ORIGINAL INVESTIGATION

Genomic rearrangements at the FRA2H common fragile sitefrequently involve non-homologous recombination eventsacross LTR and L1(LINE) repeats

Lena M. Brueckner • Evgeny Sagulenko •

Elisa M. Hess • Diana Zheglo • Anne Blumrich •

Manfred Schwab • Larissa Savelyeva

Received: 26 January 2012 / Accepted: 24 March 2012 / Published online: 5 April 2012

� Springer-Verlag 2012

Abstract Common fragile sites (cFSs) are non-random

chromosomal regions that are prone to breakage under

conditions of replication stress. DNA damage and chro-

mosomal alterations at cFSs appear to be critical events in

the development of various human diseases, especially

carcinogenesis. Despite the growing interest in under-

standing the nature of cFS instability, only a few cFSs have

been molecularly characterised. In this study, we fine-

mapped the location of FRA2H using six-colour fluores-

cence in situ hybridisation and showed that it is one of the

most active cFSs in the human genome. FRA2H encom-

passes approximately 530 kb of a gene-poor region con-

taining a novel large intergenic non-coding RNA gene

(AC097500.2). Using custom-designed array comparative

genomic hybridisation, we detected gross and submicro-

scopic chromosomal rearrangements involving FRA2H in a

panel of 54 neuroblastoma, colon and breast cancer cell

lines. The genomic alterations frequently involved different

classes of long terminal repeats and long interspersed

nuclear elements. An analysis of breakpoint junction

sequence motifs predominantly revealed signatures of

microhomology-mediated non-homologous recombination

events. Our data provide insight into the molecular struc-

ture of cFSs and sequence motifs affected by their activa-

tion in cancer. Identifying cFS sequences will accelerate

the search for DNA biomarkers and targets for individua-

lised therapies.

Introduction

In recent years, common fragile sites (cFSs) have become

of increasing interest, as their tendency to breakage has

been associated with genomic instability in different types

of disease, especially in cancer (Glover 2006; Dillon et al.

2010). CFSs are non-random chromosomal regions that

tend to undergo double-strand breakage in response to

DNA replication stress. They are present in all individuals

as a part of the normal chromosome architecture. In vivo,

cFS expression may be triggered by various endogenous

and exogenous factors, including hypoxia, chemothera-

peutics and other drugs, exposure to UV or ionising radi-

ation, pesticides, cigarette smoke, and chronic caffeine or

alcohol abuse (Dillon et al. 2010). In vitro, cFSs may be

induced using agents impairing DNA synthesis, such as

aphidicolin, an inhibitor of DNA polymerases a, d and e,which has been shown to activate most fragile sites

(Mrasek et al. 2010; Glover et al. 1984). Active cFSs are

visible microscopically as gaps and breaks in metaphase

chromosomes.

The molecular events underlying the observed fragility

at cFSs are not well understood. A deficiency in proteins

associated with the ATR DNA damage checkpoint pathway

(e.g. ATR, BRCA1, CHK1) appears to result in elevated

cFS breakage, suggesting an essential role of this pathway

in maintaining cFS stability (Casper et al. 2002; Durkin

and Glover 2007). It has been hypothesised that fragility at

cFSs, induced by DNA replication stress, results from

extended single-stranded regions of unreplicated DNA

accumulating at stalled replication forks having escaped

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00439-012-1165-3) contains supplementarymaterial, which is available to authorized users.

L. M. Brueckner � E. Sagulenko � E. M. Hess � D. Zheglo �A. Blumrich � M. Schwab � L. Savelyeva (&)

Division of Tumor Genetics, German Cancer Research Center

(DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg,

Germany

e-mail: l.savelyeva@dkfz.de

123

Hum Genet (2012) 131:1345–1359

DOI 10.1007/s00439-012-1165-3

the ATR replication checkpoint. Recently it has been

demonstrated that topoisomerase I activity is required for

cFS breakage and that polymerase–helicase uncoupling is

an initial key event in cFS instability (Arlt and Glover

2010). Although it is unclear why cFSs are particularly

sensitive to perturbations in DNA replication, there is

evidence suggesting that DNA sequences capable of

forming stable secondary structures impair replication of

these genomic regions (Zlotorynski et al. 2003; Burrow

et al. 2010). While currently the cytogenetic locations of 89

cFSs are listed in the Entrez NCBI human genome data-

base, several additional cFSs have been reported (Mrasek

et al. 2010). Despite growing evidence of their importance

in disease development, most cFSs have not yet been

investigated at the molecular level. Up to now, only nine of

them have been characterised at kilobase resolution:

FRA1E (Hormozian et al. 2007), FRA2C (Blumrich et al.

2011), FRA2G (Limongi et al. 2003), FRA3B (Wilke et al.

1994; Zimonjic et al. 1997), FRA7K (Helmrich et al. 2007),

FRA9G (Sawinska et al. 2007), FRA13A (Savelyeva et al.

2006), FRA16D (Mangelsdorf et al. 2000) and FRAXB

(Arlt et al. 2002). All of these cFSs span AT-rich genomic

regions, ranging between 300 kb and 1 Mb, that appear to

be enriched in peaks of enhanced DNA flexibility (Sch-

wartz et al. 2006). Fragility at cFSs has also been linked to

some epigenetic features, including their association with

regions of late replication (Debatisse et al. 2006) and his-

tone hypoacetylation (Jiang et al. 2009). Ectopic expres-

sion of cFS sequences has been shown to increase breakage

at the integration site, supporting the hypothesis that the

DNA sequence itself is a critical factor underlying cFS

instability (Ragland et al. 2008). In contrast to rare fragile

sites, where fragility can be attributed to either AT-rich

minisatellites or CGG repeat expansions (Sutherland

2003), no such extended repeat motifs have been identified

within cFSs. Nevertheless, all molecularly characterised

cFSs appear to be enriched in stretches of interrupted AT-

dinucleotide-rich sequences with the potential to form

secondary structures, which may thus impair replication

fork progression, in turn resulting in elevated chromosomal

breakage (Zlotorynski et al. 2003; Dillon et al. 2010).

Generally, the level of susceptibility to breakage varies

among cFSs, FRA3B at chromosome band 3p14.2 being the

most frequently expressed and best-studied cFS in the

human genome (Denison et al. 2003; Mrasek et al. 2010).

The second and third most fragile cFSs are FRA16D at

16q23.2 and FRAXB at Xp22.3, respectively, followed by a

group of other cFSs frequently observed in the human gen-

ome, including FRA2H (2q32.1), FRA1E (1p21.3), FRA6E

(6q26), FRA7K (7q22-31) and FRA7H (7q32). Normally,

cFSs are stable in somatic cells, but they are frequently

involved in chromosomal rearrangements in many different

cancers. Heterozygous and homozygous deletions appear to

be the most prevalent genomic alterations of cFS regions in

malignancies such as lung, kidney, breast, and digestive tract

cancers (Arlt et al. 2006). To date, all molecularly charac-

terised cFSs span genomic regions containing protein-cod-

ing gene sequences, and most of them encompass large

genes extending over hundreds of kilobases of genomic

DNA (Smith et al. 2007). The two most active and best-

characterised cFS genes, FHIT at FRA3B and WWOX at

FRA16D, have been demonstrated to function as tumour

suppressor genes, whose inactivation provides a selective

growth advantage to cancer cells (Lewandowska et al. 2009;

Saldivar et al. 2010). Recently, direct evidence suggesting

involvement of cFSs in generating cancer-specific rear-

rangements in human cells has been provided by Gandhi

et al. (2010). After exposure to fragile site-inducing chem-

icals, the RET, CCDC6, and NCOA4 genes, located in

FRA10C and FRA10G, respectively, undergo DNA break-

age and form rearrangements known to contribute to papil-

lary thyroid carcinoma development. DNA damage at cFSs

appears to be among the earliest events during tumouri-

genesis, resulting from oncogene-induced replication stress

in many kinds of human tumours (Halazonetis et al. 2008;

Tsantoulis et al. 2008). This assumption stems from the

observation that, in precancerous lesions, genomic altera-

tions preferentially target cFSs as those loci most sensitive to

replication stress and has led to suggesting DNA damage at

cFSs as potential biomarkers for clinical patient manage-

ment and cFS genes as targets for therapies (Lai et al. 2010).

CFS activation may also trigger gene amplification (e.g.

MET, MYC, MYCN) (Blumrich et al. 2011; Hellman et al.

2002; Cicek et al. 2009) and serve as preferred integration

sites for several oncogenic viruses, such as hepatitis B and

human papilloma viruses (Ferber et al. 2003).

Besides their involvement in somatic rearrangements

associated with tumourigenesis, cFSs also appear to con-

tribute to germline rearrangements leading to non-malig-

nant human diseases. Recently, evidence supporting this

hypothesis has emerged from sequence analysis of break-

point junctions within the PARK2 (FRA6E) and DMD

(FRAXC) cFS genes in a large number of patients with

autosomal-recessive juvenile Parkinsonism and Duchenne

and Becker muscular dystrophy, respectively (Mitsui et al.

2010). The investigated germline breakpoint sequences

shared some features with somatic breakpoints within

PARK2 and DMD in cancer cell lines, suggesting that

common mechanisms may be involved in generating both

germline and somatic rearrangements over cFS regions. As

a growing body of evidence points to cFS instability as an

important factor contributing to human disease develop-

ment, it is crucial to identify and molecularly characterise

the full repertoire of these regions in the human genome.

In this study, using the advantage of six-colour fluo-

rescence in situ hybridisation (FISH), we were able to

1346 Hum Genet (2012) 131:1345–1359

123

delineate the boundaries of the entire FRA2H region at

kilobase resolution level and to characterise the genetic

complexity of the fragile DNA sequence. We show that

FRA2H is one of the most active cFSs in the human gen-

ome. Using custom-designed 60 bp-resolution comparative

genomic hybridisation arrays (array CGH), FISH with

specific DNA probes and multicolour FISH (mFISH), we

detected gross chromosomal rearrangements in colon car-

cinoma and neuroblastoma cell lines, as well as two novel

copy number variants (CNVs) at FRA2H. Sequence anal-

ysis of breakpoint junctions revealed that DNA damage

repair at FRA2H predominantly appears to occur via non-

homologous recombination events mediated by short

microhomologies.

Materials and methods

Cell culture and fragile site induction

Colorectal carcinoma cell lines were cultured in DMEM/

Ham’s F12 medium (50:50; Biochrom, Berlin, Germany)

supplemented with 20 % foetal calf serum (FCS; PAA,

Pasching, Austria). HDC cell lines (Bruderlein et al. 1990)

were of low passage number. Epstein–Barr virus (EBV)-

transformed lymphocytes from eight healthy individuals,

neuroblastoma and breast cancer cell lines were cultivated

in RPMI 1640 medium (Lonza, Cologne, Germany) sup-

plemented with 10 % FCS. Cell line authenticity was

verified cytogenetically and/or via DNA typing (DSMZ,

Braunschweig, Germany and DKFZ Genomics and Pro-

teomics Core Facility, Heidelberg, Germany). To induce

fragile site breakage, EBV-transformed lymphocytes were

treated with 0.4 lM aphidicolin (Sigma-Aldrich, Deis-

enhofen, Germany) in 0.5 % ethanol 24 h prior to cell

harvest. Metaphase preparations were made following

standard procedures.

Bacterial artificial chromosome (BAC) and fosmid

clones

BAC and fosmid clones were obtained from the Children

Hospital Oakland Research Institute (CHORI, Oakland,

California, USA). DNA isolation was carried out following

standard phenol–chloroform extraction techniques. DNA

extracts served as probes for FISH-based fragile site

mapping and validation of array CGH data. End sequences

of BAC clones at FRA2H (RP11-334K17, RP11-400O18,

RP11-561J1, RP11-335G13, RP13-513D10, RP13-541C6,

RP11-639N24 and RP11-625P14) were determined using

T7 and SP6 standard primers (Seqlab sequencing services,

Gottingen, Germany).

Fluorescence in situ hybridisation

Six-colour FISH was performed to map the location of

FRA2H and to validate array CGH data. BAC and fosmid

DNA probes were labelled with DEAC, FITC, Cy3, Cy3.5,

Cy5 and Cy5.5-coupled dUTPs by nick-translation. The

succinimidyl-ester derivatives of the fluorescent dyes

DEAC, FITC (Molecular Probes, Eugene, Oregon, USA),

Cy3, Cy3.5, Cy5 and Cy5.5 (GE Healthcare, Freiburg,

Germany) were used for the synthesis of modified nucle-

otides. Fluorescent dye coupling to allylamine-dUTPs

(Sigma-Aldrich, Deisenhofen, Germany) was carried out as

described previously (Henegariu et al. 2000). Hybridisation

to metaphase spreads occurred overnight and followed

standard cytogenetic procedures. Hybridised slides were

counterstained with DAPI (4, 6-diamidino-2-phenylindole;

Sigma-Aldrich, Munich, Germany). Fluorescent signals

were visualised using a Leica DMRA 2 microscope and

analysed with the corresponding Leica CW 4000 FISH

software. Array CGH validation assays included a probe

control experiment on normal lymphocytes to ensure the

correct signal number and position of BAC and fosmid

clones.

Multicolour FISH

Karyotypes of each tumour cell line were obtained using

the commercially available 24XCyte multicolour FISH

probe mix (Metasystems, Altlussheim, Germany). DNA

denaturation and hybridisation to metaphase spreads fol-

lowed the manufacturer’s recommendations. Slides were

viewed with a Zeiss Axio Imager.Z1 microscope and kar-

yotypes constructed with ISIS FISH imaging software

(Metasystems, Altlussheim, Germany).

Array comparative genomic hybridisation

The custom array designs of selected fragile site regions

(Roche NimbleGen, Madison, Wisconsin, USA) used in this

study contained 730,000 60-mer oligonucleotide probes per

array yielding a resolution of approximately 60 bp. Geno-

mic DNA from lymphocytes and tumour cell lines was

isolated by phenol–chloroform extraction following stan-

dard procedures. A lymphocyte genomic DNA pool from

five healthy individuals served as reference DNA. Sample

labelling and hybridisation were carried out following the

manufacturer’s instructions (Roche NimbleGen). DNA

quality and labelling efficiency were determined using a

Nanodrop-1000 spectrophotometer (NanoDrop Technolo-

gies, Wilmington, Delaware, USA). Array slides were

scanned at a 2 lm resolution using a Roche ms-200 scan-

ner. Array CGH images were processed with NimbleGen

Hum Genet (2012) 131:1345–1359 1347

123

NimbleScan (v. 2.4) and viewed with NimbleGen Signal-

Map (v. 1.9.0.03).

PCR amplification

Specific primers on either side of each break were designed

using the Primer3 (v. 0.4.0) web interface or the Invitrogen

OligoPerfectTM

Designer software. All primers were ana-

lysed using NCBI BLAST and IDT SciTools OligoAnalyzer

3.0 software. Two negative controls were included in each

PCR run, substituting tumour DNA with normal lymphocyte

genomic DNA and water. In case of multiple PCR products,

nested PCR was carried out using a second pair of primers.

The following primers were used: HDC-133 nm: forward

50-AAGCCAGAATGCCAGCTTAT-30, reverse 50-AGGA

AAGCAAATGGGGTTTA-30; HDC-54: forward 1 50-GGC

AAATGGAATCAGTGGAT-30, forward 2 50-GAATCAA

TTAAACGAGGCTTGG-30, reverse 1 50-TTCTCCCCAT

CACTTTCAGG-30, reverse 2 50-TCCATTGCTGATACC

CTTTCTT-30; NB-69: forward 50-TGCTTGACATCCTTA

GTCATGG-30, reverse 50-CTCTTTGTGGGGATCTCTC

ATC-30; HDC-114: forward 50-CTGACTGGAACAAGCT

AGTGGGT-30, reverse 50-GGTTTGAAGAGCTGAAAT

AGCAA-30. Purified PCR products were sequenced using

GATC (Konstanz, Germany) or Seqlab (Gottingen, Ger-

many) sequencing services.

Sequence analysis

DNA sequences of FRA2H and 500 kb of the adjacent non-

fragile regions were obtained from the UCSC Genome

Browser (NCBI build 37, hg19). The positions of break-

point junction sequences were identified using the BLAT

tool on the UCSC website. The interspersed repeat com-

position was identified using RepeatMasker (v.3.3.0). To

evaluate DNA flexibility, TwistFlex, a programme mea-

suring the potential variation in the twist angle between

consecutive base pairs, was used. TwistFlex analysis was

performed with default settings; windows with values

[13.7� were considered as flexibility peaks. The EMBOSS

Needle alignment was used to calculate the extent of

sequence homology between the sequences (1,000 bp)

flanking each breakpoint. Palindromic sequences larger

than 20 nt were identified using EMBOSS palindrome and

microsatellites with more than 20 repeat units using Tan-

dem Repeats Finder.

Web resources

Database of Genomic Variants (DGV; last update: 02 Nov

2010), http://projects.tcag.ca/variation/; 1000 Genomes

Project (release 9 September 2011), http://browser.1000

genomes.org/; Ensembl human genome database (release

63, June 2011), http://www.ensembl.org/; Entrez NCBI

human genome database, http://www.ncbi.nlm.nih.gov/

gene/; Primer3, http://frodo.wi.mit.edu/primer3/input.htm;

Invitrogen OligoPerfectTM

Designer, http://tools.invitrogen.

com/content.cfm?pageid=9716; NCBI BLAST, http://www.

ncbi.nlm.nih.gov/tools/primer-blast/; IDT SciTools Oli-

goAnalyzer, http://eu.idtdna.com/analyzer/applications/

oligoanalyzer/; UCSC Genome Browser, http://genome.

ucsc.edu/; RepeatMasker, http://www.repeatmasker.org/;

TwistFlex, http://margalit.huji.ac.il/TwistFlex/; EMBOSS

Needle, http://www.ebi.ac.uk/Tools/psa/emboss_needle/

nucleotide.html; EMBOSS palindrome, http://emboss.

bioinformatics.nl/cgi-bin/emboss/palindrome; Tandem

Repeats Finder, http://tandem.bu.edu/trf/trf.html.

Results

FRA2H spans 533 kb of a gene-poor region at 2q32.1

According to the Entrez NCBI human genome database,

FRA2H has been cytogenetically assigned to chromosome

band 2q32.1, encompassing approximately 6.4 Mb of the

human genome. To determine the molecular location of

FRA2H at 100–150 kb resolution level, six-colour FISH

was carried out using 16 BAC probes. Mapping data are

summarised in Table 1 and illustrated in Fig. 1. Initially,

six differently labelled BAC probes (RP11-357A22, RP11-

262E6, RP11-1112I8, RP11-114I18 RP11-124A13 and

RP11-761I2) spaced at approximately 5 Mb, covering

about 25 Mb of 2q31.1–2q32.3, were hybridised to meta-

phase spreads of aphidicolin-treated lymphocytes from

eight healthy individuals (Fig. 1a). Of 821 investigated

metaphases, FRA2H breaks occurred in 78 cases. All

FRA2H breaks were observed between the signals of RP11-

114I18 and RP11-124A13, within a 4.4 Mb genomic region

extending from 185.5 to 189.9 Mb. To further approximate

the position of FRA2H, RP11-114I18, RP11-124A13 and

four additional BAC clones (RP11-334K17, RP11-16M14,

RP11-60J7, RP11-843D8), evenly spaced within the

defined region, were hybridised to metaphase spreads from

the same set of individuals (Fig. 1b). All FRA2H breaks (i.e.

42 breaks in 460 metaphases) were found to be located

between RP11-334K17 and RP11-16M14, defining FRA2H

to a 900 kb region (186.6–187.5 Mb). Finally, the molec-

ular location of FRA2H was fine-mapped using eight

contiguous BAC probes (RP11-334K17, RP11-400O18,

RP11-561J1, RP11-335G13, RP13-513D10, RP11-639N24,

RP11-625P14, RP11-16M14) spanning this region (Fig. 1c;

Table 1). In all investigated metaphases with FRA2H

breakage (i.e. 71 breaks in 732 metaphases), RP11-334K17

and RP11-400O18 only produced signals centromeric to the

break, while RP11-625P14 and RP11-16M14 signals were

1348 Hum Genet (2012) 131:1345–1359

123

only observed telomeric to the break. BAC clones were

considered to span the fragile region when their fluorescent

signal was detected on either side of a FRA2H break. Sig-

nals spanning FRA2H breaks were produced by RP11-

561J1 (11.3 %), RP11-335G13 (31.0 %), RP13-513D10

(53.5 %) and RP11-639N24 (4.2 %), defining the entire

FRA2H region to a size of 533 kb, encompassing the

genomic region from 186.72 to 187.25 Mb. To construct a

FRA2H physical and genetic map, the positions of the eight

BAC clones covering or bordering FRA2H were verified by

end sequencing. Alignment of the 533 kb sequence with the

UCSC human genome browser confirmed that FRA2H

maps to the 2q32.1 G-band and is located within a gene-

poor region. 84.5 % of all FRA2H breaks occurred within

RP11-335G13 and RP13-513D10, suggesting a core fragile

region of about 344 kb. Overall, our data indicate a high

sensitivity of this genomic region to replication stress, as

FRA2H breakage was detected in 9.5 % (i.e. 191 out of

2,013) of aphidicolin-treated lymphocyte metaphases.

FRA2H is the most active cFS on the long arm

of chromosome 2

The relative frequency of expression differs among cFSs in

the human genome, breakage at 3p14.2 (FRA3B) being

most commonly observed, followed by breakage at 16q23

(FRA16D), Xp22.3 (FRAXB) and 2q31–32 (FRA2H) (Glo-

ver et al. 1984; Mrasek et al. 2010). We aimed to determine

whether FRA2H, spanning the genomic region from 186.72

to 187.25 Mb, corresponds to the fourth most active cFS in

the human genome, which has previously been assigned to

2q32.1 by G-banding (Glover et al. 1984). Therefore, we

compared FRA2H fragility to five other known cFSs on the

long arm of chromosome 2 using FISH on aphidicolin-

treated lymphocytes from eight healthy individuals

(Table 2). BAC probes located near FRA2F (RP11-

1193F23), FRA2G (RP11-357A22), FRA2H (RP11-121I13),

FRA2I (RP11-388A17) and FRA2J (RP11-225M4) were

used for this purpose. Although expression patterns differed

slightly among individuals, FRA2H expression was

observed most frequently with breakage in 181 out of 2,595

metaphases overall. FRA2F with breaks in 40 metaphases

and a novel cFS at 2q12–14 with breaks in 33 metaphases

appear to be the second and third most fragile site on

chromosome arm 2q, respectively. In comparison to

FRA2H, FRA2I (23 breaks), FRA2G (17 breaks) and FRA2J

(16 breaks) seem only moderately susceptible to aphidico-

lin-induced breakage. Overall, these data indicate that

FRA2H is the most active cFS on the long arm of chro-

mosome 2 and together with previous estimates (Mrasek

et al. 2010) may thus be considered to be the fourth most

active aphidicolin-induced cFS in the human genome.

FRA2H is enriched in LTR and L1 long interspersed

nuclear elements (LINE) sequences

To determine whether FRA2H harbours any particular

sequence repeat motifs that may account for its fragility,

Table 1 FISH mapping of FRA2H

BAC clones Chromosome 2 bp

(GRCh37/hg19)

Known genes Number of fluorescent signals

C S T %C %S %T

RP11-357A22 170,227,020–170,299,104 191 0 0 100 0 0

RP11-262E6 174,894,479–175,057,417 191 0 0 100 0 0

RP11-1112I8 179,875,332–180,030,900 191 0 0 100 0 0

RP11-114I18 185,375,595–185,431,815 191 0 0 100 0 0

RP11-334K17 186,448,899–186,614,447 FSIP2, AC007966.1 120 0 0 100 0 0

RP11-400O18 186,572,178–186,717,985 FSIP2, AC007966.1 71 0 0 100 0 0

RP11-561J1 186,652,393–186,839,345 FSIP2 63 8 0 88.7 11.3 0

RP11-335G13 186,781,768–186,961,978 AC097500.2 41 22 8 57.7 31.0 11.3

RP13-513D10 186,946,516–187,125,789 3 38 30 4.2 53.5 42.3

RP11-639N24 187,108,314–187,264,292 0 3 68 0 4.2 95.8

RP11-625P14 187,251,137–187,446,479 ZC3H15 0 0 71 0 0 100

RP11-16M14 187,447,002–187,639,629 FAM171B, ITGAV 0 0 120 0 0 100

RP11-60J7 188,007,222–188,177,239 0 0 120 0 0 100

RP11-843D8 189,015,216–189,220,306 0 0 120 0 0 100

RP11-124A13 189,942,867–190,096,644 0 0 191 0 0 100

RP11-761I2 195,011,019–195,179,513 0 0 191 0 0 100

C centromeric, S spanning the break, T telomeric

Hum Genet (2012) 131:1345–1359 1349

123

we analysed the FRA2H sequence as well as 500 kb of the

non-fragile adjacent sequences with the RepeatMasker web

interface (Table 3; Supplementary Figure 1b, c). Sequen-

ces of FRA2H and its flanking regions were obtained from

the UCSC genome browser. With an AT content of 64.6 %,

the FRA2H sequence is considered to be enriched in AT

nucleotides, which are uniformly distributed along the

analysed sequence. The adjacent non-fragile sequences,

also located within the 2q32.1 G-band, have a similar AT

content. FRA2H is composed of 57.6 % interspersed

RP11-400O18

RP11-561J1 RP11-639N24

RP11-625P14

q32.1

RP11-335G13

RP13-513D10

533 kb

c

b

a

170 Mb 175 Mb 180 Mb 185 Mb 190 Mb 195 Mb2q31.1 2q31.2 2q31.3 2q32.1 2q32.2 2q32.3

RP11-357A22 RP11-262E6 RP11-1112I8 RP11-114I18 RP11-124A13 RP11-761I2

185 Mb 186 Mb 187 Mb 188 Mb 189 Mb 190 Mb

RP11-114I18 RP11-334K17 RP11-16M14 RP11-60J7

2p25.2 2p24.1 2p22.3 2p16.1 2p11.22p13.1 2q12.1 2q14.1 2q21.2 2q23.3 2q24.3 2q31.2 2q32.3 2q34 2q36.12q37.2

RP11-334K17

11.3% 31.0% 53.5% 4.2%

ZC3H15 AC018867.1 AC007966.1

FSIP2

186.6 Mb186.5 Mb 186.7 Mb 187.2 Mb186.8 Mb 186.9 Mb 187.0 Mb 187.1 Mb 187.3 Mb 187.4 Mb

RH48029 RH48586 SHGC-110012 WI-22618 SHGC-106163RH92115 D2S1875 RH119604

centromeric telomericspanning

RP11-16M14

187.5 Mb 187.6 Mb

FRA2H

ITGAV

FAM171B

D2S2967 D2S2500

AC097500.2

RP11-843D8 RP11-124A13

RP11-334K17 RP11-400O18 RP11-561J1 RP11-335G13 RP13-513D10 RP11-639N24 RP11-625P14 RP11-16M14

1350 Hum Genet (2012) 131:1345–1359

123

repeats, including 4.5 % ALU, 1.0 % MIR, 30.0 %

L1(LINE), 3.6 % L2(LINE), 14.5 % long terminal repeat

(LTR) and 3.3 % DNA elements. Percentages of ALU,

MIR, L2(LINE) and DNA elements are similar to those

estimated to represent the genomic fraction of autosomal

DNA with an AT value over 64 % (Smit 1999). In com-

parison to the genomic mean (20 %), L1(LINE) appear to

be overrepresented in FRA2H and the 500 kb region

proximal to FRA2H (30.0 and 28.1 %, respectively).

Moreover, the frequency of LTR elements appears to be

elevated with 14.5 % in FRA2H relative to an estimate of

6.8 % in the mean AT-rich fraction of the genome.

To examine whether FRA2H contains a higher number of

potentially highly flexible sequences relative to the non-

fragile adjacent regions, the sequences were analysed using

TwistFlex, a tool designed to predict the inherent flexibility

of a given sequence in relation to the DNA helix twist angle.

At a helix twist angle of 13.7�, with a value of 12.9, FRA2H

appears to have more flexibility peaks per 100 kb in com-

parison to the centromeric and telomeric flanking sequences

with values of 8.8 and 6.6, respectively (Table 3). FRA2H is

about 2.5-fold enriched in clusters of flexibility peaks,

defined as at least three flexibility peaks between which the

distance of any two adjacent peaks is B5 kb (Supplementary

Figure 1a). As the mean flexibility value for non-fragile

G-band sequences has been defined as 3.3 flexibility islands/

100 kb (Debacker et al. 2007), the number of flexibility

peaks is increased 3.9-fold in FRA2H and 2.7-fold in its

centromeric flanking sequence. At a twist angle of 16�,

however, the values do not appear to differ substantially. To

estimate the content of sequence motifs with the potential to

form unusual secondary structures, we identified the location

of palindromes ([20 nt), microsatellites ([20 repeat units)

and segmental duplications using EMBOSS palindrome and

Tandem Repeats Finder (Supplementary Figure 1a). The

FRA2H sequence contains about 2.8-fold more palindromes

than the adjacent genomic regions, but exhibits a similar

amount of microsatellite repeats. The analysis revealed

neither stretches of perfect AT repeats nor segmental

duplications within FRA2H.

Overall, the FRA2H region is AT-rich and appears to be

enriched in flexible sequences, like the majority of

molecularly characterised cFSs. Although computational

analysis showed that the entire 1.53 Mb region is AT-rich

and abundant in peaks of enhanced DNA flexibility,

FRA2H exhibits a much higher density of flexibility peaks

in comparison to the upstream and downstream sequences.

A notable feature of FRA2H is the elevated retrotransposon

LTR content, which is considerably higher than in the AT-

rich genomic mean and in other cFS sequences studied to

date (Savelyeva et al. 2006).

Table 2 Frequency of breakage at cFSs on the long arm of chromosome 2

Individual 2q12-14 FRA2F FRA2G FRA2H FRA2I FRA2 J Total 2q No. of metaphases

I 20 19 7 61 12 5 124 888

II 5 6 3 46 4 4 68 577

III 3 5 0 28 1 3 40 319

IV 1 6 2 15 2 2 28 158

V 1 1 0 1 0 1 4 113

VI 2 2 3 18 3 1 29 304

VII 1 1 0 10 1 0 13 204

VIII 0 0 2 2 0 0 4 32

Total 33 40 17 181 23 16 310 2,595

Fig. 1 FISH mapping and genomic localisation of FRA2H. a An

ideogram of chromosome 2 and a detailed presentation of the

2q31–32 region including the genomic location of six differently

labelled BAC clones are shown at the top. DAPI (left) and six-colour

FISH (right) images represent chromosomes 2 with aphidicolin-

induced FRA2H breaks from metaphase spreads of four individuals.

All breaks occurred between BAC clones RP11-114I18 and RP11-

124A13 (dotted red frame). b The genomic positions of six BAC

probes within the 185–190 Mb region selected to further approach

FRA2H are depicted at the top. A panel of chromosome 2 images with

FRA2H breaks is shown. Each chromosome 2 is presented three

times—left: DAPI; middle: FISH with RP11-114I18 and PR11-60J7;

right: FISH with RP11-334K17 and RP11-16M14. All breaks

occurred between RP11-334K17 and RP11-16M14 signals (dottedred frame). c Fine-mapping of FRA2H using contiguous BAC probes

spanning the region between RP11-334K17 and RP11-16M14. In all

investigated metaphases with FRA2H breakage RP11-334K17 and

RP11-400O18 produced signals centromeric to the break, while

RP11-625P14 and RP11-16M14 only produced signals telomeric to

the break. Breakpoint-spanning signals included those from RP11-

561J1, RP11-335G13, RP13-513D10 and RP11-639N24. The fre-

quency (%) of observed breakpoint-spanning signals is indicated for

each BAC below the FISH images. The genomic positions of the

contiguous BAC probes verified by end sequencing are displayed in a

physical map of the FRA2H region (borders indicated by red frame).

The genomic map shows that the AC097500.2 lincRNA gene is the

only known gene located within FRA2H. Known coding genes are

presented in black. FISH signals and bar colours on the physical and

genomic map correspond to fluorescent labels: DEAC dUTP (purple),

FITC dUTP (green), Cy3 dUTP (red), Cy3.5 dUTP (pink), Cy5 dUTP

(yellow), Cy5.5 dUTP (blue)

b

Hum Genet (2012) 131:1345–1359 1351

123

FRA2H is affected by intrachromosomal

rearrangements in human cancer cell lines

To determine whether FRA2H is unstable in cancer cells, 26

colorectal carcinoma, 17 neuroblastoma and 11 breast can-

cer cell lines (Supplementary Table 1) were subjected to

array CGH analysis (Fig. 2a). Custom-designed oligonu-

cleotide CGH arrays included a 1.1 Mb genomic region

(chr2: 186,290,000–187,400,000) encompassing the entire

FRA2H region and approximately 400 kb of its centromeric

and 150 kb of its telomeric flanking sequence. Array CGH

detected a number of apparently tumour-related rearrange-

ments at FRA2H in 7.4 % of tumour cell lines (i.e. in 4 out of

54). Rearrangements were most frequently observed in

colorectal cancer cell lines (i.e. in 11.5 %; 3 out of 26

samples), comprising a 300.5 kb loss in HDC-133 nm, a

133.5 kb loss in HDC-54 and a 82.5 kb loss in SW-620 sub1

cells. A gain of genomic material, affecting the entire

FRA2H sequence, was found in the NB-69 neuroblastoma

cell line. No rearrangements were detected in any of the

investigated breast cancer cell lines. To validate copy

number changes and to assess the chromosomal architecture

of the observed FRA2H rearrangements, we performed

FISH (Fig. 2b) using BAC and fosmid DNA probes (Sup-

plementary Table 2) located within or in close proximity to

each copy number alteration detected by array CGH and

mFISH (Fig. 2c). In HDC-133 nm cells, mFISH revealed

three apparently normal copies of chromosome 2, on all of

which RP11-561J1 and RP11-639N24 produced hybridisa-

tion signals, while RP11-335G13 and RP13-513D10 signals

were only visible on two out of three chromosomes, sug-

gesting an interstitial deletion. HDC-54 cells contain a

structurally normal chromosome 2 displaying three specific

BAC signals (RP11-334K17, RP11-400O18 and RP11-

335G13) and a derivative chromosome 2 homologue

showing RP11-334K17 and RP11-335G13 signals only. The

missing RP11-400O18 signal on the derivative chromosome

2 implies an interstitial deletion. SW-620 sub1 cells have a

set of four chromosomes 2, which appear to be structurally

normal on mFISH resolution level. However, FISH using

RP11-334K17, G248P81916F8 and RP11-291P02 probes

revealed that two of the chromosome 2 homologues harbour

an interstitial deletion at FRA2H as G248P81916F8 signals

were not visible on either of them. Thus, heterozygous

interstitial deletions at FRA2H were determined in all three

colorectal carcinoma cell lines with genomic losses. To

Table 3 DNA sequence

analysis of FRA2H and flanking

regions

FRA2H Non-fragile flanking regions (500 kb) Genome

Centromeric Telomeric

Flexibility peaks/100 kb

13.7� 12.9 8.8 6.6

16� 1.3 3.0 0.6

DNA repeat composition (%)

GC content 35.4 34.6 37.0 \36.0

ALUs 4.5 4.4 7.4 5.0

MIRs 1.0 0.7 2.1 1.5

L1(LINE) 30.0 28.1 21.2 20.0

L2(LINE) 3.6 2.1 4.2 3.1

LTR elements 14.5 12.3 9.7 6.8

DNA elements 3.3 5.8 4.2 2.9

Total interspersed repeats 57.6 53.4 49.5 39.4

Fig. 2 Copy number alterations at FRA2H in human cancer cell

lines. a Array CGH revealed losses of genetic material in HDC-

133 nm, HDC-54 and SW-620 sub1 cell lines and a gain in the NB-69

cell line. Dotted lines indicate the borders of FRA2H. The genomic

coordinates of oligomers are shown on the x axis, the log2 (ratio of

cancer cell line to reference DNA) on the y axis. Arrowheads on array

plots indicate the genomic position of BAC clones used to validate

array data. b FISH validation of DNA copy number alterations. In

HDC-133 nm cells, there are two normal chromosomes 2 with four

hybridisation signals and a rearranged chromosome with signals of

RP11-561J1 (purple) and RP11-639N24 (green). HDC-54 cells have

a normal chromosome 2 with three hybridisation signals and an

aberrant chromosome 2 lacking the RP11-400O18 signal (red). SW-

620 sub1 cells have four chromosomes 2, two of which do not display

G248P81916F8 signals (red). In NB-69 cells, the abnormal chromo-

some 2 displays single copy RP11-334K17 and RP11-475D24 signals

flanking the duplication. RP11-625P14 and RP11-707M15 signals are

duplicated and their orientation suggests a direct tandem duplication.

Normal chromosomes 2 are marked by white arrows; aberrant

chromosomes 2 by red arrows. c The intrachromosomal nature of

FRA2H rearrangements is confirmed by mFISH. Chromosome 2

material (purple) is marked by white arrows. Chromosome 2

translocations in HDC-54 and NB-69 cells do not involve the FRA2Hlocus. d Alignment of rearrangements to the transcriptional map of

FRA2H. FSIP2 and AC008174.3 at the centromeric border of FRA2Hare deleted in HDC-54 cells. AC097500.2 is heterozygously lost in

HDC-133 nm cells. The deletion in SW-620 sub1 cells does not affect

any known genes. In NB-69 cells, the large duplication covers the

entire FRA2H region. Known coding genes are presented in black,

rearranged regions in either green (gain) or red (loss)

c

1352 Hum Genet (2012) 131:1345–1359

123

estimate the size of the gain in the NB-69 neuroblastoma cell

line, we monitored its length across the more telomeric

portion of 2q, extending from 186.65 to 206.24 Mb. To

validate NB-69 array CGH data, we performed mFISH and

FISH using four BAC probes (RP11-334K17, RP11-

625P14, RP11-707M15 and RP11-475D24) covering the

entire length of the 19.6 Mb gain. FISH revealed a direct

tandem duplication within a large derivative chromosome

composed only of chromosome 2 material. An additional

portion of chromosome 2 was revealed by mFISH, located

HD

C-1

33nm

NB

-69

0.0

0.8

-0.8

0.0

0.8

-0.8

FRA2H

0.0

0.8

-0.8

0.0

0.8

-0.8

HD

C-5

4S

W-6

20 s

ub1

a b c

d

FRA2H

Chromosome 2 (Mb)

RP11-561J1 RP11-335G13 RP13-513D10 RP11-639N24

SW-620 sub1

187.4

186.4 186.6 186.8 187.0 187.2

HDC-54

NB-69

186.4 186.5 186.6 186.7 186.8 186.9 187.0 187.1 187.2 187.3

ZC3H15 AC018867.1

AC080125.1 AC097500.1 AC104058.1 AC093038.1 AC017071.1

AC018867.1U8 AC007966.1

FSIP2

HDC-133nm

RP11-334K17 RP11-625P14 RP11-707M15 RP11-475D24

RP11-334K17 G248P81916F8 RP11-291P02

RP11-334K17 RP11-400O18 RP11-335G13

AC008174.3 AC097500.2

Hum Genet (2012) 131:1345–1359 1353

123

on a derivative chromosome 11 with several translocations

including part of the short arm of chromosome 2 (validated

using fosmid clone G248P89404A5; data not shown). To

test whether FRA2H is involved in balanced rearrangements

undetectable by array-CGH, FISH using BAC clones RP11-

334K17 and RP11-16M14 was performed on all 54 cell

lines. Neither translocations nor inversions appeared to

affect the FRA2H region in any of the tested cancer cell

lines. Hence, all detected alterations represented simple in-

trachromosomal deletions or, in one case duplication, all

with one or two breaks within or in close proximity to

FRA2H.

To determine whether genomic rearrangements at

FRA2H are associated with damage of known genetic

elements, we aligned the detected chromosomal rear-

rangements with a transcriptional map of the 1.1 Mb

genomic region analysed by array CGH (Fig. 2d). The

transcriptional map shows that AC097500.2, a novel large

intergenic non-coding RNA (lincRNA) gene, is the only

known gene located within FRA2H. Fibrous sheath inter-

acting protein 2 (FSIP2), situated at the centromeric border

of FRA2H, is the only known protein-coding gene located

in close proximity to FRA2H. Another protein-coding gene

in the analysed region, zinc finger CCCH-type containing

15 (ZC3H15), lies approximately 650 kb telomeric to

FSIP2, in 100 kb distance to the telomeric border of

FRA2H. Apart from AC097500.2, FSIP2 and ZC3H15,

another lincRNA gene (AC007966.1) and a novel pro-

cessed transcript (AC008174.3) map within the analysed

region. The 300.5 kb deletion found in HDC-133 nm cells

appears to affect AC097500.2, while the 133.5 kb deletion

in HDC-54 cells leads to heterozygous loss of both FSIP2

and AC008174.3, as well as to partial loss of lincRNA

AC007966.1 sequences. The 82.5 kb deletion within the

centromeric portion of FRA2H in SW-620 sub1 cells does

not involve any known genes, in contrast to the large

19.6 Mb duplication in NB-69 cells, which may affect

expression of more than 80 protein-coding genes. In con-

clusion, of the investigated tumour types, colorectal cancer

appears to be most prone to FRA2H DNA copy number

alterations due to intrachromosomal recombination events.

Genomic rearrangements at FRA2H occur mainly

within L1(LINE) and LTR elements

Despite the involvement of cFSs in cancer-related chro-

mosomal recombination being well documented, there are

limited data regarding the impact of particular DNA

sequences on break formation at these regions. Taking

advantage of the 60 bp probe density on our CGH arrays,

we attempted to resolve the rearrangements found in

tumour cell lines at sequence level. To obtain the break-

point junctions, PCR was carried out on genomic DNA

from four tumour cell lines harbouring FRA2H rearrange-

ments using specific primers located on either side of each

breakpoint. PCR products and sequence data are shown in

Fig. 3. In HDC-133 nm cells, the 300,526 bp deleted

region lies within FRA2H, extending from 186,849,907 to

187,150,432 bp. Alignment with the genomic sequence

revealed a cytosine nucleotide insertion and no microho-

mology at the deletion junction. HDC-54 cells have a

133,465 bp deletion from 186,596,799 to 186,730,263 bp

of chromosome 2. The comparison of the joined DNA

fragment with the reference genomic sequence demon-

strated a 7 bp microhomology at the junction. We failed to

amplify the breakpoint junction in SW-620 sub1 cells,

owing to long stretches of repetitive elements on either side

of the deletion. In NB-69 cells, using outward facing

primers, we successfully sequenced the 19,579,975 bp

duplication extending from 186,656,022 to 206,235,996 bp

of chromosome 2, confirming a direct tandem orientation.

The duplication junction results from simple end-to-end

joining of DNA segments and is flanked by 6 bp of

microhomology. To find common DNA sequence motifs at

sites of chromosomal recombination at FRA2H, we ana-

lysed 500 bp of the upstream and downstream sequences

flanking each breakpoint. Overall, with a mean of 62.7 %,

the analysed breakpoint sequences have an AT-content

similar to the entire FRA2H sequence ranging from 51.9 %

in the sequence centromeric to the breakpoint in HDC-54

to 71 % in the sequence telomeric to the breakpoint in

HDC-133 nm cells. An examination of sequence homology

between centromeric and telomeric breakpoints using

EMBOSS Needle did not reveal a significant sequence

identity (i.e. mean value of 39.6 %) in any of the analysed

sequence pairs. A DNA repeat composition analysis using

RepeatMasker showed that both centromeric breakpoints of

the deletions in HDC-133 nm and HDC-54 cells are loca-

ted within LTRs, belonging to the ERVL–MaLR family,

and that both telomeric breakpoints lie within the L1

family of LINE repeats. Our 60 bp-resolution array CGH

data show that both the telomeric and centromeric break-

points of the FRA2H deletion in SW-620 sub1 cells lie

within ERV1 (chr2: 187,194,563–187,195,110) and ERVL

(chr2: 187,274,735–187,277,066) LTRs, respectively. In

contrast to the sequences at deletion breakpoints, both

duplication ends in NB-69 cells align to unique genomic

regions. To investigate whether the regions of high DNA

flexibility identified within FRA2H coincide with the

sequences encompassing breakpoints, we analysed them

using TwistFlex. The only flexibility peak was found

within the telomeric breakpoint of HDC-133 nm, partially

overlapping with a (TCTA)19 simple repeat spanning from

187,150,581 to 187,150,653 bp.

According to the DGV (Supplementary Table 3) and the

1000 Genomes Project, several germline CNVs have been

1354 Hum Genet (2012) 131:1345–1359

123

identified within FRA2H in the healthy population. In this

study, we have detected two novel germline CNVs in close

proximity to FRA2H (Supplementary Figure 2). Since

deletions identical at array-resolution level were observed

twice at 186,337,009–186,339,129 bp in CHLA-90 and

SK-N-FI, as well as at 187,296,433–187,302,233 bp in

HDC-90 and GI-ME-N cells among a total of 52 geneti-

cally distinct samples, we assume these variations to be of

germline origin, uncovering novel CNVs. Moreover, we

detected and sequenced a breakpoint junction of a

submicroscopic deletion of 8,067 bp (chr2: 186,740,882–

186,748,948) located within FRA2H in the HDC-114

colorectal carcinoma cell line (Fig. 3). Whether this dele-

tion represents a de novo tumour-associated chromosomal

alteration or is a relatively rare benign CNV listed in the

DGV (chr2: 186,740,927–186,747,368 l; Variation_63258

and Variation_90177), remains uncertain as further

sequence information is not provided in the database.

Sequence analysis did not show any insertions of novel

nucleotides at the junction but revealed a 4 bp

TGC AGA TG CT CAG CA T CT G CT CAGC T

T G TCTA GCAT G AGT CAT CG A CGA

T C ATG TC ACT C A C CTC TA CGCAGAGCGC

tcaacgtcc...ccacctccadeletion

deletion

deletion

AGT C A AAA C

C AA G TG AA T TTT T

T G A T T T T G A AAAA

133,465 bp

300,526 bp

8,067 bp

atggcacag...ctactttga

ctcaaaatg...aacgggaaa

CTCAGC T CAGC TGACA GTGT GATGTACA GCAT

duplication duplication

C T C T AA T

206.2 Mb

2q32.1 FRA2H 2q32.2 2q32.3 2q33.1 2q33.2 2q33.3

186.7 Mb

186.5-186.8 Mb

186.8-187.2 Mb

CTG

NB-69

500 bp

1 kb

Ly H2O

100 bp

HDC-114

500 bp

1 kb

Ly H2O

100 bp

HDC-54

500 bp

1 kb

Ly H2O

100 bp

HDC-133nm

500 bp

1 kb

Ly H2O

100 bp

LTR L1(LINE)

)ENIL(1L RTL

CTCTGA AAC G AG TAA AG

i

A

AAAATACTCA CTC C TC CTT

TTAGA TTGCCG

)ENIL(1L )ENIL(1L

CCGTCTCACC A GGA CCTGCATA

HD

C-1

33nm

HD

C-5

4N

B-6

9H

DC

-114

Fig. 3 Sequence analysis of the FRA2H breakpoint junctions. Gel

electrophoresis of breakpoint junction PCR products is shown on the

left as follows: 100 bp DNA ladder, specific tumour DNA product,

lymphocyte DNA from a healthy male (Ly) and water (H2O). PCR

product sizes range from 500 bp in HDC-114 to[1 kb in HDC-54. A

schematic representation of the rearranged regions is shown on the

right. DNA sequencing confirmed a 300.5 kb deletion in HDC-

133 nm and revealed a cytosine nucleotide insertion at the junction

(i). In HDC-54 cells, a 7 bp microhomology was detected at the

breakpoint junction of a 133.5 kb deletion. In the NB-69 cells, the

duplication junction is flanked by 6 bp of microhomology and results

from simple end-to-end joining of DNA segments. A reference

ideogram shows the original position of the duplicated regions

highlighted in pink (FRA2H sequence) and green. The 8.1 kb deletion

in HDC-114 cells located in a region listed as a CNV locus displays a

4 bp microhomology in immediate vicinity to the centromeric

breakpoint. Capital letters indicate a present sequence; lower caseletters a deleted sequence. Purple boxes highlight microhomologies.

Grey bars indicate the presence of LTR or L1(LINE) at breakpoints

Hum Genet (2012) 131:1345–1359 1355

123

microhomology in immediate vicinity to the centromeric

breakpoint. Both deletion boundaries map to regions of

L1(LINE) repeats. Thus, all eight endpoints of the

observed FRA2H deletions lie within L1(LINE) or LTR

elements, corresponding to the high abundance of these

repeats in the entire FRA2H sequence. The lack of exten-

ded homology and the presence of 4–7 bp microhomolo-

gies at the majority of junctions suggest the involvement of

non-homologous repair pathways mediated by short

microhomologies.

Discussion

In this study, we fine-mapped FRA2H delimiting the

boundaries between fragile and non-fragile genomic

regions at a BAC clone resolution of *100 kb using six-

colour FISH. We show that FRA2H maps to a gene-poor

chromosomal area and is one of the most active cFSs in the

human genome. High-density array CGH designed for

monitoring copy number changes at cFSs enabled us to

define large and submicroscopic DNA alterations at

FRA2H in cancer cells. Sequence analysis of the observed

breakpoint junctions revealed that genomic rearrangements

at FRA2H appear to result predominantly from non-

homologous recombination involving LTR and L1(LINE)

repeats.

Until now, the approximate genomic location of about

30 cFSs has been identified at megabase resolution level

(Durkin and Glover 2007). Recently, FRA2H has been

approximated to a region at chromosome bands 2q32.1 and

2q32.2 spanning about 8 Mb (Pelliccia et al. 2010). How-

ever, based on the analysis of more than 190 breaks, we

show that FRA2H is actually limited to a genomic region of

about 530 kb at 2q32.1.

All molecularly characterised cFSs span genomic

regions containing protein-coding genes, and the majority

of them are associated with extremely large genes extending

over hundreds of kilobases of genomic DNA (Smith et al.

2007). Unlike other cFSs, FRA2H encompasses a gene-poor

region, which according to the latest version of the Ensembl

genome database does not contain any protein-coding

genes. The newly annotated AC097500.2 lincRNA gene is

the only known gene residing within FRA2H. Growing

evidence suggests an important role of lincRNAs in diverse

cellular processes including regulation of gene expression

and epigenetic marks in disease pathways (Gibb et al. 2011;

Guttman et al. 2011).

The relative frequency of breakage differs among indi-

vidual cFSs, as breaks at the 20 most fragile cFSs account

for more than 80 % of all lesions observed in lymphocytes

after aphidicolin treatment (Glover et al. 1984). A recent

genome-wide screen quantifying cFS expression

frequencies revealed a broad spectrum of sensibility to

induced replication stress. FRA2H at 2q32.1 exhibited

3.9 % of breaks, thus belonging to the four most commonly

expressed cFSs in lymphocytes, preceded by FRA3B at

3p14.2 (14.2 %), FRA16D at 16q23 (7.6 %), and FRAXB at

Xp22.3 (5.5 %) (Mrasek et al. 2010). Comparing expres-

sion patterns of cFSs on the long arm of chromosome 2

using six-colour FISH, we were able to confirm that, fol-

lowing aphidicolin induction, the FRA2H genomic region

determined in this study is by far the most commonly

observed cFS on this chromosome, accounting for about

60 % of 2q breaks. Combined with previous estimates at

G-banding level, FRA2H may be considered to be the

fourth most frequently expressed cFS in human lympho-

cytes overall.

Instability at cFSs is thought to contribute to cancer

development or progression, as chromosomal breakpoint

loci found in different human cancers appear to coincide

with known cFS loci. Recently, a large-scale screen of 746

cancer cell lines demonstrated a significant amount of

homozygous deletion clusters occurring within cFS regions

(Bignell et al. 2010). As it has been demonstrated at

FRA3B, replication stress can lead to chromosomal rear-

rangements resembling somatic copy number alterations in

cancer cells (Durkin et al. 2008).

Despite the high frequency of FRA2H breakage in

aphidicolin-treated lymphocytes, we observed copy num-

ber alterations in only 4 out of 54 cancer cell lines (7.4 %).

This is a rather moderate level of recombination compared

to other active cFSs, such as FRA3B, FRA16D and FRA6E,

displaying breakage at much higher frequencies (Mitsui

et al. 2010; Lewandowska et al. 2009; Saldivar et al. 2010).

An explanation may be that FRA2H expression is high in

lymphocytes, but low in the cell types from which the

investigated tumours originated. Tissue-specific cFS

expression patterns have not yet been explored, however,

as differences in breakage susceptibility of some cFSs have

been observed between lymphocytes and fibroblasts, tis-

sue-dependent variation is to be expected (Letessier et al.

2011; Debatisse et al. 2012).

Although few genomic alterations were detected at

FRA2H, our data indicate that the frequency of FRA2H

recombination differs among tumour types. Rearrange-

ments were most frequently detected in colorectal carci-

noma cell lines. All copy number alterations found in colon

carcinoma cells were large intrachromosomal deletions

ranging from 82.5 to 300.5 kb, thus representing a major

type of genomic recombination that has also been observed

at other cFS loci (Arlt et al. 2006). FSIP2, encoding a

protein involved in fibrous sheath formation (Brown et al.

2003), is the only protein-coding gene located within the

deleted sequences. Two other genes affected by rear-

rangements, AC007966.1 and AC097500.2, are lincRNA

1356 Hum Genet (2012) 131:1345–1359

123

genes, whose function is unknown. The heterozygous sta-

tus of the deletions indicates that the affected genes retain

their function; however, the effect of haploinsufficiency

cannot be excluded.

The molecular nature of fragility at cFSs is largely

unknown, but the hypothesis that the DNA sequence itself

is a primary cause of instability at these genomic loci has

been supported by experimental data from Ragland et al.

(2008). Alternatively, epigenetic factors have been pro-

posed to play a prevalent role in FRA3B fragility (Letessier

et al. 2011), suggesting that the instability at cFSs results

from a combination of late replication and paucity of ini-

tiation events. Recently, an analysis of the replication

dynamics along FRA16C suggested that both DNA

sequences perturbing fork progression and inherent origin

paucity underlie an increased susceptibility to breakage at

cFS loci (Ozeri-Galai et al. 2011). In contrast to rare fragile

sites, cFSs are not associated with any expanded repeats

that would account for their fragility. The only DNA fea-

tures they share are a relatively high content of AT-rich

sequences and enrichment in flexibility peaks. These traits

appear to be characteristic of not only FRA2H but also the

entire 1.53 Mb region analysed in this study. However,

there appear to be local variation biases towards a poten-

tially higher flexibility at FRA2H relative to the non-fragile

adjacent sequences. The FRA2H sequence is primarily

abundant in L1(LINE) and LTR elements. A relative

L1(LINE) repeat enrichment of over 30 % has also been

observed in two other cFSs, FRA13A and FRA9G. The

most notable difference to the corresponding AT-rich

fraction of the genome is the high number of different LTR

retrotransposon sequences within FRA2H. Such elevated

LTR content reminds of another cFS, FRAXB, supporting

several reports that cFSs are preferred sites for viral inte-

gration (Ferber et al. 2003). A higher density of L1(LINE)

and LTR elements is also an intrinsic feature of the adja-

cent non-fragile sequences, in particular the centromeric

region, indicating that deviations from the mean are a

feature of a large genomic region including FRA2H rather

than the FRA2H fragile sequence alone. Several sequence

motifs potentially capable of impairing replication fork

progression and increasing chromosome fragility have been

considered to contribute to fragility in cFSs. For instance,

long stretches of AT perfect repeats, having been suggested

to cause FRA16D fragility (Zhang and Freudenreich 2007),

were not found within FRA2H.

One way to determine the genetic components within

cFSs responsible for their instability is to identify the

breakpoint junctions of chromosomal rearrangements at the

single nucleotide level. Sequences of cFS breakpoint

junctions were first obtained from aphidicolin-induced

FRA3B deletions (Durkin et al. 2008). Recently, a com-

prehensive sequence analysis of approximately 500 breaks

within the PARK2 (FRA6E) and DMD (FRAXC) cFS genes

in cancer cells and germlines showed breakpoint clustering

in specific genomic regions predominantly involving

microhomologies (Mitsui et al. 2010). In line with these

observations, our sequence data did not reveal extended

homology but 4–7 bp microhomologies in three out of four

breakpoint junctions at FRA2H.

Two main mechanisms, homologous and non-homolo-

gous recombination, have been proposed to be involved in

generating structural chromosomal rearrangements (Has-

tings et al. 2009). Involvement of non-allelic homologous

recombination (NAHR) in the formation of the observed

rearrangements is unlikely, as FRA2H breakpoint junctions

lack extended sequence homology. Non-homologous

recombination mechanisms include non-homologous end

joining (NHEJ), microhomology-mediated end joining

(MMEJ), as well as replicative pathways as described by the

fork stalling and template switching (FoSTeS) (Lee et al.

2007; McVey and Lee 2008) or the microhomology-medi-

ated break-induced replication (MMBIR) model (Hastings

et al. 2009). The deletion found in HDC-133 nm cells may

be a consequence of classical NHEJ, as this mechanism does

not require homologies to join DNA double-strand breaks

and often results in addition of single nucleotides. Micro-

homologies at the breakpoint junctions of the FRA2H

deletions in HDC-54 and SW-620 sub1 cells and the dupli-

cation in NB-69 cells indicate involvement of MMEJ or

MMBIR mechanisms, which appear to require 5–25 nucle-

otide microhomologies for effective repair (Gu et al. 2008;

Hastings et al. 2009). Combined with other studies discussed

above, our data suggest that non-homologous recombination

pathways mediated by short microhomologies play a major

role in generating and repairing cFS lesions.

A FRA2H breakpoint sequence analysis showed that all

detected deletions involve recombination between different

LTR families and L1(LINE). Several repetitive elements,

including L1(LINE) and LTR elements, are thought to

promote secondary structure formation, potentially leading

to replication fork stalling or collapse (Labib and Hodgson

2007). Thus, L1(LINE) repeats have been proposed to

constitute the molecular basis of FRA3B instability (Mimori

et al. 1999; Inoue et al. 1997). Moreover, involvement of

LTR retrotransposons in generating chromosomal rear-

rangements has been frequently observed in yeast genetic

systems (Szilard et al. 2010; Admire et al. 2006; Lemoine

et al. 2005). As all the detected FRA2H deletion endpoints

map within L1(LINE) or LTR elements, it is possible that

the high density of L1(LINE) and LTR at FRA2H favours

such recombination events.

Somatic cancer-related alterations and germline CNVs

are thought to have a common molecular basis, as similar

sequence features have been observed and replication

stress plays a role in the formation of both (Arlt et al.

Hum Genet (2012) 131:1345–1359 1357

123

2011). Whether non-homologous recombination events

across LTR and L1 elements are also involved in the for-

mation of germline CNVs at FRA2H remains uncertain as,

to our knowledge, none of the CNVs annotated in this

region have been assessed at the single nucleotide level.

Identifying DNA sequences directly involved in somatic

chromosomal rearrangements and CNVs in cFSs may

provide important clues for understanding the molecular

basis of cFS fragility. Such DNA alterations may serve as

potential biomarkers for early cancer diagnostics and as

targets for individualised therapies.

Acknowledgments The authors would like to thank Dr. Johannes

Gebert and Dr. Diego Arango del Corro for providing the SW-948

and SW-620 colorectal cancer cell lines and Dr. Wilhelm Dirks for

verifying cell line identity by DNA typing. This work was supported

by the Helmholtz-Russia Joint Research Groups (HRJRG-006) and

the Bundesministerium fur Bildung und Forschung NeuroblastomGenom Forschungs Netzwerk (BMBF NGFNPlus #01GS0896).

Conflict of interest The authors declare that they have no conflict

of interest.

References

Admire A, Shanks L, Danzl N, Wang M, Weier U, Stevens W, Hunt

E, Weinert T (2006) Cycles of chromosome instability are

associated with a fragile site and are increased by defects in

DNA replication and checkpoint controls in yeast. Genes Dev

20(2):159–173

Arlt MF, Glover TW (2010) Inhibition of topoisomerase I prevents

chromosome breakage at common fragile sites. DNA Repair

(Amst) 9(6):678–689

Arlt MF, Miller DE, Beer DG, Glover TW (2002) Molecular

characterization of FRAXB and comparative common fragile

site instability in cancer cells. Genes Chromosom Cancer

33(1):82–92

Arlt MF, Durkin SG, Ragland RL, Glover TW (2006) Common

fragile sites as targets for chromosome rearrangements. DNA

Repair (Amst) 5(9–10):1126–1135

Arlt MF, Ozdemir AC, Birkeland SR, Lyons RH Jr, Glover TW,

Wilson TE (2011) Comparison of constitutional and replication

stress-induced genome structural variation by SNP array and

mate-pair sequencing. Genetics 187(3):675–683

Bignell GR, Greenman CD, Davies H, Butler AP, Edkins S, Andrews

JM, Buck G, Chen L, Beare D, Latimer C, Widaa S, Hinton J,

Fahey C, Fu B, Swamy S, Dalgliesh GL, Teh BT, Deloukas P,

Yang F, Campbell PJ, Futreal PA, Stratton MR (2010) Signa-

tures of mutation and selection in the cancer genome. Nature

463(7283):893–898

Blumrich A, Zapatka M, Brueckner LM, Zheglo D, Schwab M,

Savelyeva L (2011) The FRA2C common fragile site maps to the

borders of MYCN amplicons in neuroblastoma and is associated

with gross chromosomal rearrangements in different cancers.

Hum Mol Genet 20(8):1488–1501

Brown PR, Miki K, Harper DB, Eddy EM (2003) A-kinase anchoring

protein 4 binding proteins in the fibrous sheath of the sperm

flagellum. Biol Reprod 68(6):2241–2248

Bruderlein S, van der Bosch K, Schlag P, Schwab M (1990)

Cytogenetics and DNA amplification in colorectal cancers.

Genes Chromosom Cancer 2(1):63–70

Burrow AA, Marullo A, Holder LR, Wang YH (2010) Secondary

structure formation and DNA instability at fragile site FRA16B.

Nucleic Acids Res 38(9):2865–2877

Casper AM, Nghiem P, Arlt MF, Glover TW (2002) ATR regulates

fragile site stability. Cell 111(6):779–789

Cicek MS, Slager SL, Achenbach SJ, French AJ, Blair HE, Fink SR,

Foster NR, Kabat BF, Halling KC, Cunningham JM, Cerhan JR,

Jenkins RB, Boardman LA, Petersen GM, Sargent DJ, Alberts

SR, Limburg PJ, Thibodeau SN (2009) Functional and clinical

significance of variants localized to 8q24 in colon cancer. Cancer

Epidemiol Biomarkers Prev 18(9):2492–2500

Debacker K, Winnepenninckx B, Ben-Porat N, FitzPatrick D, Van

Luijk R, Scheers S, Kerem B, Frank Kooy R (2007) FRA18C: a

new aphidicolin-inducible fragile site on chromosome 18q22,

possibly associated with in vivo chromosome breakage. J Med

Genet 44(5):347–352

Debatisse M, El Achkar E, Dutrillaux B (2006) Common fragile sites

nested at the interfaces of early and late-replicating chromosome

bands: cis acting components of the G2/M checkpoint? Cell

Cycle 5(6):578–581

Debatisse M, Le Tallec B, Letessier A, Dutrillaux B, Brison O (2012)

Common fragile sites: mechanisms of instability revisited.

Trends Genet 28(1):22–32

Denison SR, Simper RK, Greenbaum IF (2003) How common are

common fragile sites in humans: interindividual variation in the

distribution of aphidicolin-induced fragile sites. Cytogenet

Genome Res 101(1):8–16

Dillon LW, Burrow AA, Wang YH (2010) DNA instability at

chromosomal fragile sites in cancer. Curr Genomics 11(5):

326–337

Durkin SG, Glover TW (2007) Chromosome fragile sites. Annu Rev

Genet 41:169–192

Durkin SG, Ragland RL, Arlt MF, Mulle JG, Warren ST, Glover TW

(2008) Replication stress induces tumor-like microdeletions in

FHIT/FRA3B. Proc Natl Acad Sci USA 105(1):246–251

Ferber MJ, Thorland EC, Brink AA, Rapp AK, Phillips LA,

McGovern R, Gostout BS, Cheung TH, Chung TK, Fu WY,

Smith DI (2003) Preferential integration of human papillomavi-

rus type 18 near the c-myc locus in cervical carcinoma.

Oncogene 22(46):7233–7242

Gandhi M, Dillon LW, Pramanik S, Nikiforov YE, Wang YH (2010)

DNA breaks at fragile sites generate oncogenic RET/PTC

rearrangements in human thyroid cells. Oncogene 29(15):2272–

2280

Gibb EA, Brown CJ, Lam WL (2011) The functional role of long

non-coding RNA in human carcinomas. Mol Cancer 10:38

Glover TW (2006) Common fragile sites. Cancer Lett 232(1):4–12

Glover TW, Berger C, Coyle J, Echo B (1984) DNA polymerase

alpha inhibition by aphidicolin induces gaps and breaks at

common fragile sites in human chromosomes. Hum Genet

67(2):136–142

Gu W, Zhang F, Lupski JR (2008) Mechanisms for human genomic

rearrangements. Pathogenetics 1(1):4

Guttman M, Donaghey J, Carey BW, Garber M, Grenier JK, Munson

G, Young G, Lucas AB, Ach R, Bruhn L, Yang X, Amit I,

Meissner A, Regev A, Rinn JL, Root DE, Lander ES (2011)

lincRNAs act in the circuitry controlling pluripotency and

differentiation. Nature 477(7364):295–300

Halazonetis TD, Gorgoulis VG, Bartek J (2008) An oncogene-

induced DNA damage model for cancer development. Science

319(5868):1352–1355

Hastings PJ, Ira G, Lupski JR (2009) A microhomology-mediated

break-induced replication model for the origin of human copy

number variation. PLoS Genet 5(1):e1000327

Hellman A, Zlotorynski E, Scherer SW, Cheung J, Vincent JB, Smith

DI, Trakhtenbrot L, Kerem B (2002) A role for common fragile

1358 Hum Genet (2012) 131:1345–1359

123

site induction in amplification of human oncogenes. Cancer Cell

1(1):89–97

Helmrich A, Stout-Weider K, Matthaei A, Hermann K, Heiden T,

Schrock E (2007) Identification of the human/mouse syntenic

common fragile site FRA7K/Fra12C1—relation of FRA7 K and

other human common fragile sites on chromosome 7 to

evolutionary breakpoints. Int J Cancer 120(1):48–54

Henegariu O, Bray-Ward P, Ward DC (2000) Custom fluorescent-

nucleotide synthesis as an alternative method for nucleic acid

labeling. Nat Biotechnol 18(3):345–348

Hormozian F, Schmitt JG, Sagulenko E, Schwab M, Savelyeva L

(2007) FRA1E common fragile site breaks map within a

370 kilobase pair region and disrupt the dihydropyrimidine

dehydrogenase gene (DPYD). Cancer Lett 246(1–2):82–91

Inoue H, Ishii H, Alder H, Snyder E, Druck T, Huebner K, Croce CM

(1997) Sequence of the FRA3B common fragile region:

implications for the mechanism of FHIT deletion. Proc Natl

Acad Sci USA 94(26):14584–14589

Jiang Y, Lucas I, Young DJ, Davis EM, Karrison T, Rest JS, Le Beau

MM (2009) Common fragile sites are characterized by histone

hypoacetylation. Hum Mol Genet 18(23):4501–4512

Labib K, Hodgson B (2007) Replication fork barriers: pausing for a

break or stalling for time? EMBO Rep 8(4):346–353

Lai LA, Kostadinov R, Barrett MT, Peiffer DA, Pokholok D, Odze R,

Sanchez CA, Maley CC, Reid BJ, Gunderson KL, Rabinovitch

PS (2010) Deletion at fragile sites is a common and early event

in Barrett’s esophagus. Mol Cancer Res 8(8):1084–1094

Lee JA, Carvalho CM, Lupski JR (2007) A DNA replication

mechanism for generating nonrecurrent rearrangements associ-

ated with genomic disorders. Cell 131(7):1235–1247

Lemoine FJ, Degtyareva NP, Lobachev K, Petes TD (2005)

Chromosomal translocations in yeast induced by low levels of

DNA polymerase a model for chromosome fragile sites. Cell

120(5):587–598

Letessier A, Millot GA, Koundrioukoff S, Lachages AM, Vogt N,

Hansen RS, Malfoy B, Brison O, Debatisse M (2011) Cell-type-

specific replication initiation programs set fragility of the

FRA3B fragile site. Nature 470(7332):120–123

Lewandowska U, Zelazowski M, Seta K, Byczewska M, Pluciennik

E, Bednarek AK (2009) WWOX, the tumour suppressor gene

affected in multiple cancers. J Physiol Pharmacol 60(Suppl

1):47–56

Limongi MZ, Pelliccia F, Rocchi A (2003) Characterization of the

human common fragile site FRA2G. Genomics 81(2):93–97

Mangelsdorf M, Ried K, Woollatt E, Dayan S, Eyre H, Finnis M,

Hobson L, Nancarrow J, Venter D, Baker E, Richards RI (2000)

Chromosomal fragile site FRA16D and DNA instability in

cancer. Cancer Res 60(6):1683–1689

McVey M, Lee SE (2008) MMEJ repair of double-strand breaks

(director’s cut): deleted sequences and alternative endings.

Trends Genet 24(11):529–538

Mimori K, Druck T, Inoue H, Alder H, Berk L, Mori M, Huebner K,

Croce CM (1999) Cancer-specific chromosome alterations in the

constitutive fragile region FRA3B. Proc Natl Acad Sci USA

96(13):7456–7461

Mitsui J, Takahashi Y, Goto J, Tomiyama H, Ishikawa S, Yoshino H,

Minami N, Smith DI, Lesage S, Aburatani H, Nishino I, Brice A,

Hattori N, Tsuji S (2010) Mechanisms of genomic instabilities

underlying two common fragile-site-associated loci, PARK2 and

DMD, in germ cell and cancer cell lines. Am J Hum Genet

87(1):75–89

Mrasek K, Schoder C, Teichmann AC, Behr K, Franze B, Wilhelm K,

Blaurock N, Claussen U, Liehr T, Weise A (2010) Global

screening and extended nomenclature for 230 aphidicolin-

inducible fragile sites, including 61 yet unreported ones. Int J

Oncol 36(4):929–940

Ozeri-Galai E, Lebofsky R, Rahat A, Bester AC, Bensimon A, Kerem

B (2011) Failure of origin activation in response to fork stalling

leads to chromosomal instability at fragile sites. Mol Cell

43(1):122–131

Pelliccia F, Bosco N, Rocchi A (2010) Breakages at common fragile

sites set boundaries of amplified regions in two leukemia cell

lines K562—molecular characterization of FRA2H and locali-

zation of a new CFS FRA2S. Cancer Lett 299(1):37–44

Ragland RL, Glynn MW, Arlt MF, Glover TW (2008) Stably

transfected common fragile site sequences exhibit instability at

ectopic sites. Genes Chromosom Cancer 47(10):860–872

Saldivar JC, Shibata H, Huebner K (2010) Pathology and biology

associated with the fragile FHIT gene and gene product. J Cell

Biochem 109(5):858–865

Savelyeva L, Sagulenko E, Schmitt JG, Schwab M (2006) The

neurobeachin gene spans the common fragile site FRA13A. Hum

Genet 118(5):551–558

Sawinska M, Schmitt JG, Sagulenko E, Westermann F, Schwab M,

Savelyeva L (2007) Novel aphidicolin-inducible common fragile

site FRA9G maps to 9p22.2, within the C9orf39 gene. Genes

Chromosom Cancer 46(11):991–999

Schwartz M, Zlotorynski E, Kerem B (2006) The molecular basis of

common and rare fragile sites. Cancer Lett 232(1):13–26

Smit AF (1999) Interspersed repeats and other mementos of

transposable elements in mammalian genomes. Curr Opin Genet

Dev 9(6):657–663

Smith DI, McAvoy S, Zhu Y, Perez DS (2007) Large common fragile

site genes and cancer. Semin Cancer Biol 17(1):31–41

Sutherland GR (2003) Rare fragile sites. Cytogenet Genome Res

100(1–4):77–84

Szilard RK, Jacques PE, Laramee L, Cheng B, Galicia S, Bataille AR,

Yeung M, Mendez M, Bergeron M, Robert F, Durocher D (2010)

Systematic identification of fragile sites via genome-wide

location analysis of gamma-H2AX. Nat Struct Mol Biol

17(3):299–305

Tsantoulis PK, Kotsinas A, Sfikakis PP, Evangelou K, Sideridou M,

Levy B, Mo L, Kittas C, Wu XR, Papavassiliou AG, Gorgoulis

VG (2008) Oncogene-induced replication stress preferentially

targets common fragile sites in preneoplastic lesions. A genome-

wide study. Oncogene 27(23):3256–3264

Wilke CM, Guo SW, Hall BK, Boldog F, Gemmill RM, Chandrase-

kharappa SC, Barcroft CL, Drabkin HA, Glover TW (1994)

Multicolor FISH mapping of YAC clones in 3p14 and identi-

fication of a YAC spanning both FRA3B and the t(3;8)

associated with hereditary renal cell carcinoma. Genomics

22(2):319–326

Zhang H, Freudenreich CH (2007) An AT-rich sequence in human

common fragile site FRA16D causes fork stalling and chromo-

some breakage in S. cerevisiae. Mol Cell 27(3):367–379

Zimonjic DB, Druck T, Ohta M, Kastury K, Croce CM, Popescu NC,

Huebner K (1997) Positions of chromosome 3p14.2 fragile sites

(FRA3B) within the FHIT gene. Cancer Res 57(6):1166–1170

Zlotorynski E, Rahat A, Skaug J, Ben-Porat N, Ozeri E, Hershberg R,

Levi A, Scherer SW, Margalit H, Kerem B (2003) Molecular

basis for expression of common and rare fragile sites. Mol Cell

Biol 23(20):7143–7151

Hum Genet (2012) 131:1345–1359 1359

123