Multicolor Fluorescence In Situ Hybridization Research Articles

A case report of Pallister-Killian syndrome with an unusual mosaic supernumerary marker chromosome 12 with interstitial 12p13.1-p12.1 duplication.

Pallister-Killian syndrome (PKS) is a rare inherited disease with multiple congenital anomalies, profound intellectual disability, and the presence in the karyotype of sSMC - i(12)(p10). The frequency of PKS may be underestimated due to problems with cytogenetic diagnosis caused by tissue-specific mosaicism and usually a low percentage of peripheral blood cells containing sSMC. Such tissue-specific mosaicism also complicates a detailed analysis of the sSMC, which, along with the assessment of mosaicism in different tissues, is an important part of cytogenetic diagnosis in PKS. Unfortunately, a full-fledged diagnosis in PKS is either practically impossible or complicated. On the one hand, this is due to problems with the biopsy of various tissues (skin biopsy with fibroblast culture is most often used in practice); on the other - a low percentage of dividing peripheral blood cells containing sSMC, which often significantly complicates the analysis of its composition and organization. In the present study, a detailed analysis of sSMC was carried out in a patient with a characteristic clinical picture of PKS. A relatively high percentage of peripheral blood cells with sSMC (50%) made it possible to perform a detailed molecular cytogenetic analysis of de novo sSMC using chromosomal in situ suppression hybridization (CISS-hybridization), multicolor FISH (mFISH), multicolor chromosome banding (MCB), array CGH (aCGH), and quantitative real-time PCR (qPCR), and short tandem repeat (STR) - analysis. As a result, it was found that the sSMC is not a typical PKS derivative of chromosome 12. In contrast to the classical i(12)(p10) for PKS, the patient's cells contained an acrocentric chromosome consisting of 12p material. Clusters of telomeric repeats were found at the both ends of the sSMC. Furthemore, the results of aCGH and qPCR indicate the presence of interstitial 8.9Mb duplication at 12p13.1-p12.1 within the sSMC, which leads to different representations of DNA from different segments of 12p within cells containing sSMC. The obtained data raise the question of the instability of the sSMC and, as a consequence, the possible presence of additional rearrangements, which, in traditional cytogenetic analysis of patients with PKS, are usually described as i(12)(p10).

Characterizing Chemotherapy/Radiotherapy-Induced Genome Chaos in Hodgkin's Lymphoma Patients Using M-FISH.

Hodgkin lymphoma (HL) is one of the most common lymphomas, with an incidence of 3 per 100,000 persons. Current treatment uses a cocktail of genotoxic agents, including adriamycin, bleomycin, vinblastine, and dacarbazine (ABVD), along with or without radiotherapy. This treatment regimen has proved to be efficient in killing cancer cells, resulting in HL patients having a survival rate of >90% cancer-free survival at five years. However, this therapy does not have a specific cell target, and it can induce damage in the genome of non-cancerous cells. Previous studies have shown that HL survivors often exhibit karyotypes characterized by complex chromosomal abnormalities that are difficult to analyze by conventional banding. Multicolor fluorescence in situ hybridization (M-FISH) is a powerful tool to analyze complex karyotypes; we used M-FISH to investigate the presence of chromosomal damage in peripheral blood lymphocytes from five healthy individuals and five HL patients before, during, and one year after anti-cancer treatment. Our results show that this anti-cancer treatment-induced genomic chaos that persists in the hematopoietic stem cells from HL patients one year after finishing therapy. This chromosomal instability may play a role in the occurrence of second primary cancers that are observed in 10% of HL survivors. This chapter will describe a protocol for utilizing M-FISH to study treatment-induced genome chaos in Hodgkin's lymphoma (HL) patients, following a brief discussion.

A reliable technique for karyotyping mouse oocytes prepared by a gradual fixation/air-drying method followed by multicolour FISH.

Chromosome segregation errors during oocyte meiosis increase with age and lead to aneuploidy; hence, the mechanism has been studied extensively. The mouse is the most widely used experimental animal for this purpose. However, the lack of a reliable and efficient technique for karyotyping mouse oocytes has limited comprehensive studies of chromosome-specific segregation errors in this animal model. Here, we developed a novel karyotyping technique for mouse oocytes by applying multicolour fluorescence in situ hybridisation (FISH) to chromosome slides prepared by a gradual fixation/air-drying method, which is best suited to avoid rupture of oocyte membrane and artificial loss of chromosomes. The success rate of karyotyping meiosis I and II oocytes was about 30%, which improved to over 90% when the oocytes were 'flattened' during fixation and the chromosome specimens were denatured at 4°C. When this technique was applied to the karyotyping of meiosis II oocytes from aged female mice and from young female mice injected with colchicine, more than 80% of the oocytes were successfully karyotyped and the number of chromosomes was identified on all aberrant chromosomes. In conclusion, our technique allows for the efficient and reliable karyotyping of mouse oocytes.

Enhancer-driven 3D chromatin domain folding modulates transcription in human mammary tumor cells.

The genome is organized in functional compartments and structural domains at the sub-megabase scale. How within these domains interactions between numerous cis-acting enhancers and promoters regulate transcription remains an open question. Here, we determined chromatin folding and composition over several hundred kb around estrogen-responsive genes in human breast cancer cell lines after hormone stimulation. Modeling of 5C data at 1.8 kb resolution was combined with quantitative 3D analysis of multicolor FISH measurements at 100 nm resolution and integrated with ChIP-seq data on transcription factor binding and histone modifications. We found that rapid estradiol induction of the progesterone gene expression occurs in the context of preexisting, cell type-specific chromosomal architectures encompassing the 90 kb progesterone gene coding region and an enhancer-spiked 5' 300 kb upstream genomic region. In response to estradiol, interactions between estrogen receptor α (ERα) bound regulatory elements are reinforced. Whereas initial enhancer-gene contacts coincide with RNA Pol 2 binding and transcription initiation, sustained hormone stimulation promotes ERα accumulation creating a regulatory hub stimulating transcript synthesis. In addition to implications for estrogen receptor signaling, we uncover that preestablished chromatin architectures efficiently regulate gene expression upon stimulation without the need for de novo extensive rewiring of long-range chromatin interactions.

FISH painting for chromosome identification of aneuploid cauliflower (Brassica oleracea L. var. botrytis)

A common problem in the cultivation and breeding of cauliflower (Brassica oleracea L. var. botrytis) is the occurrence of aneuploids in offspring families. To reveal the chromosomal cause of such numerical variants, it was necessary to develop karyotype tools with which chromosomes can be easily identified. Since mitotic chromosomes in this crop are morphologically similar and lack differentiating banding patterns, we tested two Fluorescent in situ Hybridization (FISH) procedures for chromosome identification: (1) FISH painting with diagnostic repetitive DNA patterns and (2) cross-species chromosome painting. The first method consists of a five-colour FISH with 5s rDNA, 45S rDNA, and two Brassica rapa centromere-specific repeats, and a B. rapa BAC (KBrH092N02) containing a dispersed repeat of an unknown class. The second method is an advanced FISH technology based on hybridising DNA probes of a related species under adapted stringency conditions to identify their homoeologous loci. To this end, we applied four pools of BACs from Arabidopsis thaliana in a multicolour FISH for a banding pattern on the chromosomes of cauliflower (Brassica oleracea L. var. botrytis). Due to the genome triplication and various chromosome rearrangements of Brassica oleracea compared to Arabidopsis, we used MUMmer whole-genome alignment plot information to select Arabidopsis BAC pools with which all cauliflower chromosomes could be identified. In a sample of 21 plants with aberrant phenotypes, we demonstrated primary trisomy for chromosomes 1–6 and 8, and telo-trisomy for chromosomes 7 and 9. Finally, we discuss the advantages and drawbacks of the two painting methods and eventual alternatives for demonstrating numerical aberrations in the cauliflower populations.Graphical

Abstract 1214: Establishment and characterization of reversibly immortalized endometrial and ovarian epithelial cell lines using Sendai virus

Abstract Objective: We aimed to establish reversibly immortalized cell lines from human uterine and ovary cells using the Sendai virus (SeV) vector. The immortalized cells derive from normal and benign ovarian epithelial cells and endometrial epithelial cells. Furthermore, we sought to elucidate the mechanisms of carcinogenesis using the immortalized cell lines. Methods: Cells were collected at the time of surgery after obtaining patient consent. The cells used in this study were as follows: ovarian epithelial cells (normal epithelium, Ov n; normal epithelium with germline BRCA1 or BRCA2 mutation, Ov BRCA1 2; ovarian endometrioma, Ov endo; mucinous cystadenoma; Ov m), normal fallopian tube (FT) cells, and endometrial epithelium (normal epithelium, Em n). These cells were infected with temperature-sensitive SeV vectors carrying three immortalization genes, Bmi-1, hTERT, and SV40T.The presence of infection was confirmed through Green Fluorescent Protein (GFP) and Orange Fluorescent Protein (OFP). Immunoreactivity to the anti-human EpCAM antibody (a marker derived from epithelial carcinoma) in each SeV-infected cell was confirmed through flow cytometry. QH and multicolor FISH staining were performed for karyotyping of metaphase chromosomes in each cell line to determine chromosome number and structural abnormalities. Human transcriptome sequencing analysis was performed with NovaSeq 6000 (Illumina) using total RNA from each cell line. Some genes that showed significant expression in each cell line were subjected to real-time PCR (RT-PCR). Results: We established the immortalized cell lines from human uterine and ovarian tissues. SeV-infected cells exhibited GFP and OFP fluorescence, while non-infected cells did not. SeV infection allowed all primary cell lines to grow for 25 or more passages, while non-infected SeV cells lacked the proliferative capacity and showed senescence-like morphology. SeV-infected cells senesced in a temperature-dependent manner. Ov n SeV- infected cells ion causedshowed a small increase in chromosome structural abnormalities. But, Ov BRCA1 and 2 SeV-infected cells showed larger than thatand . Llong-term passaged cells did not show immune response to anti-human EpCAM antibodies in normal cells. Eleven Three genes were predominantly expressed in Ov BRCA1 and Ov BRCA2 cells and were not expressed in Ov n cells. SomeTwo out of ththe three genesem were found to be predominantly expressed in Ov endo cells compared to the expression in the Ovn cell line. Furthermore, RT-PCR results also indicated substantially higher expression of two genes in Ov BRCA1/2 cells and in Ov endo cells compared to the expression in Ov n cells. Conclusion: We succeeded in the reversible immortalization of endometrial and ovarian epithelial cells by using SeV infection. We identified several candidate genes that may be involved in the oncogenic mechanism of ovarian cancer associated with endometriosis or germline BRCA1 and BRCA2. Citation Format: Masayo Okawa, Hiroaki Komatsu, Kohei Hikino, Yuki Iida, Masayo Hosokawa, Mayumi Sawada, Akiko Kudoh, Jun Chikumi, Shinya Sato, Genki Hichiwa, Yasuhiro Kazuki, Kanako Kazuki, Fuminori Taniguchi, Mitsuo Oshimura, Tasuku Harada. Establishment and characterization of reversibly immortalized endometrial and ovarian epithelial cell lines using Sendai virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1214.

A New Multi-Color FISH Assay for Brush Biopsy-Based Detection of Chromosomal Aneuploidy in Oral (Pre)Cancer in Patients with Fanconi Anemia.

Simple SummaryPatients with Fanconi Anemia (FA) are at high risk of developing oral squamous cell carcinoma with poor prognosis. Oral brush biopsy–based cytology represents an attractive non-invasive advantage in the characterization of (pre) malignant oral lesions, but a sufficient number of abnormal cells is mandatory to make the diagnosis. The aim of this 2-phases retrospective study is to introduce a new multi-color FISH assay including a 9p21 FISH assay to determine the oral lesions that require treatment, with just a few cells. We demonstrate a good accuracy of the multicolor FISH assay if applied on oral brush biopsy-based specimens from FA patients, using different algorithms to determine chromosomal aneuploidy. Nevertheless, some false positive results were observed and the detection of a genetically altered field in the oral cavity as a possible reason for what may hamper the application of multi-color FISH is discussed.Background: Fanconi anemia (FA) is a rare inherited DNA instability disorder with a remarkably elevated risk of oral squamous cell carcinoma. These cancers can be detected with oral brush biopsy-based cytology even at early stages. This study aims to determine the diagnostic accuracy of a new multi-color fluorescent in situ hybridization (FISH) assay consisting of probes for CCND1, TERC, MYC and centromere of chromosome 6, as well as a 9p21 FISH assay consisting of probes for CDKN2A and centromere of chromosome 9 for the detection of oral (pre) malignant lesions in FA. Methods: (I) Cutoffs for the dichotomization of positive or negative multi-color FISH results are determined and (II) retrospectively validated by using archived oral brush biopsy specimens from individuals with Fanconi anemia. In addition, the specimens for cutoff determination were re-hybridized with the 9p21 FISH assay. Results: A cutoff of six or more chromosomal aneuploid cells for a positive FISH result was determined in the cutoff study on 160 biopsy specimens. The validating of this cutoff on 152 specimens showed at best a sensitivity of 87% and a specificity of 82.9%. Conclusion: Multi-color FISH is a sufficient tool to detect chromosomal aneuploidy in oral (pre) malignant lesions of individuals with Fanconi anemia. However, some false positive results may hamper the application as an adjuvant method to oral brush biopsy-based cytology in an oral cancer surveillance program.

PB1750: REARRANGEMENTS OF T-CELL RECEPTOR (TCR) LOCI IN CHILDREN WITH T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a clinically and genetically heterogeneous disease that constitutes 10%–15% of newly diagnosed pediatric ALL cases and is caused by the accumulation of genetic abnormalities that alter the mechanisms controlling normal T-cell development. The most common structural aberrations involve rearrangements of the T-cell receptor (TCR) loci – TRA/TRD (14q11) and TRB (7q34), which are found in ~25% of patients. These aberrations are the main factors initiating the events in T-ALL carcinogenesis and juxtapose TCR genes and protooncogenes that encode pivotal transcription factors, leading to their aberrant expression. The outcomes of children with T-ALL significantly improved on intensified protocols, however, ~20% of patients relapse and die owing to acquired therapy resistance. Moreover, survivors also endure the acute and long-term effects of intensive toxic chemotherapy. Aims: To analyze TCR loci rearrangements and associated genetic abnormalities in children with T-ALL treated according to BFM-based protocols, and to correlate our findings with the clinical features and the treatment responses. Methods: The bone marrow samples of 66 children diagnosed between 1996-2017 (46 boys, 20 girls, age median 7.9 years, follow-up median 86.4 months) were analyzed with cytogenomic methods. For the detection of TCR loci rearrangements and other recurrent aberrations of the genes TLX3, CDKN2A/CDKN2B and ABL1 interphase FISH (DAKO, Abbott Molecular) was used. MLPA analysis with ALL-IKZF1 probemix (MRC-Holland) was performed to detect deletions/amplifications of additional genes. Complex karyotypes were analyzed with multicolor FISH (MetaSystems) or array CGH/SNP (Agilent). Differences in OS and EFS were assessed using Kaplan-Meier method and the Mantel-Cox test. Results: Rearrangements of TCR loci were detected in 18/66 (27%) patients. Translocations involving the TRA/TRD locus were demonstrated in nine children, the TRB locus in five and the simultaneous occurrence of these aberrations in two cases, respectively. In 10/18 patients, known recurrent chromosomal translocations affecting the oncogenes TAL1 (3x), LMO2 (3x), TLX1 (2x), TAL2 (1x) and MYC (1x) were identified. In the remaining cases, TCR rearrangements were cryptic or were a part of complex karyotype. Additional chromosomal aberrations were detected in all but one patient with the deletion of CDKN2A gene being the most frequent one. Immunophenotypic data classified the patients into cortical-T (12x) and mature-T (5x) groups (1x no data). Children with aberrations of TCR loci had a significantly better prognosis - EFS (p=0.011) and OS (p=0.0074), all patients are living in the first complete remission. Summary/Conclusion: Aberrations of T-cell receptors form a genetically heterogeneous group of rearrangements, leading to aberrant expression of oncogenes involved in T-cell maturation and proliferation. They mostly occur with abnormalities of genes involved in the regulation of the cell cycle and/or signaling pathways, confirming the multistep process of T-ALL pathogenesis. In our cohort, patients with TCR rearrangements had an excellent prognosis regardless of the presence of other aberrations. Although data on larger series are clearly required, we suppose, that these children could benefit from less-intensive therapy because they may experience fewer therapeutic consequences of toxic treatment. Supported by MH CZ-DRO-VFN64165.

Establishment of a set of wheat-rye addition lines with resistance to stem rust.

Complete new wheat-rye disomic, telosomic addition lines and various chromosomal aberrations were developed and characterized by molecular cytogenetic method as novel chromosome engineering materials. A new stem rust resistance (Ug99) gene was located on 3RL. Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating fungal disease worldwide. A recently emerged great threat to global wheat production is Pgt strain Ug99 and its derivatives, which have overcome most of the commonly used resistance genes. Rye (Secale cereale L.), closely related to wheat (Triticum aestivum L.), is a significant and valuable resource of resistance genes for wheat germplasm improvement. It is of great importance and urgency to identify new resistance gene sources of rye and transfer them into wheat. In this study, two complete sets of wheat-rye addition lines were established through wide hybridization, chromosome doubling and backcrossing. A wheat-rye 3RL telosomic addition line was identified with high resistance to stem rust strain Ug99. PCR-based markers specific for the rye chromosome were developed. Furthermore, abundant chromosomal aberrations such as minichromosomes, ring chromosomes as well as centromere reduction and expansion were identified in the progeny of wheat-rye addition lines by multicolor GISH and FISH. The line carrying a novel resistance gene to stem rust can be utilized as a bridge material for wheat disease resistance breeding. The chromosomal and centromeric variation within the wheat-rye hybrids can further contribute to genetic diversity of their offspring.

Oocyte aneuploidy rates in river and swamp buffalo types (Bubalus bubalis) determined by Multi-color Fluorescence In Situ Hybridization (M-FISH)

Aneuploidy is one of the main causes of fetal and embryonic mortality in mammals. Nonetheless, its incidence in domestic ruminants has been investigated little. Indeed, no incidence data have ever been reported for water buffalo. To establish the incidence of aneuploidy in this species, we analysed in vitro matured metaphase II (MII) oocytes with corresponding first polar bodies (I PB) of the river (2n = 50) and swamp (2n = 48) buffaloes. For the first time, six river type probes (corresponding to chromosomes 1–5 and heterosome X), were tested on swamp buffalo metaphases using Multicolor-Fluorescent In Situ Hybridization (M-FISH) before their use on oocytes MII metaphases. Of the 120 total Cumulus Oocyte Complexes (COCs, 60 for each buffalo type) subjected to in vitro maturation, 104 reached the MII stage and were analysed by M-FISH. Haploid chromosome arrangement and visible I PB were observed in 89 of the oocytes (45 in river and 44 in swamp type). In the river type, the analysis revealed one oocyte was disomic for the chromosome X (2.22%). In the swamp type, one oocyte was found to be nullisomic for chromosome X (2.27%); another was found to be nullisomic for chromosome 5 (2.27%). We also observed one oocyte affected by a premature separation of sister chromatids (PSSC) on the chromosome X (2.27%). In both buffalo types, no abnormalities were detected in other investigated chromosomes. Based on merged data, the overall aneuploidy rate for the species was 3.37%. Oocytes with unreduced chromosomes averaged 1.92% across the two types, with 1.96% in river and 1.88% in swamp. The interspecies comparison between these data and cattle and pig published data revealed substantial difference in both total aneuploidy and diploidy rates. Reducing the negative impact of the meiotic segregation errors on the fertility is key to more sustainable breeding, an efficient embryo transfer industry and ex-situ bio-conservation. In this respect, additional M-FISH studies are needed on oocytes of domestic species using larger sets of probes and/or applying next generation sequencing technologies.

Molecular Cytogenetic Characterization of Novel 1RS.1BL Translocation and Complex Chromosome Translocation Lines with Stripe Rust Resistance.

Rye is the most important source for the genetic improvement of wheat. In this study, two stable wheat-rye primary 1RS.1BL translocation lines, RT855-13 and RT855-14, were selected and identified by acid polyacrylamide gel electrophoresis (A-PAGE), co-dominant PCR, and multi-color fluorescence in situ hybridization (MC-FISH) from the progeny of the crossing of the wheat cultivar Mianyang11 and a Chinese rye Weining. When more than two independent, simple reciprocal translocations are involved in a carrier, they are defined as complex chromosome translocations (CCT). The MC-FISH results also indicated that CCT occurred in RT855-13; namely that, besides 1RS.1BL translocation chromosomes, there are other two pairs of balanced reciprocal translocations. It was demonstrated that the interchange between a distal segment of 4B and long arm of 3D occurred in the RT855-13. The novel translocation chromosomes in wheat were recorded as 3DS.4BSDS and 3DL-4BSPS.4BL. Reports about CCT as a genetic resource in plant breeding programs are scarce. Both lines expressed high resistance to Puccinia striiformis f. sp. tritici, which are prevalent in China and are virulent on Yr9, and the CCT line RT855-13 retained better resistance as adult plants compared with RT855-14 in the field. Both lines, especially the CCT line RT855-13, exhibited better agronomic traits than their wheat parent, Mianyang11, indicating that both translocation lines could potentially be used for wheat improvement. The results also indicated that the position effects of CCT can lead to beneficial variations in agronomic and resistant traits, making them a valuable genetic resource to wheat breeding programs.

Cytogenetic Characterization of Complex Karyotypes by Multicolor FISH in Myelodysplastic Syndromes and Associated Acute Myeloid Leukemias

Complex karyotypes (CK) were thoroughly analyzed by using the data of multicolor FISH in 27 patients with myelo-dysplastic syndromes (MDS) and MDS-associated acute myeloid leukemias (AMLm). Despite a vast variety of identified genetic impairments, chromosomes 5, 8, and 7 appeared to be most frequently (79 %, 76 %, and 73 %, respectively) involved in rearrangements, a fact also documented in literature. In view of this, two independent cytogenetic subgroups with chromosome 5/7 and 5/7/8 rearrangements were formed. Chromosomes 5 and 7 predominantly showed unbalanced karyotype, and chromosome 8 was characterized by its combinations with trisomies. The study also revealed that complex markers, more often than the other ones, contain chromosome 7 material, which has not so far been adequately explained. At the same time, the accumulation of chromosome 8 material in CK was associated with a more favorable course of underlying disease. On the other hand, detailed structural analysis of some supercomplex CK markers affords grounds for the assertion that chromothripsis notably participates in their formation. The overall survival of MDS and AMLm patients in artificially formed joint subgroups with combinations of involved chromosomes 5/7 and 5/7/8 was significantly lower than in AMLm (p = 0.035).

High-Throughput DNA FISH (hiFISH).

High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.

Detection of cytogenetic changes and chromosomal aneuploidy with fluorescent in situ hybridization in cytological specimens of oral cancers in Fanconi anemia-Proof of concept.

ObjectivesFanconi anemia (FA) is a rare inherited DNA instability disorder with a remarkably elevated risk of neoplasia compared with the general population, mainly leukemia and squamous cell carcinoma (SCC). Two thirds of the SCCs arise in the oral cavity and are typically preceded by visible lesions. These lesions can be classified with brush biopsy‐based cytological methods regarding their risk of a malignant transformation. As a proof of concept, this study aims to investigate genetic changes and chromosomal aneuploidy using fluorescent in situ hybridization (FISH) on oral squamous cells derived from FA affected individuals.Material and MethodsFive FA oral SCC (OSCC) tumor cell lines, one FA OSCC cervical lymph node metastasis as well as tumor‐negative and atypical smears from oral brush biopsies were analyzed with FISH probes covering 5p15.2, MYC, EGFR, TERC, 9q34.1, CCND1, 9p21 and centromeres of chromosomes 3, 6, 7, 9, 11, and 17.ResultsOSCC specimens showed gains of all analyzed chromosomal regions. Chromosomal aneuploidy was observed in five of the six OSCC specimens in two multicolor FISH assays with panels of four probes each. Five out of six OSCC specimens displayed a relative deletion of 9p21. Applied on atypical brush biopsy‐based smears, chromosomal aneuploidy was detected in malignant lesions but not in the smear derived from a severe parodontitis.ConclusionsAs proof of concept, FISH was able to detect genetic changes and chromosomal aneuploidy discriminating oral cancer from noncancerous lesions in individuals with FA. This supports its application on oral brush biopsy‐based cytology.

Coexpression of Three Odorant-Binding Protein Genes in the Foreleg Gustatory Sensilla of Swallowtail Butterfly Visualized by Multicolor FISH Analysis.

Lepidopteran insects are mostly monophagous or oligophagous. Female butterflies distinguish their host plants by detecting a combination of specific phytochemicals through the gustatory sensilla densely distributed on their foreleg tarsi, thereby ensuring oviposition on appropriate host plants. In this study, to gain insight into the molecular mechanism underlying host plant recognition by the gustatory sensilla, using Asian swallowtail, Papilio xuthus, we focused on a family of small soluble ligand-binding molecules, odorant-binding proteins (OBPs), and found that three OBP genes showed enriched expression in the foreleg tarsus. Multicolor fluorescence in situ hybridization analyses demonstrated the coexpression of these three OBP genes at the bases of the foreleg gustatory sensilla. Further analyses on other appendages revealed that PxutOBP3 was exclusively expressed in the tissues which could have direct contact with the leaf surface, suggesting that this OBP gene specifically plays an important role in phytochemicals perception.

Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation.

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes.

Unbalanced translocation der(5;17) resulting in a TP53 loss as recurrent aberration in myelodysplastic syndrome and acute myeloid leukemia with complex karyotype.

A complex karyotype, detected in myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML), is associated with a reduced median survival. The most frequent chromosomal aberrations in complex karyotypes are deletions of 5q and 17p harboring the tumor suppressor gene TP53. The unbalanced translocation der(5;17) involving chromosome 5q and 17p is a recurrent aberration in MDS/AML, resulting in TP53 loss. We analyzed the karyotypes of 178 patients with an unbalanced translocation der(5;17) using fluorescence R-/G-banding analysis. Whenever possible, fluorescence in situ hybridization (FISH) (n = 138/141), multicolor FISH (n = 8), telomere length measurement (n = 9), targeted DNA sequencing (n = 13), array-CGH (n = 7) and targeted RNA sequencing (n = 2) were conducted. The der(5;17) aberration was accompanied with loss of genetic material in 7q (53%), -7 (27%), gain of 21q (29%), +8 (17%) and - 18 (16%) and all analyzed patients (n = 13) showed a (likely) pathogenic variant inTP53. The der(5;17) cohort showed significantly shortened telomeres in comparison to the healthy age-matched controls (P < .05), but there was no significant telomere shortening in comparison to MDS/AML patients with a complex karyotype without der(5;17). No fusion genes resulted from the unbalanced translocation. This study demonstrates that the unbalanced translocation der(5;17) is associated with a biallelic inactivation of TP53 due to a deletion of TP53 in one allele and a pathogenic variant of the second TP53 allele. Since the breakpoints are located within (near-) heterochromatic regions, alterations of DNA methylation or histone modifications may be involved in the generation of der(5;17).

Imaging approaches for chromosome structures.

This review describes image analyses for chromosome visible structures, focusing on the chromosome imaging system CHIAS (Chromosome Image Analyzing System). CHIAS is the first comprehensive imaging system for the analysis and characterization of plant chromosomes. A simulation method for human vision for capturing band positive regions was developed and used for the image analysis of large plant chromosomes with bands. Applying this method to C-banded Crepis chromosomes enabled recognition of band positive regions as seen by human vision. Furthermore, a new image parameter, condensation pattern was developed and successfully applied to identify small plant chromosomes such as rice and brassicas. Condensation profile (CP) derived from condensation pattern was also effective in developing quantitative chromosome maps. The result was quantitative chromosomal maps of several plants with small chromosomes, including Arabidopsis, diploid brassicas, rapeseed, rice, spinach, and sugarcane. In the final chapter, various applications of imaging techniques to the analysis of pachytene chromosomes, improved visibility of multicolor FISH images, 3D reconstruction of a human chromosome based on cross-section images obtained by a FIB/SEM, automatic extraction of chromosomal regions by machine learning, etc. are described.

Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability

BackgroundThe U937 cell line is widely employed as a research tool. It has a complex karyotype. A PICALM-MLLT10 fusion gene formed by the recurrent t(10;11) translocation is present, and the myeloid common deleted region at 20q12 has been lost from its near-triploid karyotype. We carried out a detailed investigation of U937 genome reorganisation including the chromosome 20 rearrangements and other complex rearrangements.ResultsSNP array, G-banding and Multicolour FISH identified chromosome segments resulting from unbalanced and balanced rearrangements. The organisation of the abnormal chromosomes containing these segments was then reconstructed with the strategic use of targeted metaphase FISH. This provided more accurate karyotype information for the evolving karyotype. Rearrangements involving the homologues of a chromosome pair could be differentiated in most instances. Centromere capture was demonstrated in an abnormal chromosome containing parts of chromosomes 16 and 20 which were stabilised by joining to a short section of chromosome containing an 11 centromere. This adds to the growing number of examples of centromere capture, which to date have a high incidence in complex karyotypes where the centromeres of the rearranged chromosomes are identified. There were two normal copies of one chromosome 20 homologue, and complex rearrangement of the other homologue including loss of the 20q12 common deleted region. This confirmed the previously reported loss of heterozygosity of this region in U937, and defined the rearrangements giving rise to this loss.ConclusionsCentromere capture, stabilising chromosomes pieced together from multiple segments, may be a common feature of complex karyotypes. However, it has only recently been recognised, as this requires deliberate identification of the centromeres of abnormal chromosomes. The approach presented here is invaluable for studying complex reorganised genomes such as those produced by chromothripsis, and provides a more complete picture than can be obtained by microarray, karyotyping or FISH studies alone. One major advantage of SNP arrays for this process is that the two homologues can usually be distinguished when there is more than one rearrangement of a chromosome pair. Tracking the fate of each homologue and of highly repetitive DNA regions such as centromeres helps build a picture of genome evolution. Centromere- and telomere-containing elements are important to deducing chromosome structure. This study confirms and highlights ongoing evolution in cultured cell lines.

Applications of fluorescence in situ hybridization (FISH) in genome research

Fluorescence in situ hybridization (FISH) technique enables the direct detection of DNA sequences inintact cellular materials (e.g. individual chromosomes in metaphase spreads). This review article focuses on theapplications of FISH in genome research, including validation and correction of the genome assembly from thenext-generation sequencing (NGS) projects. DNA probes for specific DNA fragments of the assembly can beobtained from PCR amplicon or cloned products using different vector systems. Localization of these probeson their respective chromosomal regions can be visualized by FISH, providing useful information to crosscheckthe assembly data. Furthermore, the recent refinements in the FISH technology including using smartpooling scheme of differently colored DNA probes, together with consecutive FISH experiments (stripping andreprobing of the same slide) are described. These advances in multicolor FISH can provide crucial linkageinformation on association of linkage groups and assembly scaffolds, resulting in so-called cytogenetic maps.Integration of the cytogenetic maps and assembly sequences assists to resolve the chromosome-level genomeassembly and to reveal new insights in genome architecture and genome evolution. Especially, comparativechromosome painting with pooled DNA probes from one reference species can be used to investigate ancestralrelationships (chromosome homeology and rearrangements) among other not-yet-sequenced species. Inaddition, FISH using DNA probes for certain specific classes of repetitive DNA elements as well as for basicchromosome structures (e.g. centromere or telomere DNA repeats, ribosomal DNA loci) can be used to studythe genome organization and karyotype differentiation. We also discussed about limitations and futureperspectives of the FISH technology.