Chromosomal Rearrangement Breakpoints Research Articles

Balanced chromosomal rearrangements implicate YIPF5 and SPATC1L in non-obstructive oligoasthenozoospermia and oligozoospermia and of a derivative chromosome 22 in recurrent miscarriage

Naturally occurring balanced, unbalanced, and complex chromosomal rearrangements have been reported to cause pathogenic genomic or genetic variants leading to infertility and recurrent miscarriage. Therefore, balanced chromosomal rearrangements were used as genomic signposts for identification of candidate genes or genomic loci associated with male infertility due to defects of spermatogenesis, or with recurrent miscarriage. In three male probands, structural chromosomal variants and copy number variants were identified at nucleotide resolution by long-insert genome sequencing approaches and Sanger sequencing. The pathogenic potential of these and affected candidate genes was assessed based on convergent genomic and genotype-phenotype correlation data. Identification of balanced chromosomal rearrangement breakpoints and interpretation in the context of their genomic background of structural and copy number variants led us to conclude that the infertility due to oligoasthenozoospermia and oligozoospermia is most likely associated with a position effect on YIPF5 and SPATC1L, respectively. In a third proband with intellectual disability and recurrent miscarriage, disruption of CAMK2B causing autosomal dominant, intellectual developmental disorder 54 and increased meiotic segregation during gametogenesis of a der(22) are responsible for the reported phenotype. Our data further support the existence of loci at 5q23 and 21q22.3 for these spermatogenesis defects and highlight the importance of the naturally occurring balanced chromosomal rearrangements for assessment of the pathogenic mechanisms. Furthermore, we show comorbidities due to the same balanced chromosomal rearrangement caused by different pathogenic mechanisms.

Identification of Recurrent Chromosome Breaks Underlying Structural Rearrangements in Mammary Cancer Cell Lines.

Cancer genomes are characterized by the accumulation of small-scale somatic mutations as well as large-scale chromosomal deletions, amplifications, and complex structural rearrangements. This characteristic is at least partially dependent on the ability of cancer cells to undergo recurrent chromosome breakage. In order to address the extent to which chromosomal structural rearrangement breakpoints correlate with recurrent DNA double-strand breaks (DSBs), we simultaneously mapped chromosome structural variation breakpoints (using whole-genome DNA-seq) and spontaneous DSB formation (using Break-seq) in the estrogen receptor (ER)-positive breast cancer cell line MCF-7 and a non-cancer control breast epithelium cell line MCF-10A. We identified concurrent DSBs and structural variation breakpoints almost exclusively in the pericentromeric region of chromosome 16q in MCF-7 cells. We fine-tuned the identification of copy number variation breakpoints on 16q. In addition, we detected recurrent DSBs that occurred in both MCF-7 and MCF-10A. We propose a model for DSB-driven chromosome rearrangements that lead to the translocation of 16q, likely with 10q, and the eventual 16q loss that does not involve the pericentromere of 16q. We present evidence from RNA-seq data that select genes, including SHCBP1, ORC6, and MYLK3, which are immediately downstream from the 16q pericentromere, show heightened expression in MCF-7 cell line compared to the control. Data published by The Cancer Genome Atlas show that all three genes have increased expression in breast tumor samples. We found that SHCBP1 and ORC6 are both strong poor prognosis and treatment outcome markers in the ER-positive breast cancer cohort. We suggest that these genes are potential oncogenes for breast cancer progression. The search for tumor suppressor loss that accompanies the 16q loss ought to be augmented by the identification of potential oncogenes that gained expression during chromosomal rearrangements.

Analysis of complex chromosomal rearrangements using a combination of current molecular cytogenetic techniques

BackgroundUsing combined fluorescence in situ hybridization (FISH) and high-throughput whole-genome sequencing (WGS) molecular cytogenetic technology, we aim to analyze the junction breakpoints of complex chromosome rearrangements (CCR) that were difficult to identify by conventional karyotyping analysis and further characterize the genetic causes of recurrent spontaneous abortion.ResultsBy leveraging a combination of current molecular techniques, including chromosome karyotype analysis, FISH, and WGS, we comprehensively characterized the extremely complex chromosomal abnormalities in this patient with recurrent spontaneous abortions. Here, we demonstrated that combining these current established molecular techniques is an effective and efficient workflow to identify the structural abnormalities of complex chromosomes and locate the rearrangement of DNA fragments.ConclusionsIn conclusion, leveraging results from multiple molecular and cytogenetic techniques can provide the most comprehensive genetic analysis for genetic etiology research, diagnosis, and genetic counseling for patients with recurrent spontaneous abortion and embryonic abortion.

Whole-Genome Mate Pair Sequencing Reflex Test to Characterize Chromosome Rearrangements in Hematologic Neoplasia

Chromosomal alterations such as translocations, inversions and deletions involving one or more breaks are common in hematologic malignancies. Mate pair sequencing (MPseq) is highly informative when a chromosome rearrangement of uncertain significance is identified by conventional chromosome or fluorescence in situ hybridization (FISH) studies and characterization of the genes affected could be helpful, but an alternative clinical assay is not available for this purpose. MPseq utilizes a modified library preparation method to generate paired end sequencing from long fragments of genomic DNA, resulting in higher sensitivity and lower cost compared to conventional Next Generation Sequencing (NGS) for detecting structural variation. Precise molecular characterization by MPseq of clinically significant genes at or near the breakpoints of chromosome rearrangements can provide tremendous diagnostic, prognostic and predictive value, and this assay has already demonstrated its potential to offer targeted therapeutic treatment opportunities for patients with hematologic malignancies.Of the 33 MPseq tests that have been performed following abnormal chromosome or FISH studies of a hematologic malignancy, 15 were for lymphoid and 18 were for myeloid disorders. Each case had a chromosome alteration detected by routine chromosome or by FISH studies. In all cases, multiple partner genes or just one partner gene was known and the MPseq analysis was limited to the region targeted by the rearrangement. Sanger sequencing confirmed each abnormal result identified by MPseq. Of these 33 samples, 26 were found to have direct concordance between MPseq and the targeted rearrangement, 4 demonstrated concordance but the rearrangement was found to be more complex than appreciated from conventional cytogenetic studies and in 3 cases MPseq did not identify the abnormality likely due to the presence of a low level clone and/or as a result of the repetitive nature of the DNA near the rearrangement. Of the 30 concordant cases, MPseq identified in 21 of these a clinically relevant gene at the breakpoint resulting in identification of a fusion gene or deletion of a potentially significant gene within the rearrangement. In 5 cases, the gene at the breakpoint was predicted to be influenced by a position effect such as translocation to an immunoglobulin locus. In all 26 cases where a clinically significant gene was identified, MPseq provided diagnostic, prognostic and/or predictive value which had not been appreciated with conventional cytogenetic studies alone.MPseq was critical for the identification of fusion genes allowing for the classification of Philadelphia Chromosome (Ph)-Like B-ALL. In one example of adult B-ALL, an inv(1)(q25q41) was identified by chromosomes and subsequent MPseq confirmed a chimeric gene comprised of the 5' end of RCSD1 fused to the tyrosine kinase domain of ABL2 resulting in an RCSD1-ABL2 fusion. A second example of relapsed pediatric B-ALL identified an ETV6 rearrangement by FISH and subsequent MPseq confirmed a chimeric gene composed of exons 1-4 of ETV6 and exons 15-20 including the tyrosine kinase domain of NTRK3 . Each fusion identified may be targetable by tyrosine kinase inhibitors. MPseq was also valuable in identification of fusion genes in patients with myeloid malignancies. In one example of infant AML, FISH studies identified a cryptic NUP98 rearrangement and subsequent MPseq confirmed a chimeric gene comprised on exons 1-13 of NUP98 fused to exons 25-28 of the KDM5A gene. This fusion is a known recurrent rearrangement associated with non-Down syndrome acute megakaryocytic leukemia. In summary, by presenting our initial clinical experience along with case examples, we demonstrate that MPseq represents an NGS technology that has the potential to revolutionize the diagnosis of hematologic malignancies and provide an opportunity to advance precision medicine. DisclosuresNo relevant conflicts of interest to declare.

Topologically associating domains and their role in the evolution of genome structure and function in Drosophila.

Topologically associating domains (TADs) were recently identified as fundamental units of three-dimensional eukaryotic genomic organization, although our knowledge of the influence of TADs on genome evolution remains preliminary. To study the molecular evolution of TADs in Drosophila species, we constructed a new reference-grade genome assembly and accompanying high-resolution TAD map for D. pseudoobscura. Comparison of D. pseudoobscura and D. melanogaster, which are separated by ∼49 million years of divergence, showed that ∼30%–40% of their genomes retain conserved TADs. Comparative genomic analysis of 17 Drosophila species revealed that chromosomal rearrangement breakpoints are enriched at TAD boundaries but depleted within TADs. Additionally, genes within conserved TADs show lower expression divergence than those located in nonconserved TADs. Furthermore, we found that a substantial proportion of long genes (>50 kbp) in D. melanogaster (42%) and D. pseudoobscura (26%) constitute their own TADs, implying transcript structure may be one of the deterministic factors for TAD formation. By using structural variants (SVs) identified from 14 D. melanogaster strains, its three closest sibling species from the D. simulans species complex, and two obscura clade species, we uncovered evidence of selection acting on SVs at TAD boundaries, but with the nature of selection differing between SV types. Deletions are depleted at TAD boundaries in both divergent and polymorphic SVs, suggesting purifying selection, whereas divergent tandem duplications are enriched at TAD boundaries relative to polymorphism, suggesting they are adaptive. Our findings highlight how important TADs are in shaping the acquisition and retention of structural mutations that fundamentally alter genome organization.

Resolving Breakpoints of Chromosomal Rearrangements at the Nucleotide Level Using Sanger Sequencing.

Novel cytogenetic tools are increasingly based on genome sequencing for detecting chromosomal abnormalities. Different sequence-based techniques optimized for diagnosis of structural variants can be useful for narrowing down the localization of breakpoints of chromosomal abnormalities, but do not offer nucleotide resolution of breakpoints for proper interpretation of gene disruption. This protocol presents the characterization of structural variants at nucleotide resolution using Sanger sequencing after low-pass large-insert genome sequencing or other long-molecule methods. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Primer design for junction amplification at translocations and inversions Basic Protocol 2: Amplification of derivative chromosomes using a long-range polymerase Alternate Protocol: Amplification of derivative chromosomes using a hot-start polymerase Basic Protocol 3: Preparation of DNA for Sanger sequencing Basic Protocol 4: Interpretation and reporting of breakpoints based on Sanger sequencing.

Non-Random Distribution of Reciprocal Translocation Breakpoints in the Pig Genome.

Balanced chromosome rearrangements are one of the main etiological factors contributing to hypoprolificacy in the domestic pig. Amongst domestic animals, the pig is considered to have the highest prevalence of chromosome rearrangements. To date over 200 unique chromosome rearrangements have been identified. The factors predisposing pigs to chromosome rearrangements, however, remain poorly understood. Nevertheless, here we provide empirical evidence which sustains the notion that there is a non-random distribution of chromosomal rearrangement breakpoints in the pig genome. We sought to establish if there are structural chromosome factors near which rearrangement breakpoints preferentially occur. The distribution of rearrangement breakpoints was analyzed across three level, chromosomes, chromosome arms, and cytogenetic GTG-bands (G-banding using trypsin and giemsa). The frequency of illegitimate exchanges (e.g., reciprocal translocations) between individual chromosomes and chromosome arms appeared to be independent of chromosome length and centromere position. Meanwhile chromosome breakpoints were overrepresented on some specific G-bands, defining chromosome hotspots for ectopic exchanges. Cytogenetic band level factors, such as the length of bands, chromatin density, and presence of fragile sites, were associated with the presence of translocation breakpoints. The characteristics of these bands were largely similar to that of hotspots in the human genome. Therefore, those hotspots are proposed as a starting point for future molecular analyses into the genomic landscape of porcine chromosome rearrangements.

Whole genome paired-end sequencing elucidates functional and phenotypic consequences of balanced chromosomal rearrangement in patients with developmental disorders

BackgroundBalanced chromosomal rearrangements associated with abnormal phenotype are rare events, but may be challenging for genetic counselling, since molecular characterisation of breakpoints is not performed routinely. We used next-generation sequencing...

Genome-wide analysis of genomic alterations induced by oxidative DNA damage in yeast.

Oxidative DNA damage is a threat to genome stability. Using a genetic system in yeast that allows detection of mitotic recombination, we found that the frequency of crossovers is greatly elevated when cells are treated with hydrogen peroxide (H2O2). Using a combination of microarray analysis and genomic sequencing, we mapped the breakpoints of mitotic recombination events and other chromosome rearrangements at a resolution of about 1 kb. Gene conversions and crossovers were the two most common types of events, but we also observed deletions, duplications, and chromosome aneuploidy. In addition, H2O2-treated cells had elevated rates of point mutations (particularly A to T/T to A and C to G/G to C transversions) and small insertions/deletions (in/dels). In cells that underwent multiple rounds of H2O2 treatments, we identified a genetic alteration that resulted in improved H2O2 tolerance by amplification of the CTT1 gene that encodes cytosolic catalase T. Lastly, we showed that cells grown in the absence of oxygen have reduced levels of recombination. This study provided multiple novel insights into how oxidative stress affects genomic instability and phenotypic evolution in aerobic cells.

The Development of Cytogenetic Maps for Malaria Mosquitoes.

Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic basis of a Plasmodium transmission. Physical mapping of genome sequences to polytene chromosomes significantly improves reference assemblies. High-resolution cytogenetic maps are essential for anchoring genome sequences to chromosomes as well as for studying breakpoints of chromosome rearrangements and chromatin protein localization. Here we describe a detailed pipeline for the development of high-resolution cytogenetic maps using polytene chromosomes of malaria mosquitoes. We apply this workflow to the refinement of the cytogenetic map developed for Anopheles beklemishevi.

Cryptic breakpoint identified by whole-genome mate-pair sequencing in a rare paternally inherited complex chromosomal rearrangement

BackgroundPrecise characterization of apparently balanced complex chromosomal rearrangements in non-affected individuals is crucial as they may result in reproductive failure, recurrent miscarriages or affected offspring.Case presentationWe present a family, where the non-affected father and daughter were found, using FISH and karyotyping, to be carriers of a three-way complex chromosomal rearrangement [t(6;7;10)(q16.2;q34;q26.1), de novo in the father]. The family suffered from two stillbirths, one miscarriage, and has a son with severe intellectual disability. In the present study, the family was revisited using whole-genome mate-pair sequencing. Interestingly, whole-genome mate-pair sequencing revealed a cryptic breakpoint on derivative (der) chromosome 6 rendering the rearrangement even more complex. FISH using a chromosome (chr) 6 custom-designed probe and a chr10 control probe confirmed that the interstitial chr6 segment, created by the two chr6 breakpoints, was translocated onto der(10). Breakpoints were successfully validated with Sanger sequencing, and small imbalances as well as microhomology were identified. Finally, the complex chromosomal rearrangement breakpoints disrupted the SIM1, GRIK2, CNTNAP2, and PTPRE genes without causing any phenotype development.ConclusionsIn contrast to the majority of maternally transmitted complex chromosomal rearrangement cases, our study investigated a rare case where a complex chromosomal rearrangement, which most probably resulted from a Type IV hexavalent during the pachytene stage of meiosis I, was stably transmitted from a fertile father to his non-affected daughter. Whole-genome mate-pair sequencing proved highly successful in identifying cryptic complexity, which consequently provided further insight into the meiotic segregation of chromosomes and the increased reproductive risk in individuals carrying the specific complex chromosomal rearrangement. We propose that such complex rearrangements should be characterized in detail using a combination of conventional cytogenetic and NGS-based approaches to aid in better prenatal preimplantation genetic diagnosis and counseling in couples with reproductive problems.

Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress.

Oncogene-induced DNA replication stress contributes critically to the genomic instability present in cancer1–4. However, elucidating how oncogenes deregulate DNA replication has been impeded by the difficulty in mapping replication initiation sites on the human genome. In this study, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 or MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, prior to all genic regions having been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-strand break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

Identification and Use of Personalized Genomic Markers for Monitoring Circulating Tumor DNA.

Digital PCR techniques are ideally suited for accurately quantifying trace amounts of target DNA sequences, such as tumor-derived mutant DNA that is present in the blood circulation of patients with cancer. Here, we describe an approach marrying low-coverage whole-genome sequencing of tumor tissues, to enumerate chromosomal rearrangement breakpoints, together with droplet digital PCR (ddPCR)-based personalized rearrangement assays to cost-effectively monitor circulating tumor DNA levels at multiple time-points during the clinical course. The method is generally applicable to essentially any cancer patient, as all cancers harbor unstable genomes, and may have uses for measuring minimal residual disease, response to therapy, and early detection of metastasis.

3C-PCR: a novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants.

Recent advances in molecular cytogenetics highlight the importance of noncoding structural variation in human disease. Genomic rearrangements can disrupt chromatin architecture, leading to long-range alterations in gene expression. With increasing ability to assess distal gene dysregulation comes new challenges in clinical interpretation of rearrangements. While haplotyping methods to determine compound heterozygosity in a single gene with two pathogenic variants are established, such methods are insufficient for phasing larger distances between a pathogenic variant and a genomic rearrangement breakpoint. Herein, we present an inexpensive and efficient proximity ligation-based method called 3C-PCR for phasing chromosomal rearrangement breakpoints with distal allelic variants. 3C-PCR uses canonical chromosome conformation capture (3C) libraries for targeted distal phasing by implementing a novel nested PCR strategy with primers anchored across the rearrangement breakpoints and subsequent Sanger sequencing. As a proof of concept, 3C-PCR was used to phase a highly variable region 1.3Mb upstream of a chromosomal rearrangement breakpoint in a balanced translocation. We found that the nested PCR approach amplified the derivative chromosome substrate exclusively and identified the same haplotype by Sanger sequencing reliably. Given its efficacy and versatility, 3C-PCR is ideal for use in phasing chromosomal rearrangement breakpoints with allelic variants located at a genomic distance over a megabase.

Effects of Gene Dose, Chromatin, and Network Topology on Expression in Drosophila melanogaster.

Deletions, commonly referred to as deficiencies by Drosophila geneticists, are valuable tools for mapping genes and for genetic pathway discovery via dose-dependent suppressor and enhancer screens. More recently, it has become clear that deviations from normal gene dosage are associated with multiple disorders in a range of species including humans. While we are beginning to understand some of the transcriptional effects brought about by gene dosage changes and the chromosome rearrangement breakpoints associated with them, much of this work relies on isolated examples. We have systematically examined deficiencies of the left arm of chromosome 2 and characterize gene-by-gene dosage responses that vary from collapsed expression through modest partial dosage compensation to full or even over compensation. We found negligible long-range effects of creating novel chromosome domains at deletion breakpoints, suggesting that cases of gene regulation due to altered nuclear architecture are rare. These rare cases include trans de-repression when deficiencies delete chromatin characterized as repressive in other studies. Generally, effects of breakpoints on expression are promoter proximal (~100bp) or in the gene body. Effects of deficiencies genome-wide are in genes with regulatory relationships to genes within the deleted segments, highlighting the subtle expression network defects in these sensitized genetic backgrounds.

DNA recombination. Recombination initiation maps of individual human genomes.

DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.

Mutational showers during carcinogenesis

Mutational showers during carcinogenesis

Clustered Mutations in Yeast and in Human Cancers Can Arise from Damaged Long Single-Strand DNA Regions

Mutations are typically perceived as random, independent events. We describe here nonrandom clustered mutations in yeast and in human cancers. Genome sequencing of yeast grown under chronic alkylation damage identified mutation clusters that extend up to 200 kb. A predominance of "strand-coordinated" changes of either cytosines or guanines in the same strand, mutation patterns, and genetic controls indicated that simultaneous mutations were generated by base alkylation in abnormally long single-strand DNA (ssDNA) formed at double-strand breaks (DSBs) and replication forks. Significantly, we found mutation clusters with analogous features in sequenced human cancers. Strand-coordinated clusters of mutated cytosines or guanines often resided near chromosome rearrangement breakpoints and were highly enriched with a motif targeted by APOBEC family cytosine-deaminases, which strongly prefer ssDNA. These data indicate that hypermutation via multiple simultaneous changes in randomly formed ssDNA is a general phenomenon that may be an important mechanism producing rapid genetic variation.

Detection of gene expression changes at chromosomal rearrangement breakpoints in evolution

BackgroundWe study the relation between genome rearrangements, breakpoints and gene expression. Genome rearrangement research has been concerned with the creation of breakpoints and their position in the chromosome, but the functional consequences of individual breakpoints remain virtually unknown, and there are no direct genome-wide studies of breakpoints from this point of view. A question arises of what the biological consequences of breakpoint creation are, rather than just their structural aspects. The question is whether proximity to the site of a breakpoint event changes the activity of a gene.ResultsWe investigate this by comparing the distribution of distances to the nearest breakpoint of genes that are differentially expressed with the distribution of the same distances for the entire gene complement. We study this in data on whole blood tissue in human versus macaque, and in cerebral cortex tissue in human versus chimpanzee. We find in both data sets that the distribution of distances to the nearest breakpoint of "changed expression genes" differs little from this distance calculated for the rest of the gene complement. In focusing on the changed expression genes closest to the breakpoints, however, we discover that several of these have previously been implicated in the literature as being connected to the evolutionary divergence of humans from other primates.ConclusionsWe conjecture that chromosomal rearrangements occasionally interrupt the regulatory configurations of genes close to the breakpoint, leading to changes in expression.

Using Bacterial Artificial Chromosomes in Leukemia Research: The Experience at the University Cytogenetics Laboratory in Brest,France

The development of the bacterial artificial chromosome (BAC) system was driven in part by the human genome project in order to construct genomic DNA libraries and physical maps for genomic sequencing. The availability of BAC clones has become a valuable tool for identifying cancer genes. We report here our experience in identifying genes located at breakpoints of chromosomal rearrangements and in defining the size and boundaries of deletions in hematological diseases. The methodology used in our laboratory consists of a three-step approach using conventional cytogenetics followed by FISH with commercial probes, then BAC clones. One limitation to the BAC system is that it can only accommodate inserts of up to 300 kb. As a consequence, analyzing the extent of deletions requires a large amount of material. Array comparative genomic hybridization (array-CGH) using a BAC/PAC system can be an alternative. However, this technique has limitations also, and it cannot be used to identify candidate genes at breakpoints of chromosomal rearrangements such as translocations, insertions, and inversions.