Molecular analysis of a constitutional complex genome rearrangement with 11 breakpoints involving chromosomes 3, 11, 12, and 21 and a ∼0.5-Mb submicroscopic deletion in a patient with mild mental retardation

Complex chromosome rearrangements (CCRs) are extremely rare but often associated with mental retardation, congenital anomalies, or recurrent spontaneous abortions. We report a de novo apparently balanced CCR involving chromosomes 3 and 12 and a two-way translocation between chromosomes 11 and 21 in a woman with mild intellectual disability, obesity, coarse facies, and apparent synophrys without other distinctive dysmorphia or congenital anomalies. Molecular analysis of breakpoints using fluorescence in situ hybridization (FISH) with region-specific BAC clones revealed a more complex character for the CCR. The rearrangement is a result of nine breaks and involves reciprocal translocation of terminal chromosome fragments 3p24.1→pter and 12q23.1→qter, insertion of four fragments of the long arm of chromosome 12: q14.1→q21?, q21?→q22, q22→q23.1, and q23.1→q23.1 and a region 3p22.3→p24.1 into chromosome 3q26.31. In addition, we detected a ～0.5-Mb submicroscopic deletion at 3q26.31. The deletion involves the chromosome region that has been previously associated with Cornelia de Lange syndrome (CdLS) in which a novel gene NAALADL2 has been mapped recently. Other potential genes responsible for intellectual deficiency disrupted as a result of patient’s chromosomal rearrangement map at 12q14.1 (TAFA2), 12q23.1 (METAP2), and 11p14.1 (BDNF).     

De novo 9-break-event in one chromosome 21 combined with a microdeletion in 21q22.11 in a mentally retarded boy with short stature

We report on a moderately mentally retarded 12-year-old boy of short stature showing the most complex chromosomal rearrangement (CCR) within a single chromosome ever described. A de novo derivative chromosome 21 was recognized in GTG-banding shortly after birth. However, the nature of the rearrangement remained obscure up to the application of the chromosome 21-specific centromere-near multicolor-FISH (subcenM-FISH) probe set and of six selected locus-specific probes along chromosome 21. An unbalanced 9-break-event was uncovered with breakpoints in 21p13, 21p13-->12, 21q11.2, 21q21.1, 21q22.11, 21q22.11, 21q22.12, 21q22.22 and 21q22.3. A deletion of 21q22.11 was detected by application of the BAC probe bk249H10. The karyotype can be described as 46,XY,der(21)(:p13-->p1213::q22.3-->q22.22:: q11.2-->p1213::q11.2-->q21.1::q22.11-->q21.1::q22.12--> q22.22::p13-->p13). The clinical signs can either be due to gene inactivation in connection with structural changes at the break and fusion regions, to the building of new fusion genes within the CCR and/or to the deletion of genes in 21q22.11.     

复杂染色体重排的女性携带者与她健康的女儿

一例新生复杂染色体重排的女性携带者（complex chromosome rearrangement ,CCR）,易位涉及1号、5号和12号染色体。病人因2次自然流产而要求进行外周血淋巴细胞G显带核型分析。最初G显带核型疑为46,XX,t(1;5;12)(1pter→1q25::12q24→12qter;5qter→5p11::1q25→1qter;12pter→12q24::5p11→5pter).经荧光原位杂交（FISH）技术检测,证实患者的核型为46,XX,t(1;5;12)(1pter→1q23::12q22→12qter;5qter→5p11::1q25→1qter;12pter→12q22::1q23→1q25::5p11→5pter).7年后病人再次妊娠,并拒绝产前诊断。女婴足月分娩,生长发育正常。核型为46,XX。比较以前报告的女性复杂易位携带者与我们报告的病例可以认为,CCR并不总是表现为自然流产或分娩畸形儿,仍有机会生出正常的孩子。Abstract: We reported in the paper one case of a de novo complex chromosomal rearrangement (CCR) involving three different chromosomes,1, 5 and 12. Two pregnancies of the female carrier over three years resulted in two spontaneous abortions. Initial cytogenetic analysis of her peripheral lymphocyte by G banding suspected a karyotype 46,XX,t(1;5;12)(1pter →1q25::12q24→12qter;5qter→ 5p11::1q25→1qter;12pter →12q24::5p11→5pter). Fluorescense in -situ hybridization (FISH) was used to confirm the karyotype 46,XX,t(1;5;12)(1pter→1q23::12q22→12qter;5qter→5p11::1q25→1qter;12pter→12q22::1q23→1q25::5p11→5pter). Seven years later she was pregnant again and refused to have prenatal diagnosis. The fetus is normal both in phenotype and karyotype。Comparing previously reported female CCR carriers with the case, we conclude that female CCR carriers may not always present spontaneous abortion or have offspring with congenital malformation and can have chance to get a healthy child.     

A de novo (1;2;3;15;18) chromosome rearrangement with six nonreciprocal translocations

A de novo complex chromosome rearrangement (CCR) found in a phenotypically abnormal boy was characterized by G-bands, FISH with subtelomere probes, and M-FISH. The G-banding analysis revealed involvement of chromosomes 1, 2, 3, 15, and 18 with (at least) eight breakpoints, five nonreciprocal translocations (1q --> 2q --> 8q --> 15q --> 2p --> 1q), and a 3p insertion into the der(2); there was also a presumptive deletion of 1q41. The 5 derivatives were described as follows: der(1)(1pter --> 1q32.3?::2p21--> 2pter),der(2)(1qter --> 1q42?::2q24.2 --> 2p21::3p13 --> 3p26::15q15 --> 15qter),der(3)(3qter --> 3p13:),der(15)(15pter --> 15q15::18q11 --> 18qter),der(18)(18pter --> 18q11::2q24.2 --> 2qter). The molecular assays confirmed the segmental composition of each derivative and documented the localization of most relevant telomeres. In addition to the novelty of the 1, 2, 3, 15 and 18 combination, this CCR may also be unique in the sense that it represents a cluster of 6 nonreciprocal transpositions regardless of the occurrence (or lack thereof) of secondary unbalances. Finally, there appears to be an excess of CCRs in fetuses conceived by intracytoplasmic sperm injection.     

Characterization of a de novo complex chromosome rearrangement (CCR) involving chromosomes 2 and 12, associated with mental retardation and impaired speech development

We report on a currently six-year-old patient with a de novo complex chromosome rearrangement (CCR) involving chromosomes 2 and 12. A translocation 2;12 that appeared to be reciprocal after standard banding turned out to be a complex event with seven breaks after molecular cytogenetic analyses. Array CGH analysis showed no imbalances at the breakpoints but revealed an additional microdeletion of about 80 kb on chromosome 11. The same deletion was also present in the phenotypically normal father. The patient showed relatively mild mental retardation, defined mainly as impaired speech development (orofacial dyspraxia) and psychomotor retardation. In addition, mild dysmorphic facial features like hypertelorism, a prominent philtrum and down-turned corners of the mouth were observed. We narrowed down all breakpoint regions to about 100 kb, using a panel of mapped bacterial artificial chromosome (BAC) clones for fluorescence in situ hybridization (FISH). BACs spanning or flanking all seven breakpoints were identified and no chromosomal imbalances were found consistent with the array CGH results. Our investigations resulted in the following karyotype: 46,XY,t(2;12)(2pter-->2p25.3::2p23.3-->2p25.2::2p23.3-->2p14::2q14.3-->2p14::2q14.3-->2q14.3::12q 12-->12qter;12pter-->12q12::2p25.3-->2p25.2::2q14.3-->2qter).     

Proximal trisomy 13q and distal monosomy 8p in a dysmorphic and mentally retarded patient with an isodicentric chromosome 13q and a 13q/8p translocation chromosome

A 40 year-old dysmorphic and mentally retarded female is reported with a de novo unbalanced chromosomal rearrangement (karyotype: 46,XX,der(8)t(8;13)(p23;q123),idic(13)(pter-->q123: q123-->pter) resulting in an isodicentric chromosome 13 and a double aneusomy including partial trisomy 13 (13pter-q123) and distal monosomy 8p (8pter-p23). The main clinical findings consist of developmental/mental retardation, behavioural disturbances and minor congenital defects, not consistent with the clinical pattern of either of the two aneusomies. A mechanism for the chromosome rearrangement is proposed and the absence of specific physical findings in the present patient is discussed in the light of the available literature data.     

De novo balanced complex chromosome rearrangement (CCR) involving chromosome 8, 11 and 16 in a boy with mild developmental delay and psychotic disorder

Congenital Complex Chromosome rearrangements (CCRs) compatible with life are rare in humans. We report a de novo CCR involving chromosomes 8, 11 and 16 with 4 breakpoints in a patient with mild dysmorphic features, acquisition delay and psychotic disorder. Conventional cytogenetic analysis revealed an apparently balanced 8;16 translocation. Further FISH analysis with WCP 8 and WCP 16 probes revealed the presence of a third chromosome involved in the translocation. The multicolour karyotype confirmed the complexity of the rearrangement and showed that the derivative chromosome 8 was composed of 3 distinct segments derived from chromosomes 8, 16 and 11. The breakpoints of this complex rearrangement were located at 8q21, 11q14, 11q23 and 16q12. Comparative genomic hybridization (CGH) and array-CGH were performed to investigate the possibility of any genomic imbalance as a result of the complex rearrangement. No imbalance was detected by these two techniques. Our study showed: i) the necessity to confirm reciprocal translocations with FISH using painting probes, particularly when the karyotype resolution is weak; ii) the usefulness of multicolour karyotype for the characterization of structural chromosomal rearrangements, particularly when they are complex; iii) the usefulness of CGH and array-CGH in cases of abnormal phenotype and apparently balanced rearrangement in order to explore the breakpoints and to detect additional imbalances.     

Fc receptor gene translocation in a t(1;19) pre-B ALL cell line

We have recently mapped the human FCGR2 gene to chromosome 1 bands q23-q24. In situ hybridization of FCGR2 cDNA with a cell line containing a t(1;19)(g23;p13) derived from a patient with pre-B ALL has allowed a more accurate localization of this gene to chromosome 1 band q23. Furthermore, this study indicated a splitting of the FCGR2 gene or gene cluster by the t(1;19). However, Southern analysis showed no genetic rearrangement when compared with a karyotypically normal Epstein-Barr virus (EBV)-transformed cell line from the same patient. This suggests that the translocation breakpoint does not occur within the coding region of this gene.     

Double partial trisomy of 6p23-pter and 9pter-q21.2 in a neonate resulting from 4:2 meiotic segregation of a maternal complex t(6;7;9)(p23;p15;q21.2) translocation

We report, a newborn presenting multiple congenital abnormalities with karyotype; 47,XY,der(7)t(6;7)(pter-p23::p15-->qter),+der(9)t(7;9)(pter-->p15::q21.2--> pter)t(6;7;9)(p23;p15;q21.2)mat[20]. The mother and her phenotypically normal daughter were carriers of a complex chromosomal rearrangement with karyotypes; 46,XX,t(6;7;9)(p23;p15;q21.2)[20]. Paternal chromosomes were normal. In our case the extra derivative chromosome was the result of a 4:2 segregation of the chromosomes involved in translocation during oogenesis. Double partial trisomy in newborns resulting from 4:2 segregation is a rare event, and double partial trisomies of the 6p23-pter and trisomy 9pter-q22 regions have not reported to date.     

Meiotic recombination and spatial proximity in the etiology of the recurrent t(11;22)

Although balanced translocations are among the most common human chromosomal aberrations, the constitutional t(11;22)(q23;q11) is the only known recurrent non-Robertsonian translocation. Evidence indicates that de novo formation of the t(11;22) occurs during meiosis. To test the hypothesis that spatial proximity of chromosomes 11 and 22 in meiotic prophase oocytes and spermatocytes plays a role in the rearrangement, the positions of the 11q23 and 22q11 translocation breakpoints were examined. Fluorescence in situ hybridization with use of DNA probes for these sites demonstrates that 11q23 is closer to 22q11 in meiosis than to a control at 6q26. Although chromosome 21p11, another control, often lies as close to 11q23 as does 22q11 during meiosis, chromosome 21 rarely rearranges with 11q23, and the DNA sequence of chromosome 21 appears to be less susceptible than 22q11 to double-strand breaks (DSBs). It has been suggested that the rearrangement recurs as a result of the palindromic AT-rich repeats at both 11q23 and 22q11, which extrude hairpin structures that are susceptible to DSBs. To determine whether the DSBs at these sites coincide with normal hotspots of meiotic recombination, immunocytochemical mapping of MLH1, a protein involved in crossing over, was employed. The results indicate that the translocation breakpoints do not coincide with recombination hotspots and therefore are unlikely to be the result of meiotic programmed DSBs, although MRE11 is likely to be involved. Previous analysis indicated that the DSBs appear to be repaired by a mechanism similar to nonhomologous end joining (NHEJ), although NHEJ is normally suppressed during meiosis. Taken together, these studies support the hypothesis that physical proximity between 11q23 and 22q11--but not typical meiotic recombinational activity in meiotic prophase--plays an important role in the generation of the constitutional t(11;22) rearrangement.     

Human chromosome hot points

Summary Peripheral lymphocytes from 16 healthy adults, 9 pregnant women, and 3 fragile X syndrome patients were cultured in Eagle's minimum essential medium without folic acid (MEM-FA). The addition of 2mM, 4mM, or 8mM uridine 24h or 72h prior to harvest resulted in increases of chromosome gaps or breaks, especially at hot points 3p14, 16q23-24, and at fragile site Xq27. Pregnant women showed higher frequencies of 3p14 breaks and total chromosome breaks than men and non-pregnant women. The other chromosome regions, such as 6q26, 7q23, 7q35, 6p25, Xp22, 14q23 and 11p13, also frequently showed gaps or breaks. The results indicated that the unbalance of nucleotide pools was one of the causes of chromosome breakages. The higher frequencies of chromosome gaps and breaks under the condition of thymidylate stress may be due to the misincorporation of uracil instead of thymine into DNA.     

A complex chromosome rearrangement involving chromosome 8, 11, and 12 analyzed by conventional cytogenetic investigations, fluorescence in situ hybridisation, and spectral karyotyping.

We report on a 29-year-old woman with a history of five spontaneous abortions and a balanced complex chromosome rearrangement (CCR) involving break points between chromosomes 8, 11, and 12. Fluorescence in situ hybridisation (FISH) in combination with giemsa trypsin banding techniques were essential for the identification of the breakpoints. In addition, the results were confirmed by 24-colour FISH using the spectral karyotyping system (SKY). The karyotype was 46,XX,t(8;11;12)(8qter-->8p10::12p10-->12pter;11pter--> 11q14::8p10-->8pter;12qter-->12p10::11q14-->11qter). Application of SKY facilitated detection of all three chromosomes involved and supported the localisation of the breakpoints by a single time and sample saving investigation.     

Langer-Giedion syndrome,in a child with complex structural aberration of chromosome 8

A patient with typical features of the Langer-Giedion syndrome (tricho-rhino-phalangeal syndrome, type II) is described. In the karyotype an interstitial deletion of the long arm of chromosome 8 (band 8q22) was observed as the result of a complex rearrangement of chromosomes 1 and 8: 46,XY inv(8)(q23 leads to q242), del(8)(q221 leads to q223), ins(8;1) (q221;p321 p341;q242). Previously reported cases of Langer-Giedion syndrome with deletion of 8q are compared with the present one.     

Complex chromosomal rearrangement involving chromosomes 1, 4 and 22 in an infertile male: case report and literature review

Here we describe a rare case of anapparently balanced karyotypeof 46, XY, t(1;22;4)(p22.3;q11.1;q31.1) in a infertile male with oligoastenoteratozoospermia (OAT). He was the second patient with complex chromosomal rearrangement (CCR) referred to our center because of infertility. Wealso review reportson 24 males carrying CCRs with spermatogenesis failure or a malformed child, to provide information on the reproductive outcome of male CCR carriers.     

A, 1;6, translocation associated with congenital glaucoma and cleft lip and palate.

A translocation of a part of the long arm of a chromosome No. 1 onto the long arm of a chromosome No. 6 was observed in a 2 1/2-year-old boy with mental retardation, harelip, cleft palate and congenital glaucoma. Different banding methods revealed that the translocation t(1;6)(q23;q27) apparently was balanced. The conncection between the patients' symptoms and the chromosomal rearrangement might be fortuitous or produced by the chromosome aberration.     

Karyotype evolution in the bone marrow of a patient with Fanconi anemia: breakpoints in clonal anomalies of this disease

A 21-year-old Fanconi anemia patient developed refractory anemia. Laboratory studies revealed a transitory increased platelet count and a typical del(5q). Bone marrow karyotyping showed a -6, +der(6)t(1;6)(q12;p25) rearrangement and, two years later, a mosaic -6, +der(6),t(1:6)(q12;p25)/-2, +der 2), t(1;2)(q12;q37) constitution. The chromosome mechanism operating in this patient is discussed.     

Molecular involvement of the pvt-1 locus in a gamma/delta T-cell leukemia bearing a variant t(8;14)(q24;q11) translocation.

A highly malignant human T-cell receptor (TCR) gamma/delta+ T-cell leukemia was shown to have a productive rearrangement of the TCR delta locus on one chromosome 14 and a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on the other chromosome 14. Chromosome walking coupled with pulsed-field gel electrophoretic (PFGE) analysis determined that the TCR J delta 1 gene fragment of the involved chromosome was relocated approximately 280 kb downstream of the c-myc proto-oncogene locus found on chromosome band 8q24. This rearrangement was reminiscent of the Burkitt's lymphoma variants that translocate to a region identified as the pvt-1 locus. Sequence comparison of the breakpoint junctions of interchromosomal rearrangements in T-cell leukemias involving the TCR delta-chain locus revealed novel signal-like sequence motifs, GCAGA(A/T)C and CCCA(C/G)GAC. These sequences were found on chromosome 8 at the 5' flanking site of the breakpoint junction of chromosome 8 in the TCR gamma/delta leukemic cells reported here and also on chromosome 1 in T-cell acute lymphocytic leukemia patients carrying the t(1;14)(p32;q11) rearrangement. These results suggest that (i) during early stages of gamma delta T-cell ontogeny, the region 280 kb 3' of the c-myc proto-oncogene on chromosome 8 is fragile and accessible to the lymphoid recombination machinery and (ii) rearrangements to both 8q24 and 1p32 may be governed by novel sequence motifs and be subject to common enzymatic mechanisms.     

Non-random distribution of Alu-family repeats in human chromosomes

A phage lambda recombinant clone containing at least 8 Alu-family repeats (AFRs) has been isolated from a human genomic library, and DNA from the phage was used as a probe for in situ hybridization on G-banded human metaphase chromosomes of healthy donors and leukemic patients. Some chromosome bands show prominent clusters of silver grains in all individuals examined: 1p34, 1q23, 2q21–22, 10p14, 11p14, 10q21 and 11q14. The data suggest non-random distribution of AFRs in the human genome.     

NOTCH2 Is Neither Rearranged nor Mutated in t(1;19) Positive Oligodendrogliomas

The combined deletion of 1p and 19q chromosomal arms is frequent in oligodendrogliomas (OD) and has recently been shown to be mediated by an unbalanced t(1;19) translocation. Recent studies of 1p/19q co-deleted OD suggest that the NOTCH2 gene is implicated in oligodendrocyte differentiation and may be involved in this rearrangement. The objective of the present study was to analyze the NOTCH2 locus either as a chromosomal translocation locus that may be altered by the 1p/19q recurrent rearrangement or as a gene that may be inactivated by a two hit process. We performed an array-CGH analysis of 15 ODs presenting 1p/19q co-deletion using a high-density oligonucleotide microarray spanning 1p and 19q pericentromeric regions with 377 bp average probe spacing. We showed that the 1p deletion extends to the centromere of chromosome 1 and includes the entire NOTCH2 gene. No internal rearrangement of this gene was observed. This strongly suggests that the t(1;19) translocation does not lead to an abnormal NOTCH2 structure. The analysis of the entire NOTCH2 coding sequence was performed in four cases and did not reveal any mutation therefore indicating that NOTCH2 does not harbor genetic characteristics of a tumor suppressor gene. Finally, the detailed analysis of chromosome 19 pericentromeric region led to the identification of two breakpoint clusters at 19p12 and 19q11–12. Interestingly, these two regions share a large stretch of homology. Together with previous observations of similarities between chromosome 1 and 19 alphoid sequences, this suggests that the t(1;19) translocation arises from complex intra and interchromosomal rearrangements.This is the first comprehensive deletion mapping by high density oligo-array of the 1p/19q co-deletion in oligodendroglioma tumors using a methodological approach superior to others previously applied. As such this paper provides clear evidence that the NOTCH2 gene is not physically rearranged by t(1;19) translocation of oligodendroglioma tumors.     

Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.