Cryptic terminal chromosome rearrangements in colorectal carcinoma cell lines detected by subtelomeric FISH analysis.

Epithelial tumour karyotypes are often difficult to study by standard cytogenetic methods because of poor chromosome preparation quality and the high complexity of their genomic rearrangements. Subtelomeric fluorescence in situ hybridisation (FISH) has proved to be a useful method for detecting cryptic constitutional chromosomal rearrangements but little is known about its usefulness for tumour cytogenetic analysis. Using a combination of chromosome banding, multicolour karyotyping and subtelomeric FISH, five colorectal cancer cell lines were characterised. The resulting data were compared to results from previous studies by comparative genomic hybridisation and spectral karyotyping or multicolour FISH. Subtelomeric FISH made it possible to resolve several highly complex chromosome rearrangements, many of which had not been detected or were incompletely characterised by the other methods. In particular, previously undetected terminal imbalances were found in the two cell lines not showing microsatellite instability.

Chromosomal translocations fusing the BCL6 gene to different partner loci are recurrent in primary central nervous system lymphoma and may be associated with aberrant somatic hypermutation or defective class switch recombination.

Primary central nervous system lymphomas (PCNSLs) are diffuse large B cell lymphomas confined to the brain. Only minimal data exist on chromosomal aberrations underlying PCNSLs. We studied 41 PCNSLs by fluorescence in situ hybridization for breakpoints affecting the BCL6 locus in chromosomal band 3q27. Of 37 cases evaluable, 14 (38%) carried a breakpoint in the BCL6 locus. Two of these showed juxtaposition of BCL6 to the IGH locus. In 4 cases, the BCL6 breakpoints were cloned using long-distance inverse polymerase chain reaction. All breakpoints were located within the BCL6 major translocation cluster. The translocation partners were the IGH gene in 14q32.33, the IGL gene in 22q11.22, and the histone 1 H4I gene in 6p22.1. In the fourth case, a deletion in 3q leads to loss of an 837-kb fragment extending from the first intron of BCL6 to the third intron of the lipoma-preferred partner (LPP) gene. This deletion may bring the BCL6 gene under the control of regulatory elements of the LPP gene or the miRNA-28 gene located in intron 4 of LPP. DNA sequence analysis of the junctional sequences provided evidence that aberrant class switch recombination or somatic hypermutation may be involved in the generation of BCL6 translocations.

Molecular analysis of an idic(Y)(qter -->p11.32::p11.32-->qter) chromosome from a female patient with a complex karyotype.

A female patient with a structurally abnormal idic(Y) (p11.32) chromosome was studied using fluorescence in situ hybridization and PCR to define the precise position of the breakpoint. The patient had a complex mosaic karyotype with eight cell lines and at least two morphologically distinct derivatives from the Y chromosome. The rearrangement was a result of a meiosis I exchange between sister chromatids at the pseudoautosomal region, followed by centromere misdivision at meiosis II. Due to instability of the dicentric Y chromosome, new cell lines later arose because of mitotic errors occurring during embryonic development. Physical examination revealed a normal female phenotype without genital ambiguity, a normal uterus and rudimentary gonads which were surgically removed.

FISH mapping of i(7q) in acute leukemias and myxoid liposarcoma reveals clustered breakpoints in 7p11.2: implications for formation and pathogenetic outcome of the idic(7)(p11.2).

Isochromosome 7q - i(7q) - is seen in a wide variety of hematologic malignancies and solid tumors, often as a secondary change to a characteristic primary translocation. Despite its high frequency, nothing is known about the formation and the pathogenetic outcome of this abnormality. To address these issues, we performed a detailed fluorescence in situ hybridization (FISH) investigation of four acute lymphoblastic leukemias, one acute myeloid leukemia, and two myxoid liposarcomas with i(7q). Using FISH with bacterial artificial chromosomes (BACs) mapping between 7p12.2 and 7q11.2, the breakpoints (BPs) in all seven cases were shown to cluster to an approximately 340 kb segment at 7p11.2, covered by the overlapping BAC probes RP11-760D2 and RP11-10F11. Thus, the i(7q) should formally be designated idic(7) (p11.2). In one of the cases, FISH with fosmids could narrow down the BP further to an 80-kb sequence delineated by G248P81983A10 and G248P8793H7. No known genes are located in the 340-kb BP cluster region, indicating that the idic(7)(p11.2) does not result in a fusion or deregulation of genes in this segment. The pathogenetically important outcome is thus likely to be an altered gene expression because of copy number changes. The clustering of breakpoints might be due to frequent intrachromosomal duplicons in the BP region.

The pattern of gene amplification is determined by the chromosomal location of hairpin-capped breaks.

DNA palindromes often colocalize in cancer cells with chromosomal regions that are predisposed to gene amplification. The molecular mechanisms by which palindromes can cause gene amplification are largely unknown. Using yeast as a model system, we found that hairpin-capped double-strand breaks (DSBs) occurring at the location of human Alu-quasipalindromes lead to the formation of intrachromosomal amplicons with large inverted repeats (equivalent to homogeneously staining regions in mammalian chromosomes) or extrachromosomal palindromic molecules (equivalent to double minutes [DM] in mammalian cells). We demonstrate that the specific outcomes of gene amplification depend on the applied selection, the nature of the break, and the chromosomal location of the amplified gene relative to the site of the hairpin-capped DSB. The rules for the palindrome-dependent pathway of gene amplification defined in yeast may operate during the formation of amplicons in human tumors.

ATM stabilizes DNA double-strand-break complexes during V(D)J recombination.

The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.

Saccharomyces cerevisiae Mer2, Mei4 and Rec114 form a complex required for meiotic double-strand break formation.

In budding yeast, at least 10 proteins are required for formation of the double-strand breaks (DSBs) that initiate meiotic recombination. Spo11 is the enzyme responsible for cleaving DNA and is found in a complex that also contains Ski8, Rec102, and Rec104. The Mre11/Rad50/Xrs2 complex is required for both DSB formation and DSB processing. In this article we investigate the functions of the remaining three proteins--Mer2, Mei4, and Rec114--with particular emphasis on Mer2. The Mer2 protein is present in vegetative cells, but it increases in abundance and becomes phosphorylated specifically during meiotic prophase. Mer2 localizes to distinct foci on meiotic chromosomes, with foci maximally abundant prior to the formation of synaptonemal complex. If DSB formation is blocked (e.g., by a spo11 mutation), dephosphorylation of Mer2 and its dissociation from chromosomes are delayed. We have also found that the Mei4 and Rec114 proteins localize to foci on chromosomes and these foci partially colocalize with each other and with Mer2. Furthermore, the three proteins co-immunoprecipitate. Mer2 does not show significant colocalization with Mre11 or Rec102 and Mer2 does not co-immunoprecipitate with Rec102. We propose that Mer2, Mei4, and Rec114 form a distinct complex required for DSB formation.

Breakpoint locations within chromosomes 1, 2, and 4 of patients with increased radiosensitivity.

The exposure to low LET-radiation leads to a relative homogeneous distribution of initial damage at the DNA. Subsequent repair and post-repair mechanisms might lead to a selection of specific breakpoint locations along chromosomes. Cells from patients with increased radiosensitivity may have more specific breakpoints due to impaired repair mechanisms. We tested whether cells from patients with increased radiosensitivity had an increase in specific breakpoint clusters. Structural chromosomal aberrations of in vitro irradiated lymphocytes from 11 healthy individuals and another 3 patients with increased radiosensitivity were examined. The chromosome pairs 1, 2, and 4 were treated using the three-color FISH technique. The breakpoints were analyzed by means of computerized imaging software. In total, 1752 chromosomal breakpoints had been considered, 498 from healthy individuals, and 1254 from patients with increased radiosensitivity. For both groups there was a non-homogeneous breakpoint distribution along the chromosomes and a trend towards increased breaks in the telomere-proximal region. Also, both groups had distinct locations with increased breaks. No evidence for significant breakpoint patterns across all patients with increased radiosensitivity was found.

Complex chromosomal rearrangements in patients with chronic myeloid leukemia.

During progression of chronic myeloid leukemia (CML) from the chronic to the accelerated phase and/or blast crisis, clonal evolution with nonrandom secondary aberrations such as +8, +Ph, i(17q), +19, -Y, +21, +17, and -7 is frequently observed. Complex chromosomal rearrangements (CCR) are rather rare, and the significance and frequency of different anomalies are poorly understood. The aim of this study was to determine the chromosomes and chromosomal regions which are involved in CCR during progression of the disease and the frequency of nonrandom changes. Conventional cytogenetics, FISH, and multicolor FISH (mFISH) were used to study karyotypes of 18 CML patients with CCR ascertained by G-banding. Most often involved in CCR were chromosomes 2 (x6); 3, 7, and 17 (x5); 1 and 4 (x4); and 5, 6, 11, and 12 (x3); regions 1q, 2q, 5q, 7p, and 17p; and breakpoints 17p11.2 (x3) and 7p15 (x2). There were no recurrent complex translocations. The present findings demonstrate the very high instability of the genome of malignant cells at the chromosomal level. Precise determination of breakpoints involved in CCR can give new dimension to the understanding of genetic mechanisms which play role in progression of malignant disease.

IGH switch breakpoints in Burkitt lymphoma: exclusive involvement of noncanonical class switch recombination.

Most chromosomal t(8;14) translocations in sporadic Burkitt lymphomas (BL) are mediated by immunoglobulin class switch recombination (CSR), yet all tumors express IgM, suggesting an incomplete or exclusively monoallelic CSR event. We studied the exact configuration of both the nontranslocated IGH allele and the MYC/IGH breakpoint by applying a combination of low- and high-resolution methods (interphase FISH, DNA fiber FISH, long-distance PCR, and Southern blotting) on 16 BL. IGH class switch events involving the nontranslocated IGH allele were not observed. Thirteen cases had MYC/IGH breakpoints in or nearby IGH switch (S) sites, including five at Smu, three at Sgamma and five at Salpha. All eight translocations with a breakpoint at Sgamma or Salpha were perfectly reciprocal, without deletion of Cmu-Cdelta or other CH elements. Internal Smu deletions claimed to be a marker for CSR activity and implicated in stabilization of IgM expression were found in BL but did not correlate with downstream translocation events. This study shows that switch breakpoints in sporadic BL are exclusively resolved by a noncanonical recombination mechanism involving only one switch region.

Targeted disruption of FANCC and FANCG in human cancer provides a preclinical model for specific therapeutic options.

BACKGROUND & AIMS: How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. METHODS: We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. RESULTS: FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. CONCLUSIONS: Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.

Array-based comparative genomic hybridization facilitates identification of breakpoints of a novel der(1)t(1;18)(p36.3;q23)dn in a child presenting with mental retardation.

Monosomy of distal 1p36 represents the most common terminal deletion in humans and results in one of the most frequently diagnosed mental retardation syndromes. This deletion is considered a contiguous gene deletion syndrome, and has been shown to vary in deletion sizes that contribute to the spectrum of phenotypic anomalies seen in patients with monosomy 1p36. We report on an 8-year-old female with characteristics of the monosomy 1p36 syndrome who demonstrated a novel der(1)t(1;18)(p36.3;q23). Initial G-banded karyotype analysis revealed a deleted chromosome 1, with a breakpoint within 1p36.3. Subsequent FISH and array-based comparative genomic hybridization not only confirmed and partially characterized the deletion of chromosome 1p36.3, but also uncovered distal trisomy for 18q23. In this patient, the duplicated 18q23 is translocated onto the deleted 1p36.3 region, suggesting telomere capture. Molecular characterization of this novel der(1)t(1;18)(p36.3;q23), guided by our clinical array-comparative genomic hybridization, demonstrated a 3.2 Mb terminal deletion of chromosome 1p36.3 and a 200 kb duplication of 18q23 onto the deleted 1p36.3, presumably stabilizing the deleted chromosome 1. DNA sequence analysis around the breakpoints demonstrated no homology, and therefore this telomere capture of distal 18q is apparently the result of a non-homologous recombination. Partial trisomy for 18q23 has not been previously reported. The importance of mapping the breakpoints of all balanced and unbalanced translocations found in the clinical laboratory, when phenotypic abnormalities are found, is discussed.

The CT-element of the c-myc gene does not predispose to chromosomal breakpoints in Burkitt's lymphoma.

BACKGROUND: Chromosomal translocations are causally related to the development of many tumors. In Burkitt's lymphoma, abnormalities involving the c-myc gene are essential. The CT-element of the c-myc promoter adopts non-B-conformation in vivo and in vitro, and therefore provides a potential fragile site. METHODS: We have developed a LM-PCR-based approach to test if chromosomal breakpoints indeed cluster in this region. RESULTS: Amplifying both, wild-type as well as the translocated c-myc gene by LM-PCR, it was shown that chromosomal breakpoints did not cluster within the CT-element. CONCLUSIONS: Therefore, the CT-element is not especially susceptible to the formation of breakpoints leading to chromosomal translocations in Burkitt's lymphoma.

Structural damage to meiotic chromosomes impairs DNA recombination and checkpoint control in mammalian oocytes.

Meiosis in human oocytes is a highly error-prone process with profound effects on germ cell and embryo development. The synaptonemal complex protein 3 (SYCP3) transiently supports the structural organization of the meiotic chromosome axis. Offspring derived from murine Sycp3(-)(/)(-) females die in utero as a result of aneuploidy. We studied the nature of the proximal chromosomal defects that give rise to aneuploidy in Sycp3(-)(/)(-) oocytes and how these errors evade meiotic quality control mechanisms. We show that DNA double-stranded breaks are inefficiently repaired in Sycp3(-)(/)(-) oocytes, thereby generating a temporal spectrum of recombination errors. This is indicated by a strong residual gammaH2AX labeling retained at late meiotic stages in mutant oocytes and an increased persistence of recombination-related proteins associated with meiotic chromosomes. Although a majority of the mutant oocytes are rapidly eliminated at early postnatal development, a subset with a small number of unfinished crossovers evades the DNA damage checkpoint, resulting in the formation of aneuploid gametes.

Increase of homologous recombination frequency in vascular tissue of Arabidopsis plants exposed to salt stress.

Here we analyzed the influence of salt stress on plant genome stability. Homologous recombination events were detected in transgenic Arabidopsis plants that carried in their genome a beta-glucuronidase recombination marker. Recombination events were scored as blue sectors using a stereo microscope. Exposure to 50 mM salt resulted in a 3.0-fold increase in recombination frequency. To analyze the organ and tissue specificity of recombination events, we examined cross-sections of leaves, stems and roots. We found that nearly 30% of recombination events in plants grown under normal conditions and nearly 50% of events in plants grown on salt were undetected by the conventional method. Most of the recombination events represented a cluster/group of cells (12 on average), although events with single cells were also detected. Recombination events were very frequent in leaf mesophyll cells. On average, individual recombination events located on leaves contained more cells than events located on roots or stems. Analysis of recombination events in cross-sectioned tissue of salt-treated plants revealed a shift in the distribution of recombination events towards the vascular tissue. We discuss the significance of the finding for plant stress physiology.

Multicolor banding detects a complex three chromosome, seven breakpoint unbalanced rearrangement in an ICSI-derived fetus with multiple abnormalities.

We describe a fetus from an intracytoplasmic sperm injection (ICSI) pregnancy with severe facial clefts, receding jaw, preauricular skin tags, postaxial hexadactyly, bi-lobed right lung, supernumerary cranial bone, and dilated lateral ventricles of the brain. Using a combination of G-banding, fluorescence in situ hybridization (FISH), whole chromosome paints (WCPs), subtelomere probes, and multicolor banding (MCB), the karyotype was found to include a de novo unbalanced highly complex chromosome rearrangement (hCCR) involving chromosomes 3, 12, and 15 with seven breakpoints, and including monosomy for two separate regions of chromosome 12.

The DNA sequence, annotation and analysis of human chromosome 3.

After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

DNA damage repair: anytime, anywhere?

Regulation of the DNA damage response is tightly connected to transcription and replication. These DNA transacting processes share common factors and use similar strategies to exert their function. However, unlike replication and transcription, DNA repair systems may be required anywhere, and at any time, whenever DNA damage occurs in the cell nucleus. This raises questions concerning the spatiotemporal organization of genome caretaking. Currently, quantitative live cell imaging techniques combined with methods to induce local DNA damage in a small region of the nucleus are contributing substantially to unravelling the molecular mechanisms underlying the cellular response to DNA damage.

[Increased risk of malignancies in heterozygotes in families of patients with Nijmegen breakage syndrome]. / Zvýsené riziko malignit u heterozygotu v rodinách pacientu s Nijmegen breakage syndromem.

BACKGROUND: The autosomal recessive chromosomal instability and hyperradiosensitivity Nijmegen breakage syndrome (NBS) in consequence of a mutation in the NBSI gene at 8q21 is associated with high occurrence of lymphoreticular malignancies due to deficient DNA reparation (double strand breaks). In the Slavic population the majority of patients are homozygotes of the so-called "Slavic mutation" 657de15 in exon 6. Increased occurrence of malignant solid tumors (1) in families of NBS patients has been described already prior to the identification of the responsible gene, and the increased risk of malignancies in heterozygotes was thus hypothetical. METHODS AND RESULTS: The possibility of discerning mutation carriers in families from normal homozygotes enables verification of that hypothesis. Through molecular genetics investigations of grandparents and immediate relatives, we have been successful in determining the genotype in 79 of 112 grandparents in 28 families of our 39 patients and 54 their parents and siblings. A single family had affected children in consequence of compound heterozygosity of the 657de15 and R215W mutations in the same exon of the NBSI gene. The proband's families were investigated genealogically and data on relatives were obtained over four generations. Obtained data were repeatedly supplemented and objectively verified in church books and in healthcare documentation. Seven families have been followed up for 20-30 years, six families for 10-20 years, and 15 families for 1-10 years. Out of 28 families we were successful in examining the genotype of both grandparents in 18 families, there having been revealed one non-paternity; in five families only one of the grandparents has been examined; in five families we were not successful in examining any grandparent. Among 40 grandparents - normal homozygotes, there has appeared a malignancy in three (7.4 %), while among 39 heterozygotes of mutation 657de15 in the NBSI gene malignancies were documented in 15 (38,2 %). Mean age of NBS heterozygotes at manifestation of malignancy was 59.3 year (range 47-72 years), in the group of homozygotes it was 52.6 years (range 44-62 years). Nine grandparents died of malignancy prior to the discovery of the NBSI gene and their genotype has been deduced genealogically in seven on the basis of the genotype in the sponse and children, in two from preserved DNA. Out of that number, from three grandparents that had died of malignancies we were successful in obtaining neoplastic tissue for molecular genetics investigation, aimed at LOH or amplification of the NBS1 gene. In another seven grandparents - heterozygotes, malignancies were manifested after determination of their genotype by DNA analysis, and consequently also from tumor tissue that has been obtained from three of them for molecular genetic investigation. CONCLUSIONS: The age distribution and socio-economic status of both groups of grandparents did not differ, the sex ratio was slightly shifted towards females in the group of homozygotic grandparents (22 females and 18 males), and in the group of heterozygotes it was towards males (21 males and 18 females). The sex ratio between heterozygotic grandparents with malignancies was likewise shifted towards the male gender (11 males and 4 females), in the group of homozygotic grandparents malignancy affected one male and two females. As verified in healthcare and church books documentation, the occurrence of malignancies was significantly more frequent among grandparents heterozygotic for NBS1 mutation than in healthy homozygotes. Among sibs of grandparents and great-grandparents was found significant difference in frequency of malignancies in heterozygotes (5/18 = 27,7 %) and healthy homozygotes (2/36 = 5,5 %), too.

Combined spectral karyotyping, comparative genomic hybridization, and in vitro apoptyping of a panel of Burkitt's lymphoma-derived B cell lines reveals an unexpected complexity of chromosomal aberrations and a recurrence of specific abnormalities in chemoresistant cell lines.

The comprehensive cytogenetic profiles of a panel of 10 Burkitt's lymphoma (BL)-derived B cell lines, designated Akata, BL-28, BL-41, Daudi, DG-75, Mutu I, Mutu III, Namalwa, Rael, and Ramos, respectively, are reported herein. The unique origin of each cell line was established using multiplex quantitative fluorescence polymerase chain reaction (QF-PCR). Spectral karyotyping (SKY) revealed a large number of structural and numerical chromosomal aberrations, many of which had not been previously identified or resolved by conventional G-banding techniques. Notably, whereas all 10 cell lines harbored the hallmark translocation t(8;14)(q24;q32), no other common structural aberrations were identified, although translocations involving chromosomes 3, 13, and 17 were frequently seen. Moreover, analysis of chromosomal breakpoints by comparative genomic hybridization (CGH) revealed a number of recurring aberrations, such as gain of chromosomes 7 and 20, gains of regions at 2p, 3q, 13q and 16q, and losses at 3p, 4q and 17p. In addition, apoptyping (i.e. determination of in vitro responses to apoptosis stimulation) of the cell lines suggested specific association patterns between karyotypic changes (e.g. translocations involving 17p, and gains of portions of chromosomes 7 and 20) and resistance to the chemotherapeutic agent, etoposide. The current molecular cytogenetic characterization of 10 BL cell lines has thus identified several novel sites of rearrangements; moreover, the combined karyotyping and functional assessment (apoptyping) of these cell lines serves to enhance their utility in future studies aimed at gene discovery and gene function.