16.2 and 11p15.4 connected by dotted lines are >94% sequence identical. The translocation breakpoints in patient 3
are located in the homologous LCRs indicated by the vertical arrows, implying a NAHR-based recombination mechanism. (C) Ethidium bromide stained
agarosegelimageofthe;9-kb t(4;11) patient3-specificjunction fragmentamplified bylong-range PCR withprimersharboring trans-morphisms specific
for each 4p16.2 and 11p15.4 LCR (lane 2). Lane 1 represents the DNA marker with the 10-kb band indicated to the left. Lane 3 represents a negative
control. (D) The NAHR cross-over site for patient 3 is located in a 130-kb subunit with 94.7% DNA sequence identity. UCSC Genome Browser view of the
homologous LCR blocks of the same orientation in the chromosome regions 4p16.2 (top) and 11p15.4 (bottom) indicated by the gray bars. The black
arrows indicate the NAHR site for patient 3 determined by sequence analysis. (E) DNA sequence alignment of the PCR amplified translocation junction
fragment in patient 3 (middle sequence). The NAHR site was narrowed to a 24-bp segment (red rectangle) with 100% DNA sequence identity between
chromosomes 4 (top) and 11 (bottom). Blue nucleotides indicate alignment with the chromosome 11 sequence, red nucleotides indicates alignment
with the chromosome 4 sequence, purple nucleotides indicate SNPs, and trans-morphic mismatches are indicated by black dots above or below the
Identification of the LCR pairs acting as potential substrates for interchromosomal NAHR, resulting in the t(4;11) formation. (A)CMAprofileof
Ou et al.
Phenotypic features of WHS, BWS, and RSS compared to those found in the presented and reported patients
et al. (2008)
et al. (2006)
et al. (2007)
et al. (2009)
Origin of the t(4;11)c
Prenatal onset growth deficiency
Postnatal growth retardation
High arched eyebrows
Broad nasal bridge
Short upper lip and philtrum
Ear creases/ear pits
Advanced bone age
Low nasal root
aDesignated as patient 4 in our study.
bDesignated as patient 4U in our study.
cP, Paternal; M, maternal; Nl, normal.
Recurrent translocations by NAHR
This poor genome resolution renders translocation breakpoint
assignments by karyotype analysis reported in the literature in-
herently inaccurate and unreliable for precise breakpoint map-
ping. Historically, FISH analysis using tiling BAC clones within the
predicted translocation junction chromosome bands has been
used to identify the clone that spans the breakpoint region. Cur-
rently, the focus of most of the studies investigating reciprocal
translocation breakpoints utilizes array technology to determine
whether an apparent balanced translocation by GTG banding
analysis is indeed balanced (De Gregori et al. 2007; Fantes et al.
2008; Schluth-Bolard et al. 2009) rather than breakpoint identifi-
cation per se.
Of the published 31 balanced translocations in which
breakpoints have been mapped, none had homologous LCRs at
the breakpoint regions (Baptista et al. 2008). These balanced rear-
rangementswouldnot be detectedby array CGHanalysis.Thefine
mapping of balanced translocation breakpoint regions performed
was focused predominately in segments containing genes, thus
less likely to be associated with LCRs (Baptista et al. 2008). We
believe the apparent discrepancy between our analysis and that
published by Baptista et al. (2008) is due to the fact that the
translocations in our database were unbalanced with breakpoints
mapping in the distal portions of the chromosome arms that are
enriched with interchromosomal LCRs (Linardopoulou et al.
2005). Telomeric imbalances that are smaller in size are less likely
to be embryonically lethal and therefore may be viable. Trans-
locations resulting in large imbalances are likely to be embryoni-
cally lethal, whereas translocations with small imbalances can be
viable. Potentially, several thousands of translocation breakpoints
may need to be mapped at high-resolution to assess reliable rep-
Recently, seven t(4;11)(p16.2;p15.4) cases with clustered
breakpoints from six unrelated families have been reported (Russo
et al. 2006; Mikhail et al. 2007; South et al. 2008; Thomas et al.
2009) (Table 1). The clinical features of the t(4;11) patients with 4p
monosomy and 11p trisomy in these studies represent a unique
BWS or RSS, depending on the parental origin of the duplicated
chromosome 11 (Table 2). The WHS phenotypic spectrum was
observed more often than BWS (Russo et al. 2006; Mikhail et al.
2007; South et al. 2008; Thomas et al. 2009).
We describe the results of molecular cytogenetic and clinical
analyses in three novel unrelated subjects and two published cases
from one family (South et al. 2008) with an unbalanced trans-
location der(4)t(4;11)(p16.2;p15.4), resulting in segmental 4p mono-
somy and 11p trisomy with the translocation breakpoints mapping
in the same LCR paralogues. Our high-resolution SNP array studies
clearly demonstrated deletion of the WHS critical region and du-
plication of the BWS/RSS critical region in patients 1–4U. Family
histories and/or MS-MLPA studies revealed paternal origin for the
aberrationsinpatients1,3,and4U andmaternaloriginin patients
2 and 4.
Although seven t(4;11)(p16.2;p15.4) cases with clustered
breakpoints have been described, the specific breakpoints and
DNA sequence of the junction on 4p16.2 and 11p15.4 have not
been well characterized. Our genomic analysis of the breakpoint
regions revealed the 204-kb homologous LCR portion of >94%
interchromosomal DNA sequence identity. All analyzed trans-
location breakpoints mapped within the homologous subunits,
suggesting that NAHR between the LCRs located on chromosome
4p16.2 and 11p15.4 is the likely mechanism for their formation.
This hypothesis was further substantiated by breakpoint se-
quencing of two selected translocations, t(4;11) and t(8;12). As
anticipated the breakpoints mapped to the ‘‘recurrent translo-
cation map’’ identified LCR substrates.
Some of the other predicted recurrent translocations, how-
ever,may be underrepresentedsincederivativechromosomes with
longer segments of imbalance are more likely to be incompatible
with life. High-resolution genome analyses of additional balanced
and unbalanced translocations will be required to further confirm
the utility of our ‘‘recurrent translocation map.’’
It is also likely that both balanced and unbalanced trans-
locations are under-ascertained when studied by karyotype anal-
have a GTG-negative (light) banding pattern, the reciprocal ex-
change of chromosomal material at subtelomeres is likely to be
cryptic. Human subtelomeric regions have been completely se-
quenced and it has been shown that the subtelomeric segmental
duplicated region (also known as subtelomeric repeats) in humans
make up 25% of the most distal 500 kb and 81% of the most distal
100 kb in human genome (Riethman 2008). These duplicated
segments predispose to different types of genomic rearrangements
(Linardopoulou et al. 2005). We find that 162 LCRs map to the
most distal 100 kb on each chromosome, and 506 LCRs map to
the most distal 500 kb. Interestingly, 22.97% of the breakpoints
from the 85 unbalanced rearrangements are located within the
first 5 Mb from each end of the chromosomes (Supplemental
Table 2). These results support the hypothesis that segmental
duplications in subtelomeric regions mediate translocations by
interchromosomal NAHR mechanisms.
DNA sequencing analysis in one patient allowed us to narrow
the NAHR sites to the 24-bp regions between chr4:3,940,888–
paralogous directly oriented (centromere to telomere direction)
LCRs of ;130 kb in length and 94.7% DNA sequence identity
LCRs. Nonhomologous chromosomes are shown in black and white with
the centromeres shown as circles. The arrows indicate the orientation of
the LCRs. Only interchromosomal LCRs located in the same orientation
on the same chromosomal arms (i.e., q-arm to q-arm) (A), or those in
In contrast, LCRs located on the same chromosomal arm in opposite ori-
entation (B) or on different chromosomal arms in the same orientation (D)
would lead to unstable dicentric or acentric chromosomes, resulting in
chromosome breakage or loss, respectively. Note: Both HR substrate ori-
entation (direct versus inverted) and chromosomal arm location (p versus
q), required for viable interchromosomal recombinant products.
Potential outcomes of interchromosomal NAHR mediated by
Ou et al.
40 Genome Research
located in olfactory receptor (OR) gene clusters. As much as half of
the members of the OR gene family (;852 genes) intercept copy
number variation regions suggesting NAHR plays a major role in
remodeling of the OR gene family (Young et al. 2008). In addition,
the translocation map shows 25% of these OR genes intercept
the first 5 Mb of the subtelomeric regions from each end of the
The subtelomeric LCRs are very polymorphic and their
structures differ between different individuals and populations,
likely as a result of gene conversion events. The complexity of the
among different population is still largely unknown. For example,
the 17q21.31 microdeletion apparently resulted from a meiotic
recombination between the H1 and the inversion-bearing H2
haplotype, which is carried at a frequency of ;20% in populations
of European ancestry (Stefansson et al. 2005). However, the fre-
quency of this inversion polymorphism has yet to be determined
in other populations. Furthermore, both structural and nucleotide
sequence diversity within LCRs (i.e., paralogous sequence and
structural variations) were observed in the 24-kb-long Charcot-
Marie-Tooth disease type 1A, CMT1A-REP, LCRs that sponsors
deletion and duplication of this genomic region (Lindsay et al.
2006), high frequency of retroelement insertions, accelerated se-
quence evolution after duplication, and extensive paralogous
gene conversion were observed. These findings were consistent
with the recent observation that repetitive elements such as
LINE-1 and Alu may also contribute significantly to structural
variations(Beck et al. 2010; Ewing andKazazian 2010; Huang et al.
2010; Iskow et al. 2010; Lupski 2010). Additionally, the observa-
tion of thousands of new structural variants with sizes ranging
from kilobases to megabases using single molecule analysis only
of the human genome (Conrad et al. 2010a,b; Pang et al. 2010; Park
et al. 2010; Teague et al. 2010). The landscape and impact of these
genomic variants that are individually rare butcollectively common
in the human population remains to be explored.
We show that the interchromosomal LCR harboring the olfac-
tory receptor gene cluster in 11p15.4 is a novel genomic instability
region that mediates the relatively common recurrent constitutional
non-Robertsonian translocation t(4;11)(p16.2;p15.4) by NAHR. We
identified the interchromosomal LCRs that can potentially mediate
chromosomes to construct a computationally derived ‘‘recurrent
translocation map’’ and provide experimental evidence by virtue
of t(8;12) breakpoint mapping to support the predictions. Our
findingssuggestinterchromosomalLCR-mediatedNAHR may be a
major mechanism for recurrent constitutional translocation for-
mation, in particular within the subtelomeric regions.
represented by dotted lines and distribution divided into four groups based on the size of LCR. To create this plot we circularized the genome using polar
coordinates. We then connected points between a pair of chromosomes linked by LCRs satisfying our size sequence identify criteria (see Supplemental
Table 3). The midpoints of the LCRs were used to identify each segment with a single location on each chromosome. The red dotted lines indicate the
translocations identified in our patient database, while the green dotted lines represent the olfactory receptor LCRs. (A) The size of LCR ranges from 5030
to 9935 basesinthe first25%. (B)Thesize ofLCRs rangefrom 9936 to16,593 basesforthe second 25%ofLCRs.(C)Thesize ofLCRsrange from16,594 to
31,678 bases for the third 25% of LCRs. (D) The size of LCRs range from 31,679 to 754,003 bases for the final 25% of LCRs.
Recurrent translocation map. A global genomic view of interchromosomal LCR pairs with >5 kb in size and >94% DNA sequence identity
Recurrent translocations by NAHR
of DNA from patients 5 (left) and 6 (right) tested on Version 8 OLIGO array (left) revealed a 7.9-Mb deletion of chromosome bands 8p23.1-pter and an
8.2-Mb duplication of chromosome bands 12p13.31-pter. The results of FISH analysis (right) with the probe RP11-440E12 (patient 5; red) or VIJTYAC14
(patient 6; green) specific for 12p33.33 indicate the presence of chromosome 12 material on the der(8), whereas the results of FISH analysis with probe
RP11-1001A23 (patient 5; red) or D8S504 (patient 6; green) specific for the chromosome region 8p23 show the deletion on chromosome 8. (B) Two
t(8;12) cases mapped this to NAHR substrate pair. Summary of the sequence similarity BLAST2 analysis of an ;200-kb sequence surrounding the 8p23.1
and 12p13.31 breakpoint regions (bottom). (C) Ethidium bromide stained agarose gel image of the patient 5–specific ;12-kb t(8;12) junction fragment
amplified by long-range PCR with primers harboring trans-morphisms specific for each 8p23.1 and 12p13.31 LCR (lane 2). (Lane 1) The DNA marker with
the 10-kb band indicated to the left. (Lane 3) A negative control. (D) The NAHR crossover site for patient 5 is located in a 7.7-kb subunit with 95.2% DNA
sequenceidentity. UCSCGenome Browser viewof thehomologous LCR blocks in the8p23.1 (top)and 12p13.31 (bottom)chromosomeregions.(E)DNA
sequence alignment of the PCR amplified translocation junction fragment for patient 6 (middle sequence). The NAHR site was narrowed to a 55-bp
segment (red rectangle) with 100% sequence identity between chromosomes 8 (top, red) and 12 (bottom, blue).
Identification of the LCR pairs acting as potential substrates for interchromosomal NAHR resulting in the t(8;12) formation. (A) CMA profiles
42 Genome Research
The distributions of 1143 LCR potential substrate pairs with relation to chromosome position. For each chromosome, the distribution of the LCR (x-axis) is plotted against the frequency of
the LCR (y-axis) along the entire chromosome. The red dotted vertical lines on both ends of the chromosome represent the first and last 5 Mb of each chromosome; the yellow line represents the
centromeric region as aligned with the karyogram on the bottom of each graphic. The red hash marks underneath the plots depict the density of the LCRs, while the green bar represents the distribution
of the 170 breakpoint regions from our genome-wide unbalanced translocation data.
Recurrent translocations by NAHR
Informed consents approved by the Institutional Review Board for
obtained for further delineation of the breakpoints and publica-
tion of photographs.
We obtained clinical information for three patients with the
t(4;11) translocation, designated as patients 1 to 3. The Supple-
mentary Notes contain detailed clinical information for these pa-
tients. We also obtained DNA from two other reported patients
from a family with a similar t(4;11) translocation (South et al.
2008) (patient 2 and patient 3), designated here as patient 4 and
Chromosome microarray analysis
Blood samples were obtained from patients and their family
members referred to the Medical Genetics Laboratories at Baylor
College of Medicine for chromosomal microarray analysis (CMA).
Samples from patients 1 and 2 were analyzed on the CMAV5 BAC
array, and patient 3 on the CMAV6 OLIGO array.
Version 5 BAC array contained 853 BAC/PAC clones designed
to cover genomic regions of 75 known genomic disorders, all 41
subtelomeric regions, and 43 pericentromeric regions. Version
6 BAC array consisted of 1472 BAC/PAC clones, covering ;150
pericentromeric regions with backbone coverage of every chromo-
some at the 650-band level of cytogenetic resolution (http://
www.bcm.edu/geneticlabs/?pmid=16207). The BAC microarrays
were designed and manufactured at Medical Genetics Laboratories
as previously described (Cheung et al. 2005). The procedures for
DNA digestion, labeling, and hybridization as well as data analysis
were performed as described (Lu et al. 2007). The BAC emulated
Version 6 OLIGO array was comprised of ;42,460 oligonucleo-
tides representing 1400 BAC clones. The 42.46 K oligonucleotides
(oligos) were selected from initial testing of 105,000 oligos derived
from the Agilent eArray library with strict selection criteria and
removal of repetitive sequences to ensure optimal performance
with greater dynamic range (Ou et al. 2008). This targeted 42.46 K
OLIGO array (V6 OLIGO) corresponds to genomic regions covered
by the V6 BAC arrays and was manufactured in a 4 3 44 K format
with an average of 28–30 oligos per region previously covered by
a single BAC clone. The procedures for DNA digestion, labeling,
and hybridization as well as data analysis were performed as pre-
viously described (Probst et al. 2007).
Affymetrix Genome-Wide SNP Array 6.0 arrays (Affymetrix,
Inc.) were employed to define the breakpoints on chromosomes 4,
8, 11, and 12. Analysis was performed according to the Genome-
Wide Human SNP Nsp/Sty Assay kit 5.0/6.0 protocol provided by
the supplier. The arrays were scanned using a GeneChip Scanner
3000 7G (Affymetrix, Inc.) and results were analyzed using Geno-
typing Console version 2.1 software.
Cytogenetic and FISH analyses
GTG-banded chromosome analysis was performed using standard
protocols. FISH was performed using standard procedures with
BAC clones labeled by nick translation with SpectrumOrange or
SpectrumGreen (Abbot). BAC clones specific for human chromo-
some regions 4p16.2 and 11p15.4, as well as 8p23.1 and 12p13.31
for confirmation of the CMA findings, were selected from UCSC
Genome Browser (http://www.genome.ucsc.edu).
Long-range PCR and DNA sequencing
Long-range PCR primers were designed to harbor at least three
nucleotides specific for one LCR on chromosome 4p16.2 or 8p23.1
and in the other primer for 11p15.4 or 12p13.31, respectively, to
allowpreferential amplificationofthepredicted chimericfragment
nonhomologous chromosomes, but not the fragment of the origi-
nal LCRs. The primers were designed using Primer 3 software
(http://frodo.wi.mit.edu/primer3). Amplification of 8–15-kb frag-
ments was performed using Takara LATaq polymerase (Takara Bio),
following the manufacturer’s protocol. Briefly, we used 25-mL re-
actionmixtures containing 100 ng ofgenomic DNA, 0.4mM dNTP
(each), 0.2 mM primers (each), and 1.25 U of Taq polymerase. PCR
conditions were: 94°C for 1 min, followed by 30 cycles at 94°C for
30 sec, 68°C for 12 min, and 72°C for 10 min. The PCR products
were treated with ExoSAP-IT (USB) to remove unconsumed dNTPs
and primers, and bidirectionally sequenced using the dye-termi-
nator method (Lone Star Labs) with the primers used to amplify
these DNA fragments and primers specific for both paralogous LCR
copies to map the NAHR sites within the PCR products.
The genomic sequences defined by coordinates identified
in the aCGH experiments, were downloaded from the UCSC
Genome Browser (genome build GRCh37/hg19) and assembled
and compared to the sequence from the junction fragments us-
ing the Sequencher V4.8 software (Gene Codes). Interspersed
repeat sequences were identified using RepeatMasker (http://
Methylation-specific multiplex ligation-dependent
Methylation-specific multiplex ligation-dependent probe ampli-
fication (MS-MLPA) analysis (Nygren et al. 2005; Scott et al. 2008)
was performed in patients 2, 4, and 4U using a commercially
available SALSA kit ME030 B1 (MRC-Holland). The ME030 B1 kit
contains five HhaI sensitive probes in the DMR1 (differentially
methylated region 1) and four in the DMR2 (differentially meth-
ylated region 2) imprinted 11p15 regions. In addition, it includes
17 probes that cover H19, IGF2, KCNQ1, and CDKN1C, and 19
reference probes located in other parts of the genome for a total of
45 probes. Analyses were performed according to the manufac-
turer’s protocol. Briefly, 200 ng of DNA was denatured and hy-
bridized to MLPA probes. The reaction was split into two aliquots.
One aliquot was processed as a standard MLPA reaction for the
copy number analysis. The restriction enzyme HhaI was added
to the ligation reaction of the second aliquot. HhaI recognizes
unmethylated DNA-probe hybrids, therefore only methylated
were separated by capillary electrophoresis using an ABI 3730xl
genetic analyzer (Applied Biosystems). Data were visually in-
spected and analyzed using GeneMarker software (SoftGenetics)
for copy number alteration and methylation pattern.
Bioinformatics and in silico sequence analysis
We used the segmental duplications database from the University
of Washington (Eichler laboratory, http://humanparalogy.gs.
on human genome build 36 (NCBI36/hg18), to obtain the co-
ordinates and sequence identities of the known LCRs (Bailey et al.
2001). There are a total of 15,605 computationally determined in-
terchromosomal LCRs with >1 kb in size and with >90% sequence
identity occurring in ;3%–4% of the human genome (Eichler
et al. 2001). A subset of these LCRs with characteristics consisting
of: (1) location on the same chromosomal arm in the same ori-
entation, (2) location on different chromosomal arms in opposite
orientation, (3) >5 kb in size, and (4) >94% sequence identity
Ou et al.
were computationally identified to derive a circle shaped global
view genomic map of potential NAHR mediated recurrent, non-
homologous, interchromosomal translocations.
Genomic sequences of the breakpoint regions were down-
loaded from the NCBI (http://www.ncbi.nlm.nih.gov) and UCSC
websites. The alignment of two given sequences was performed
and assembledusingthe NCBI BLAST2 (http://www.ncbi.nlm.nih.
We thank the patients and their families for participation in these
studies. This work was supported in part by NINDS grant R01
NS058529 to J.R.L.
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