Fragile Sites Research Articles

Next-generation sequencing analysis of embryos from mosaic patients undergoing in vitro fertilization and preimplantation genetic testing

To investigate the effects of parental mosaicism on their preimplantation embryos. Case series. An institute for reproductive and stem cell engineering. Sixty-eight mosaic couples. Assisted reproduction with preimplantation genetic testing (PGT). Karyotypes, embryo-related chromosomal abnormalities, and PGT results. A total of 209 embryos were obtained from 68 mosaic couples, and 153 (73.21%) of 209 of the total embryos were obtained from 55 mosaic couples with abnormal sex chromosome numbers. Of these 153 embryos, 2 (1.31%) had an abnormal copy number of X chromosome, 1 had mosaicism with 46,XN,+X(mosaic, 40%), 1 (0.65%) had an extra Y chromosome, 3 (1.96%) exhibited both X chromosomal and autosomal abnormalities, and 4 (2.61%) exhibited de novo X chromosome structural abnormalities. A total of 56 (26.79%) of 209 embryos were obtained from mosaic couples (n = 13) with abnormal autosomal structures. Notably, of these 56 embryos, 5 (8.93%) had a 16q21-q24.3 copy number abnormality related to the parental karyotype, with a fragile site at 16q22; 5 (7.14%) exhibited 46,XX,dup(8p23.1-8p11.21) and 46,XY,del(8p22-8p11.21), which were related to the parental karyotype; and 10 (17.86%) were de novo chromosome abnormalities. Our data demonstrate that the risk of embryo-related chromosome abnormalities in mosaic patients with abnormal sex chromosomes is very low. Therefore, PGT may not need to be recommended for mosaic patients with abnormal copy numbers of sex chromosomes, especially for patients with financial difficulties. By contrast, the mosaic patients with structural abnormalities of autosomes may have a relatively high risk of abnormal embryos with an unbalanced segment of the involved chromosomes. Thus, PGT is highly recommended for mosaic patients with autosomal structure abnormalities, especially those with a fragile site at 16q22.

The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development.

The Pol32 protein is one of the universal subunits of DNA polymerase δ (Pol δ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol δ localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol δ would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32’s role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism.

ERCC1/XPF Is Important for Repair of DNA Double-Strand Breaks Containing Secondary Structures

SummaryThe structure-specific endonuclease ERCC1/XPF plays an important role in nucleotide excision repair and interstrand cross-link repair. In this study, we identified new functions of ERCC1/XPF in DNA double-strand break (DSB) repair. We found that the conserved function of ERCC1/XPF to remove non-homologous sequences at DSBs is a rate-limiting step for homologous recombination in mammalian cells, and more importantly, we uncovered an indispensable role of ERCC1/XPF in repair of DSBs containing DNA secondary structures, including the structure-prone AT-rich DNA sequences derived from common fragile sites and G-quadruplexes (G4s). We also demonstrated a synthetic lethal interaction of XPF with DNA translocase FANCM that is involved in removing DNA secondary structures. Furthermore, inactivation of XPF sensitizes FANCM-deficient cells to G4-interacting compounds. These results suggest an important function of ERCC1/XPF in protecting DNA secondary structures and provide a rationale for targeted treatment of FANCM-deficient tumors through inhibition of XPF.

Permeability and Surface Hardness Surveying of Stone Damaged by Ballistic Impact

Recent instances of the destruction of cultural assets in conflict zones have demonstrated the need to develop methods which will allow for the assessment of damage to heritage stone in the field. In particular, non-destructive methods would be invaluable when working on sites damaged by contemporary ballistics. Permeability (TinyPerm 3) and surface hardness (Equotip) surveys of stone damaged by 7.62 × 39 mm (AK-47) projectiles were undertaken to determine the ability of these methods to identify the spatial distribution of damage patterns such as shear faces and surface fractures. Results demonstrate the ability of surface hardness surveys to distinguish between non-impacted surfaces of the target stone and surfaces which shattered/sheared upon impact. Whilst spatial distribution analysis (“heat mapping”) of Equotip data did not correlate directly with surface fractures, permeability data heat maps were found to be indicative of surface fracture distribution. The data suggests that compaction of the stone matrix at the impact crater results in a lesser reduction of hardness in this area relative to the wider damaged surface. Surveys of impacted stone using the methods outlined here can identify damage patterns that are not visible to the naked eye, thus aiding in damage identification on fragile sites.

Exposure index of methyl isocyanate (MIC) gas disaster and a comprehensive spectrum of cytogenetic analysis after 30 years.

Severity of clinical expression and high mortality could not facilitate establishing exposure index/association following MIC disaster in Bhopal. Mortality-based exposure stratification was critiqued by the International Medical Commission on Bhopal (IMCB). IMCB stratified exposure considering distance as surrogate at 2km intervals after 10years. The first follow-up cytogenetic screening of the pre-screened survivors after 30years has demonstrated chromosome abnormalities (CA). Exposure stratification was attempted considering cytogenetic screening conducted during 1986-1988. Elevation of CA appeared proportional to exposure status and authenticated the initial mortality-based stratification. The one-on-one comparison of the previous and present cytogenetics has described the individual response to MIC exposure over 30years. Chi-square test has been carried out for checking the cytogenetic changes at the individual level statistically, which revealed that differences of chromosomal aberrations collected immediately post-disaster and 30years later are nonsignificant. The prominence of interindividual variation was noticed in general. The impact of overall exposure was higher in males. Constitutional abnormalities in 8.5% of the study population, including translocation, inversion, deletion, fragile sites, etc., necessitate screening of blood-linked members. The incidence of acrocentric association was prominent in the study population. Normal karyotype in children born to severely exposed parents with congenital anomalies indicates necessity of molecular karyotyping and/or screening of mutations. The study highlights follow-up of the health of the index cases at shorter (3-6months) intervals. This comprehensive spectrum of cytogenetic report highlights immediate post-disaster chromosomal aberrations, the changes that occurred over 30years in conjunction with other environmental factors at the individual level, constitutive genomic aberrations, polymorphic variations, and chromosomal patterns in congenitally malformed children of the survivors, which collectively indicate the possibility of acquisition/persistence of stable aberrations in MIC-exposed lymphocytes through interaction with environmental/biological confounders.

Design and implementation of an augmented reality application for rock art visualization in Cova dels Cavalls (Spain)

Abstract Prehistoric rock art paintings, specifically rock-shelters exposed to environmental and anthropogenic factors, are usually faint and severely damaged, being them difficult to identify and understand by visitors. Augmented Reality (AR) supplements reality with virtual information superimposed onto the real world. This sensor-based technology in smartphones/tablets can improve the paintings experience displaying the 2D digital tracings overlapped onto the real scene (rock with faint paintings). This paper presents an AR application (app) developed in Cova dels Cavalls that shows a recreation of a possible original composition full of motifs with descriptive information to improve current guided tour user experiences. This case study aims to evaluate the rock art AR app targeting non-expert visitors as a means of improving rock art knowledge and sensibility of a fragile archaeological UNESCO Work Heritage site. To achieve this, a variety of participants with different backgrounds and interests tested the AR app on site and answered a complete questionnaire about the use of AR mobile apps. Overall, the results showed great acceptance of this AR app, mainly because in addition to adding new information interactively, it helps to identify the rock art motifs, as well as to recognise them quickly, improving their understanding.

Transcriptional silencing of centromere repeats by heterochromatin safeguards chromosome integrity.

The centromere region of chromosomes consists of repetitive DNA sequences, and is, therefore, one of the fragile sites of chromosomes in many eukaryotes. In the core region, the histone H3 variant CENP-A forms centromere-specific nucleosomes that are required for kinetochore formation. In the pericentromeric region, histone H3 is methylated at lysine 9 (H3K9) and heterochromatin is formed. The transcription of pericentromeric repeats by RNA polymerase II is strictly repressed by heterochromatin. However, the role of the transcriptional silencing of the pericentromeric repeats remains largely unclear. Here, we focus on the chromosomal rearrangements that occur at the repetitive centromeres, and highlight our recent studies showing that transcriptional silencing by heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres in fission yeast. Inactivation of the Clr4 methyltransferase, which is essential for the H3K9 methylation, increased GCRs with breakpoints located in centromeric repeats. However, mutations in RNA polymerase II or the transcription factor Tfs1/TFIIS, which promotes restart of RNA polymerase II following its backtracking, reduced the GCRs that occur in the absence of Clr4, demonstrating that heterochromatin suppresses GCRs by repressing the Tfs1-dependent transcription. We also discuss how the transcriptional restart gives rise to chromosomal rearrangements at centromeres.

WWOX Inhibits Metastasis of Triple-Negative Breast Cancer Cells via Modulation of miRNAs.

Triple-negative breast cancer (TNBC) is a heterogeneous, highly aggressive, and difficult to treat tumor type. The tumor suppressor WWOX spans FRA16D, a common fragile site that is commonly altered in breast cancer. Despite recent progress, the role of WWOX in TNBC metastasis is unknown. Here we report that WWOX inactivation correlates with advanced stages of TNBC and that its levels are frequently altered in TNBC cells. Ectopic restoration of WWOX in WWOX-negative TNBC cells inhibited metastasis while its depletion in WWOX-positive TNBC cells promoted metastasis. WWOX was a negative regulator of c-MYC, which regulated miR-146a expression and consequently fibronectin levels, contributing to an epithelial status of the cell. Treatment of TNBC cells with anti-miR-146a rescued the WWOX antimetastatic phenotype. Moreover, overexpression of MYC in WWOX-expressing TNBC cells overrode WWOX effects on miR-146a and fibronectin levels. Altogether, our data uncover an essential role for WWOX in antagonizing TNBC progression and highlight its potential use as a biomarker for metastasis. SIGNIFICANCE: These findings highlight the mechanism by which the tumor suppressor WWOX regulates metastasis of triple-negative breast cancer.See related commentary by Sharma, p. 1746.

High BrdU Sensitivity of Passeriformes Chromosomes: Conservation of BrdU-Sensitive Fragile Sites on Their Z Chromosomes during Evolution

Amongst 15 bird species, representative of 7 orders, recurrent breakages evocating the presence of fragile sites were detected in the chromosomes of the 5 species belonging to Passeriformes. These breaks appeared when 5-bromodeoxyuridine (BrdU) was added to the cell culture medium at a dose inefficient for inducing chromosome structure alterations in other birds and mammals. They involved, similarly in male and female, 3 loci on the Z chromosome of 3 Turdus species (Turdidae). Labeling by BrdU antibody confirmed the correlation between BrdU incorporation into DNA and breakage, especially around and in the sites of breakage. Thus, 3 BrdU-sensitive fragile sites were present in the Z chromosomes of these birds. Three fragile sites were also detected at different locations in the Z chromosomes of the European robin (Erithacus rubecula, Muscicapidae), suggesting that a structural rearrangement occurred during the evolution of Turdidae and Muscicapidae. Chromosome banding confirmed this interpretation. Finally, in the more distantly related species Parus major (Paridae), the almost acrocentric Z chromosome displayed a single BrdU-sensitive fragile site in its short arm, and the W appeared to be pulverized by BrdU incorporation. Although it cannot be excluded that the BrdU-sensitive fragile sites may be involved in rearrangements, their conservation in many species, and possibly all Passeriformes, provides evidence that they do not constitute a pejorative character during evolution.

HumCFS: a database of fragile sites in human chromosomes

BackgroundFragile sites are the chromosomal regions that are susceptible to breakage, and their frequency varies among the human population. Based on the frequency of fragile site induction, they are categorized as common and rare fragile sites. Common fragile sites are sensitive to replication stress and often rearranged in cancer. Rare fragile sites are the archetypal trinucleotide repeats. Fragile sites are known to be involved in chromosomal rearrangements in tumors. Human miRNA genes are also present at fragile sites. A better understanding of genes and miRNAs lying in the fragile site regions and their association with disease progression is required.ResultHumCFS is a manually curated database of human chromosomal fragile sites. HumCFS provides useful information on fragile sites such as coordinates on the chromosome, cytoband, their chemical inducers and frequency of fragile site (rare or common), genes and miRNAs lying in fragile sites. Protein coding genes in the fragile sites were identified by mapping the coordinates of fragile sites with human genome Ensembl (GRCh38/hg38). Genes present in fragile sites were further mapped to DisGenNET database, to understand their possible link with human diseases. Human miRNAs from miRBase was also mapped on fragile site coordinates. In brief, HumCFS provides useful information about 125 human chromosomal fragile sites and their association with 4921 human protein-coding genes and 917 human miRNA’s.ConclusionUser-friendly web-interface of HumCFS and hyper-linking with other resources will help researchers to search for genes, miRNAs efficiently and to intersect the relationship among them. For easy data retrieval and analysis, we have integrated standard web-based tools, such as JBrowse, BLAST etc. Also, the user can download the data in various file formats such as text files, gff3 files and Bed-format files which can be used on UCSC browser.Database URL:http://webs.iiitd.edu.in/raghava/humcfs/

Sequence and Nuclease Requirements for Breakage and Healing of a Structure-Forming (AT)n Sequence within Fragile Site FRA16D

Common fragile sites (CFSs) are genomic regions thatdisplay gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied an ∼300-bp subregion (Flex1) of human CFS FRA16D in yeast and found that it recapitulates characteristics of CFS fragility in human cells. Flex1 fragility is dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication. Fragility at Flex1 is initiated by structure-specific endonuclease Mus81-Mms4 acting together with the Slx1-4/Rad1-10 complex, whereas Yen1 protects Flex1 against breakage. Sae2 is required for healing of Flex1 after breakage. Our study shows that breakage within a CFS can be initiated by nuclease cleavage at forks stalled at DNA structures. Furthermore, our results suggest that CFSs are not just prone to breakage but also are impaired in their ability to heal, and this deleterious combination accounts for their fragility.

A Virtual Reality Platform for Analyzing Remote Archaeological Sites.

This paper describes a Virtual Reality (VR) prototype developed to help archaeologists and other stakeholders explore and analyse archaeological data in a more immersive context. We describe a VR reconstruction of Pleito Cave, a fragile world class rock-art site with accessibility limitation. Key stakeholders are identified and a prototype is described that provides a VR platform for visualizing and interacting with complex archaeological data (gathered from techniques such as decorrelation stretch and X-ray fluorescence) virtually ‘in situ’, in a way that would not be possible at the real site. The prototype allows multiple remote users to interact with the cave together remotely providing opportunities for collaborative interpretation and analysis of archaeological data. We also present a survey-based evaluation in which both archaeologists and Native American stakeholders indicate positive responses for measures of both engagement and value.

Using fragile sites of chromosomes and chromosomal instability of peripheral blood lymphocytes to predict the development of endometrial cancer

Using fragile sites of chromosomes and chromosomal instability of peripheral blood lymphocytes to predict the development of endometrial cancer

Sequence and Nuclease Requirements for Breakage and Healing of a Structure-Forming (AT)n Sequence within Fragile Site FRA16D

Common fragile sites (CFSs) are genomic regions that display gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied a ~300 basepair subregion (Flex1) of human CFS FRA16D in yeast, and found it recapitulated characteristics of CFS fragility in human cells. Flex1 fragility was dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication. Fragility at Flex1 is initiated by structure-specific endonuclease Mus81-Mms4, likely acting within an Slx1-4- Rad1-10 complex, while Yen1 protects Flex1 against breakage. Sae2 is required for healing of Flex1 after breakage. Our study shows that breakage within a CFS can be initiated by nuclease cleavage of forks stalled at DNA structures. Furthermore, our results suggest that CFSs are not just prone to breakage but also impaired in their ability to heal, and this deleterious combination accounts for their fragility.

Complex structural rearrangements are present in high-grade dysplastic Barrett\u2019s oesophagus samples

BackgroundOesophageal adenocarcinoma (EAC) incidence is increasing and has a poor survival rate. Barrett’s oesophagus (BE) is a precursor condition that is associated with EAC and often occurs in conjunction with chronic gastro-oesophageal reflux, however many individuals diagnosed with BE never progress to cancer. An understanding of the genomic features of BE and EAC may help with the early identification of at-risk individuals.MethodsIn this study, we assessed the genomic features of 16 BE samples using whole-genome sequencing. These included non-dysplastic samples collected at two time-points from two BE patients who had not progressed to EAC over several years. Seven other non-dysplastic samples and five dysplastic BE samples with high-grade dysplasia were also examined. We compared the genome profiles of these 16 BE samples with 22 EAC samples.ResultsWe observed that samples from the two non-progressor individuals had low numbers of somatic single nucleotide variants, indels and structural variation events compared to dysplastic and the remaining non-dysplastic BE. EAC had the highest level of somatic genomic variations. Mutational signature 17, which is common in EAC, was also present in non-dysplastic and dysplastic BE, but was not present in the non-progressors. Many dysplastic samples had mutations in genes previously reported in EAC, whereas only mutations in CDKN2A or in the fragile site genes appeared common in non-dysplastic samples. Rearrangement signatures were used to identify a signature associated with localised complex events such as chromothripsis and breakage fusion-bridge that are characteristic of EACs. Two dysplastic BE samples had a high contribution of this signature and contained evidence of localised rearrangements. Two other dysplastic samples also had regions of localised structural rearrangements. There was no evidence for complex events in non-dysplastic samples.ConclusionsThe presence of complex localised rearrangements in dysplastic samples indicates a need for further investigations into the role such events play in the progression from BE to EAC.

The role of fork stalling and DNA structures in causing chromosome fragility.

Alternative non-B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single-stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT-rich and break under replication stress, yet the molecular basis for their fragility is still being investigated. Over the past several years, new evidence has linked both the formation of secondary structures and transcription to fork stalling and fragility of CFSs. How these two events may synergize to cause fragility and the role of nuclease cleavage at secondary structures in rare and CFSs are discussed here. We also highlight evidence for a new hypothesis that secondary structures at CFSs not only initiate fragility but also inhibit healing, resulting in their characteristic appearance.

PREFACE.

Genes, Chromosomes and CancerVolume 58, Issue 5 p. 257-259 PREFACE PREFACE Kay Huebner, Corresponding Author Kay Huebner kay.huebner@osumc.edu orcid.org/0000-0002-2848-1382 Search for more papers by this author Kay Huebner, Corresponding Author Kay Huebner kay.huebner@osumc.edu orcid.org/0000-0002-2848-1382 Search for more papers by this author First published: 22 December 2018 https://doi.org/10.1002/gcc.22722Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume58, Issue5Special Issue: The intersection of chromosome fragile sites and genome instabilityMay 2019Pages 257-259 RelatedInformation

Environmental Assessment in Neotropical Watersheds: A Multi-Factorial Approach

This study evaluated the environmental conditions of two watersheds selected on the basis of similarities in land use. The environmental conditions of the watersheds were analyzed using two geoprocessing methods and in situ evaluations by applying the rapid assessment protocol (RAP). Geospatial image processing was used to analyze land use, slope, soil classification, and rainfall, while RAP was used to evaluate the connectivity and size of riparian corridors. The results revealed varied uses of land with the landscape matrix in both watersheds, composed of agriculture, pasture, and urban centers. Urban centers were defined as spots and riparian zones as corridors. The analysis of environmental fragility considering all the geospatial variables, classified both watersheds as having medium fragility. The most fragile sites were the urban centers, which had a high slope and degraded riparian zone. Onsite assessments have shown that corridors do not have the size required by legislation; they are fragmented and unstructured, and they contain exotic species. We also propose that a multi-factorial approach be used to evaluate watersheds, associating geospatial assessments and onsite analyses that consider the limitations pointed out in each protocol. This reduces sampling errors and shows the actual state of conservation in riparian zones.

IGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity

Genome engineering methods have advanced greatly with the development of programmable nucleases, but methods for quantifying on- and off-target cleavage sites and associated deletions remain nascent. Here, we report an improvement of the GUIDE-seq method, iGUIDE, which allows filtering of mispriming events to clarify the true cleavage signal. Using iGUIDE, we specify the locations of Cas9-guided cleavage for four guide RNAs, characterize associated deletions, and show that naturally occurring background DNA double-strand breaks are associated with open chromatin, gene dense regions, and chromosomal fragile sites. iGUIDE is available from https://github.com/cnobles/iGUIDE.

Genomic Instability in Cancer II: 4N-Skewed (90°) Reductive Division via Fragile Sites to Fitness Increase for Solid and Hematological Cancer Beginnings

The objective herein was to connect the ontogeny process of diplochromosomal, amitotic, 4n-skewed division-system, to cytogenetic deficiency lesions in satellite, repetitive DNAs, especially in the chromosomal fragile sites, some 100 distributed over the genome. These latter studies had shown that chemical induced replication-stress led to un-replicated lesions in these fragile sites, which from inaccurate repair processes caused genomic instability. In the chain of events of the ontogeny process to the special tetraploidy, it was proposed that primary damaged human cells could undergo replication stress from repair-process present during cell replication, a suggestion verified by X-ray damaged cells producing the unstable fragile sites (see text). The cancer-importance for therapy is recognition of cell cycle change for the 4n derivative fitness-gained, diploid progeny cells. An open question is whether RB controlling G1 to S-period is mutated at this suggested tumorigenesis initiating phase, and if so, with what consequences for therapy. The fragile site studies further showed that repair of repetitive DNAs could produce two types of genomic changes: single gene mutations and CNVs, which were here shown to be chromosomally located on “borders” to repairing satellite lesions. This genomic placement was found to correspond to mutations identified in tumor sequencing (p53, Rb, MYC), favoring a bad luck location for their cancer “mutational nature”. The CNVs in cancers, are here seen as molecular expressions of long-known cytogenetic HSRs and DMs also with demonstrated origin from amplifications of single genes. Over-expression of oncogenes was hinted of being from duplications, but Drosophila genetics demonstrated the opposite, gene inactivation. The reduced eye-size from dominant, BAR-Ultra-Bar-eye phenotypes, was caused by duplications, inactivating the genetic system for eye-size. The finding of CNVs showing “evasion” of the immune system suggests, inactivation of immune-determining genetics. Since mutated genes on borders to satellite DNAs are a fact in hematological cancers, the 4n-skewed division-system is suggested to replace debated leukemogenesis with fitness-gain from molecular mutations. For these cancers the question is how normal bone marrow cells attain genomic damage for special tetraploidy, which was referred to studies of cells moving in artificial marrow-like substrate, needing serious attention.