Chromosome Fragility Research Articles

Prognostic significance of mutation type and chromosome fragility in Fanconi anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by high phenotypic and genotypic heterogeneity, and extreme chromosome fragility. To better understand the natural history of FA, identify genetic risk and prognostic factors, and develop novel therapeutic strategies, the Spanish Registry of Patients with FA collects data on clinical features, chromosome fragility, genetic subtypes, and DNA sequencing with informed consent of participating individuals. In this article, we describe the clinical evolution of 227 patients followed up for up to 30 years, for whom our data indicate a cumulative cancer incidence of 86% by age 50. We found that patients with lower chromosome fragility had a milder malformation spectrum and better outcomes in terms of later-onset hematologic impairment, less severe bone marrow failure, and lower cancer risk. We also found that outcomes were better for patients with mutations leading to mutant FANCA protein expression (genetic hypomorphism) than for patients lacking this protein. Likewise, prognosis was consistently better for patients with biallelic mutations in FANCD2 (mainly hypomorphic mutations) than for patients with biallelic mutations in FANCA and FANCG, with the lack of the mutant protein in patients with biallelic mutations in FANCG contributing to their poorer outcomes. Our results regarding the clinical impact of chromosome fragility and genetic hypomorphism suggest that mutant FA proteins retain residual activity. This finding should encourage the development of novel therapeutic strategies aimed at partially or fully enhancing mutant FA function, thereby preventing or delaying bone marrow failure and cancer in patients with FA. Clinical Trial Registration number: NCT06490510.

C9orf72 expansion creates the unstable folate-sensitive fragile site FRA9A

Abstract The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions (C9orf72Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immuno-stimulatory or damaged DNA is unknown. Here, we show C9orf72Exp in pre-symptomatic and ALS-FTD patient cells and brains cause the folate-sensitive chromosomal fragile site, FRA9A. FRA9A centers on > 33kb of C9orf72 as highly-compacted chromatin embedded in an 8.2Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. C9orf72Exp cells show chromosomal instability, heightened global- and Chr9p-enriched sister-chromatid exchanges, truncated-Chr9s, acentric-Chr9s and Chr9-containing micronuclei, providing endogenous sources of damaged and immunostimulatory DNA. Cells from one C9orf72Exp patient contained highly-rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72Exp repeat instability and chromosomal fragility are sensitive to folate-deficiency. Age-dependent repeat instability, chromosomal fragility, and chromosomal instability can be transferred to CNS and peripheral tissues of transgenic C9orf72Exp mice, implicating C9orf72Exp as the source. Our results highlight unappreciated effects of C9orf72 expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere.

Experimental in vivo models for understanding inter-molecular interactions and preventing multifactor disease development

The increased spontaneous fragility in the centromere regions of the chromosomes predisposes to abnormal cellular division due to abnormal interaction with the mitotic spindle, which leads to number chromosomal aberrations. The abnormal structure of the microtubule-associated proteins (MAPs) could also be among the internal factors, predisposing to these pathologies. On the other hand, the increased frequency of the spontaneous chromosomal fragility suggests abnormal structure of the respective chromosomal/chromatin components and thus an increased risk about gene mutations and structural chromosomal aberrations. The spontaneous chromosomal fragility was tested by light microscopy observation of metaphases from peripheral blood lymphocytes of 8 patients with polygene pathologies (neoplasms, neuro-degenerative diseases, and neuro-psychiatric disorders) and of 18 healthy controls. In the tested patients was established significantly higher frequency of the spontaneous chromosomal fragility, including in the centromere chromosomal regions, compared to the controls. Different inter-molecular interactions, which could prevent the pathology appearance, were investigated by appropriate experimental in vivo models. As a whole, in the most cases, the presented data showed significantly lower average titers of anti-ganglioside antibodies and gangliosides in the samples from myocardium and liver than in the samples from brain and pancreas. By taking into consideration that the myocardium and liver are known as the most enriched of GSH anatomic organs, the presented data could be explained with the literature messages of higher amounts of de novo-synthesized free GSH tri-peptide in the same two organs. Moreover, a possibility about the production of antibodies/immunoglobulins by non-lymphoid types of cells, tissues, and organs in appropriate conditions was shown. Because the so-produced antibodies are out of the germinative centers in the specialized lymphoid tissues and organs, the control of their functions by small ions and molecules as gangliosides is very important. In the current study, the attention was particularly directed to the iteractions with the participation of gangliosides, the reduced form of the tri-peptide glutathione (GSH), and the protein HACE1. Besides the tumor-suppressor activity of protein HACE1, also its neuroprotective influence was proven, namely by interactions with specific molecules.

C9orf72 expansion creates the unstable folate-sensitive fragile site FRA9A.

The hyper-unstable Chr9p21 locus, harbouring the interferon gene cluster, oncogenes and C9orf72, is linked to multiple diseases. C9orf72 (GGGGCC)n expansions ( C9orf72 Exp) are associated with incompletely penetrant amyotrophic lateral sclerosis, frontotemporal dementia and autoimmune disorders. C9orf72 Exp patients display hyperactive cGAS-STING-linked interferon immune and DNA damage responses, but the source of immuno-stimulatory or damaged DNA is unknown. Here, we show C9orf72 Exp in pre-symptomatic and ALS-FTD patient cells and brains cause the folate-sensitive chromosomal fragile site, FRA9A. FRA9A centers on >33kb of C9orf72 as highly-compacted chromatin embedded in an 8.2Mb fragility zone spanning 9p21, encompassing 46 genes, making FRA9A one of the largest fragile sites. C9orf72 Exp cells show chromosomal instability, heightened global- and Chr9p-enriched sister-chromatid exchanges, truncated-Chr9s, acentric-Chr9s and Chr9-containing micronuclei, providing endogenous sources of damaged and immunostimulatory DNA. Cells from one C9orf72 Exp patient contained highly-rearranged FRA9A-expressing Chr9 with Chr9-wide dysregulated gene expression. Somatic C9orf72 Exp repeat instability and chromosomal fragility are sensitive to folate-deficiency. Age-dependent repeat instability, chromosomal fragility, and chromosomal instability can be transferred to CNS and peripheral tissues of transgenic C9orf72 Exp mice, implicating C9orf72 Exp as the source. Our results highlight unappreciated effects of C9orf72 expansions that trigger vitamin-sensitive chromosome fragility, adding structural variations to the disease-enriched 9p21 locus, and likely elsewhere.

Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.

Evaluation of DNA damage through cytogenetic approach in smokers and vapers with and without nicotine compared with control group

The use of tobacco and electronic cigarettes is harmful to health and can cause genetic damage, increasing the risk of cancer and other serious diseases. Although e-cigarettes contain fewer toxic chemicals than conventional tobacco, recent studies have shown that the vapor caused by burning produced by e-cigarettes can also be toxic and carcinogenic. Various studies have found that those exposed to tobacco and vaping have significantly higher levels of damage to their DNA in different types of cells and tissues. Evidence has accumulated that e-cigarette vaping can alter cellular functions and DNA itself, increasing the risk of cancer and aging. The present work evaluatesthe cytogenetic damage in individuals exposed to conventional cigarette vapors by burning with nicotine and vapors without nicotine, compared with a non-smoking population. The study included participants with an average age of 30 years (+/-10), with a majority of men representing 70% of the sample. The Chi-square test found no significant statistical differences between the men and women exposed (p<0.05). The results of chromosomal fragility found in the four groups studied (control group, conventional smokers, vapers with nicotine and vapers without nicotine) showed breaks and gaps in one or both of the chromatids in all exposed individuals, with highly significant statistical differences (p<0.001) compared to the unexposed control group. No statistically significant differences were found between the group of conventional smokers and the vapers with and without nicotine, nor between the two types of vapers (p>0.05). In conclusion, cytogenetic evidence of DNA damage produced by vaping is shown in the same proportions as a normal cigarette. This will significantly impact public health, which must be considered in preventive actions. Keywords: cigarettes, vaping, chromosome breakage, DNA damage

Evaluation of DNA damage through cytogenetic approach in smokers and vapers with and without nicotine compared with control group

The use of tobacco and electronic cigarettes is harmful to health and can cause genetic damage, increasing the risk of cancer and other serious diseases. Although e-cigarettes contain fewer toxic chemicals than conventional tobacco, recent studies have shown that the vapor caused by burning produced by e-cigarettes can also be toxic and carcinogenic. Various studies have found that those exposed to tobacco and vaping have significantly higher levels of damage to their DNA in different types of cells and tissues. Evidence has accumulated that e-cigarette vaping can alter cellular functions and DNA itself, increasing the risk of cancer and aging. The present work evaluatesthe cytogenetic damage in individuals exposed to conventional cigarette vapors by burning with nicotine and vapors without nicotine, compared with a non-smoking population. The study included participants with an average age of 30 years (+/-10), with a majority of men representing 70% of the sample. The Chi-square test found no significant statistical differences between the men and women exposed (p<0.05). The results of chromosomal fragility found in the four groups studied (control group, conventional smokers, vapers with nicotine and vapers without nicotine) showed breaks and gaps in one or both of the chromatids in all exposed individuals, with highly significant statistical differences (p<0.001) compared to the unexposed control group. No statistically significant differences were found between the group of conventional smokers and the vapers with and without nicotine, nor between the two types of vapers (p>0.05). In conclusion, cytogenetic evidence of DNA damage produced by vaping is shown in the same proportions as a normal cigarette. This will significantly impact public health, which must be considered in preventive actions. Keywords: cigarettes, vaping, chromosome breakage, DNA damage

Evaluation of DNA damage through cytogenetic approach in smokers and vapers with and without nicotine compared with control group

The use of tobacco and electronic cigarettes is harmful to health and can cause genetic damage, increasing the risk of cancer and other serious diseases. Although e-cigarettes contain fewer toxic chemicals than conventional tobacco, recent studies have shown that the vapor caused by burning produced by e-cigarettes can also be toxic and carcinogenic. Various studies have found that those exposed to tobacco and vaping have significantly higher levels of damage to their DNA in different types of cells and tissues. Evidence has accumulated that e-cigarette vaping can alter cellular functions and DNA itself, increasing the risk of cancer and aging. The present work evaluatesthe cytogenetic damage in individuals exposed to conventional cigarette vapors by burning with nicotine and vapors without nicotine, compared with a non-smoking population. The study included participants with an average age of 30 years (+/-10), with a majority of men representing 70% of the sample. The Chi-square test found no significant statistical differences between the men and women exposed (p<0.05). The results of chromosomal fragility found in the four groups studied (control group, conventional smokers, vapers with nicotine and vapers without nicotine) showed breaks and gaps in one or both of the chromatids in all exposed individuals, with highly significant statistical differences (p<0.001) compared to the unexposed control group. No statistically significant differences were found between the group of conventional smokers and the vapers with and without nicotine, nor between the two types of vapers (p>0.05). In conclusion, cytogenetic evidence of DNA damage produced by vaping is shown in the same proportions as a normal cigarette. This will significantly impact public health, which must be considered in preventive actions. Keywords: cigarettes, vaping, chromosome breakage, DNA damage

In Vivo Cytotoxic, Genotoxic and Radiosensitizing Effects of Clofarabine.

ClFdA is a second-generation antineoplastic agent that has demonstrated significant anticancer activity, particularly against acute lymphoblastic leukemia and has been shown to have radiosensitizing activity. The aim of the study was to explore the genotoxic, cytotoxic and radiosensitizing effects of clofarabine (ClFdA) on bone marrow cells (BMCs), normoblasts and leukocytes of mice in vivo. Cytotoxicity was determined by the reduction in reticulocytes (RET), and genotoxicity was determined by the induction of micronucleated reticulocytes (MN-RET) in the peripheral blood and by DNA break induction in leukocytes determined by single-cell gel electrophoresis (SCGE). The radiosensitizing capacity of ClFdA was determined in leukocytes and BMCs by SCGE. Two mechanisms of MN-RET induction were identified according to the antecedents, that could be due to inhibition of DNA synthesis and demethylation of G-C regions, and subsequent chromosome fragility. ClFdA cytotoxicity causes two contiguous peaks, an early peak that seems to inhibit MN-RET induction and a second peak that seems to be caused by ribonucleotide reductase (RR) and/or DNA synthesis inhibitions. ClFdA induced early DNA damage in noncycling leukocytes, and also radiosensitizes leukocytes immediately after treatment. ClFdA-ionizing radiation (IR) causes two time-dependent episodes of DNA damage, the latest after 80 min triggers a major breakage of DNA. In terms of the number of damaged cells, leukocytes and BMCs are similarly sensitive to ionizing radiation; BMCs are slightly more sensitive than leukocytes to ClFdA, but BMCs are doubly sensitive to combined treatment. ClFdA causes early DNA damage and radiosensitivity in non-proliferating leukocytes, which rules out the most favored hypotheses of the participation of RR and DNA polymerase inhibition.

Diagnostic Utility of Proteogenomic Analysis for Inherited Bone Marrow Failure Syndrome

Background: Inherited bone marrow failure syndrome (IBMFS)-a heterogeneous group of disorders characterized by marked cytopenia in one hematopoietic cell lineage-may progress to pancytopenia and make the patient susceptible to hematological malignancies or solid tumors. No clinically useful screening tests have been established for IBMFS, with a few exceptions, including the chromosome fragility test in Fanconi anemia (FA) and telomere length measurement in dyskeratosis congenita (DC). Therefore, high-throughput sequencing is widely used for not only clinical diagnosis of IBMFS but also screening applications; however, a significant number of patients remain genetically undiagnosed. Recent technological advances in in-depth data-independent acquisition proteomic analysis have enabled comprehensive quantitative analysis of >9000 proteins, including very low-abundant proteins. Integration of proteomic analysis with genomic information has been performed in brain tumors and acute leukemia, showing the potential to bridge the gap between genetic information and clinical validation; however, to our knowledge, proteogenomics in IBMFS has not been fully evaluated thus far. Herein, an omics analysis of patients with various IBMFS was performed to assess the diagnostic utility of in-depth proteomic techniques. Methods: We performed an omics analysis of the discovery cohort of 60 patients with IBMFS for in-depth proteomic analysis, targeted capture DNA sequencing, and RNA sequencing. For non-targeted proteomic analysis, digested peptides from the peripheral blood mononuclear cell samples were processed using a Q Exactive HF-X (Thermo Fisher Scientific, Waltham, MA, USA) data-independent acquisition mass spectrometer. Furthermore, diagnostic proteomic analyses targeting several IBMFS-related proteins were performed on a developmental cohort (417 samples; IBMFS [n = 229], aplastic anemia [n = 75], myelodysplastic syndrome/acute myeloid leukemia [n = 113], and healthy controls [n = 27]) using an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific) equipped with an EVOSEP ONE system (EVOSEP, Odense, Denmark). This study was approved by the ethics committee of the Nagoya University Graduate School of Medicine. Results: Non-targeted proteomic analysis was performed on 74 samples obtained from 60 patients with IBMFS and 14 healthy controls. Unsupervised clustering identified eight independent proteomic clusters (C1-C8), with ribosomal pathway-associated protein expression specifically downregulated in C1 and C2. Patients with Diamond-Blackfan anemia, Shwachman-Diamond syndrome (SDS), FA, ADH5/ALDH2 deficiency, and DC were enriched in C1, C2/C8, C3/C4, C6, and C7 clusters, respectively. In particular, among the 74 samples, four patients with ADH5/ALDH2 deficiency showed significantly reduced ADH5 protein expression, whereas the remaining patients showed normal expression, with 100% diagnostic concordance. In addition, 6 of 74 samples had significantly decreased SBDS protein expression, including two patients with monoallelic pathogenic variants in the SBDS gene. This suggested that proteomic analysis may play a complementary role to genetic analysis. To provide a large-scale rapid screening system for IBMFS in a practical clinical setting, targeted proteomic analysis was performed using a small panel of ADH5, SBDS, and GAPDH proteins in a developmental cohort of 417 patients. We demonstrated the significantly decreased SBDS protein expression levels in patients with SDS ( P = 4.98 × 10 -9). ADH5 protein expression levels were also significantly reduced in ADH5/ALDH2 deficiency ( P = 1.66 × 10 -6). Regarding the diagnostic tests, the sensitivity and specificity values were 85.7% and 93.4% for SDS, and 100.0% and 97.5% for ADH5/ALDH2 deficiency, respectively, indicating the sufficient performance to identify patients who required early diagnosis and therapeutic intervention. Conclusion: We performed the first proteogenomic analysis of patients with IBMFS and identified eight independent proteomic clusters associated with IBMFS subtypes and characteristic pathways. Furthermore, the clinical application of targeted proteomic assays constructed from these results may help diagnose and screen IBMFS, including SDS and ADH5/ALDH2 deficiency, for which appropriate clinical screening tests are lacking.

Massive contractions of myotonic dystrophy type 2-associated CCTG tetranucleotide repeats occur via double-strand break repair with distinct requirements for DNA helicases.

Myotonic dystrophy type 2 (DM2) is a genetic disease caused by expanded CCTG DNA repeats in the first intron of CNBP. The number of CCTG repeats in DM2 patients ranges from 75 to 11,000, yet little is known about the molecular mechanisms responsible for repeat expansions or contractions. We developed an experimental system in Saccharomyces cerevisiae that enables the selection of large-scale contractions of (CCTG)100 within the intron of a reporter gene and subsequent genetic analysis. Contractions exceeded 80 repeat units, causing the final repetitive tract to be well below the threshold for disease. We found that Rad51 and Rad52 are involved in these massive contractions, indicating a mechanism that uses homologous recombination. Srs2 helicase was shown previously to stabilize CTG, CAG, and CGG repeats. Loss of Srs2 did not significantly affect CCTG contraction rates in unperturbed conditions. In contrast, loss of the RecQ helicase Sgs1 resulted in a 6-fold decrease in contraction rate with specific evidence that helicase activity is required for large-scale contractions. Using a genetic assay to evaluate chromosome arm loss, we determined that CCTG and reverse complementary CAGG repeats elevate the rate of chromosomal fragility compared to a short-track control. Overall, our results demonstrate that the genetic control of CCTG repeat contractions is notably distinct among disease-causing microsatellite repeat sequences.

Identification of a robust DNA methylation signature for Fanconi anemia

Fanconi anemia (FA) is a clinically variable and genetically heterogeneous cancer-predisposing disorder representing the most common bone marrow failure syndrome. It is caused by inactivating predominantly biallelic mutations involving >20 genes encoding proteins with roles in the FA/BRCA DNA repair pathway. Molecular diagnosis of FA is challenging due to the wide spectrum of the contributing gene mutations and structural rearrangements. The assessment of chromosomal fragility after exposure to DNA cross-linking agents is generally required to definitively confirm diagnosis. We assessed peripheral blood genome-wide DNA methylation (DNAm) profiles in 25 subjects with molecularly confirmed clinical diagnosis of FA (FANCA complementation group) using Illumina's Infinium EPIC array. We identified 82 differentially methylated CpG sites that allow to distinguish subjects with FA from healthy individuals and subjects with other genetic disorders, defining an FA-specific DNAm signature. The episignature was validated using a second cohort of subjects with FA involving different complementation groups, documenting broader genetic sensitivity and demonstrating its specificity using the EpiSign Knowledge Database. The episignature properly classified DNA samples obtained from bone marrow aspirates, demonstrating robustness. Using the selected probes, we trained a machine-learning model able to classify EPIC DNAm profiles in molecularly unsolved cases. Finally, we show that the generated episignature includes CpG sites that do not undergo functional selective pressure, allowing diagnosis of FA in individuals with reverted phenotype due to gene conversion. These findings provide a tool to accelerate diagnostic testing in FA and broaden the clinical utility of DNAm profiling in the diagnostic setting.

Scurvy: A treatable forgotten fatal differential diagnosis and potential etiology of leukemia and aplastic anemia in pediatric population.

Scurvy is a rare nutritional deficiency disease which is less likely to be suspected and it mostly lead to delayed diagnosis. It can present with features which can mislead clinicians to misdiagnose the condition as leukemia or aplastic anemia. This can subject patients to the wrong management which leads to poor outcome and increased preventable morbidity and mortality. Vitamin C deficiency is still prevalent among pediatric population even in the modern days and should no longer be considered as historical condition. Chromosomal fragility has been greatly accounted for the development of leukemia and aplastic anemia secondary to various triggers. The role of vitamin C toward DNA stability, prevention, and control of mutations have been documented. Vitamin C plays a vital role in hematopoiesis by controlling regulation and prevent dysfunction of hematopoietic stem cells. Scurvy deficiency has been a silent growing clinical problem which needs a high index of suspicion for a clinician to pick it. It should be considered as one among potential differential diagnosis of leukemia and aplastic anemia especially in the pediatric population. History of any dietary restriction should be obtained and addressed properly. Serum vitamin C should be among the essential laboratory workout in diagnosis of both leukemia and aplastic anemia. All patients suspected to have such conditions should be screened and supplemented for vitamin C deficiency irrespective of positive confirmatory test results of leukemia or aplastic anemia since the probability of co-occurrence is likely also. Moreover, studies should be conducted to explore the clinical link, if any, between vitamin C deficiency or insufficiency and development of leukemia and aplastic anemia among the pediatric population given its physiological and genomic role in hematopoiesis. Furthermore, the potential pharmacological therapeutic use of vitamin C in treatment of leukemia and aplastic anemia should be determined clinically.

Phenotype reversion as "natural gene therapy" in Fanconi anemia by a gene conversion event.

Somatic mosaicism appears as a recurrent phenomenon among patients suffering from Fanconi anemia (FA), but its direct prognostic significance mostly remains an open question. The clinical picture of FA mosaic subjects could indeed vary from just mild features to severe hematologic failure. Here, we illustrate the case of a proband whose FA familiarity, modest signs (absence of hematological anomalies and fertility issues), and chromosome fragility test transition to negative overtime were suggestive of somatic mosaicism. In line with this hypothesis, genetic testing on patient's peripheral blood and buccal swab reported the presence of the only FANCA paternal variant (FANCA:c.2638C>T, p. Arg880*) and of both parental alleles (the additional FANCA:c.3164G>A, p. Arg1055Gln), respectively. Moreover, the SNP analysis performed on the same biological specimens allowed us to attribute the proband's mosaicism status to a possible gene conversion mechanism. Our case clearly depicts the positive association between somatic mosaicism and the proband's favorable clinical course due to the occurrence of the reversion event at the hematopoietic stem cell level. Since this condition concerns only a limited subgroup of FA individuals, the accurate evaluation of the origin and extent of clonality would be key to steer clinicians toward the most appropriate therapeutic decision for their FA mosaic patients.

Role of the telomere abnormalities and chromosome fragility on the risk of second malignant tumor in papillary thyroid cancer patients

Role of the telomere abnormalities and chromosome fragility on the risk of second malignant tumor in papillary thyroid cancer patients

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.

Response of the Endometrial Mesenchymal Stem Cell Genome to the Procedure of Long-Term Cryopreservation

Information about the effect of cryopreservation on functions and the genetic of cells of different genesis is not unambiguous and is in the process of accumulation. This work is aimed at studying the effect of long-term storage (7 years) of human endometrial mesenchymal stem cells (eMSCs) in the frozen state on the stability of their genome in vitro. The results showed destabilization of the karyotype structure in the descendants of cells after their thawing, namely, aneupolyploidization, increased fragility of chromosomes, resulting in a huge pool of aberrant chromosomes, and impaired condensation in homologues. Chromosomal breakds in centromeric regions offen accompanied by the preservation of genetic material in the form of independent chromosomes. Almost all chromosomes of the set were involved in the process of destabilization of the eMSCs cell genome. It has been shown that the procedure of long-term cryopreservation can become an inducer of premature cellular aging of eMSCs after their thawing. Comparison of the data obtained with the results of karyotyping of transformed Chinese hamster cells that underwent a similar procedure led to the conclusion that cryopreservation for biological systems can be a stress that induces genetic defects of various types at the karyotype level. The response of the genome of cells of different origin to the same conditions of cryopreservation may differ.

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

Dicentric chromosome breakage in Drosophila melanogaster is influenced by pericentric heterochromatin and occurs in nonconserved hotspots.

Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster, we find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of 3 different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all 3 chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the 3 chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment vary significantly between the 3 chromosomes and are correlated with the origin of the centromere and the amount of pericentric heterochromatin. We suggest that different centromere strengths could account for this.

Clinical and cytogenetic profile of Fanconi anemia diagnosed after implementation of mitomycin C cytogenetic test in the state of Pernambuco, Brazil

IntroductionFanconi anemia (FA) is a rare autosomal recessive disease characterized by chromosomal instability and increased predisposition to malignancy. The diagnosis of FA requires clinical evaluation, confirmation of chromosomal fragility and/or analysis of genetic mutations. Therefore, this study aims to identify the clinical profile of patients with FA in the state of Pernambuco, Brazil. MethodWe analyzed 100 individuals referred from the major hematology and bone marrow (BM) transplant centers in the state of Pernambuco, Brazil, between the years 2018 and 2022. The diagnosis of FA was performed using the mitomycin C chromosomal fragility test, clinical data and classical and molecular cytogenetic analyses. ResultsWe enrolled a total of 16 patients with FA to comprise this study. Most of these individuals (87.5%) came from the Agreste and Sertão regions of Pernambuco. We observed a slight female prevalence of FA (1.3:1). The primary clinical and laboratory findings were café au lait spots (62.5%) and bone abnormalities (53%, mainly thumb deformities [40%]). We performed BM cytogenetic analysis for eight patients – seven showed no chromosomal abnormalities and one presented the karyotype 47,XY,+21 [15]. ConclusionsOur results are important to promote public health measures for the early diagnosis of FA, as well as to foster the engagement of a multidisciplinary group in the treatment of this disease.