R-loops and regulatory changes in chronologically ageing fission yeast cells drive non-random patterns of genome rearrangements

Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins.

P–576 Preimplantation genetic testing for consanguineous couple carrying an identical reciprocal translocation: clinical, ethical considerations and quandaries

Study question Which ethical and clinical aspects should be considered for preimplantation genetic testing PGT-SR strategy management for a consanguineous couple carrying the same reciprocal translocation? Summary answer PGT-SR management required specific probe designs to distinguish chromosomal patterns of balanced embryos, leading to complex transfer choices that required an adapted genetic counseling. What is known already Reciprocal translocation is a classic case in PGT-SR management, since all balanced embryos are transferable without distinguish between normal and balanced embryos. In accordance with the several recommendations, professionals calculated the reproductive risk related to the abnormalities and established an appropriate genetic counseling. However, an extreme case, such as the same reciprocal translocation carried by both members of a couple complicates PGT-SR management at all levels. Mainly, the genetics counseling around balanced embryo transfers. To date, only one study has reported a similar case, however, genetic counseling and the choice of embryos to be transferred have been poorly documented Study design, size, duration This study reports an extremely rare case of a couple (26-year-old woman and 29-year-old man) who was referred to our PGT center of the Montpellier University Hospital after 4 spontaneous miscarriages. The couple, first degree cousins in whom both partners are carrying the same reciprocal translocation 46,XX/XY,t(3;18) t(q26.1;q12.1). The patients were informed of the investigations and gave their consent before participation in the study. Participants/materials, setting, methods Peripheral blood of each member was investigated by FISH to caracterize chromosomal breakpoints. Secondly, a theoretical estimation of different segregation products to find a normal or balanced embryos were performed considering the extreme complexity of the case. Finally, an adapted PGT-SR probe strategy was conceived and proposed to a couple. Choices of balanced embryos to transfer were detailed to ensure that the patient is aware of risks and potential benefits. Main results and the role of chance In this particular case where both members of the couple are carrying the same reciprocal translocation, the chance of finding a normal or balanced embryo was further lowered 2% (4/196). It is estimated that the couple would produce 1 normal embryo and 15 balanced embryos. Diagnostic was possible on 16 biopsied embryos on day 3. Probe signal interpretations revealed four balanced embryos. Two embryos were proposed for a transferred on day 4. These balanced embryos had a different probe patterns, the first balanced embryo was normal and the second balanced embryo resulting from an adjacent–1 segregation mode presented an uniparental disomy (UPD). Limitations, reasons for caution ESHRE recommendations were established for common chromosomal rearrangements. In specific cases, limitations are strongly related to the complexity of the human genome. In this study, the choice of the embryos to be transferred depended entirely to our knowledge of phenotypic consequences of a homozygous gene alterations involved in chromosomal breakpoints. Wider implications of the findings: Professionals were confronted with requests to transfer balanced embryos with a partial/complete UPD or a balanced double translocation homozygote to improve the transfer rate from 3/196 of balanced combinations to 16/196. Dilemma between risks and benefits were considered for counseling to ensure an informed decision-making by patients. Trial registration number NA

Recurring Translocations in Barrett’s Esophageal Adenocarcinoma

Barrett’s esophagus (BE) is a premalignant metaplasia in patients with chronic gastroesophageal reflux disease (GERD). BE can progress to esophageal adenocarcinoma (EA) with less than 15% 5-year survival. Chromosomal aneuploidy, deletions, and duplication are early events in BE progression to EA, but reliable diagnostic assays to detect chromosomal markers in premalignant stages of EA arising from BE are lacking. Previously, we investigated chromosomal changes in an in vitro model of acid and bile exposure-induced Barrett’s epithelial carcinogenesis (BEC). In addition to detecting changes already known to occur in BE and EA, we also reported a novel recurring chromosomal translocation t(10:16) in the BE cells at an earlier time point before they undergo malignant transformation. In this study, we refine the chromosomal event with the help of fluorescence microscopy techniques as a three-way translocation between chromosomes 2, 10, and 16, t(2:10;16) (p22;q22;q22). We also designed an exclusive fluorescent in situ hybridization for esophageal adenocarcinoma (FISH-EA) assay that detects these chromosomal breakpoints and fusions. We validate the feasibility of the FISH-EA assay to objectively detect these chromosome events in primary tissues by confirming the presence of one of the fusions in paraffin-embedded formalin-fixed human EA tumors. Clinical validation in a larger cohort of BE progressors and non-progressors will confirm the specificity and sensitivity of the FISH-EA assay in identifying malignant potential in the early stages of EA.

In BRCA1 and BRCA2 breast cancers, chromosome breaks occur near herpes tumor virus sequences

Inherited mutations in BRCA1 and BRCA2 genes increase risks for breast, ovarian, and other cancers. Both genes encode proteins for accurately repairing chromosome breaks. If mutations inactivate this function, broken chromosome fragments get lost or reattach indiscriminately. These mistakes are characteristic of hereditary breast cancer. We tested the hypothesis that mistakes in reattaching broken chromosomes preferentially occur near viral sequences on human chromosomes. We tested millions of DNA bases around breast cancer breakpoints for similarities to all known viral DNA. DNA around breakpoints often closely matched the Epstein-Barr virus (EBV) tumor variants HKHD40 and HKNPC60. Almost all breakpoints were near EBV anchor sites, EBV tumor variant homologies, and EBV-associated regulatory marks. On chromosome 2, EBV binding sites accounted for 90% of breakpoints (p<0.0001). On chromosome 4, 51/52 inter-chromosomal breakpoints were close to EBV variant sequences. Five viral anchor sites at critical genes were near breast cancer breakpoints. Twenty-five breast cancer breakpoints were within 1.25% of breakpoints in model EBV cancers. EBV-like sequence patterns around breast cancer breakpoints resemble gene fusion breakpoints in model EBV cancers. All BRCA1 and BRCA2 breast cancers had mutated genes essential for immune responses. Because of this immune compromise, herpes viruses can attach and produce nucleases that break chromosomes. Alternatively, anchored viruses can retard break repairs, whatever the causes. The results imply proactive treatment and prevention of herpes viral infections may benefit BRCA mutation carriers.

Application of Chromosome Conformation Capture Method for Detection MYC/TRD Chromosomal Translocation in Leukemia Cell Line

Background: Chromosomal breakpoints are the most common cause of hereditary diseases and cancers. Today, many standard clinical methods such as cytogenetic and PCR based techniques are used which have limitation regarding detection resolution. Chromosome conformation capture is a method for detecting gene proximity and chromosomal rearrangements. Materials and Methods: In this study, SKW3 cell line was used for detecting t(8;14)(q24;q11) using a 3Cbased technique. SKW3 cell line was used for 3C library preparation. For Inverse PCR, two regions were selected in upstream and downstream of the viewpoint locus on chromosome 8-MYC gene based on EcoRI restriction sites. The captured sequence with intra-chromosomal interaction between chr8-c-MYC and chr14-TRD was selected for the translocation PCR primer design. Results: The DNA fragment captured in 3C PCR showed a specific TRD sequence translocated downstream of the MYC gene. Translocation PCR demonstrated the existence of (8; 14) (q24; q11) MYC /TRD in both library and genomic DNA. Conclusion: This result demonstrated 3C- based method could be used as a useful low-cost easy operating technique in chromosomal rearrangements detection. In this study, the integration of whole genome library monitoring and PCR method was used as a high- through put method in chromosomal breakpoints detection.

Centromere Repeats: Hidden Gems of the Genome

Satellite DNAs are now regarded as powerful and active contributors to genomic and chromosomal evolution. Paired with mobile transposable elements, these repetitive sequences provide a dynamic mechanism through which novel karyotypic modifications and chromosomal rearrangements may occur. In this review, we discuss the regulatory activity of satellite DNA and their neighboring transposable elements in a chromosomal context with a particular emphasis on the integral role of both in centromere function. In addition, we discuss the varied mechanisms by which centromeric repeats have endured evolutionary processes, producing a novel, species-specific centromeric landscape despite sharing a ubiquitously conserved function. Finally, we highlight the role these repetitive elements play in the establishment and functionality of de novo centromeres and chromosomal breakpoints that underpin karyotypic variation. By emphasizing these unique activities of satellite DNAs and transposable elements, we hope to disparage the conventional exemplification of repetitive DNA in the historically-associated context of ‘junk’.

Variation and diversity of the breakpoint sequences on 4AL for the 4AL/5AL translocation in Triticum

The translocation of 4AL/5AL in Triticum, which occurred before the differentiation of T. urartu and einkorn, is an important chromosomal rearrangement. Recently, the first identification of breakpoint sequence on 4AL for this translocation provides the opportunity to analyze the variation and diversity of breakpoints in Triticum. In this study, the breakpoint regions of 52 accessions from 21 species were isolated and further characterized. The sequences were divided into 12 types based on their lengths, which ranged from 2009 to 2552 bp. Cluster analysis showed that they were further divided into three groups. Interesting evolutionary relationships among a few of the species were observed and discussed. Multiple sequence alignment of the 52 sequences made it possible to detect 13 insertion and deletion length polymorphisms (InDels) and 101 single nucleotide polymorphisms (SNPs). Furthermore, several species- or accession-specific SNPs or InDels were also identified. Based on BLAST analysis of the conserved sequences, the breakpoint was narrowed down to a 125 bp fragment. Taken together, the results obtained in this study enrich our understanding of chromosomal breakpoints and will be useful for the identification of other breakpoints in wheat.

GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes

Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs) of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large) series of tumor samples. ‘GeneBreak’ is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS). First, ‘GeneBreak’ collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, ‘GeneBreak’, is implemented in R (www.cran.r-project.org) and is available from Bioconductor (www.bioconductor.org/packages/release/bioc/html/GeneBreak.html).

Detection of Chromosomal Translocation in Hematologic Malignancies by a Novel DNA-Based Looped Ligation Assay (LOLA)

BACKGROUND Disease-defining chromosomal translocations are seen in various neoplasms, especially in lymphomas and leukemias. Translocation detection at the DNA level is often complicated by chromosomal breakpoints that are distributed over very large regions. We have developed a ligation-based assay [the looped ligation assay (LOLA)] to detect translocations from diseases with multiple widely spaced breakpoint hot spots. METHODS Oligonucleotide sets that probe breakpoints of IGH-BCL2 (immunoglobulin heavy–apoptosis regulator) in follicular lymphoma (FL), MYC-IGH (MYC proto-oncogene, bHLH transcription factor–immunoglobulin heavy) in Burkitt lymphoma (BL) and BCR-ABL1 (RhoGEF and GTPase activating protein–ABL proto-oncogene 1, non-receptor tyrosine kinase) in chronic myelogenous leukemia (CML) were designed. DNA from cell lines with these translocations was mixed with oligonucleotides in a single-step ligation reaction followed by PCR amplification. Detection was by capillary electrophoresis. We also tested peripheral blood from 16 CML patients and frozen tissue from 17 FL cases, and the results were compared to reverse transcription (RT)-PCR (CML) or fluorescent in situ hybridization (FISH) and δ-PCR (FL). RESULTS LOLA produced signals of the expected sizes for the cell lines. Normal control DNA yielded no signals. A dilution series yielded translocation-specific peaks at dilutions as low as 1%. Signal intensity was log linear to the DNA concentration (R2 = 0.94). Furthermore, we were able to detect a LOLA peak in DNA from 53.3% of FL patients and 87.5% of CML patients. The concordance between LOLA, FISH, and δ-PCR in FL was also excellent. CONCLUSIONS Our results indicate that LOLA is a simple method that is useful for DNA-based detection of translocations in challenging situations, particularly where the breakpoints are not tightly clustered. The assay also has the added benefit of permitting rapid mapping of the breakpoints.