American Journal of Medical Genetics 124A:10–18 (2004)

Molecular Cytogenetic Analysis of ComplexChromosomal Rearrangements in Patients WithMental Retardation and Congenital Malformations:Delineation of 7q21.11 Breakpoints

Stefan Vermeulen,* Bjorn Menten, Nadine Van Roy, Heidi Van Limbergen,Anne De Paepe, Geert Mortier, and Frank SpelemanCenter for Medical Genetics, 0K5, Ghent University Hospital, De Pintelaan 185, Ghent, Belgium

Constitutional de novo complex chromoso-mal rearrangements (CCRs) are a rare find-ing in patients with mild to severe mentalretardation. CCRs pose a challenge to theclinical cytogeneticist: generally CCRs areassumed to be the cause of the observedphenotypic abnormalities, but the complexnature of these chromosomal changes oftenhamper the accurate delineation of thechromosomal breakpoints and the identifi-cation of possible imbalances. In a first steptowards a more detailed molecular cytoge-netic characterization of CCRs, we studiedfour de novo CCRs using multicolor fluores-cent in situ hybridization (M-FISH), com-parative genomic hybridization (CGH), andFISH with region specific probes. Thesemethods allowed a more refined character-ization of the breakpoints in three of the fourCCRs. The occurrence of 7q breakpoints inthree out of these four CCRs and in 30% ofreported CCRs suggested preferential invol-vement of this chromosomal region in theformation of CCRs. Further analysis of these7q breakpoints revealed a 2 Mb deletion at7q21.11 in one patient and involvement of thesame region in a cryptic insertion in a secondpatient. This particular region contains atleast 5 candidate genes for mental retarda-tion. The other patient had a breakpoint

more proximal to this region. The presentdata together with these from the literatureprovide evidence that a region within7q21.11 may be prone to breakage and for-mation of CCRs. � 2003 Wiley-Liss, Inc.

KEY WORDS: complex chromosomal rear-rangements; 7q21.11

INTRODUCTION

Chromosomal abnormalities are a frequent cause ofmental retardation and congenital abnormalities. Asmall subset of these patients present with an appar-ently balanced de novo complex chromosomal rearran-gement (CCR). CCRs are defined as structuralrearrangements with more than two breakpoints andexchange of genetic material between two or morechromosomes [Pai et al., 1980]. CCRs can be groupedaccording to thenumber of breaks [Kousseff et al., 1993],the type of rearrangements [Kausch et al., 1988], thepreferential occurrence of inter- or intra-chromosomalrearrangements [Lurie et al., 1994], and the de novo orfamilial occurrence of CCRs [Kleczkowska et al., 1982].More than 100 constitutional CCRs have been docu-mented (for review see Batanian and Eswara, 1998,Table I). De novo CCRs are associated with mentalretardation and congenital abnormalities. In mostpatients it is assumed that the observed phenotypicanomalies are the result of submicroscopic deletions orduplications or alternatively disruption, activation, orinactivation of genes located at or near one or morebreakpoints. So far this hypothesis has not been provenby molecular analysis. Due to the complex and uniquenature of these rearrangements nouniform strategyhasbeen used to investigate CCRs. Until now, only a smallnumber of CCRs were investigated at the molecularcytogenetic level with M-FISH or SKY [Schrock et al.,1997; Haddad et al., 1998; Ogilvie et al., 1998; Phelanet al., 1998; Jalal and Law, 1999; Peschka et al., 1999;Bayani and Squire, 2001]. M-FISH [Speicher et al.,1996], SKY, spectral karyotyping [Schrock et al., 1996],

Grant sponsor: The Fund for Scientific Research—Flanders(Belgium) (Research Grant); Grant numbers: 1.5.183.02,G.0200.03.

*Correspondence to: Dr. Sc. Stefan Vermeulen, Center forMedical Genetics, Ghent University Hospital 0K5, De Pintelaan185, 9000 Ghent, Belgium. E-mail: Stefan.Vermeulen@ugent.be

Received 16 July 2002; Accepted 16 April 2003

DOI 10.1002/ajmg.a.20378

� 2003 Wiley-Liss, Inc.

TABLE

I.Overview

ofCCRsnot

Rev

iewed

intheArticle

ofBatanianandEsw

ara

[1998]

Chromosom

esinvolved

Breakpoints

Technolog

yused

Transm

ission

Referen

ce

12;16

12p13;16q22

FIS

Hdenov

oCalabrese

etal.[1998]

1;7;10;21

7q31;1p21;21q11.2;1p21;10q11.2;7q21;7q31;21q21;7q21;10q11.2

FIS

Hdenov

oCurottiet

al.[1999]

2;11;22

2q13;11q23;22q11.2

ISH

Familial

Fuster

etal.[1997]

1;2;5

1q42.3;5q23.2;5q21.2;2q33;2q35

FIS

HFamilial

Gibsonet

al.[1997]

1;4;16

1p36;16p13;4q34

FIS

HJoh

annessonet

al.[1997]

1;6;7;15;Y

Yq12;7p22;1p36.1;1p32;6q21;1p34;6q23

FIS

Hdenov

oJoy

ceet

al.[1999a]

4;10;12

4p12;12q11.2;10q11;10q25.2

M-FIS

HLukusa

etal.[1998]

3;4;10;17

3p22.2;10q11.22;4q25;10q26.3;17q25.3

FIS

HOgilvie

etal.[1998]

2;3;8

2q23;3q13.2;2q33;8q13

FIS

HFamilial

Madanet

al.[1997]

2;5;16;17

2q37.3;17q25.3;5q21.2;16q22.3;16q13;5q22;5q31.1;5q33.3

FIS

HMaseratiet

al.[1999]

2;3;4;13

2q14.2;13q34;3p12.2;4q23;2q21.1

FIS

Hdenov

oMercier

etal.[1996]

2;16;7

2q33;16q24;inv(7)(7p15q11.23)

denov

oCotteret

al.[1996]

6;12;14;16

6p21.1;16q22;6q15;12q12;6q21;14q22;16q12;6q25;12q12

FIS

Hdenov

oPhelanet

al.[1998]

6;7;18;21

6q22;6q25;7q21.3;7q32.1;18p11.21;18q21.3;21q21.3

FIS

H,SKY

Familial

Rothlisb

erger

etal.[1999]

1;4;10

1q21.3;4q27;10q26.1

FIS

HFamilial

Sawickaet

al.[1998]

9;10;11

9p22;11?q21;10p11.2;11q21

DU/TR

COLFIS

HFamilial

Stankiewiczet

al.[1997]

1;2;4;11

2q11.2;1p13.1;1q25;4q31.1;4q33;4q35.1;11q23;11p13;11p11.11;11q13.1

denov

oTupleret

al.[1992]

1;5;11

5q31;1p31.3;1q44;11q23

CIS

SVermaet

al.[1993]

5;7;11

5p15.1;7q31.2;5p15.3;11q13.3

FIS

HWallersteinet

al.[1996]

3;6;15

3q29;15q26;6q26

FIS

HFamilial

Wieczorek

etal.[1998]

5;16;22

5q31.3;16q12.1;22q11.2

Xuet

al.[1997]

1;8;9

1p31;8q21.1;8p23;9q34

FIS

HFamilial

Zahed

etal.[1998]

5;21;7;11;14

5q22;7p22;11q21;14q11.2;21q22

denov

oHou

geG.,personalcommunication

1;2;3;4;8

1p36.1;2q31.1;2q32;2q33.1;3p25.3;4q21.3;4q22.2;4q22.3;4q24;

8q24.1;8q24.13;8q24.22

FIS

H,SKY,M-FIS

H,

multicolor

banding

FIS

H

denov

oHou

geG.,personalcommunication

3;7;10

3q23;7p15.3;10p11.23;10q25.3

Case

96,www.bwhpatholog

y.org/dgap

3;7;11

3q23;3q27;7q21.3;11q21;

Case

14,www.bwhpatholog

y.org/dgap

2;7

7q32;7q35;2p12;2q31

Case

44,www.bwhpatholog

y.org/dgap

or multipaint FISH [Joyce et al., 1999b] allowed a moreaccurate characterization of CCRs at the cytogeneticlevel but has limits (5–10 Mb) in detecting smallduplications or deletions [Lee et al., 2001]. Here, wedescribe the combined application of M-FISH and CGHin the study of four de novo CCRs in order to accuratelycharacterize the nature of the rearrangements. Subse-quently, we investigated the observed 7q breakpointswith bacterial artificial chromosome (BAC) probes tomap the breakpoints in further detail.

MATERIALS AND METHODS

G-Banding

Karyotypingwasperformedonshort term lymphocytecultures from peripheral blood with G-banding. Karyo-types were described according to the guidelines of theISCN [1995]. The study was approved by the ethicalcommittee of Ghent University Hospital (Ghent, Bel-gium) under project 2000/80.

M-FISH

For M-FISH, the ‘‘24 Xcyte’’ M-FISH probe kit andsoftware were applied (MetaSystems, Altlussheim,Germany; http://www.metasystems.de). The probe mix-ture was prepared by combinatorial labeling of de-generated oligonucleotide primer polymerase chainreaction (DOP-PCR) amplified microdissected chromo-somes using biotin and four fluorochromes: fluoresceinisothiocyanate (FITC), Spectrum Orange (SO), TexasRed (TR), and diethylcoumarine (DEAC). Biotinylatedprobes were detected with avidin-Cy5. M-FISH wasperformed following the manufacturer instructions.Metaphase slides were pre-treated with RNAse andpepsin. Slides were denatured with 70% formamide/2� SSCP at 808C for 5 min. The probe mixture wasdenatured at 758C for 5 min, incubated at 378C for30 min, and subsequently applied to the slides under an18�18 mm coverslip. After 4 days of hybridization,slides were washed three times for 5 min with 50%formamide/2� SSC (pH 7.3–7.5) at 428C, followed bythree washes in 2� SSC (428C). For counterstaining4,6-diamidino-2-phenylindole dihydrochloride (DAPI,Rocche Molecular Biochemicals, Brussels, Belgium)was added to the antifade reagents [(Vectashield, Vectorlaboratories, Burlingame, CA). A Zeiss Axioplan epi-fluorescence microscope with an eight-position filterwheel [Carl Zeiss Jena GmbH, Jena, Germany; http://www.zeiss.de) and a black and white high-resolutioncamera were used for capturing images for eachdifferent fluorochrome. The following single-band passfilters were used: DAPI (filterset no. 01; Carl Zeiss,excitation: 359 nm, emission: 441 nm), FITC (filtersetno. 09;Carl Zeiss, excitation: 490nm, emission: 525nm),Cy3 (filterset no. 15; Carl Zeiss, excitation: 575 nm,emission: 605 nm), Cy5 (filterset no. 26; Carl Zeiss,excitation: 640 nm, emission: 705 nm), Texas Red(filterset no. 00;Carl Zeiss, excitation: 590nm, emission:615 nm), and DEAC (aqua chroma 31036v2; excitation:436 nm, emission: 480 nm; Chroma Technology Corp,

Brattleboro, VT). TheM-FISHmodule in the ISIS imageanalysis software from MetaSystems was used toprocess the images. On the basis of a color definitiontable, pseudocolors were assigned to each individualchromosome resulting in the unambiguous identifica-tion of the rearrangements. M-FISH from patient 2 wasperformed on Epstein–Barr virus-immortalized lym-phoblastoid cells.

Comparative Genomic Hybridization

CGHwas performed as described previously [VanRoyet al., 1997]. Briefly, metaphases stained with DAPIwere recorded prior to hybridization using a Leitz DMmicroscope equipped with a high-sensitivity integratedmonochromeCCD camera (Sony IMAC-CCDS30). Afterhybridization the images were processed with the ISIS-CGH software (MetaSystems). For each patient, 10–20metaphase cells were analyzed. For evaluation of CGHdata, average ratio profiles with fixed limits at 1.25 and0.75 and standard deviation limits (the width of theconfidence interval being three times the standarddeviation) as well as individual ratio profiles wereanalyzed. A chromosomal region was considered to beover-represented (gain) respectively under-represented(loss) if the average ratio profile crossed the standarddeviation limit. As a control, normal to normal hybridi-zations were performed.

FISH With Region Specific Probes

Fluorescence in situ hybridization (FISH) was per-formed with YAC, BAC, PAC, and plasmid probeslabeled either with digoxigenin or biotin. 7q locus spe-cific RPCI-BAC probes (see Table II) were obtainedthrough screening of the October 2000, December 2000,and April 2001 Freeze (http://genome.ucsc.edu/) of thehuman genome project. The following plasmid probeswere used for the centromeres of the respective chromo-somes:D7Z1, centromere 7; pa3.5, centromere 3;D10Z1,centromere 10; pBS4D, centromere 2; pZ4.1, centromere4 and centromere 9. CEP12 for centromere 12 wasobtained from Vysis (Downers Grove, IL). For the sub-telomeric regions the following probes were used: YACclones; TYAC75 (2pter); TYAC162 (3qter); 946-a-3(4qter); 626-g-11 (7pter); TYAC109 (7qter); TYAC95(10pter); TYAC93 (10qter); PAC clone; 483G12 (12pter);BAC clone; GS-820-M16 (14qter) kindly provided by Dr.A. Jauch, Laboratory of Human genetics, Heidelberg,Germany. Telvysion 14q (14qtel) was purchased fromVysis. The following YAC probes for chromosome 2pspecific bands were used; 713-g-9 (2p23); 830-d-1 (2p21-22); 783-f-7 (2p13), and 850-a-4 (2p12).

RESULTS

Clinical Findings

Patient 1 was born at term to healthy, non-consan-guineous parents. The pregnancy was complicated bymaternal fever due to a respiratory infection in the fifthmonth of pregnancy. His birth weight was 2,600 g,length 48.5 cm, and head circumference 30.5 cm

12 Vermeulen et al.

(P10¼32 cm). After birth, microcephaly was noted.Evaluation at the age of 4 months revealed generalizedhypertonia with increased deep tendon reflexes, andpsychomotor delay. He did not follow objects, showedhyperacusis, and had a history of feeding problems.Head circumference was 36.5 cm. CT-scan of the brainrevealed punctiform calcifications around the occipitalhorns of the lateral ventricles, periventricular leuko-malacia, and small cerebellum.

Patient 2 was born at 37 weeks gestation fromhealthy, non-consanguineous parents. Prenatal ultra-sound showed polyhydramnios. The delivery was un-eventful.His birthweightwas 2,700g, length50 cm, andhead circumference 36 cm. After birth, an oesophagealatresia was documented. In addition, physical examina-tion revealed preaxial polydactyly of the right thumb, anon-functional and hypoplastic right thumb (‘‘floatingthumb’’), a single palmar crease in the left hand, andpartial cutaneous syndactyly of the second and thirdtoes. In the neonatal period, ultrasound examination ofbrain and abdomen did not reveal any abnormalities.Echocardiographic evaluation showed a normal struc-ture and function of the heart.

Patient 3 was born at term after an uncomplicatedpregnancy. The parents were healthy and non-consan-guineous.His birthweightwas3,500gand length51 cm.The delivery and postnatal course were uneventful. Theboy sat alone at 10 months and started walking at

19 months. From the beginning, the parents noticed apoor balance and coordination during walking. Aroundthe age of 3 years, a delay in speechdevelopment becameevident. Because of the psychomotor delay (IQ¼65), hewas referred to special education school. Physicalexamination at the age of 6 years and 9months revealeda height of 126 cm (P75), weight of 25 kg (P50–P75), andhead circumference of 55.8 cm (P97¼54.5 cm). Moststrikingwere themacrocephalywith prominent occiput,hypertelorismwith antimongoloid slant of the palpebralfissures, retrognathia, large mouth, widely spacedteeth, and pectus excavatum. Neurologic examinationshowed mild hypotonia with dystonic postures, dysme-tria, and tremor of the hands. The deep tendon reflexeswere normal and pathologic reflexes absent.MRI scan ofthe brain did not reveal abnormalities.

Patient 4 was born at 37 weeks gestation after anuncomplicated pregnancy. His birth weight was 3,450 gand length50 cm.Deliverywasnormal. Thefirstmonthsof postnatal life were complicated by feeding pro-blems and recurrent ear infections. He sat alone at12 months and started walking at 2 years of age.Because of a delay in the psychomotor developmentand the relatively large skull, a MRI scan of the brainwas performed at the age of 2 years and 8months but noabnormalities were found. Physical examination at theage of 3 years revealed a weight of 15.5 kg (P50–P75),length of 96.5 cm (P25), and head circumference of

TABLE II. Chromosomal Position on the Various Derivatives of 7q BAC Clones in the ThreePatients With 7q Involvement in Their CCRs

BAC-clone Localization

Patienta

2 3 4

t(4;7;14) t(2;7;14) t(7;10;12)

RP11-89A20 7q11.23 der (7) der (7) der (7)(q)RP11-261L16b 7q11.23 der (7) der (7) der (7)(p)RP11-129J21b 7q11.23 der (4) der (7) der (7)(p)RP11-5B9b 7q21.11 der (4) der (7) der (7)(p)RP11-163O5b 7q21.11 der (4) der (7) der (7)(p)RP4-802A9b 7q21.11 der (4) der (7) der (7)(p)RP5-897G10b 7q21.11 der (4) D der (10)(p)RP11-665O4b 7q21.11 der (4) D der (10)(p)RP11-618A7 7q21.11 der (4) D der (10)(p)RP11-242J14 7q21.11 der (4) D der (10)(p)RP11-543D8b 7q21.11 der (4) D der (10)(p)RP4-789N1b 7q21.11 der (4) D der (10)(p)RP4-649P17b 7q21.11 der (4) D der (10)(p)RP11-796I6b 7q21.11 der (4) der (7) der (10)(p)RP11-727N2b 7q21.11 der (4) der (7)c der (7)(q)RP11-175K20b 7q21.11 der (4) der (7)c der (7)(q)RP11-638A9b 7q21.11 der (4) der (7) der (7)(q)RP11-91M13 7q21 der (4) der (7) der (7)(q)RP11-2074H8 7q21.2-7q21.3 der (4) der (7) der (7)(q)RP11-10D8 7q22.1 der (4) der (7) der (7)(q)RP11-91F7 7q31.1 der (4) der (14) der (7)(q)RP11-51M22 7q31.2 der (4) der (14) der (7)(q)RP11-2039F11 7q31.3 der (4) der (14) der (7)(q)RP11-2051H12 7q34 der (4) der (14) der (7)(q)RP11-2027I18 7q36 der (4) der (14) der (7)(q)

aThe respective derivative chromosomes where the probes hybridized are indicated.bProbes mapping on the draft sequence of the human genome.cProbes not hybridizing next to each other on derivative 7 (see Fig. 2); D, deletion on the derivative 7.

Chromosomal Rearrangements in Patients 13

52.3 cm (P98¼53 cm). The most striking clinicalfeatures were large skull, frontal bossing with recedingfrontal hairline, sparse eyebrows and eyelashes, largenose, and small mouth. Neurologic evaluation revealedapoor balance.Psychometric testingat the age of 3 years8 months revealed a developmental age of 2 years4months (IQ¼ 67). Speech assessments revealed severearticulation problems and better receptive than expres-sive language skills.

Molecular Cytogenetic Findings

Partial G-banded karyotypes of the CCR of the fourpatients are shown in Figure 1. The karyotypes from theparents of all four patients were normal indicatingthat the CCRs described here arose de novo. M-FISHanalysis allowed to revise the karyotype in three out offour patients.

In patient 1 the initial karyotype 46,XY,der(3)t(3;12)(3pter!3q13.2::12q12!12qter)der(10)t(3;10)(10pter!10q25::3q13.2!3qter)der(12)t(10;12)(12pter!12q12::10q25!10qter) was confirmed by M-FISH. No addi-tional breakpoints or translocations with other chro-mosomeswere observed.FISHwith subtelomeric probesshowed that the orientation of the translocated chromo-some segments was preserved.

In patient 2 the breakpoints inferred from G-bandingwere revised after M-FISH. M-FISH confirmed thepresence of chromosome 14 material in the derivativechromosome 7 but in addition revealed part of chromo-some 4 in this derivative. The breakpoint on derivativechromosome 7 had been originally assigned to 7q31.2

due to the similarity in the G-banding pattern ofsegment 4q12!4q21.3 when compared to 7q11.2!7q22 (see also Fig. 1). Hence, instead of three breaks,four breaks were present. FISH with subtelomericprobes for the translocated chromosome arms showedthat on the derivative chromosomes the orientations ofthe translocated segments were retained with respectto the centromere. FISH with region specific probesfor 7q allowed to localize the 7q breakpoint betweenBAC clone RP11-261L16 and RP11-129J21 bothlocated within band 7q11.23. The karyotype descrip-tion was modified as follows: 46,XY,der(4)t(4;7)(4pter!4q12::7q11.23!7qter) der(7)t(4;7;14)(7pter!7q11.23::4q12!4q21.3::14q24.1!14qter)der(14)t(4;14)(14pter!14q24.1::4q21.3!4qter).

In patient 3, G-banding showed chromosomal rear-rangements involving chromosome 2, 7, and 14. Theposition of the missing part of the derivative 2 could notbe determined by G-banding. M-FISH showed that thisfragment was inserted into the derivative 7 with thebreakpoint located at 7q21.2. FISH with region specificprobes for 2p indicated that probe 783-f-7 (2p13) wastranslocated to the derivative 7 whereas the probeslocated at 2p23, 2p21-22, and 2p12 were retained on thederivative 2. FISH with region specific probes for 7qrevealed adeletionwithin band7q21.11 (Fig. 2). The twoprobes RP11-727N2 and RP11-175K20 immediatelyflanking the deleted region were present on the deri-vative 7 but located respectively proximal and distal tothe inserted chromosome 2 segment. The second break-point resulting from the translocationwith chromosome14 was localized between probe RP11-10D8 (7q22.1) on

Fig. 1. Partial G-banding karyotypes (panel A), M-FISH partial karyotypes in 24 color mode (panel B), and schematic overview (panel C) of thedescribed complex chromosomal rearrangements represents the breakpoints and the observed derivative chromosomes for patient 1, 2, 3, and 4 respectively.

14 Vermeulen et al.

the derivative chromosome 7 and RP11-91F7 (7q31.1,Table II). The extended karyotype could be writtenas follows: 46,XY,der(2)(2pter!2p13::2p11.2!2qter)der(7)t(2;7;14)(7pter!7q21.11::7q21.11!7q21.11::2p11.2!2p13::7q21.11!7q31.1::14q24.1!14qter)del(7)(q21.11q21.11)der(14)t(7;14)(14pter!14q24.1::7q32!7qter).In patient 4, a three-way translocation was assumed

but the fate of the translocated chromosome 7 segmentcould not be determined by G-banding. G-banding, M-FISH, and FISH with subtelomeric probes indicatedthat the observed complex rearrangement was theresult of two apparently independent rearrangements:a pericentric inversion of chromosome 7 and a simplereciprocal translocation between chromosome 10 and12. However, mapping of the chromosome 7 inversionbreakpoints showed, unexpectedly, the translocation ofprobes RP11-665O4, RP11-618A7, RP11-242J14, andRP11-796I6 to the derivative chromosome 10 at band10p13. FISH with a centromere probe for chromosome10 (Fig. 2) confirmed these observations. The 7q in-version breakpoint was mapped to 7p22 and 7q21.2.The karyotype was revised as: 46,XY,inv(7)(7pter!7p22::7q21.11!7p22::7q21.11!7qter)der(10)t(7;10;12)(12qter!12q21.3::7q21.11!7q21.11::10p13!10qter)der(12)t(10;12)(12pter!12q21.3::10p13!10pter).

CGH analysis including a detailed analysis of theindividual CGH profiles in the breakpoint regionsrevealed no deletions or duplications in the investigatedpatients (data not shown). Moreover, the deletion found

in patient 3 was not detectable by CGH, most probablydue to its small size (<2 Mb).

DISCUSSION

CCRs are defined as chromosomal rearrangementswithmore than two breakpoints betweenmore than twochromosomes [Pai et al., 1980]. Such complex rearran-gements are often difficult to characterize due to themonochrome nature of conventional banding techni-ques. However, the accurate description of the chromo-somal rearrangements and the detection of imbalancesresulting in the altered expression of genes located at ornear the breakpoints could provide significant informa-tion for themolecularmechanisms causing the observedphenotype. The advent of new molecular cytogenetictechniques such as M-FISH provides a solution to thisdifficulty [Schrock et al., 1997; Phelan et al., 1998; Leeet al., 2001].

In a first step towards the molecular characterizationof CCRs, we performed M-FISH, FISH with regionspecific probes, and CGH. We have demonstrated thatthe combination of M-FISH and FISH with regionspecific probes allowed a more accurate description ofchromosomal rearrangements in three out of four pa-tients (Fig. 1). A translocation between chromosomes 4,7, and 14 was shown to be accompanied by a crypticinsertion of part of chromosome4 into7q11.23. Similarlyin the CCR involving chromosomes 2, 7, and 14, a chro-mosome 2 short arm segment was inserted into the

Fig. 2. FISH with locus specific probes indicated a translocation inpatient 2 (panel A), a deletion and an insertion in patient 3 (panels B, C,and D), and a cryptic translocation in patient 4 (panels E and F). Panel A(probe RP11-261L16, 7q11.23, SG, and probe RP11-129J21, 7q11.23, SO)shows the translocation of RP11-129J21 to the derivative chromosome 2 inpatient 2. Panel B (probe RP11-5B9 (7q21.11) in spectrum green (SG) andprobe RP11-618A7 (7q21.11) in spectrum orange (SO) shows a deletion inpatient 3. Panel C (probe RP11-727N2, 7q21.11, SG, and probe RP11-

175K20, 7q21.11, SO) shows an insertion on the derivative chromosome 7 inpatient 3. Panel D (probe RP11-91M13, 7q21, SO, and 783-f-7, 2p13, SG)shows that the insertion in the derivative chromosome 7 of patient 3 is from2p13. Panel E (probe RP11-5B9, 7q21.11, SG, and probe RP11-618A7,7q21.11, SO) and panel F (probe RP11-618A7, 7q21.11, SO, and centromere10 probe in SG) show the translocation of probe RP11-618A7 to thederivative chromosome 10 in patient 4.

Chromosomal Rearrangements in Patients 15

derivative chromosome 7 at position 7q21.11. Theseresults confirm that the number of breakpoints and thecomplexity of the CCRs are generally underestimatedand that they can be described more accurately with M-FISH [Schrock et al., 1997; Haddad et al., 1998; Ogilvieet al., 1998; Phelan et al., 1998; Jalal and Law, 1999;Joyce et al., 1999b; Peschka et al., 1999; Bayani andSquire, 2001] even though M-FISH does not allowthe detection of intra chromosomal rearrangements[Lee et al., 2001]. A further increase in resolution in theanalysis of CCRs and the detection of resulting imbal-ancesmay be obtained through differentmore advancedmulticolor approaches such as combined binary ratiolabeling-fluorescence in situ hybridization (COBRA)[Wiegant et al., 2000], multicolor banding [Chudobaet al., 1999], or CGH-arrays [Solinas-Toldo et al., 1997;Pinkel et al., 1998; Pollack et al., 1999; Albertson et al.,2000; Snijders et al., 2001; Wessendorf et al., 2002].However, none of the above mentioned methods doesallow accurate fine mapping of the breakpoints whichneeds to bedonewith region specificprobes.CGHdidnotprovide evidence for imbalances in the investigatedCCRs. Although the resolution of CGH is limited toapproximately 5–10Mb [VanGele et al., 1997;Kirchhoffet al., 2000; Brecevic et al., 2001], two small deletions intwo other de novo CCRs were described with highresolutionCGH[Kirchhoff et al., 2000]. The size of one ofthe deletions was 5 Mb and both of the deletions werelocated at one of the CCR breakpoints.

The second part of this study focused on thecharacterization of the 7q breakpoints which occurredin three of the four CCRs. A search of the literatureshowed that 30% of CCRs had chromosome 7 break-points indicating a preferential involvement of chromo-some 7 (Table I and Batanian and Eswara, 1998).Indeed, when taking into account the length of thechromosomes, nearly two foldmore breakpoints inCCRsper centiMorgan per chromosome (3.7 chromosomebreaks per 10Mb) occur in contrast to the other chromo-somes (average 1.9 chromosome breaks per 10 Mb). Ahigher number of breakpoints per centiMorganwas onlyobserved for chromosome 21 (5.3 chromosome breaksper 10Mb). In order to investigate possible clustering ofbreakpoints and to look for submicroscopic imbalances,region specific probes covering the long arm of chromo-some 7 were tested. In one patient the breakpoints weremapped within band 7q21.11. They were the result of a2p insertion that was shown to be accompanied by asubmicroscopic deletion at 7q21.11 of 2 Mb. Interest-ingly this same 7q21.11 region was inserted into at(10;12) in another patient without detectable loss orgain at the 7q breakpoint region (Fig. 3). The genomicregion at 7q21.11 contains five candidate genes whichare either involved in neuronal development or highlyexpressed in brain tissue. SEMA3E is a protein highlysimilar to murine semaphorin H and may be involvedin neuronal growth cone guidance [Eckhardt andMeyerhans, 1998]. SEMA3A or semaphorin 3A is a che-moattractant for cortical apical dendrites [Polleux et al.,2000]. GRM3, the glutamate receptor metabotrophicgene, is aneurotransmitter receptor [Makoff et al., 1996;Scherer et al., 1996]. Glutamate receptorsmediate most

of the excitatory neurotransmission in the mammalianbrain and participate in processes of synaptic plasticityand efficacy in learning and memory. DMTF, the genefor cyclin Myb-like protein, is preferentially expressedin the human brain [Makoff et al., 1996; Bodner et al.,1999]. Finally, the ADAM22 metalloproteinase, ishighly expressed in the brain and may function as anintegrin ligand in the brain [Poindexter et al., 1999].Obviously, further studies are needed to determine thepossible involvement of any of these genes in theobserved phenotypes. Also, since much more break-points were involved in this complex translocation,other candidate genes on other chromosomal regionspossibly contribute to the phenotype of these patients.

Altogether these results indicate that the combinationof the above-mentioned techniques result in a moreaccurate description of CCRs. We and others haveshown that CCRs are often characterized erroneouslydue to the monochrome nature of conventional bandingtechniques. We also demonstrated a preferential 7qinvolvement in the formation of the various breakpointsfound in these CCRs. In this respect, it is of interest thatsegmental duplications are particularly enriched inchromosome 7 [Bailey et al., 2002]. In contrast to thisobservation, however, we have no arguments that ahigher number of breaks occur in simple translocations.Another line of evidence, albeit observed in malignantcells, comes from the observation of increased genomic

Fig. 3. Ideogram of chromosome 7 according to ISCN 95. Filled dots tothe right of the ideogram represent chromosome breaks found in theliterature. Open dots represent the breakpoints found in the patientsdescribed in this report. The numbers in superscript indicate the respectivepatients in whom the breakpoints were found. The open triangle representsthe deletion found in patient 3.

16 Vermeulen et al.

instability of 7q in combination with other karyotypicchanges [Luna-Fineman et al., 1995; Liang et al., 1998].Further analysis at the sequence level of these break-points should clarify whether simple or complex re-peats or segmental duplications are indeed involved inthe formation of these chromosomal rearrangements[Emanuel and Shaikh, 2001] and could clarify theirpreferential involvement in CCRs but not 7q arrange-ments in simple translocations. In addition, furtherdetailed molecular analysis on 7q21.11 may provideimportant clues to the regulation of one of the above-mentioned genes as well as point to the underlyingmechanisms resulting in CCRs.

ACKNOWLEDGMENTS

We thank C. Vantieghem, C. Maes, and M. Yoruk forexpert technical assistance.Wewould also kindly like tothank Dr. A. Jauch from the Laboratory of HumanGenetics, Heidelberg, Germany for providing us withmost of the subtelomeric YACs, BACs, and PACs used inthis study andDr.M.Rocchi from theUniversity ofBari,Italy for providing us with the YAC clones whichmapped to chromosome 2. We would like to thank TheWellcome Trust Sanger Institute (Hinxton, Cambridge,UK) for providing us with most of the BAC probes forchromosome 7q.

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