Chromosomal abnormalities are a frequent cause of mental retardation and congenital abnormalities. A small subset of these patients present with an apparently balanced de novo complex chromosomal rearrangement (CCR). CCRs are defined as structural rearrangements with more than two breakpoints and exchange of genetic material between two or more chromosomes [Pai et al., 1980]. CCRs can be grouped according to the number of breaks [Kousseff et al., 1993], the type of rearrangements [Kausch et al., 1988], the preferential occurrence of inter- or intra-chromosomal rearrangements [Lurie et al., 1994], and the de novo or familial occurrence of CCRs [Kleczkowska et al., 1982]. More than 100 constitutional CCRs have been documented (for review see Batanian and Eswara, 1998, Table I). De novo CCRs are associated with mental retardation and congenital abnormalities. In most patients it is assumed that the observed phenotypic anomalies are the result of submicroscopic deletions or duplications or alternatively disruption, activation, or inactivation of genes located at or near one or more breakpoints. So far this hypothesis has not been proven by molecular analysis. Due to the complex and unique nature of these rearrangements no uniform strategy has been used to investigate CCRs. Until now, only a small number of CCRs were investigated at the molecular cytogenetic level with M-FISH or SKY [Schröck et al., 1997; Haddad et al., 1998; Ogilvie et al., 1998; Phelan et al., 1998; Jalal and Law, 1999; Peschka et al., 1999; Bayani and Squire, 2001]. M-FISH [Speicher et al., 1996], SKY, spectral karyotyping [Schröck et al., 1996], or multipaint FISH [Joyce et al., 1999b] allowed a more accurate characterization of CCRs at the cytogenetic level but has limits (5–10 Mb) in detecting small duplications or deletions [Lee et al., 2001]. Here, we describe the combined application of M-FISH and CGH in the study of four de novo CCRs in order to accurately characterize the nature of the rearrangements. Subsequently, we investigated the observed 7q breakpoints with bacterial artificial chromosome (BAC) probes to map the breakpoints in further detail.
Table I. Overview of CCRs not Reviewed in the Article of Batanian and Eswara 
Karyotyping was performed on short term lymphocyte cultures from peripheral blood with G-banding. Karyotypes were described according to the guidelines of the ISCN . The study was approved by the ethical committee of Ghent University Hospital (Ghent, Belgium) under project 2000/80.
For M-FISH, the “24 Xcyte” M-FISH probe kit and software were applied (MetaSystems, Altlussheim, Germany; http://www.metasystems.de). The probe mixture was prepared by combinatorial labeling of degenerated oligonucleotide primer polymerase chain reaction (DOP-PCR) amplified microdissected chromosomes using biotin and four fluorochromes: fluorescein isothiocyanate (FITC), Spectrum Orange (SO), Texas Red (TR), and diethylcoumarine (DEAC). Biotinylated probes were detected with avidin-Cy5. M-FISH was performed following the manufacturer instructions. Metaphase slides were pre-treated with RNAse and pepsin. Slides were denatured with 70% formamide/2× SSCP at 80°C for 5 min. The probe mixture was denatured at 75°C for 5 min, incubated at 37°C for 30 min, and subsequently applied to the slides under an 18 × 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 42°C, followed by three washes in 2× SSC (42°C). For counterstaining 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Rocche Molecular Biochemicals, Brussels, Belgium) was added to the antifade reagents [(Vectashield, Vector laboratories, Burlingame, CA). A Zeiss Axioplan epifluorescence microscope with an eight-position filter wheel [Carl Zeiss Jena GmbH, Jena, Germany; http://www.zeiss.de) and a black and white high-resolution camera were used for capturing images for each different fluorochrome. The following single-band pass filters were used: DAPI (filterset no. 01; Carl Zeiss, excitation: 359 nm, emission: 441 nm), FITC (filterset no. 09; Carl Zeiss, excitation: 490 nm, emission: 525 nm), 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: 590 nm, emission: 615 nm), and DEAC (aqua chroma 31036v2; excitation: 436 nm, emission: 480 nm; Chroma Technology Corp, Brattleboro, VT). The M-FISH module in the ISIS image analysis software from MetaSystems was used to process the images. On the basis of a color definition table, pseudocolors were assigned to each individual chromosome resulting in the unambiguous identification of the rearrangements. M-FISH from patient 2 was performed on Epstein–Barr virus-immortalized lymphoblastoid cells.
Comparative Genomic Hybridization
CGH was performed as described previously [Van Roy et al., 1997]. Briefly, metaphases stained with DAPI were recorded prior to hybridization using a Leitz DM microscope equipped with a high-sensitivity integrated monochrome CCD camera (Sony IMAC-CCD S30). After hybridization the images were processed with the ISIS-CGH software (MetaSystems). For each patient, 10–20 metaphase cells were analyzed. For evaluation of CGH data, average ratio profiles with fixed limits at 1.25 and 0.75 and standard deviation limits (the width of the confidence interval being three times the standard deviation) as well as individual ratio profiles were analyzed. A chromosomal region was considered to be over-represented (gain) respectively under-represented (loss) if the average ratio profile crossed the standard deviation limit. As a control, normal to normal hybridizations were performed.
FISH With Region Specific Probes
Fluorescence in situ hybridization (FISH) was performed with YAC, BAC, PAC, and plasmid probes labeled either with digoxigenin or biotin. 7q locus specific RPCI-BAC probes (see Table II) were obtained through screening of the October 2000, December 2000, and April 2001 Freeze (http://genome.ucsc.edu/) of the human genome project. The following plasmid probes were used for the centromeres of the respective chromosomes: D7Z1, centromere 7; pα3.5, centromere 3; D10Z1, centromere 10; pBS4D, centromere 2; pZ4.1, centromere 4 and centromere 9. CEP12 for centromere 12 was obtained from Vysis (Downers Grove, IL). For the subtelomeric regions the following probes were used: YAC clones; TYAC75 (2pter); TYAC162 (3qter); 946-a-3 (4qter); 626-g-11 (7pter); TYAC109 (7qter); TYAC95 (10pter); TYAC93 (10qter); PAC clone; 483 G12 (12pter); BAC clone; GS-820-M16 (14qter) kindly provided by Dr. A. Jauch, Laboratory of Human genetics, Heidelberg, Germany. Telvysion 14q (14qtel) was purchased from Vysis. The following YAC probes for chromosome 2p specific bands were used; 713-g-9 (2p23); 830-d-1 (2p21-22); 783-f-7 (2p13), and 850-a-4 (2p12).
Table II. Chromosomal Position on the Various Derivatives of 7q BAC Clones in the Three Patients With 7q Involvement in Their CCRs
Patient 1 was born at term to healthy, non-consanguineous parents. The pregnancy was complicated by maternal fever due to a respiratory infection in the fifth month of pregnancy. His birth weight was 2,600 g, length 48.5 cm, and head circumference 30.5 cm (P10 = 32 cm). After birth, microcephaly was noted. Evaluation at the age of 4 months revealed generalized hypertonia with increased deep tendon reflexes, and psychomotor delay. He did not follow objects, showed hyperacusis, and had a history of feeding problems. Head circumference was 36.5 cm. CT-scan of the brain revealed punctiform calcifications around the occipital horns of the lateral ventricles, periventricular leukomalacia, and small cerebellum.
Patient 2 was born at 37 weeks gestation from healthy, non-consanguineous parents. Prenatal ultrasound showed polyhydramnios. The delivery was uneventful. His birth weight was 2,700 g, length 50 cm, and head circumference 36 cm. After birth, an oesophageal atresia was documented. In addition, physical examination revealed preaxial polydactyly of the right thumb, a non-functional and hypoplastic right thumb (“floating thumb”), a single palmar crease in the left hand, and partial cutaneous syndactyly of the second and third toes. In the neonatal period, ultrasound examination of brain and abdomen did not reveal any abnormalities. Echocardiographic evaluation showed a normal structure and function of the heart.
Patient 3 was born at term after an uncomplicated pregnancy. The parents were healthy and non-consanguineous. His birth weight was 3,500 g and length 51 cm. The delivery and postnatal course were uneventful. The boy sat alone at 10 months and started walking at 19 months. From the beginning, the parents noticed a poor balance and coordination during walking. Around the age of 3 years, a delay in speech development became evident. Because of the psychomotor delay (IQ = 65), he was referred to special education school. Physical examination at the age of 6 years and 9 months revealed a height of 126 cm (P75), weight of 25 kg (P50–P75), and head circumference of 55.8 cm (P97 = 54.5 cm). Most striking were the macrocephaly with prominent occiput, hypertelorism with antimongoloid slant of the palpebral fissures, retrognathia, large mouth, widely spaced teeth, and pectus excavatum. Neurologic examination showed mild hypotonia with dystonic postures, dysmetria, and tremor of the hands. The deep tendon reflexes were normal and pathologic reflexes absent. MRI scan of the brain did not reveal abnormalities.
Patient 4 was born at 37 weeks gestation after an uncomplicated pregnancy. His birth weight was 3,450 g and length 50 cm. Delivery was normal. The first months of postnatal life were complicated by feeding problems and recurrent ear infections. He sat alone at 12 months and started walking at 2 years of age. Because of a delay in the psychomotor development and the relatively large skull, a MRI scan of the brain was performed at the age of 2 years and 8 months but no abnormalities were found. Physical examination at the age of 3 years revealed a weight of 15.5 kg (P50–P75), length of 96.5 cm (P25), and head circumference of 52.3 cm (P98 = 53 cm). The most striking clinical features were large skull, frontal bossing with receding frontal hairline, sparse eyebrows and eyelashes, large nose, and small mouth. Neurologic evaluation revealed a poor balance. Psychometric testing at the age of 3 years 8 months revealed a developmental age of 2 years 4 months (IQ = 67). Speech assessments revealed severe articulation problems and better receptive than expressive language skills.
Molecular Cytogenetic Findings
Partial G-banded karyotypes of the CCR of the four patients are shown in Figure 1. The karyotypes from the parents of all four patients were normal indicating that the CCRs described here arose de novo. M-FISH analysis allowed to revise the karyotype in three out of four 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 additional breakpoints or translocations with other chromosomes were observed. FISH with subtelomeric probes showed that the orientation of the translocated chromosome segments was preserved.
In patient 2 the breakpoints inferred from G-banding were revised after M-FISH. M-FISH confirmed the presence of chromosome 14 material in the derivative chromosome 7 but in addition revealed part of chromosome 4 in this derivative. The breakpoint on derivative chromosome 7 had been originally assigned to 7q31.2 due to the similarity in the G-banding pattern of segment 4q12→4q21.3 when compared to 7q11.2→7q22 (see also Fig. 1). Hence, instead of three breaks, four breaks were present. FISH with subtelomeric probes for the translocated chromosome arms showed that on the derivative chromosomes the orientations of the translocated segments were retained with respect to the centromere. FISH with region specific probes for 7q allowed to localize the 7q breakpoint between BAC clone RP11-261L16 and RP11-129J21 both located within band 7q11.23. The karyotype description 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 rearrangements involving chromosome 2, 7, and 14. The position of the missing part of the derivative 2 could not be determined by G-banding. M-FISH showed that this fragment was inserted into the derivative 7 with the breakpoint located at 7q21.2. FISH with region specific probes for 2p indicated that probe 783-f-7 (2p13) was translocated to the derivative 7 whereas the probes located at 2p23, 2p21-22, and 2p12 were retained on the derivative 2. FISH with region specific probes for 7q revealed a deletion within band 7q21.11 (Fig. 2). The two probes RP11-727N2 and RP11-175K20 immediately flanking the deleted region were present on the derivative 7 but located respectively proximal and distal to the inserted chromosome 2 segment. The second breakpoint resulting from the translocation with chromosome 14 was localized between probe RP11-10D8 (7q22.1) on the derivative chromosome 7 and RP11-91F7 (7q31.1, Table II). The extended karyotype could be written as 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 segment could not be determined by G-banding. G-banding, M-FISH, and FISH with subtelomeric probes indicated that the observed complex rearrangement was the result of two apparently independent rearrangements: a pericentric inversion of chromosome 7 and a simple reciprocal translocation between chromosome 10 and 12. However, mapping of the chromosome 7 inversion breakpoints showed, unexpectedly, the translocation of probes RP11-665O4, RP11-618A7, RP11-242J14, and RP11-796I6 to the derivative chromosome 10 at band 10p13. FISH with a centromere probe for chromosome 10 (Fig. 2) confirmed these observations. The 7q inversion 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 the individual CGH profiles in the breakpoint regions revealed no deletions or duplications in the investigated patients (data not shown). Moreover, the deletion found in patient 3 was not detectable by CGH, most probably due to its small size (<2 Mb).
CCRs are defined as chromosomal rearrangements with more than two breakpoints between more than two chromosomes [Pai et al., 1980]. Such complex rearrangements are often difficult to characterize due to the monochrome nature of conventional banding techniques. However, the accurate description of the chromosomal rearrangements and the detection of imbalances resulting in the altered expression of genes located at or near the breakpoints could provide significant information for the molecular mechanisms causing the observed phenotype. The advent of new molecular cytogenetic techniques such as M-FISH provides a solution to this difficulty [Schröck et al., 1997; Phelan et al., 1998; Lee et al., 2001].
In a first step towards the molecular characterization of CCRs, we performed M-FISH, FISH with region specific probes, and CGH. We have demonstrated that the combination of M-FISH and FISH with region specific probes allowed a more accurate description of chromosomal rearrangements in three out of four patients (Fig. 1). A translocation between chromosomes 4, 7, and 14 was shown to be accompanied by a cryptic insertion of part of chromosome 4 into 7q11.23. Similarly in the CCR involving chromosomes 2, 7, and 14, a chromosome 2 short arm segment was inserted into the derivative chromosome 7 at position 7q21.11. These results confirm that the number of breakpoints and the complexity of the CCRs are generally underestimated and that they can be described more accurately with M-FISH [Schröck et al., 1997; Haddad et al., 1998; Ogilvie et al., 1998; Phelan et al., 1998; Jalal and Law, 1999; Joyce et al., 1999b; Peschka et al., 1999; Bayani and Squire, 2001] even though M-FISH does not allow the detection of intra chromosomal rearrangements [Lee et al., 2001]. A further increase in resolution in the analysis of CCRs and the detection of resulting imbalances may be obtained through different more advanced multicolor approaches such as combined binary ratio labeling-fluorescence in situ hybridization (COBRA) [Wiegant et al., 2000], multicolor banding [Chudoba et 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 does allow accurate fine mapping of the breakpoints which needs to be done with region specific probes. CGH did not provide evidence for imbalances in the investigated CCRs. Although the resolution of CGH is limited to approximately 5–10 Mb [Van Gele et al., 1997; Kirchhoff et al., 2000; Brecevic et al., 2001], two small deletions in two other de novo CCRs were described with high resolution CGH [Kirchhoff et al., 2000]. The size of one of the deletions was 5 Mb and both of the deletions were located at one of the CCR breakpoints.
The second part of this study focused on the characterization of the 7q breakpoints which occurred in three of the four CCRs. A search of the literature showed that 30% of CCRs had chromosome 7 breakpoints indicating a preferential involvement of chromosome 7 (Table I and Batanian and Eswara, 1998). Indeed, when taking into account the length of the chromosomes, nearly two fold more breakpoints in CCRs per centiMorgan per chromosome (3.7 chromosome breaks per 10 Mb) occur in contrast to the other chromosomes (average 1.9 chromosome breaks per 10 Mb). A higher number of breakpoints per centiMorgan was only observed for chromosome 21 (5.3 chromosome breaks per 10 Mb). In order to investigate possible clustering of breakpoints and to look for submicroscopic imbalances, region specific probes covering the long arm of chromosome 7 were tested. In one patient the breakpoints were mapped within band 7q21.11. They were the result of a 2p insertion that was shown to be accompanied by a submicroscopic deletion at 7q21.11 of 2 Mb. Interestingly this same 7q21.11 region was inserted into a t(10;12) in another patient without detectable loss or gain at the 7q breakpoint region (Fig. 3). The genomic region at 7q21.11 contains five candidate genes which are either involved in neuronal development or highly expressed in brain tissue. SEMA3E is a protein highly similar to murine semaphorin H and may be involved in neuronal growth cone guidance [Eckhardt and Meyerhans, 1998). SEMA3A or semaphorin 3A is a chemoattractant for cortical apical dendrites [Polleux et al., 2000]. GRM3, the glutamate receptor metabotrophic gene, is a neurotransmitter receptor [Makoff et al., 1996; Scherer et al., 1996]. Glutamate receptors mediate most of the excitatory neurotransmission in the mammalian brain and participate in processes of synaptic plasticity and efficacy in learning and memory. DMTF, the gene for cyclin Myb-like protein, is preferentially expressed in the human brain [Makoff et al., 1996; Bodner et al., 1999]. Finally, the ADAM22 metalloproteinase, is highly expressed in the brain and may function as an integrin ligand in the brain [Poindexter et al., 1999]. Obviously, further studies are needed to determine the possible involvement of any of these genes in the observed phenotypes. Also, since much more breakpoints were involved in this complex translocation, other candidate genes on other chromosomal regions possibly contribute to the phenotype of these patients.
Altogether these results indicate that the combination of the above-mentioned techniques result in a more accurate description of CCRs. We and others have shown that CCRs are often characterized erroneously due to the monochrome nature of conventional banding techniques. We also demonstrated a preferential 7q involvement in the formation of the various breakpoints found in these CCRs. In this respect, it is of interest that segmental duplications are particularly enriched in chromosome 7 [Bailey et al., 2002]. In contrast to this observation, however, we have no arguments that a higher number of breaks occur in simple translocations. Another line of evidence, albeit observed in malignant cells, comes from the observation of increased genomic instability of 7q in combination with other karyotypic changes [Luna-Fineman et al., 1995; Liang et al., 1998]. Further analysis at the sequence level of these breakpoints should clarify whether simple or complex repeats or segmental duplications are indeed involved in the formation of these chromosomal rearrangements [Emanuel and Shaikh, 2001] and could clarify their preferential involvement in CCRs but not 7q arrangements in simple translocations. In addition, further detailed molecular analysis on 7q21.11 may provide important clues to the regulation of one of the above-mentioned genes as well as point to the underlying mechanisms resulting in CCRs.
We thank C. Vantieghem, C. Maes, and M. Yoruk for expert technical assistance. We would also kindly like to thank Dr. A. Jauch from the Laboratory of Human Genetics, Heidelberg, Germany for providing us with most of the subtelomeric YACs, BACs, and PACs used in this study and Dr. M. Rocchi from the University of Bari, Italy for providing us with the YAC clones which mapped to chromosome 2. We would like to thank The Wellcome Trust Sanger Institute (Hinxton, Cambridge, UK) for providing us with most of the BAC probes for chromosome 7q.