Multicolor Banding Research Articles

Evaluation of SureFISHtm DNA-FISH Probes for Validation of Array-CGH Aberrations

Evaluation of SureFISHtm DNA-FISH Probes for Validation of Array-CGH Aberrations

Inv dup del(10q): Identification by fluorescence in situ hybridization and array comparative genomic hybridization in a fetus with two concurrent chromosomal rearrangements

ObjectiveTo present molecular cytogenetic characterization of an inverted duplication with terminal deletion of 10q, or inv dup del(10q) in a fetus with two concurrent chromosomal rearrangements. Materials, Methods and ResultsA 39-year-old woman underwent amniocentesis at 20 weeks of gestation because of advanced maternal age. Amniocentesis revealed a der(10) with additional material at the end of the long arm of chromosome 10, a der(9) and a der(22). Parental karyotypes were normal. A de novo unbalanced complex chromosomal rearrangement (CCR) was diagnosed by conventional cytogenetics, but the breakpoints could not be defined. The pregnancy was subsequently terminated, and a malformed fetus was delivered with facial dysmorphism. Postnatal analysis of fetal tissues using spectral karyotyping, fluorescence in situ hybridization, multicolor banding, and array-comparative genomic hybridization identified an inv dup del(10q) with an inverted duplication of 10q25.1→q26.2 and a terminal deletion of 10q26.2→qter, and a balanced reciprocal translocation between chromosomes 9 and 22. Microsatellite analysis determined a paternal origin of the inv dup del(10q). The karyotype of the fetus was 46,XX,t(9;22)(p23;q13),der(10)del(10)(q26.2) dup(10)(q26.2q25.1)dn. ConclusionA de novo inv dup del(10q) can be associated with a concurrent de novo balanced reciprocal translocation and should be differentiated from an unbalanced CCR by molecular cytogenetic techniques.

A unique cytogenetic abnormality, t(2;7)(p13.1;p21.3), in a Philadelphia-positive chronic myeloid leukemia

The Philadelphia (Ph) chromosome is present in more than 90% of patients suffering from chronic myeloid leukemia (CML). It is the product of a reciprocal translocation between the long arms of chromosomes 9 and 22, resulting in the transfer of the 3' portion of the proto-oncogene ABL from 9q34 to the 5' portion of the breakpoint cluster region (BCR) on 22q11. Currently, most CML cases are treated with Imatinib and variant rearrangements are thought to have no specific prognostic significance, although the events of therapy resistance have not yet been studied. In this study we report a novel case of CML exhibiting an uncommon t(2;7)(p13.1;p21.3) besides t(9;22)(q34;q11). This unusual translocation has been characterized by fluorescence in situ hybridization (FISH) and array-proven multicolor banding (aMCB), the latter being extremely significant in characterizing breakpoint regions in detail. The underlying mechanisms and prognostic implications of this cytogenetic abnormality are discussed in this study.

A chronic myeloid leukemia case with a unique variant Philadelphia translocation: t(9;22;21)(q34;q11;p12).

The so-called Philadelphia (Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) patients. Approximately, 5-10% of these patients show complex translocations involving a third chromosome in addition to chromosomes 9 and 22. Since at present the majority of CML cases are treated with imatinib, variant rearrangements do not exhibit specific prognostic significance. However, events of therapy resistance remain to be studied. In this study, we report a unique case of CML exhibiting an uncommon t(21;22)(p12;q11). This translocation has been characterized by fluorescence in situ hybridization (FISH) and array-proven multicolor banding (aMCB). Using specific probes for the BCR and ABL genes, results of FISH showed a three-way variant Philadelphia translocation (9;22;21)(q34;q11;p12) with a BCR/ABL fusion residing on the der(22) and the 3'BCR region translocated on the short arm of the derivative chromosome 21. In addition, the aMCB technique is significant in the detection of the breakpoints of genetic changes. The underlying mechanisms and prognostic significance of these changes are discussed.

Genotype–phenotype correlation of a 5q22.3 deletion associated with craniofacial and limb defects

We describe here a newborn with a de novo 22.6Mb interstitial deletion of chromosome 5q22.3. The clinical findings included brachycephaly, a high forehead, hypertelorism with prominent eyes, low-set ears, clenched hands, club feet, a prominent coccyx with hair, ambiguous genitalia, inguinal hernia, heart defect and severe failure to thrive. This case had a more severe phenotype, compared with the previous reports of interstitial 5q syndrome. High resolution multicolor banding and array comparative genomic hybridization (array CGH) analysis delineated the breakpoints at 5q22.3 and 5q31.2. There were no obvious candidate genes for the specific correlation with the phenotypes except a PITX1 gene associated with the phenotype of club feet. Further cumulative data based on the molecular approach are needed to establish the genotype–phenotype correlation and to understand the role and influence of the genes in the interstitial 5q syndrome.

Intrachromosomal Changes and Genomic Instability in Site-Specific Microbeam-Irradiated and Bystander Human-Hamster Hybrid Cells

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.

264 Genomic imbalances in 73 patients with MDS and complex karyotypes

We analyzed complex chromosomal aberrations in 37 adult patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) using classical cytogenetic method, FISH with locus-specific probes, multicolor FISH (mFISH) and multicolor banding (mBAND). Unbalanced structural aberrations, leading to a gain or loss of chromosomal material, were frequently observed in bone marrow cells. In 30 patients (81.1%) loss or rearrangement of chromosome 5, 7 and/or 11 was found. The most frequent numerical change was trisomy 8 as expected (detected in six patients—16.2%) and the most frequent breakpoints 5q13, 5q33, 7q31, 10p12, 11q23, 12p13, 17p11 and 21q22 were determined.

Association of Inter- and Intrachromosomal Exchanges with the Distribution of Low- and High-LET Radiation-Induced Breaks in Chromosomes

To study the effects of low- and high-linear energy transfer (LET) radiation on break locations within a chromosome, we exposed human epithelial cells in vitro to (137)Cs γ rays at both low and high dose rates, secondary neutrons at a low dose rate, and 600 MeV/u iron ions at a high dose rate. Breakpoints were identified using multicolor banding in situ hybridization (mBAND), which paints chromosome 3 in 23 different colored bands. For all four radiation scenarios, breakpoint distributions were found to be different from the predicted distribution based on band width. Detailed analysis of chromosome fragment ends involved in inter- or intrachromosomal exchanges revealed that the distributions of fragment ends participating in interchromosomal exchanges were similar between the two low-LET radiation dose rates and between the two high-LET radiation types, but the distributions were less similar between low- and high-LET radiations. For fragment ends participating in intrachromosomal exchanges, the distributions for all four radiation scenarios were similar, with clusters of breaks found in three regions. Analysis of the locations of the two fragment ends in chromosome 3 that joined to form an intrachromosomal exchange demonstrated that two breaks with a greater genomic separation can be more likely to rejoin than two closer breaks, indicating that chromatin folding can play an important role in the rejoining of chromosome breaks. Comparison of the breakpoint distributions to the distributions of genes indicated that the gene-rich regions do not necessarily contain more breaks. In general, breakpoint distributions depend on whether a chromosome fragment joins with another fragment in the same chromosome or with a fragment from a different chromosome.

Integrated Cytogenetic and High-Resolution Array CGH Analysis of Genomic Alterations Associated with MYCN Amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.

Characterization of sSMC by FISH and molecular techniques

Small supernumerary marker chromosome (sSMC) is a structurally altered additional chromosome that may not be explicitly clarified by conventional karyotyping alone. About one third of sSMC carriers have abnormal phenotypes and its clinical correlation is difficult, especially in prenatal studies. The present study was aimed at characterizing 19 sSMC identified in 15 patients with dysmorphic features with or without multiple congenital anomalies, conspicuous family history, short stature and/or ambiguous genitalia. All the sSMC were primarily identified by routine cytogenetics studies (performed with banding techniques) from peripheral blood except in one patient, where amniotic fluid was used. All sSMCs were further characterized by array-CGH (using 44 K oligonucleotide probe) and/or fluorescence in situ hybridization (FISH) using multicolor banding (MCB), centromere specific multicolor FISH (cenM-FISH), subcentromere-specific multicolor FISH (subcenM-FISH), micro-dissection and/or reverse FISH. This report demonstrates the worth of advanced molecular (cyto)genetic techniques in characterizing sSMC, their utility in genotype–phenotype correlation and risk of clinical presentation.

Molecular cytogenetic analysis and clinical manifestations of a case with de novo mosaic ring chromosome 7

AimClinical and molecular cytogenetic investigations of a newborn girl exhibiting facial dysmorphism with developmental delay.MethodsPhenotypic evaluation was first applied to examine the proband's developmental status. Computed tomography and colour transcranial Doppler were used then to investigate her brain structure and function. Subsequently, chromosomal abnormalities were examined by karyotyping and fluorescent in situ hybridization was performed to investigate size of fragments lost at the two distal ends of the ring chromosome 7. In addition, multicolour banding was applied to rule out structural rearrangement occurs in between the ring chromosome 7.ResultsThe proband was born with mosaic supernumerary ring chromosome 7, without a normal karyotype detected in the peripheral blood lymphocytes. The distal arm of chromosome 7p (at least 255 kb from the telomere) was part of an extra ring chromosome 7. In addition, the distal arm of 7q, at least 8 kb from the telomere, was missing. There was no other chromosomal rearrangement detected by multicolour banding.InterpretationThis is the 19th reported case of complete ring chromosome 7 mosaicism and the first survived case with mosaic supernumerary ring 7 without a normal karyotype detected in the peripheral lymphocytes.

Complex translocation involving four chromosomes in a novel Philadelphia-positive chronic myeloid leukemia case

The so-called Philadelphia (Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. Approximately 5-10% of these patients show complex translocations involving a third chromosome in addition to chromosomes 9 and 22. Since the majority of CML cases are currently treated with imatinib, variant rearrangements in general have no specific prognostic significance, although the mechanisms involved in resistance to therapy have yet to be investigated. This study evalutated a CML case with complex chromosomal aberrations not previously observed. A four chromosome translocation involving chromosomal regions such as 12q24.2-24.31 and 16p11.2 besides 9q34 and 22q11 were characterized in detail by array-proven multicolor banding (aMCB). A beneficial response to imatinib was noted in the patient.

MBAND analysis for high- and low-LET radiation-induced chromosome aberrations: A review

During long-term space travel or cancer therapy, humans are exposed to high linear energy transfer (LET) energetic heavy ions. High-LET radiation is much more effective than low-LET radiation in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, and cytogenetic damage can be utilized as a biomarker for radiation insults. Epidemiological data, mainly from survivors of the atomic bomb detonations in Japan, have enabled risk estimation from low-LET radiation exposures. The identification of a cytogenetic signature that distinguishes high- from low-LET exposure remains a long-term goal in radiobiology. Recently developed fluorescence in situ hybridization (FISH)-painting methodologies have revealed unique endpoints related to radiation quality. Heavy-ions induce a high fraction of complex-type exchanges, and possibly unique chromosome rearrangements. This review will concentrate on recent data obtained with multicolor banding in situ hybridization (mBAND) methods in mammalian cells exposed to low- and high-LET radiations. Chromosome analysis with mBAND technique allows detection of both inter- and intrachromosomal exchanges, and also distribution of the breakpoints of aberrations.

Secondary abnormalities involving 1q or 13q and poor outcome in high stage Burkitt leukemia/lymphoma cases with 8q24 rearrangement at diagnosis

Classical Burkitt lymphoma/leukemia (BL/L) presenting L3 morphology is found in 1% of childhood ALL. Recently, it has been described that secondary abnormalities could influence the prognosis of these patients. However, little information is available on these cytogenetic abnormalities and their prognostic importance in BL/L. Here, we report four new childhood BL/L cases associated with duplication within 1q or 13q, which exhibited a very unfavorable therapeutic response. We performed both classical and molecular cytogenetic analysis by multicolor chromosome banding of the secondary abnormalities involving the long arms of chromosome 1 or 13. These patients were previously treated with BFM-90 protocol. All of them died during or after the initial treatment. Here, for the first time, the exact breakpoints of the derivative chromosomes involved were determined at the cytogenetic level as 1q21 and 13q33 each.

Evidence for Correlation of Fragile Sites and Chromosomal Breakpoints in Carriers of Constitutional Balanced Chromosomal Rearrangements

A molecular cytogenetic study of 251 cases with balanced chromosomal rearrangements detected due to infertility of unclear origin or in prenatal diagnostics with a later normal outcome was done. Balanced translocations (127 cases), inversions (105 cases), insertions (three cases), balanced complex rearrangements (four cases), or derivative chromosomes leading to no imbalance (12 cases), were studied by multicolor banding (MCB) and/or subcentromeric multicolor fluorescence in situ hybridization (subcenM-FISH). Five-hundred and twenty-nine break-events were characterized by molecular cytogenetics. Only 150 of these were unique breakpoints, the remainder were observed between two and 10 times. According to the results obtained, there was cytogenetic co-localization of fragile site (FS) in ~71% of the studied 529 break-events. Nine selected cases with evidence for breakpoints within FS were further analyzed by FS-specific bacterial artificial chromosome (BAC) probes; only one did not show a co-localization. Further detailed molecular analysis will be necessary to characterize the mechanisms and genetic basis for this phenomenon.

B lymphoblastic leukemia with ETV6 amplification

We presente a case of acute lymphoblastic leukemia caused by ETV6 amplification. Although the cytogenetic result revealed complex karyotype, multicolor fluorescence in situ hybridization and high-resolution multicolor banding supported amplification of a gene on 12p13. Fluorescence in situ hybridization with ETV6 probe confirmed the amplification. ETV6 generally plays as tumor-suppressor gene in leukemia. Their expression is decreased or missed by deletion or mutation. Otherwise, ETV6 protein overexpression was verified in this case by immunohistochemistry. Any translocation or mutation involving ETV6 was not detected. This experience strongly supports the hypothesis that the amplification of ETV6 is a possible mechanism of leukeogenesis as oncogene.

Methods of molecular cytogenetics for studying interphase chromosomes in human brain cells

One of the main genetic factors determining the functional activity of the genome in somatic cells, including brain nerve cells, is the spatial organization of chromosomes in the interphase nucleus. For a long time, no studies of human brain cells were carried out until high-resolution methods of molecular cytogenetics were developed to analyze interphase chromosomes in nondividing somatic cells. The purpose of the present work was to assess the potential of high-resolution methods of interphase molecular cytogenetics for studying chromosomes and the nuclear organization in postmitotic brain cells. A high efficiency was shown by such methods as multiprobe and quantitative fluorescence in situ hybridization (Multiprobe FISH and QFISH), ImmunoMFISH (analysis of the chromosome organization in different types of brain cells), and interphase chromosome-specific multicolor banding (ICS-MCB). These approaches allowed studying the nuclear organization depending on the gene composition and types of repetitive DNA of specific chromosome regions in certain types of brain cells (in neurons and glial cells, in particular). The present work demonstrates a high potential of interphase molecular cytogenetics for studying the structural and functional organizations of the cell nucleus in highly differentiated nerve cells. Analysis of interphase chromosomes of brain cells in the normal and pathological states can be considered as a promising line of research in modern molecular cytogenetics and cell neurobiology, i. e., molecular neurocytogenetics.

A rare chronic myeloid leukemia case with Philadelphia chromosome, BCR-ABL e13a3 transcript and complex translocation involving four different chromosomes

The so-called Philadelphia (Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) patients. Around 5-10% of these patients show complex translocations involving other chromosomes in addition to and/or besides chromosomes 9 and 22. CML cases with fusion transcripts, such as e13a3, in which ABL exon 3 rather than exon 2 has fused to BCR, are extremely rare. Such reported cases with the e13a3 transcript showed the Ph chromosome on karyotype analysis. This study reported a rare Ph chromosome-positive CML case with new complex chromosomal aberrations and an e13a3 BCR-ABL transcript that has yet to be established. A four-chromosome translocation involving chromosomal regions 12p11.2, 19q13.3, 9q34.1 and 22q11.2, besides a trisomy 8 and a derivative chromosome 12, were identified using high resolution multicolor banding. Reverse transcription polymerase chain reaction products showed the presence of BCR-ABL fusion transcript e13a3, and this signifies the major BCR breakpoint. The significance of the observed rearrangements and their possible role in the progression of CML was investigated.

Complex rearrangements between chromosomes 6, 10, and 11 with multiple deletions at breakpoints

Here we report on a girl with minor facial anomalies, cleft palate, seizures, microcephaly, psychomotor retardation, and a congenital heart defect. Complex of cytogenetic methods [GTG-banding, spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), multicolor banding (mBAND), and comparative genomic hybridization (array CGH)] showed complex chromosomal rearrangements (CCRs) involving chromosomes 6, 10, and 11 and 4 deletions at the breakpoints. Her father had an unrelated translocation between chromosomes 3 and 16, suggesting the possibility of an autosomal dominant trait that predisposes to complex synapses and recombination between multiple chromosomes during meiosis. This study demonstrates the power of combining available chromosome analysis technologies in resolving CCR.

MBAND analysis of chromosome aberrations in human epithelial cells induced by γ-rays and secondary neutrons of low dose rate

Human risks from chronic exposures to both low- and high-LET radiation are of intensive research interest in recent years. In the present study, human epithelial cells were exposed in vitro to γ-rays at a dose rate of 17 mGy/h or secondary neutrons of 25 mGy/h. The secondary neutrons have a broad energy spectrum that simulates the Earth's atmosphere at high altitude, as well as the environment inside spacecrafts like the Russian MIR station and the International Space Station (ISS). Chromosome aberrations in the exposed cells were analyzed using the multicolor banding in situ hybridization (mBAND) technique with chromosome 3 painted in 23 colored bands that allows identification of both inter- and intrachromosome exchanges including inversions. Comparison of present dose responses between γ-rays and neutron irradiations for the fraction of cells with damaged chromosome 3 yielded a relative biological effectiveness (RBE) value of 26 ± 4 for the secondary neutrons. Our results also revealed that secondary neutrons of low dose rate induced a higher fraction of intrachromosome exchanges than γ-rays, but the fractions of inversions observed between these two radiation types were indistinguishable. Similar to the previous findings after acute radiation exposures, most of the inversions observed in the present study were accompanied by other aberrations. The fractions of complex type aberrations and of unrejoined chromosomal breakages were also found to be higher in the neutron-exposed cells than after γ-rays. We further analyzed the location of the breaks involved in chromosome aberrations along chromosome 3, and observed hot spots after γ-ray, but not neutron, exposures.