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Fanconi anemia is a rare disease, with a prevalence of 1-5 per million inhabitants; It is caused by the pathogenic variants, usually biallelic, in one of the 22 FANC genes that participate in the FA/BRCA, DNA repair pathway. Accurate diagnosis is crucial for appropriate and timely management and genetic counseling. This can be done through the diepoxybutane or mitomycin C challenge chromosomal aberration test, which is the gold standard for the diagnosis of this disease. This cytogenetic study can be performed in various types of tissues as long as there are cells undergoing mitosis, such as peripheral blood lymphocytes, bone marrow cells, fibroblasts and amniocytes. Both laboratory settings for diagnosis and chromosome analysis can be complex and need to be tightly controlled and interpreted by highly specialized personnel. In this article, we present a step by step guide describing the cytogenetic protocol successfully used in a mexican laboratory, for more than 30 years. [ABSTRACT FROM AUTHOR]

DNA double‐strand breaks (DSBs) are highly cytotoxic lesions, and unrepaired or misrepaired DSBs can lead to various human diseases, including immunodeficiency, neurological abnormalities, growth retardation, and cancer. Nonhomologous end joining (NHEJ) is the major DSB repair pathway in mammals. Ku70 and Ku80 are DSB sensors that facilitate the recruitment of downstream factors, including protein kinase DNA‐dependent protein kinase, catalytic subunit (DNA‐PKcs), structural components [X‐ray repair cross‐complementing protein 4 (XRCC4), XRCC4‐like factor (XLF), and paralogue of XRCC4 and XLF (PAXX)], and DNA ligase IV (LIG4), which complete DNA repair. DSBs also trigger the activation of the DNA damage response pathway, in which protein kinase ataxia‐telangiectasia mutated (ATM) phosphorylates multiple substrates, including histone H2AX. Traditionally, research on NHEJ factors was performed using in vivo mouse models and murine cells. However, the current knowledge of the genetic interactions between NHEJ factors in human cells is incomplete. Here, we obtained genetically modified human HAP1 cell lines, which lacked one or two NHEJ factors, including LIG4, XRCC4, XLF, PAXX, DNA‐PKcs, DNA‐PKcs/XRCC4, and DNA‐PKcs/PAXX. We examined the genomic instability of HAP1 cells, as well as their sensitivity to DSB‐inducing agents. In addition, we determined the genetic interaction between XRCC4 paralogues (XRCC4, XLF, and PAXX) and DNA‐PKcs. We found that in human cells, XLF, but not PAXX or XRCC4, genetically interacts with DNA‐PKcs. Moreover, ATM possesses overlapping functions with DNA‐PKcs, XLF, and XRCC4, but not with PAXX in response to DSBs. Finally, NHEJ‐deficient HAP1 cells show increased chromosomal and chromatid breaks, when compared to the WT parental control. Overall, we found that HAP1 is a suitable model to study the genetic interactions in human cells.

DNA double‐strand breaks (DSBs) are highly cytotoxic lesions, and unrepaired or misrepaired DSBs can lead to various human diseases, including immunodeficiency, neurological abnormalities, growth retardation, and cancer. Nonhomologous end joining (NHEJ) is the major DSB repair pathway in mammals. Ku70 and Ku80 are DSB sensors that facilitate the recruitment of downstream factors, including protein kinase DNA‐dependent protein kinase, catalytic subunit (DNA‐PKcs), structural components [X‐ray repair cross‐complementing protein 4 (XRCC4), XRCC4‐like factor (XLF), and paralogue of XRCC4 and XLF (PAXX)], and DNA ligase IV (LIG4), which complete DNA repair. DSBs also trigger the activation of the DNA damage response pathway, in which protein kinase ataxia‐telangiectasia mutated (ATM) phosphorylates multiple substrates, including histone H2AX. Traditionally, research on NHEJ factors was performed using in vivo mouse models and murine cells. However, the current knowledge of the genetic interactions between NHEJ factors in human cells is incomplete. Here, we obtained genetically modified human HAP1 cell lines, which lacked one or two NHEJ factors, including LIG4, XRCC4, XLF, PAXX, DNA‐PKcs, DNA‐PKcs/XRCC4, and DNA‐PKcs/PAXX. We examined the genomic instability of HAP1 cells, as well as their sensitivity to DSB‐inducing agents. In addition, we determined the genetic interaction between XRCC4 paralogues (XRCC4, XLF, and PAXX) and DNA‐PKcs. We found that in human cells, XLF, but not PAXX or XRCC4, genetically interacts with DNA‐PKcs. Moreover, ATM possesses overlapping functions with DNA‐PKcs, XLF, and XRCC4, but not with PAXX in response to DSBs. Finally, NHEJ‐deficient HAP1 cells show increased chromosomal and chromatid breaks, when compared to the WT parental control. Overall, we found that HAP1 is a suitable model to study the genetic interactions in human cells. [ABSTRACT FROM AUTHOR]

Air pollution is a dominant environmental exposure factor with significant health consequences. Unexpectedly, research in a heavily polluted region of the Czech Republic, with traditional heavy industry, revealed repeatedly the lowest frequency of micronuclei in the season with the highest concentrations of air pollutants including carcinogenic benzo[a]pyrene (B[a]P). Molecular findings have been collected for more than 10 years from various locations of the Czech Republic, with differing quality of ambient air. Preliminary conclusions have suggested adaptation of the population from the polluted locality (Ostrava, Moravian-Silesian Region (MSR)) to chronic air pollution exposure. In this study we utilize the previous findings and, for the first time, investigate micronuclei (MN) frequency by type: (i) centromere positive (CEN+) MN, representing chromosomal losses, and (ii) centromere negative (CEN-) MN representing chromosomal breaks. As previous results indicated differences between populations in the expression of XRCC5 , a gene involved in the non-homologous end-joining (NHEJ) repair pathway, possible variations in epigenetic settings in this gene were also investigated. This new research was conducted in two seasons in the groups from two localities with different air quality levels (Ostrava (OS) and Prague (PG)). The obtained new results show significantly lower frequencies of chromosomal breaks in the OS subjects, related to the highest air pollution levels (p < 0.001). In contrast, chromosomal losses were comparable between both groups. In addition, significantly lower DNA methylation was found in 14.3% of the analyzed CpG loci of XRCC5 in the population from OS. In conclusion, the epigenetic adaptation (hypomethylation) in XRCC5 involved in the NHEJ repair pathway in the population from the polluted region, was suggested as a reason for the reduced level of chromosomal breaks. Further research is needed to explore the additional mechanisms, including genetic adaptation. [Display omitted] • The association of chronic air pollution exposure with chr. damage was evaluated. • The unexpectedly reduced MN frequency linked with high air pollution was explored. • A decreased frequency of chromosomal breaks was observed. • Simultaneous hypomethylation in XRCC5 was detected. • Epigenetic modification of XRCC5 could contribute to reduced chromosomal damage. [ABSTRACT FROM AUTHOR]

Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics were similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose-dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment.

Background and Aims Acute lymphoblastic leukemia (ALL) is the most common cancer in the pediatric population; ∼80% of the cases show some translocation. Translocations that result in ALL are due to chromosome breaks. However, the exact mechanisms that cause these breaks have not been well studied. A detailed search of the breakpoints associated with ALL reported in the NCBI database shows that some are concentrated in limited regions of the chromosome, whereas others are scattered throughout. Therefore, the objective of this study was to identify the structural factors involved in chromosomal breaks in ALL. Methods We performed several bioinformatic studies on the sequences where chromosomal breakpoints have been reported in search of rearrangements: areas of high similarity, thermodynamic stability, composition and conformation of the DNA. Results Certain factors may influence chromosome breaks and are capable of predicting the propensity towards these types of events. Conclusions These findings may be useful in the design of molecular techniques able to detect these changes in ALL. [ABSTRACT FROM AUTHOR]

Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment. [ABSTRACT FROM AUTHOR]

Abstract: A role for pro-inflammatory cytokines in inflammation-related cancers has been suggested, but mechanisms are not defined. Here, we demonstrate that treatment of HeLa cells with TNFα increases chromosomal aberration. In contrast, IL-1β did not increase, but rather decreased chromosomal aberration. TNFα and IL-1β increased the production of H2O2 to similar levels in cells, suggesting that increased production of reactive oxygen species might not be the premier factor involved. Reducing H2O2 through overexpression of catalase or treatment of cells with NAC or BHA did not have an effect on TNF-induced chromosomal aberration. TNFα-induced NO production has been implicated in DNA damage. Inhibiting NO did not reduce TNF-induced chromosomal aberration. Inhibiting IKK, JNK, and p38 kinase as well as caspases decreased TNF-induced chromosomal aberration, and a correlation between TNF-induced apoptosis and CA generation was not found. Single-strand DNA breaks give rise to double-strand breaks, which then results in chromosomal breaks, when replication forks reach the single-strand breaks during S-phase. In cells progressing through S-phase, TNFα activation of IKK, JNK, and p38 is significantly reduced. However, these kinases were activated by IL-1β in S-phase. The possibility that these pathways, in a TNF-specific manner, may regulate either the generation of single- and double-strand breaks or their repair, thereby resulting in increased chromosomal aberration, is discussed. [Copyright &y& Elsevier]

The frequency of pathogenic large chromosome rearrangements detected in patients with different Mendelian diseases is truly diverse and can be remarkably high. Chromosome breaks could arise through different known mechanisms. Congenital PAX6-associated aniridia is a hereditary eye disorder caused by mutations or chromosome rearrangements involving the PAX6 gene. In our recent study, we identified 11p13 chromosome deletions in 30 out of 91 probands with congenital aniridia or WAGR syndrome (characterized by Wilms' tumor, Aniridia, and Genitourinary abnormalities as well as mental Retardation). The loss of heterozygosity analysis (LOH) was performed in 10 families with de novo chromosome deletion in proband. In 7 out of 8 informative families, the analysis revealed that deletions occurred at the paternal allele. If paternal origin is not random, chromosome breaks could arise either (i) during spermiogenesis, which is possible due to specific male chromatin epigenetic program and its vulnerability to the breakage-causing factors, or (ii) in early zygotes at a time when chromosomes transmitted from different parents still carry epigenetic marks of the origin, which is also possible due to diverse and asymmetric epigenetic reprogramming occurring in male and female pronuclei. Some new data is needed to make a well-considered conclusion on the reasons for preferential paternal origin of 11p13 deletions. [ABSTRACT FROM AUTHOR]