Chromosome Breakage Research Articles

Congenital Telangiectatic Erythema: Scoping Review.

Congenital telangiectatic erythema (CTE), also known as Bloom syndrome, is a rare autosomal recessive disorder characterized by below-average height, a narrow face, a red skin rash occurring on sun-exposed areas of the body, and an increased risk of cancer. CTE is one of many genodermatoses and photodermatoses associated with defects in DNA repair. CTE is caused by a mutation occurring in the BLM gene, which causes abnormal breaks in chromosomes. We aimed to analyze the existing literature on CTE to provide additional insight into its heredity, the spectrum of clinical presentations, and the management of this disorder. In addition, the gaps in current research and the use of artificial intelligence to streamline clinical diagnosis and the management of CTE are outlined. A literature search was conducted on PubMed, DOAJ, and Scopus using search terms such as "congenital telangiectatic erythema," "bloom syndrome," and "bloom-torre-machacek." Due to limited current literature, studies published from January 2000 to January 2023 were considered for this review. A total of 49 sources from the literature were analyzed. Through this scoping review, the researchers were able to identify several publications focusing on Bloom syndrome. Some common subject areas included the heredity of CTE, clinical presentations of CTE, and management of CTE. In addition, the literature on rare diseases shows the potential advancements in understanding and treatment with artificial intelligence. Future studies should address the causes of heterogeneity in presentation and examine potential therapeutic candidates for CTE and similarly presenting syndromes. This review illuminated current advances in potential molecular targets or causative pathways in the development of CTE as well as clinical features including erythema, increased cancer risk, and growth abnormalities. Future studies should continue to explore innovations in this space, especially in regard to the use of artificial intelligence, including machine learning and deep learning, for the diagnosis and clinical management of rare diseases such as CTE.

Diagnosis status and genetic characteristics analysis of Fanconi anemia in China

Objective: To analyze the clinical and molecular diagnostic status of Fanconi anemia (FA) in China. Methods: The General situation, clinical manifestations and chromosome breakage test and genetic test results of 107 pediatric FA cases registered in the Chinese Blood and Marrow Transplantation Registry Group (CBMTRG) and the Chinese Children Blood and Marrow Transplantation Registry Group (CCBMTRG) from August 2009 to January 2022 were analyzed retrospectively. Children with FANCA gene variants were divided into mild and severe groups based on the type of variant, and Wilcoxon-test was used to compare the phenotypic differences between groups. Results: Of the 176 registered FA patients, 69 (39.2%) cases were excluded due to lack of definitive genetic diagnosis results, and the remaining 107 children from 15 hospitals were included in the study, including 70 males and 37 females. The age at transplantation treatment were 6 (4, 9) years. The enrolled children were involved in 10 pathogenic genes, including 89 cases of FANCA gene, 7 cases of FANCG gene, 3 cases of FANCB gene, 2 cases of FANCE gene and 1 case each of FANCC, FANCD1, FANCD2, FANCF, FANCJ, and FANCN gene. Compound heterozygous or homozygous of loss-of-function variants account for 69.2% (72/104). Loss-of-function variants account for 79.2% (141/178) in FANCA gene variants, and 20.8% (37/178) were large exon deletions. Fifty-five children (51.4%) had chromosome breakage test records, with a positive rate of 81.8% (45/55). There were 172 congenital malformations in 80 children.Café-au-Lait spots (16.3%, 28/172), thumb deformities (16.3%,28/172), polydactyly (13.9%, 24/172), and short stature (12.2%, 21/172) were the most common congenital malformations in Chinese children with FA. No significant difference was found in the number of congenital malformations between children with severe (50 cases) and mild FANCA variants (26 cases) (Z=-1.33, P=0.185). Conclusions: FANCA gene is the main pathogenic gene in children with FA, where the detection of its exon deletion should be strengthened clinically. There were no phenotypic differences among children with different types of FANCA variants. Chromosome break test is helpful to determine the pathogenicity of variants, but its accuracy needs to be improved.

Molecular characterization of TCF3::PBX1 chromosomal breakpoints in acute lymphoblastic leukemia and their use for measurable residual disease assessment

The translocation t(1;19)(q23;p13) with the resulting chimeric TCF3::PBX1 gene is the third most prevalent recurrent chromosomal translocation in acute lymphoblastic leukemia and accounts for 3–5% of cases. The molecular background of this translocation has been incompletely studied, especially in adult cases. We characterized the chromosomal breakpoints of 49 patients with TCF3::PBX1 and the corresponding reciprocal PBX1::TCF3 breakpoints in 15 cases at the molecular level, thus providing an extensive molecular overview of this translocation in a well-defined study patient population. Breakpoints were found to be remarkably clustered not only in TCF3 but also in PBX1. No association with DNA repeats or putative cryptic recombination signal sequence sites was observed. A simplified detection method for breakpoint identification was developed and the feasibility of patient-specific chromosomal break sites as molecular markers for detecting measurable residual disease (MRD) was explored. A highly sensitive generic real-time PCR for MRD assessment using these breakpoint sequences was established that could serve as a useful alternative to the classical method utilizing rearranged immune gene loci. This study provides the first extensive molecular data set on the chromosomal breakpoints of the t(1;19)/TCF3::PBX1 aberration in adult ALL. Based on the obtained data a generic MRD method was developed that has several theoretical advantages, including an on average higher sensitivity and a greater stability of the molecular marker in the course of disease.

Mitochondrial superoxide dismutase Sod2 suppresses nuclear genome instability during oxidative stress.

Oxidative stress can damage DNA and thereby contribute to genome instability. To avoid an imbalance or overaccumulation of reactive oxygen species (ROS), cells are equipped with antioxidant enzymes that scavenge excess ROS. Cells lacking the RecQ-family DNA helicase Sgs1, which contributes to homology-dependent DNA break repair and chromosome stability, are known to accumulate ROS, but the origin and consequences of this oxidative stress phenotype are not fully understood. Here, we show that the sgs1 mutant exhibits elevated mitochondrial superoxide, increased mitochondrial mass, and accumulation of recombinogenic DNA lesions that can be suppressed by antioxidants. Increased mitochondrial mass in the sgs1Δ mutant is accompanied by increased mitochondrial branching, which was also inducible in wildtype cells by replication stress. Superoxide dismutase Sod2 genetically interacts with Sgs1 in the suppression of nuclear chromosomal rearrangements under paraquat (PQ)-induced oxidative stress. PQ-induced chromosome rearrangements in the absence of Sod2 are promoted by Rad51 recombinase and the polymerase subunit Pol32. Finally, the dependence of chromosomal rearrangements on the Rev1/Pol ζ mutasome suggests that under oxidative stress successful DNA synthesis during DNA break repair depends on translesion DNA synthesis.

The abscission checkpoint senses chromatin bridges through Top2α recruitment to DNA knots.

In response to chromatin bridges, the abscission checkpoint delays completion of cytokinesis to prevent chromosome breakage or tetraploidization. Here, we show that spontaneous or replication stress-induced chromatin bridges exhibit "knots" of catenated and overtwisted DNA next to the midbody. Topoisomerase IIα (Top2α) forms abortive Top2-DNA cleavage complexes (Top2ccs) on DNA knots; furthermore, impaired Top2α-DNA cleavage activity correlates with chromatin bridge breakage in cytokinesis. Proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated double-strand DNA ends on DNA knots; in turn, Rad17 promotes local recruitment of the MRN complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin breakage. In contrast, dicentric chromosomes that do not exhibit knotted DNA fail to activate the abscission checkpoint in human cells. These findings are the first to describe a mechanism by which the abscission checkpoint detects chromatin bridges, through generation of abortive Top2ccs on DNA knots, to preserve genome integrity.

Replication stress causes delayed mitotic entry and chromosome 12 fragility at the ANKS1B large neuronal gene in human induced pluripotent stem cells.

Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.

Evaluation of Genotoxic and Cytotoxic Effects of Gluten in Male Albino Mice

Gluten is a protein commonly found in daily diets in the form of wheat, barley, rye, and other grains. It serves as the structural component in flour, providing the binding qualities that maintain the shape and texture of food items. This study aimed to investigate the genotoxic and cytotoxic effects of gluten on bone marrow chromosomes and DNA of male albino mice. The animals were divided into four groups: a control group, a negative control group that received an oral dose of 0.02M glacial acetic acid, and two groups that were treated with gluten dissolved in 0.02M glacial acetic acid at doses of 1.5 g/kg and 3.0 g/kg body weight. The treated animals received oral doses with non-consecutively three times a week for a period of four weeks. The study evaluated chromosomal aberrations in the bone marrow, micronucleus test, and DNA damage using the comet assay. The results of the study showed that treatment with 1.5 and 3.0g/kg body weight of gluten induced chromosomal aberrations and damage in DNA content, with an increase in the severity of effects at a higher dose of gluten. The chromosomal aberrations seen included deletion, fragment, centromeric attenuation, centric fusion, ring formation, end to end association, chromosomal gap, beaded chromosomes, and polyploidy. The micronucleus test revealed toxicity in the bone marrow, as shown by appearance of micronuclei in polychromatic erythrocytes and a reduction in the ratio of polychromatic erythrocytes. The comet assay showed a significant increase of DNA damage in the tail length of the comet cells. This study concluded that the treatment with gluten has detrimental effects on the bone marrow chromosomes and DNA of mice, as demonstrated by the increased chromosomal aberrations, micronuclei, and DNA damage observed in the treated mice. So, the use of gluten should be within an acceptable and safe range.

Inactivating TDP2 missense mutation in siblings with congenital abnormalities reminiscent of fanconi anemia

Mutations in TDP2, encoding tyrosyl-DNA phosphodiesterase 2, have been associated with a syndromal form of autosomal recessive spinocerebellar ataxia, type 23 (SCAR23). This is a very rare and progressive neurodegenerative disorder described in only nine patients to date, and caused by splice site or nonsense mutations that result in greatly reduced or absent TDP2 protein. TDP2 is required for the rapid repair of DNA double-strand breaks induced by abortive DNA topoisomerase II (TOP2) activity, important for genetic stability in post-mitotic cells such as neurons. Here, we describe a sibship that is homozygous for the first TDP2 missense mutation (p.Glu152Lys) and which presents with clinical features overlapping both SCAR23 and Fanconi anemia (FA). We show that in contrast to previously reported SCAR23 patients, fibroblasts derived from the current patient retain significant levels of TDP2 protein. However, this protein is catalytically inactive, resulting in reduced rates of repair of TOP2-induced DNA double-strand breaks and cellular hypersensitivity to the TOP2 poison, etoposide. The TDP2-mutated patient-derived fibroblasts do not display increased chromosome breakage following treatment with DNA crosslinking agents, but both TDP2-mutated and FA cells exhibit increased chromosome breakage in response to etoposide. This suggests that the FA pathway is required in response to TOP2-induced DNA lesions, providing a possible explanation for the clinical overlap between FA and the current TDP2-mutated patients. When reviewing the relatively small number of patients with SCAR23 that have been reported, it is clear that the phenotype of such patients can extend beyond neurological features, indicating that the TDP2 protein influences not only neural homeostasis but also other tissues as well.

Cytogenetic findings in Polish patients with suspected Fanconi anemia.

The high sensitivity of cells of Fanconi anemia (FA) patients to DNA cross-linking agents (clastogens), such as mitomycin C (MMC), was used as a screening tool in Polish children with clinical suspicion of FA. The aim of the study was to compare chromosome fragility between 3 groups, namely non-FA, possible mosaic FA and FA patients. The study included 100 children with hematological manifestations and/or congenital defects characteristic of FA, and 100 healthy controls. Blood samples obtained from participants were analyzed using an MMC-induced chromosomal breakage test. Patients with clinical suspicion of FA were divided into 3 subgroups based on the MMC test results, namely FA, possible mosaic FA and non-FA. Thirteen out of 100 patients had a true FA cellular phenotype. The mean value of MMC-induced chromosome breaks/cell for FA patients was higher than for non-FA patients (6.67 ±3.92 compared to 0.23 ±0.18). In addition, the percentage of cells with spontaneous aberrations was more than 9 times higher in FA patients than in non-FA patients. Our results confirmed that the MMC sensitivity test distinguishes between individuals affected by FA, those with possible somatic mosaicism, and patients with bone marrow failure for other reasons, who were classified as non-FA in the first diagnostic step. However, a definitive differential diagnosis requires follow-up mutation testing and chromosome breakage analysis of skin fibroblasts.

Biological response to the continuous occupational exposure to antineoplastic drugs and radionuclides

Purpose Antineoplastic drugs and radioiodine are recognized occupational risk factors affecting the genetic material of exposed persons. To assess cytogenetic damage and evaluate the presence of chromosomal instability during occupational exposure, a biomonitoring study was performed using a chromosomal aberration assay and a cytokinesis-block micronucleus (CBMN) test. Materials and Methods Blood samples from 314 healthy donors divided into 3 groups (control, exposed to antineoplastic drugs and exposed to radioiodine) were collected and cytogenetically analyzed. Results There was an increase in almost all analyzed parameters registered in the exposed persons. Chromatid breaks were higher in the subjects exposed to antineoplastic drugs, while dicentrics and premature centromere division (PCD) parameters were higher in nuclear medicine workers. The total number of micronuclei was higher in both groups of the exposed. The correlation analysis indicated the association of dicentrics, acentrics, chromosome and chromatid break with PCDs in both groups of the exposed, and micronuclei and nucleoplasmic bridges with PCDs in the subjects exposed to radioiodine. The discriminant analysis marked off PCD1-5 as the best predictor of exposure. Age, sex, sampling season and duration of exposure significantly influenced the analyzed parameters, while smoking habits did not show any influence. Conclusion Based on the observed results, premature centromere division can be considered a valuable parameter of genotoxic risk for individuals occupationally exposed to low doses of ionizing radiation.

Chromosomal aberrations induced by Mitomycin C in canine lymphocytes

Chromosomal aberrations comprise an abnormal number of chromosomes as well as changes in the structure of the chromosomes. Aberrations in the structure of chromosomes, such as gaps and breaks in chromatids and chromosomes, acentric fragments, telomeric associations, deletions, early chromatid-separation and large scale effects, such as pulverized metaphases, and sticky metaphases. In this study, the effect of Mitomycin C, a recognized clastogen on human lymphocytes, was assayed in cultures of canine lymphocytes, an animal species that is flourishing in comparative medicine. Blood cultures were performed on samples from a male and a female dog. Cytotoxicity using the mitotic index and genotoxicity testing were performed with 0.25 µg/ml Mitomycin C. The total chromosomal aberrations were significantly higher due to the effect of Mitomycin C (P=0.0247). The number of chromatid breaks nearly quintupled, while pulverized metaphases were found to be six times more frequent, and endoduplicated cells were three times higher than in negative control cultures. The quantitatively most relevant chromosomal aberration was the presence of sticky metaphases, related to adverse effects in chromatin proteins. The potential use of canine lymphocytes for chromosomal aberration assay is emphasized in the evaluation or re-evaluation of the genotoxic in vitro effect of xenobiotics, to evidence chromosomal damage.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity

DNA single‐strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair‐defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5‐chloro‐2′‐deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA‐defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

Laser Ablation-Generated Crystalline Selenium Nanoparticles Prevent Damage of DNA and Proteins Induced by Reactive Oxygen Species and Protect Mice against Injuries Caused by Radiation-Induced Oxidative Stress.

With the help of laser ablation, a technology for obtaining nanosized crystalline selenium particles (SeNPs) has been created. The SeNPs do not exhibit significant toxic properties, in contrast to molecular selenium compounds. The administration of SeNPs can significantly increase the viabilities of SH-SY5Y and PCMF cells after radiation exposure. The introduction of such nanoparticles into the animal body protects proteins and DNA from radiation-induced damage. The number of chromosomal breaks and oxidized proteins decreases in irradiated mice treated with SeNPs. Using hematological tests, it was found that a decrease in radiation-induced leukopenia and thrombocytopenia is observed when selenium nanoparticles are injected into mice before exposure to ionizing radiation. The administration of SeNPs to animals 5 h before radiation exposure in sublethal and lethal doses significantly increases their survival rate. The modification dose factor for animal survival was 1.2. It has been shown that the introduction of selenium nanoparticles significantly normalizes gene expression in the cells of the red bone marrow of mice after exposure to ionizing radiation. Thus, it has been demonstrated that SeNPs are a new gene-protective and radioprotective agent that can significantly reduce the harmful effects of ionizing radiation.

Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast.

A targeted double-strand break introduced into the genome of Saccharomyces cerevisiae is repaired by the relatively error-prone nonhomologous end joining (NHEJ) pathway when homologous recombination is not an option. A zinc finger nuclease cleavage site was inserted out-of-frame into the LYS2 locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5' overhangs. Repair events that destroyed the cleavage site were identified either as Lys+ colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys+ events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol ζ and Pol η. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required translesion synthesis polymerases as well as the exonuclease activity of the replicative Pol δ DNA polymerase. Survivors were equally split between NHEJ events and 1.2 or 11.7 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1-Rad10 endonuclease for the removal of presumptive 3' tails. Finally, NHEJ was more efficient in nongrowing than in growing cells and was most efficient in G0 cells. These studies provide novel insights into the flexibility and complexity of error-prone DSB repair in yeast.

CYTOGENETIC VARIABILITY OF THE HUTSUL BREED OF HORSES

Goal. Study of features of karyotypic variability of Hutsul horses. Methods. Cytogenetic analysis, micronucleus test. Results. The average rate of genomic disorders (aneuploidy) was 10.8%, with a range of values of this variability of 8.8%–11.1% in animals of the LLC "Krai Neba" and the National Nature Park "Hutsulshchyna" with a statistically insignificant difference between the mean values. The multiple increase in the number of chromosomes, polyploidy, was in the range of 1.1%–2.0%, which does not exceed the species of this variability. The magnitude of the asynchronous divergence of the centromeric regions of the chromosomes was 2.0%–6.0%, which corresponds to the spontaneous level for horses as a whole (2.2%–9.1%). Structural chromosome abnormalities (chromosomal breaks) in the horses of the Krai Neba LLC farm were not detected, and in the animals of the Hutsulshchyna National Nature Park they were 1.1%. The range of variability of lymphocytes with a micronucleus for animals of two farms was 4.0–4.2‰. The number of dinuclear lymphocytes (6.2‰) exceeded the mitotic index (4.3‰) with an insignificant difference in the average values, which indicates the absence of radionuclide contamination of the territory. Conclusions. The average number of aneuploid cells – 10.8%, polyploid – 1.4%, asynchronous divergence of the centromeric regions of chromosomes – 3.9% and the proportion of chromosomal breaks – 1.1%. The proportion of lymphocytes with a micronucleus – 4.0–4.2–4, dinuclear lymphocytes – 6.2‰ and the mitotic index – 4.3‰, which indicates the stability of the karyotype of the studied horses.

Nuclear Technology in Horticulture: Boosting Productivity, Controlling Pests, Conserving Water

Horticulture faces significant challenges due to the destruction of crops by pests and diseases, both before and after harvesting, resulting in a significant reduction in crop production. Nuclear technology has emerged as a valuable tool for enhancing agricultural productivity. Despite the common association of nuclear technology with energy and weaponry, its use in Horticulture is diverse and multifaceted. Physical mutagens such as nuclear radiation, including gamma rays, X-rays, and UV light, as well as particle radiation, can be employed to induce chromosomal breakages, cross-linking of DNA strands, nucleotide deletion, and substitution. UV rays can be used to irradiate cell suspensions and pollen grains in the early or late uninucleate phases. Through the application of nuclear technology, agricultural productivity can be improved by increasing crop yields, controlling pests and diseases, and enhancing water quality. Food preservation can also be achieved through food irradiation, where ionizing radiation is applied to target pathogens to break their DNA bonds. Nuclear technology can also be utilized to induce beneficial mutations in crops through genetic modification, sterilization-based pest control, and water usage management. In conclusion, nuclear technology presents an innovative and effective solution for addressing challenges in Horticulture, thus contributing significantly to global food security.

P-719 Evaluation of genetic risk of apparently balanced chromosomal rearrangement carriers by breakpoint characterization

Abstract Study question What are breakpoint characteristics of ABCR carriers? What are the genetic risks of ABCR carriers caused by chromosome rearrangements? Summary answer We analyzed the characteristics of ABCR's breakpoints and found that heterogeneous deletion of some AD disease-related genes may not result in clinical phenotype. What is known already ABCRs are common chromosomal abnormalities. People with ABCRs generally lack any visible abnormal features; however, during childbirth, the abnormal pairing and missegregation of homologous chromosomes can lead to the production of unbalanced gametes, resulting in spontaneous abortion or the birth of children with chromosomal disorders. Prenatal diagnosis or preimplantation diagnosis can effectively prevent the birth of children with unbalanced chromosome rearrangements, but no substantial data has been collected to report on the characteristics and potential genetic risks of the chromosome breakage caused by the rearrangement. Study design, size, duration This is a retrospective study of 2441 breakpoints in 1219 ABCR carriers who underwent Preimplantation Genetic Testing (PGT) at the Reproductive and Genetic Hospital of Citic-Xiangya from August 1, 2016 to December 31, 2021. Participants/materials, setting, methods All patients in this study sought PGT treatment due to primary or secondary infertility caused by ABCRs. To characterize the chromosome breakpoints, the MicroSeq technique, which involves chromosome microdissection and next generation sequencing, was employed. Additionally, gap-junction PCR and Sanger sequencing were utilized to further validate some of the breakpoints. Main results and the role of chance In this study, we retrospectively analyzed 2441 chromosome rearrangement breakpoint MicroSeq localization data from 1219 ABCR carriers, including 2364 breakpoint regions in 1182 reciprocal translocations, 68 breakpoint regions in 34 inversions, and 9 breakpoint regions in 3 insertional translocations. We not only identified 8q24.13, 11q11.23, and 22q11.21 as previously reported hotspots of chromosomal balanced rearrangement breaks, but also identified the 10 Mb region of 12q24.13-q24.3 as a possible rare region of balanced rearrangement breaks. the average range of breakpoints identified by MicroSeq technology was only within 3.8 Kb, which clarified 95.82% (2339/2441) of balanced chromosomal rearrangements as to whether they broke genes or not. We identified 960 genes that were interrupted, of which 53 genes with autosomal dominant mode of inheritance (AD) were interrupted. Clinical phenotypic evaluation of these patients carrying interrupted AD genes identified that some of the AD genetic interruptions may not have a clinical phenotype. Limitations, reasons for caution MicroSeq cannot precisely pinpoint breakpoints to the gene or single base level. Moreover, carriers of ABCRs were not comprehensively assessed for other clinical characteristics, and the alteration in function of the known Haploinsufficiency gene could not be confirmed. Additionally, the position effect of the breakpoints has not been evaluated. Wider implications of the findings This investigation offers a reference for evaluating the pathogenicity and genetic risks of protein-truncation mutations in some AD genes. Trial registration number Not applicable

O-305 Next-generation sequencing of a cohort of 100 patients with diminished ovarian reserve reveals an etiology in 27% of cases and may predict the fertility prognosis

Abstract Study question Identify the genetic cause of a cohort of patients with unexplained Diminished ovarian reserve (DOR) by next-generation sequencing and compare with pregnancy outcome. Summary answer A high-yield positive genetic diagnosis was obtained: 27% of cases. Defects of genes involved in DNA repair/meiosis appeared to have an unfavorable prognosis. What is known already Ten percent of women undergoing Medically Assisted Procreation-MAP have a DOR defined by an AMH level <1.2 and an antral follicle count (AFC) <5. However, most causes of DOR are unknown. There is no known criteria of success in MAP. Primary ovarian insufficiency (POI) corresponds to a complete cessation of ovarian function in 1-4% of women under 40 years. We have very recently shown in a large cohort of POI that a custom-made target next-generation sequencing (NGS) POI panel allowed a genetic diagnosis in 30% of unexplained POI and leads to personalized medicine (Heddar et al., EBioMedicine. 2022 doi: 10.1016/j.ebiom.2022.104246). Study design, size, duration Prospective genetic study of a cohort of 100 patients with undergoing MAP using a custom-made NGS-POI panel comprising 88 validated POI-causing genes. These patients were for 77% European, 20% North African and 3% Asian. The classification of the variants detected were based on the American College of Medical Genetic criteria 2015. Only pathogenic and likely pathogenic variants were used for diagnosis. Candidate gene studies were performed in negative patients. Participants/materials, setting, methods One hundred patients aged 16-35 with DOR, normal karyotype and FMR1 gene, were studied with the NGS POI panel. The segregation of the variants in the available families was performed by Sanger sequencing. Cytogenetic studies of chromosomal fragility was performed if necessary. The outcome of the pregnancy (spontaneous or induced) was recorded. A correlation between the result of the genetic study and the outcome of the pregnancy has been performed. Main results and the role of chance One family included two sisters, one with POI and the other with DOR, highlighting the proximity of the two syndromes. A high positive genetic yield was found: 27% of cases. Genes were involved 1) in follicular growth and gonadal development (SOX8, AR, NOBOX, BMPR1A, BMPR1B, GNAS) (35%) 2) DNA repair (BNC1, ERCC6, BRCA1, ATM) (31%) 3) metabolism and mitochondrial functions (POLG, STAR) (12%), 4) cellular aging (24%), in particular the LMNA gene. In a patient with isolated DOR, we identified for the first time, bi-allelic truncating variants of BRCA1, a major breast/ovarian cancer susceptibility gene. Cytogenetic studies revealed increased chromosomal breaks with radial figures typical of Fanconi Anemia (FA). However there was no sign of FA or cancer in the patient and family. This observation is reminiscent of the family with isolated POI and biallelic mutations of BRCA2 (Heddar et al, J Med Genet 2021; DOI: 10.1136/jmedgenet-2019-106672). Among the 27 patients with an established genetic diagnosis, six were able to obtain a pregnancy. However, no pregnancies were achieved in the DNA repair gene suggesting an unfavorable prognosis for this gene family. On the other hand, patients with mutations of other gene families seem to have a better prognosis of fertility. Limitations, reasons for caution The size of the cohort should be implemented to confirm these results. The issues of pregnancy are not available for the whole cohort. Wider implications of the findings This is the first genetic study of a cohort of DOR and the first implication of BRCA1 in isolated DOR. It shows i) the need for genetic studies of DOR ii) the genetic link between POI and DOR iii) NGS study could give information on the fertility prognosis of DOR. Trial registration number not applicable

Structure and function of the RAD51B-RAD51C-RAD51D-XRCC2 tumour suppressor.

Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3,are predisposed to breast, ovarian and prostate cancers1-10 and the cancer-prone syndrome Fanconi anaemia11-13. The BRCA2 tumour suppressor protein-the product of BRCA2-is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects14,15, but the precise molecular roles of RAD51 paraloguesin fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B-RAD51C-RAD51D-XRCC2 complex (BCDX2), revealing that RAD51C-RAD51D-XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments-which are essential for recombinational DNA repair-in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance.

Integrator facilitates RNAPII removal to prevent transcription-replication collisions and genome instability

DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.