Figure 2 - uploaded by Ruth Mackinnon
Content may be subject to copyright.
A representative HEL G-banded karyotype with the der(8)t(4;8;13) (arrow). The karyotype is missing a chromosome 13, the der(10)t(2;10;18), and the dup(21). The der(10) (left) and dup(21) are shown in insets, each paired with a normal homologue
Source publication
The human erythroleukaemia (HEL) cell line has a highly rearranged genome. We matched whole chromosome analysis with cytogenomic microarray data to build a detailed description of these rearrangements.
We used a combination of single nucleotide polymorphism array and multiple fluorescence in situ hybridization approaches, and compared our array dat...
Context in source publication
Citations
... Clustering using scATAC-seq data alone resulted in discrete cell populations (Fig. 1f) that were annotated on the basis of differential gene accessibility and unique mitochondrial variants, while retaining high scATAC-seq data quality (Extended Data Fig. 2b (Fig. 1g). CNV scores at chromosome 9, amplified in JAK2 V617F homozygous mutant HEL cells 25 , orthogonally validated successful genotyping (Extended Data Fig. 2j). The proportion of genotyped cells was positively correlated with the JAK2 copy number (Extended Data Fig. 2k), while genotyping accuracy remained constant (Extended Data Fig. 2l). ...
... b, Accessibility-based UMAP for original GoT-ChA protocol for CA46 (grey), HEL (gold), OCI-AML3 (violet) and SET-2 (green) cells. c, Accessibility-based UMAP for multiplexing-adapted GoT-ChA protocol (Methods) for cell lines from b. d, Differential gene accessibility markers (FDR < 0.05, Log 2 FC > 1. 25; Wilcoxon rank sum test followed by Benjamini-Hochberg correction) used for cell line identification. e, UMAP coloured by GoT-ChA JAK2 V617 genotypes of each cell as wild type (WT, blue), mutant (MUT, red), or not assignable (NA, grey) for original GoT-ChA (left) and multiplexed-adapted GoT-ChA (right). ...
In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1–3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT–ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT–ChA to CD34⁺ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT–ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
... Centromeres are vital for genetic stability and inheritance [56]. Research on centromeres is limited, as they are not typically studied [57][58][59]. Although complex involvement of chromosome 7 centromeric regions in chromoanagenesis has not been reported in AML/MDS, studies in the Cryptococcus species demonstrated that multiple DNA double-strand breaks (DSBs) at centromere-specific retrotransposons can lead to the formation of multiple interchromosomal rearrangements (chromothripsis-like events) [60]. ...
Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach’s value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival.
... By correcting for background noise, we achieved genotyping of the TP53 R248 locus in 49.5% of all cells with an accuracy of 99.7% (Fig. 1f). As an additional orthogonal validation of our genotyping, we compared wildtype and mutant cell CNV scores 36 inferred from the chromatin accessibility profiles (see materials and methods) across chromosome 9, for which the TP53 R248 wildtype HEL cell line carries an amplification [47][48][49] . Consistent with our observed genotyping, wildtype cells as defined by GoT-ChA genotyping exhibited an increased CNV score relative to mutant cells, further confirming successful single-cell genotyping (Fig. 1g). . ...
... In the SET-2 heterozygous cell line, 64.7% of cells were successfully genotyped; 34.3% of genotyped cells were correctly identified as heterozygous, confirming bi-allelic capture, with the remaining genotyped cells split between homozygous wildtype and mutant assignments, suggesting incomplete capture of the heterozygous genotype (Fig. 1l, right panel, note that SET-2 cells have a 3:1 ratio of mutated:wildtype alleles (Extended Data Fig. 2k), likely underlying the higher fraction of mutated versus wildtype calls in non-heterozygous genotype classification). CNV scores for homozygous wildtype and mutant cells along the chromosome 9 amplification present in the JAK2 V617F homozygous mutant HEL cell line [47][48][49] orthogonally validated successful genotyping of the JAK2 V617 locus (Fig. 1m). We note that variation in genotyping efficiency across cell lines is likely related to the copy number variation for the JAK2 locus 47,50,51 . ...
In normal somatic tissue differentiation, changes in chromatin accessibility govern priming and commitment of precursors towards cellular fates. In turn, somatic mutations can disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wildtype cells. To chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed Genotyping of Targeted loci with single-cell Chromatin Accessibility (GoT-ChA). This high-throughput, broadly accessible platform links genotypes to chromatin accessibility at single-cell resolution, across thousands of cells within a single assay. We applied GoT-ChA to CD34 ⁺ cells from myeloproliferative neoplasm (MPN) patients with JAK2 V617F -mutated hematopoiesis, where the JAK2 mutation is known to perturb hematopoietic differentiation. Differential accessibility analysis between wildtype and JAK2 V617F mutant progenitors revealed both cell-intrinsic and cell state-specific shifts within mutant hematopoietic precursors. An early subset of mutant hematopoietic stem and progenitor cells (HSPCs) exhibited a cell-intrinsic pro-inflammatory signature characterized by increased NF-κB and JUN/FOS transcription factor motif accessibility. In addition, mutant HSPCs showed increased myeloid/erythroid epigenetic priming, preceding increased erythroid and megakaryocytic cellular output. Erythroid progenitors displayed aberrant regulation of the γ-globin locus, providing an intrinsic epigenetic basis for the dysregulated fetal hemoglobin expression observed in MPNs. In contrast, megakaryocytic progenitors exhibited a more specialized inflammatory chromatin landscape relative to early HSPCs, with increased accessibility of pro-fibrotic JUN/FOS transcription factors. Notably, analysis of myelofibrosis patients treated with JAK inhibitors revealed an overall loss of mutant-specific phenotypes without modifying clonal burden, consistent with clinical responses. Finally, expansion of the multi-modality capability of GoT-ChA to integrate mitochondrial genome profiling and cell surface protein expression measurement enabled genotyping imputation and discovery of aberrant cellular phenotypes. Collectively, we show that the JAK2 V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner. We envision that GoT-ChA will thus serve as a foundation for broad future explorations to uncover the critical link between mutated somatic genotypes and epigenetic alterations across clonal populations in malignant and non-malignant contexts.
... This is especially true for the long terminal repeat retrotransposons (LTR-RTs), which are the most prevalent repeats in plant genomes, and their proliferation could result in genome bloating (Elert, 2014). Other repeat regions, such as centromeres, are of central importance to chromosome stability (Mackinnon et al., 2013). SDs, similarly, influence genome structure and produce numerous repeat genes, thus serving as hotbeds for genomic rearrangements followed by gene innovation and rapid gain-offunction adaptions (Zhang et al., 1998;Han et al., 2009;Marques-Bonet et al., 2009). ...
... The subspecies indica is the most widely cultivated rice subspecies, due to several desirable traits, and, interestingly, has a substantially larger genome than another widely cultivated subspecies, japonica (Du et al., 2017). Recent studies have shown that TEs, centromeres, and SDs all have strong evolutionary effects on the genome (Zhang et al., 1998;Han et al., 2009;Marques-Bonet et al., 2009;Mackinnon et al., 2013;Elert, 2014). However, the prevalence of these repeat sequences in plant genomes hinders the assembly of high-quality genomes, as they make it considerably difficult for current sequencing technologies and algorithms to produce chromosome-level assemblies without gaps. ...
The ultimate goal of genome assembly is a high-accuracy gapless genome. Here we report a new assembly pipeline to produce a gapless genome for the indica rice cultivar Minghui 63. The 397.71 Mb final assembly is composed of 12 contigs with a contig N50 size of 31.93 Mb. All chromosomes are now gapless, with each chromosome represented by a single contig. BUSCO evaluation showed that gene regions of our assembly have the highest completeness among the 16 high-quality rice genomes. Compared with the japonica rice, the indica genome has more transposable elements (TEs) and segmental duplications (SDs), the latter of which produce many duplicated genes that can affect plant traits through dose effect or sub-/neo-functionalization. The insertion of TEs can also affect the expression of duplicated genes, which may drive the evolution of these genes. We also found the expansion of NBS-LRR disease resistance genes and cZOGT growth-related genes in SDs, suggesting that SDs contribute to the adaptive evolution of rice disease resistance and developmental processes. Our findings suggest that active TEs and SDs together provide synergistic contributions to rice genome evolution.
... In addition, Ma et al. show that RBM15 affects Notch signaling by binding to RBPJk (recombination signal binding protein for immunoglobulin kappa J region), a transcription factor critical for Notch signaling (80). RBM15 has cell type-specific stimulatory and inhibitory effects on Notch signaling in transient transfection assays (88). ...
Myocardin-related transcription factor A (MRTFA) is a coactivator of serum response factor (SRF), a transcription factor that participates in several critical cellular functions including cell growth and apoptosis. MRTFA couples transcriptional regulation to actin cytoskeleton dynamics and the transcriptional targets of the MRTFA–SRF complex include genes encoding cytoskeletal proteins as well as immediate early genes. Previous work has shown that MRTFA promotes the differentiation of many cell types, including various types of muscle cells and hematopoietic cells, and MRTFA’s interactions with other protein partners broaden its cellular roles. However, despite being first identified as part of the recurrent t(1;22) chromosomal translocation in acute megakaryoblastic leukemia (AMKL), the mechanisms by which MRTFA functions in malignant hematopoiesis have yet to be defined. In this review, we provide an in-depth examination of the structure, regulation, and known functions of MRTFA with a focus on hematopoiesis. We conclude by identifying areas of study that merit further investigation.
... bloating (Elert, 2014). Other repeat regions, such as centromeres, are of central 10 importance to chromosome stability (Mackinnon et al., 2013). SDs are similarly 11 influential on genome structure due to their production of numerous repeat genes, and 12 thus serve as hotbeds for genomic rearrangements followed by gene innovation and 13 rapid gain-of-function adaptations ( improved the continuity of assembly. ...
The ultimate goal of genome assembly is a high-accuracy gapless genome. Here we report a new assembly pipeline which we have used to produce a gapless genome for the indica rice cultivar Minghui 63. The 395.82 Mb final assembly is composed of 12 contigs with a contig N50 size of 31.82 Mb. All chromosomes are now gapless, with each chromosome represented by a single contig. This is the first gapless genome assembly achieved for higher plants or animals. BUSCO evaluation showed that gene regions of our assembly have higher completeness than the current rice reference genome (IRGSP-1.0). Compared with japonica rice, indica has more transposable elements (TEs) and segmental duplications (SDs), the latter of which produce many duplicated genes that can affect plant traits through dose effect or sub-/neo-functionalization. The insertion of TEs can also affect the expression of duplicated genes, which may drive evolution of these genes. We also found the expansion of NBS-LRR disease resistance genes and cZOGT growth-related genes in SDs, suggesting that SDs contribute to the adaptative evolution of rice disease resistance and developmental processes. Our findings suggest that active TEs and SDs together provide synergistic effects to promote rice genome evolution.
... Background MacGrogan et al. [1] published a study of several acute myeloid leukaemia (AML) cell lines with loss of heterozygosity (LOH) at 20q12, to delineate the common deleted region found in myeloid malignancies [1]. We have carried out a detailed characterisation of the genomes of two of these cell lines, HEL [2] and U937, using a molecular cytogenomics approach. As well as confirming the del (20)(q12) reported in the karyotypes, these studies demonstrate the combined use of different molecular methods to characterise chromosome rearrangements in detail. ...
... We described centromere capture events for the first time, in complex unbalanced karyotypes, where acentric segments from one or more chromosomes were preserved by joining to a centromere from a different chromosome [2,18], and this concept was also later reported by Garsed et al. [19]. A centromere is necessary for stable inheritance and survival of a chromosome formed by the repair of broken chromosome segments Table 3 The U937 karyotype ...
... We have previously described four centromere capture events: in two unbalanced translocations in the cell line HEL [18] and in two anachromosomes (chromosomes produced by chromothripsis) in a case of AML [2]. The present study identifies a further example of centromere capture: acentric segments from chromosomes 16 and 20 were identified in an abnormal chromosome, which had a centromere from chromosome 11. ...
Background
The 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.
Results
SNP 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.
Conclusions
Centromere 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.
... Background MacGrogan et al. [1] published a study of several acute myeloid leukaemia (AML) cell lines with loss of heterozygosity (LOH) at 20q12, to delineate the common deleted region found in myeloid malignancies [1]. We have carried out a detailed characterisation of the genomes of two of these cell lines, HEL [2] and U937, using a molecular cytogenomics approach. As well as con rming the del (20)(q12) reported in the karyotypes, these studies demonstrate the combined use of different molecular methods to characterise chromosome rearrangements in detail. ...
... We described centromere capture events for the rst time, in complex unbalanced karyotypes, where acentric segments from one or more chromosomes were preserved by joining to a centromere from a different chromosome [2,18], and this concept was also later reported by Garsed et al. [19]. A centromere is necessary for stable inheritance and survival of a chromosome formed by the repair of broken chromosome segments [18]. ...
... We have previously described four centromere capture events: in two unbalanced translocations in the cell line HEL [18] and in two anachromosomes (chromosomes produced by chromothripsis) in a case of AML [2]. The present study identi es a further example of centromere capture: acentric segments from chromosomes 16 and 20 were identi ed in an abnormal chromosome, which had a centromere from chromosome 11. ...
Background
The 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.
Results
SNP 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.
Conclusions
Centromere 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.
... Whole-arm rearrangements have been observed in patients with pure erythroid leukemia. 31 Further molecular assessment of these abnormalities will hopefully provide additional insight into PEL with possible therapeutic implications. ...
Per the revised fourth edition World Health Organization classification of acute myeloid leukemia, pure erythroid leukemia is now the sole type of acute erythroid leukemia. The diagnosis of this rare entity is often challenging and the cytologic overlap with non-neoplastic (eg, megaloblastic anemia) and neoplastic entities (eg, other types of acute leukemia and non-hematopoietic malignancies) warrants a significant degree of clinical, laboratory, immunophenotypic, and genetic investigation. Given the limited number of reports of this rare and diagnostically challenging entity, we report detailed clinicopathologic characteristics from 15 patients, the largest series thus far, of primary de novo pure erythroid leukemia to provide further diagnostic insights into this entity and reveal strategies for making the diagnosis. We found that de novo pure erythroid leukemia is a disease of adults (median age 68 years), exhibits a striking male predominance, is universally associated with an abnormal karyotype and has an exceedingly poor overall median survival of 1.4 months. Given the general inability of immunophenotypic markers to discriminate neoplastic from non-neoplastic erythroid proliferations, key features identified in this study to help establish the diagnosis of pure erythroid leukemia and exclude mimickers include circulating pronormoblasts, clear-cut dysplasia in erythroid, granulocytic, and/or megakaryocytic lineage, utilization of a broad immunophenotypic panel, TP53 immunohistochemical positivity, and identification of a complex, often highly complex, karyotype. Given the gravity of a diagnosis of de novo pure erythroid leukemia, it should be rendered with utmost confidence.
... For the comparison of methods aforementioned, the human erythroleukaemia (HEL) cell line was used because MacKinnon et al. [11] have shown by FISH that HEL has two large, rearranged chromosomes positive for multiple nucleolar organiser regions and three rearranged chromosomes that contain centromere DNA sequences from two different chromosomes. HEL has been widely used for cell biology and differentiation studies in addition to the extensive data generated from a variety of techniques namely whole chromosome painting, single nucleotide polymorphism (SNP) array, OncoMap sequencing, mFISH, multicolour chromosome banding (M-BAND) and targeted FISH [11]. ...
... For the comparison of methods aforementioned, the human erythroleukaemia (HEL) cell line was used because MacKinnon et al. [11] have shown by FISH that HEL has two large, rearranged chromosomes positive for multiple nucleolar organiser regions and three rearranged chromosomes that contain centromere DNA sequences from two different chromosomes. HEL has been widely used for cell biology and differentiation studies in addition to the extensive data generated from a variety of techniques namely whole chromosome painting, single nucleotide polymorphism (SNP) array, OncoMap sequencing, mFISH, multicolour chromosome banding (M-BAND) and targeted FISH [11]. ...
... However, through methods Table 1 List of antibodies targeting components of active centromere. Antibodies were first tested on non-fixative treated cells to determine optimal concentration or dilution for immunocytochemistry and then tested on cells stored in methanol-acetic acid fixative As reported in MacKinnon et al. [11], three derivative chromosomes, namely der(4;20)t(4;11;20) and another two that resulted from whole arm translocations between two chromosomes, der(5;17) and der(10;19), contain centromere sequences originated from two different chromosomes. We were able to detect the presence of two active centromeres based on CENP-C signals on at least one der(10;19) chromosome in 2 out of 6 metaphase spreads with method C [ Fig. 3c (20) which could not be tested by MacKinnon et al. [11]. ...
The centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. However, in cancer and constitutional disorders, the presence of more than one active centromere on a chromosome may be a contributing factor to chromosome instability and could also have predictive value in disease progression, making the detection of properly functioning centromeres important. Thus far, antibodies that are widely used for functional centromere detection mainly work on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. Hence, we aimed to identify antibodies that would recognise active centromere antigens on methanol-acetic acid fixed cells.
A panel of active centromere protein antibodies was tested and we found that a rabbit monoclonal antibody against human CENP-C recognises the active centromeres of cells fixed in methanol-acetic acid. We then tested and compared combinations of established methods namely centromere fluorescence in situ hybridisation (cenFISH), centromere protein immunofluorescence (CENP-IF) and multicolour FISH (mFISH), and showed the usefulness of CENP-IF together with cenFISH followed by mFISH (CENP-IF-cenFISH-mFISH) with the aforementioned anti-CENP-C antibody. We further demonstrated the utility of our method in two cancer cell lines with high proportion of centromere defects namely neocentromere and functional dicentric.
We propose the incorporation of the CENP-IF-cenFISH-mFISH method using a commercially available rabbit monoclonal anti-CENP-C into established methods such as dicentric chromosome assay (DCA), prenatal karyotype screening in addition to constitutional and cancer karyotyping. This method will provide a more accurate assessment of centromere abnormality status in chromosome instability disorders.
