Multicolor Banding Research Articles

High definition cytogenetics and oligonucleotide aCGH analyses of cisplatin‐resistant ovarian cancer cells

Array comparative genomic hybridization (aCGH) is a key platform to assess cancer genomic profiles. Many structural genomic aberrations cannot be detected by aCGH alone. We have applied molecular cytogenetic analyses including spectral karyotyping, multicolor banding, and fluorescence in situ hybridization with aCGH to comprehensively investigate the genomic aberrations associated with cisplatin resistance in A2780 ovarian cancer cells. A2780 is a well-established model of chemotherapeutic resistance with distinct karyotypic abnormalities in the parental and cisplatin-resistant cells. Cytogenetic analysis revealed that two unbalanced translocations, der(8)t(1;8) and der(X)t(X;1), and loss of chromosome 13 were present only in the resistant line. Our aCGH analyses detected imbalances affecting an additional 10.59% of the genome in the cisplatin-resistant cells compared with the parental. DNA copy number changes included deletions at 1p10-p22.1, 8p23.3, and Xq13.1-pter, and a duplication of 8q11.22-q23. Cryptic genomic aberrations associated with concurrent localized changes of specific gene expression included a homozygous deletion of 0.38 Mb at 1p21.3 adjacent to SNX7, and an insertional transposition of 0.85 Mb from 13q12.12 into chromosome 22. This latter rearrangement led to an overexpression of four contiguous genes that flanked one of the breakpoint regions in chromosome 13. Furthermore, 17 genes showed differential expression correlating with genomic gain or loss between the resistant and parent lines, validated by a second expression array platform. These results highlight the integration of comprehensive profiling to determine relationships of genomic aberrations and genes associated with an in vitro drug resistance model in ovarian cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Further delineation of complex chromosomal rearrangements in fertile male using multicolor banding

BackgroundComplex chromosomal rearrangements (CCRs) are defined as structural chromosomal rearrangements with at least three breakpoints and exchange of genetic material between two or more chromosomes. Complex chromosomal translocations are rarely seen in the general population but the frequency of occurrence is anticipated to be much higher due balanced states with no phenotypic presentation. Here, we report a severely mentally retarded fertile male patient in whom further delineation of CCR involving chromosomes 1, 4 and 2 was carried out by using high resolution multicolor banding (MCB) technique. As a FISH based novel chromosome banding approach, high resolution MCB allows for the differentiation of chromosome region specific areas at band and subband levels.ResultsCytogenetic studies using high resolution banding of the proband necessitated further delineation of the breakpoints because of their uncertainty: 46,XY,t(1;4;2)(p21~31;q31.3;q31). After using high resolution MCB based on microdissection derived region-specific libraries, the exact nature of chromosomal rearrangements for chromosomes 1, 2 and 4 were revealed and these breakpoints were located on 1p31.1, 1q24.3 and 4q31.3 giving rise to a balanced situation.ConclusionFurther delineations are certainly required to provide detailed information about the relationship between balanced CCRs and their phenotypes in order to offer proper counseling to the families concerned. Carriers must be investigated with high resolution banding and molecular cytogenetic techniques to determine the exact locations of the breakpoints. High resolution MCB is an alternative and an efficient method to other FISH based chromosome banding techniques and can serve in clarifying the nature of CCR.

Chromosome distribution in human sperm – a 3D multicolor banding-study

BackgroundNuclear architecture studies in human sperm are sparse. By now performed ones were practically all done on flattened nuclei. Thus, studies close at the in vivo state of sperm, i.e. on three-dimensionally conserved interphase cells, are lacking by now. Only the position of 14 chromosomes in human sperm was studied.ResultsHere for the first time a combination of multicolor banding (MCB) and three-dimensional analysis of interphase cells was used to characterize the position and orientation of all human chromosomes in sperm cells of a healthy donor. The interphase nuclei of human sperm are organized in a non-random way, driven by the gene density and chromosome size.ConclusionHere we present the first comprehensive results on the nuclear architecture of normal human sperm. Future studies in this tissue type, e.g. also in male patients with unexplained fertility problems, may characterize yet unknown mechanisms of infertility.

Three Unusual but Cytogenetically Similar Cases With up to Five Different Cell Lines Involving Structural and Numerical Abnormalities of Chromosome 18

We report two prenatal and two postnatal diagnosed cases (the latter monozygotic twins) with ring chromosomes after GTG banding. All four, de novo r(18), cases turned out to be more complex after application of high-resolution molecular cytogenetics techniques such as use of fluorescence in situ hybridization, centromeric probes, multicolor banding, and locus-specific probes for chromosome 18. All four cases are mosaics involving chromosome 18 in up to five different cell lines, including 46,r(18); 46,dr(18); 47,r(18)x2; 46,mar(18); and 45,-18. Mosaicism sharing both numerical and structural anomalies is rare, but rings often appear as mosaics due to their mitotic instability. Overall, patients with ring chromosome 18 usually share clinical features of 18q- syndrome and, less frequently, those of 18p- syndrome. High-resolution molecular cytogenetics techniques were useful in the characterization of cases with dynamic mosaicism and in establishing the relationship between loss or gain of chromosomal material and the phenotype.

The schizophrenia brain exhibits low-level aneuploidy involving chromosome 1

Objective Genetic instability manifested as loss or gain of whole chromosomes (aneuploidy) is a newly described feature of the human brain. Aneuploidy in the brain was hypothesized to be involved in schizophrenia pathogenesis. To gain further insights into the relationship between aneuploidy in the brain and schizophrenia pathogenesis, a molecular-cytogenetic study of chromosome 1 aneuploidy was performed. Methods Interphase multiprobe fluorescence in situ hybridization (FISH) with quantitative FISH (QFISH) and interphase chromosome-specific multicolor banding (ICS-MCB) were used to define aneuploidy rate in 12 unaffected and 12 schizophrenia brains. Results In the unaffected brain ( n = 12; 22,794 cells analyzed), average frequencies of stochastic chromosome 1 loss and gain were 0.3% (95%CI 0.2–0.4%) and 0.3% (95%CI 0.2–0.4%), respectively. The threshold level for stochastic chromosome gain and loss (the mean + 3SD) in the normal brain was 0.7%. Average rate of aneuploidy in the schizophrenia brain ( n = 12; 28,482 cells analyzed) was 0.9% (95%CI 0.3–1.5%) for chromosome 1 loss and 0.9% (95%CI 0.2–1.7%) for chromosome 1 gain. Significantly increased level of mosaic aneuploidy involving chromosome 1 was revealed in two schizophrenia brains (3.6% and 4.7% of cells with chromosome 1 loss and gain, respectively). Stochastic aneuploidy rate for chromosome 1 in the schizophrenia brain without two outliers ( n = 10) reached 0.6% (95%CI 0.3–0.9%) for loss and 0.5% (0.2–0.9%) for gain and was higher than in controls ( P = 0.005 and P = 0.001, respectively). Conclusions Our findings support the hypothesis suggesting that subtle genomic imbalances manifesting as low-level mosaic aneuploidy may contribute to schizophrenia pathogenesis.

Distal partial trisomy 1q: report of two cases and a review of the literature

We report on two cases with partial trisomy 1q syndrome. One case was a mid-trimester fetus with multiple malformations that was prenatally diagnosed with a de novo distal partial trisomy 1q. Prenatal ultrasound at 24th gestational week demonstrated the presence of cleft lip and palate, increased biparietal diameter and decreased abdominal circumference. Cytogenetic analysis (GTG banding) and subsequent fluorescence in situ hybridization (FISH) using whole chromosome paint 1 and multicolor banding (MCB) demonstrated an aberrant karyotype 46,XY,dup(1)(q31q43 approximately 44). The second case was a newborn male infant with multiple congenital malformations. He had a derivative chromosome 18 as a result of a maternal insertion involving chromosomes 1 and 18. Further analyses including MCB showed his karyotype as 46,XY,ins(18;1)(q22;q23q31.1 approximately 32). The present cases and a review of the literature suggest that partial trisomy of the long arm of chromosome 1 is a distinct clinical entity.

MBAND Analysis of Chromosomal Aberrations in Human Epithelial Cells Exposed to Low- and High-LET Radiation

Energetic heavy ions pose a potential health risk to astronauts who have participated in extended space missions. High-LET radiation is much more effective than low-LET radiation in the induction of biological effects, including cell inactivation, genetic mutations, cataracts and cancer. Most of these biological end points are closely correlated with chromosomal damage, which can be used as a biomarker for radiation damage. Multicolor banding in situ hybridization (mBAND) has proven to be highly useful for the study of intrachromosomal aberrations, which have been suggested as a biomarker of exposure to high-LET radiation. To investigate biological signatures of radiation quality and the complexity of intrachromosomal aberrations, we exposed human epithelial cells in vitro to (137)Cs gamma rays or iron ions (600 MeV/nucleon) and collected chromosomes using a premature chromosome condensation technique. Aberrations in chromosome 3 were analyzed using mBAND probes. The results of our study confirmed the observation of a higher incidence of inversions for high-LET radiation. However, detailed analysis of the inversion type revealed that both iron ions and gamma rays induced a low incidence of simple inversions. Half of the inversions observed in the low-LET-irradiated samples were accompanied by other types of intrachromosome aberrations, but few inversions were accompanied by interchromosome aberrations. In contrast, iron ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges.

T(11;19)(q21;p12∼p13.11) and MECT1- MAML2 fusion transcript expression as a prognostic marker in infantile lung mucoepidermoid carcinoma

Background Primary pulmonary mucoepidermoid carcinomas (MECs) are the third most frequent pulmonary malignant neoplasm in children, and new molecular diagnostics may prove useful in determining the biologic course of such tumors. Methods We analyzed the presence of a balanced t(11;19)(q21; p12~p13.11) and the MECT1- MAML2 fusion transcript in a 9-year-old girl with mucoepidermoid lung carcinoma using conventional cytogenetics, fluorescence in-situ hybridization, spectral karyotyping, high-resolution multicolor banding, and reverse transcriptase–polymerase chain reaction. Results We confirmed the t(11;19)(q21; p12∼p13.11) in the tumor. Molecular analysis of the translocation breakpoint confirmed the presence of the MECT1-MAML2 fusion transcript postulated to lead to an altered cyclic adenosine monophosphate signaling in MEC. Conclusions Our data concur with previously reported cases, in which t(11;19) appears to be the primary chromosomal aberration for pulmonary MEC in children, and that the MECT1- MAML2 fusion transcript is associated with a better prognosis in MEC tumors.

Molecular cytogenetic analysis of complex karyotypes with derivative chromosome der(1)t(1;5) found in two patients with myeloid leukemia

Two patients with myeloid hematological malignancies were examined with conventional cytogenetic analysis, and complex chromosomal aberrations were found. Multicolor FISH (mFISH) was used to clarify these rearrangements. A similar translocation between chromosomes 1 and 5 was revealed in both patients. To specify breakpoints, high-resolution multicolor banding (mBAND) for chromosome 1 and chromosome 5 was performed. The breakpoints were resolved as der(1)t(1;5)(p21;p12) in patient 1 and as der(1)t(1;5)(p21;p11) in patient 2. The breakpoint on chromosome 1 was the same in both patients, in the 1p21 region. This region contains the colony stimulating factor 1 gene ( CSF1), which may play a role in tumorigenesis of myeloid tissue.

Maternally inherited duplication of chromosome 7, dup(7)(p11.2p12), associated with mild cognitive deficit without features of Silver–Russell syndrome

We report on a familial duplication in the short arm of chromosome 7, dup(7)(p11.2p12), present in three generations. The duplication was identified by GTG-banding and fluorescence in situ hybridization (FISH) with a whole chromosome 7 DNA painting probe that verified that the duplicated material originated from chromosome 7. The multicolor banding (mBAND) was used to refine the breakpoint assignment. The duplication identified in the proband was also present in her son and mother. All three carriers have mild cognitive deficiencies. Interstitial duplications of the short arm of chromosome 7, although relatively uncommon, have been described in association with a variety of clinical features, including mental retardation of varying severity. Duplication of the p11.2p13 region on chromosome 7 was reported in association with Silver-Russell syndrome (SRS), and an overlapping dup(7)(p11.2p14.1)dn was described in an individual with autistic disorder. Furthermore, a potentially overlapping maternally transmitted inverted duplication, dup(7)(p13p12.2), was reported in patients with cognitive delay. These observations and the phenotype of our duplication carriers suggest that partial trisomy of the proximal 7p region causes cognitive deficiency. The maternal origin of the duplication is of special interest in light of genomic imprinting and implication of the 7p11-p13 region in the SRS etiology. Locus-specific FISH targeting a growth factor receptor binding protein 10 (GRB10), the strong candidate for SRS residing at 7p12.2, showed that it is not duplicated in our patients. Our study helps refine the SRS critical region on 7p and extends our understanding of the clinical manifestations associated with 7p duplications.

Detailed characterization of 7q deletions by multicolor banding (mBAND) in marginal zone cell lymphoma

High-resolution multicolor banding (mBAND) analysis was applied to precisely fine-map the genomic extent of 7q deletions in a series of 26 marginal zone lymphoma patients displaying the abnormality on conventional karyotypes. Using this approach, the breakpoints and the extent of deletions revealed by conventional banding techniques had to be re-defined in 70% of cases. Although no common minimal region of deletion was delineated, mBAND demonstrated the involvement of the 7q32 region in more than 90% of cases. In addition, unsuspected translocations and intrachromosomal changes could be identified in four cases. Taken together, these data demonstrate that mBAND represents an alternative cytogenetic tool in the comprehensive analysis of chromosome aberrations in hematologic malignancies, allowing rapid screening and precise delineation of structural rearrangements of a defined chromosome. This also confirms the localization in the vicinity of band 7q32 of putative candidate gene(s) involved in the pathogenic development of the disease.

Molecular cytogenetic characterization of eight small supernumerary marker chromosomes originating from chromosomes 2, 4, 8,18, and 21 in three patients

Small supernumerary marker chromosomes (sSMCs) are a morphologically heterogeneous group of additional structurally abnormal chromosomes that cannot be identified unambiguously by conventional banding techniques alone. Molecular cytogenetic methods enable detailed characterization of sSMCs; however, in many cases interpretation of their clinical significance is problematic. The aim of our study was to characterize precisely sSMCs identified in three patients with dysmorphic features, psychomotor retardation and multiple congenital anomalies. We also attempted to correlate the patients' genotypes with phenotypes by inclusion of data from the literature. The sSMCs were initially detected by G-banding analysis in peripheral blood lymphocytes in these patients and were subsequently characterized using multicolor fluorescence in situ hybridization (M-FISH), (sub)centromere-specific multicolor FISH (cenM-FISH, subcenM-FISH), and multicolor banding (MCB) techniques. Additionally, the sSMCs in two patients were also studied by hybridization to whole-genome bacterial artificial chromosome (BAC) arrays (array-CGH) to map the breakpoints on a single BAC clone level. In all three patients, the chromosome origin, structure, and euchromatin content of the sSMCs were determined. In patient RS, only a neocentric r(2)(q35q36) was identified. It is a second neocentric sSMC(2) in the literature and the first marker chromosome derived from the terminal part of 2q. In the other two patients, two sSMCs were found, as M-FISH detected additional sSMCs that could not be characterized in G-banding analysis. In patient MK, each of four cell lines contained der(4)(:p11.1-->q12:) accompanied by a sSMC(18): r(18)(:p11.2-->q11.1::p11.2-->q11.1:), inv dup(18)(:p11.1-->q11.1::q11.1-->p11.1:), or der(18) (:p11.2-->q11.1::q11.1-->p11.1:). In patient NP, with clinical features of trisomy 8p, three sSMCs were characterized: r(8)(:p12-->q11.1::q11.1-->p21:) der(8) (:p11.22-->q11.1::q11.1-->p21::p21-->p11.22:) and der(21)(:p11.1-->q21.3:). The BAC array results confirmed the molecular cytogenetic results and refined the breakpoints to the single BAC clone resolution. However, the complex mosaic structure of the marker chromosomes derived from chromosomes 8 and 18 could only be identified by molecular cytogenetic methods. This study confirms the usefulness of multicolor FISH combined with whole-genome arrays for comprehensive analyses of marker chromosomes.

Interphase chromosome-specific multicolor banding (ICS-MCB): A new tool for analysis of interphase chromosomes in their integrity

Biomedical research of interphase chromosomes in their integrity is hindered by technical limitations. We introduce a technology using microdissection-based engineering of DNA probes and fluorescence multicolor chromosome banding that allows studying interaphase chromosome organization, numbers and rearrangements in somatic cells.

Bilateral periventricular nodular heterotopia, severe learning disability, and epilepsy in a male patient with 46,XY,der(19)t(X;19) (q11.1–11.2;p13.3)

Periventricular nodular heterotopia (PNH) is a rare neuronal migration disorder in which immature neurons fail to undergo a directed migration from the ventricular and subventricular zones to the cerebral cortex. Classic PNH occurs predominantly in females and is associated with periods of epilepsy and near-normal intelligence. One gene associated with PNH was mapped to chromosome Xq28. PNH with learning disability is reported in 15 male patients with several syndromes and various congenital abnormalities such as craniosynostosis, frontonasal malformation, and agenesis of the corpus callosum. We present a 26-year-old male patient who was followed up with the diagnosis of epilepsy from the age of 1 year. Additionally the patient had severe learning disability, obesity, and hypogonadism. Imaging of his brain demonstrated PNH. Klinefelter syndrome was clinically suspected, and analysis of his chromosomes revealed a karyotype 46,XY,der(19)t(X;19) (q11.1-11.2;p13.3). Molecular techniques, such as subtelomere-specific fluorescent in-situ hybridization and multicolour banding, were also used. The same translocation was demonstrated in his mother and his maternal grandmother. This family might help to explain the gene localization of X-linked recessive PNH. In our patient, PNH is associated with familial (X;19) translocation. To our knowledge, this unique combination has not been reported in the medical literature.

Structural aberrations of chromosome 7 revealed by a combination of molecular cytogenetic techniques in myeloid malignancies

In bone marrow cells of 33 patients with myelodysplastic syndrome and acute myeloid leukemia, structural rearrangements of chromosome 7 were found with conventional G-banding: 8 with deletions 7q and 25 with translocations. In 29 of the patients, complex karyotypes were confirmed using multicolor fluorescence in situ hybridization (mFISH). Commercial probes (Abbot Molecular) were used for 7q22, 7q31, and 7q35, the regions most frequently deleted in myeloid malignancies. In three cases without deletions, high-resolution multicolor banding (mBAND) for chromosome 7 revealed other aberrations. Five groups of chromosomal rearrangements were established: (a) deletion 7q as a sole aberration (2 cases), (b) deletion 7q and complex karyotypes (6 cases), (c) combined translocations and deletions of 7q (17 cases), (d) combined translocation and deletion 7p (5 cases), and (e) translocation of chromosomes 7 without deletion 7p or 7q (3 cases). Deletions of all three FISH-screened regions were the most frequent, with heterogeneous breakpoints. The region 7p13.2∼p15.2 was most commonly deleted. Most of the deletions were cryptic, not detectable with conventional cytogenetics. Aberrations of chromosome 7 are associated with a very poor outcome; survival time in our cohort was short (median 7 months).

Chromosome aberrations induced by dual exposure of protons and iron ions

During space travel, astronauts will be exposed to protons and heavy charged particles. Since the proton flux is high compared to HZE particles, on average, it is assumed that a cell will be hit by a proton before it is hit by an HZE ion. Although the effects of individual ion species on human cells have been investigated extensively, little is known about the effects of exposure to mixed beam irradiation. To address this, we exposed human epithelial cells to protons followed by HZE particles and analyzed chromosomal damage using the multicolor banding in situ hybridization (mBAND) procedure. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of intra-chromosomal aberrations (inversions and deletions within a single painted chromosome) as well as inter-chromosomal aberrations (translocation to unpainted chromosomes). Our results indicated that chromosome aberration frequencies from exposures to protons followed by Fe ions did not simply decrease as the interval between the two exposures increased, but peak when the interval was 30 min.

Correlating breakage-fusion-bridge events with the overall chromosomal instability and in vitro karyotype evolution in prostate cancer

Chromosomal instability (CIN) is thought to underlie the generation of chromosomal changes and genomic heterogeneity during prostatic tumorigenesis. The breakage-fusion-bridge (BFB) cycle is one of the CIN mechanisms responsible for characteristic mitotic abnormalities and the occurrence of specific classes of genomic rearrangements. However, there is little detailed information concerning the role of BFB and CIN in generating genomic diversity in prostate cancer. In this study we have used molecular cytogenetic methods and array comparative genomic hybridization analysis (aCGH) of DU145, PC3, LNCaP, 1532T and 1542T to investigate the in vitro role of BFB as a CIN mechanism in karyotype evolution. Analysis of mitotic structures in all five prostate cancer cell lines showed increased frequency of anaphase bridges and nuclear strings. Structurally rearranged dicentric chromosomes were observed in all of the investigated cell lines, and Spectral Karyotyping (SKY) analysis was used to identify the participating rearranged chromosomes. Multicolor banding (mBAND) and aCGH analysis of some of the more complex chromosomal rearrangements and associated amplicons identified inverted duplications, most frequently involving chromosome 8. Chromosomal breakpoint analysis showed there was a higher frequency of rearrangement at centromeric and pericentromeric genomic regions. The distribution of inverted duplications and ladder-like amplifications was mapped by mBAND and by aCGH. Adjacent spacing of focal amplifications and microdeletions were observed, and focal amplification of centromeric and end sequences was present, particularly in the most unstable line DU145. SKY analysis of this line identified chromosome segments fusing with multiple recipient chromosomes (jumping translocations) identifying potential dicentric sources. Telomere free end analysis indicated loss of DNA sequence. Moreover, the cell lines with the shortest telomeres had the most complex karyotypes, suggesting that despite the expression of telomerase, the reduced telomere length could be driving the observed BFB events and elevated levels of CIN in these lines.

A report of pure 7p duplication syndrome and review of the literature

We report on a case of a 9-month-old female infant with a direct duplication of the 7p13-p22.1 chromosome region diagnosed by combining conventional cytogenetic, FISH, and multicolor banding (MCB) studies. Traditional G-banding detected a partial 7p duplication, which was further demonstrated to be entirely of chromosome 7 origin by using a whole chromosome paint for chromosome 7, and derived from 7p13-p22.1 by MCB. The infant presented with characteristic dysmorphic features, psychomotor retardation, and generalized hypotonia. The phenotypic manifestations of partial 7p trisomy with or without other chromosome involvement are briefly reviewed. Our observations in combination with other cases confirm that 7p trisomy due to dir dup(7p) can be regarded as a defined chromosome syndrome.

An exceptional complex chromosomal rearrangement (CCR) with eight breakpoints involving four chromosomes (1;3;9;14) in an azoospermic male with normal phenotype

A 27-year-old man was referred for chromosome analysis due to infertility caused by azoospermia. Chromosome analysis by conventional karyotyping, multicolour FISH (M-FISH) and multicolour banding (MCB) analysis revealed an apparently balanced translocation between chromosomes 1, 3, 9 and 14 as well as an additional inverted insertion of 3q material with a total of eight breakpoints. Due to the diversity of theoretically unbalanced products of meiotic recombination in this exceptional complex chromosomal rearrangement a successful result of assisted reproduction seems unlikely.

Application of Multicolor Banding for Identification of Complex Chromosome 18 Rearrangements

Multicolor chromosome banding (mBAND) is a recently developed technique that allows the delineation of chromosomal regions with a resolution of a few megabase pairs. The resolution of mBAND is slightly below that of conventional chromosome banding; however, the color bands have a great value in identifying chromosomal abnormalities, particularly complex chromosome rearrangements, and intrachromosome exchanges (ie, inversions, deletions, duplications, and insertions). These abnormalities cannot be defined easily by conventional cytogenetic analysis or chromosome paint. In this report, we present the application of the mBAND analysis for identification of complex intrachromosome rearrangements of chromosome 18 in a child with dysmorphic features.