Chromothripsis and Kataegis Induced by Telomere Crisis.

Telomere crisis occurs during tumorigenesis when depletion of the telomere reserve leads to frequent telomere fusions. The resulting dicentric chromosomes have been proposed to drive genome instability. Here, we examine the fate of dicentric human chromosomes in telomere crisis. We observed that dicentric chromosomes invariably persisted through mitosis and developed into 50-200 µm chromatin bridges connecting the daughter cells. Before their resolution at 3-20 hr after anaphase, the chromatin bridges induced nuclear envelope rupture in interphase, accumulated the cytoplasmic 3' nuclease TREX1, and developed RPA-coated single stranded (ss) DNA. CRISPR knockouts showed that TREX1 contributed to the generation of the ssDNA and the resolution of the chromatin bridges. Post-crisis clones showed chromothripsis and kataegis, presumably resulting from DNA repair and APOBEC editing of the fragmented chromatin bridge DNA. We propose that chromothripsis in human cancer may arise through TREX1-mediated fragmentation of dicentric chromosomes formed in telomere crisis.

Dicentric chromosomes are resolved through breakage and repair at their centromeres.

Chromosomes with two centromeres provide a unique opportunity to study chromosome breakage and DNA repair using completely endogenous cellular machinery. Using a conditional transcriptional promoter to control the second centromere, we are able to activate the dicentric chromosome and follow the appearance of DNA repair products. We find that the rate of appearance of DNA repair products resulting from homology-based mechanisms exceeds the expected rate based on their limited centromere homology (340 bp) and distance from one another (up to 46.3 kb). In order to identify whether DNA breaks originate in the centromere, we introduced 12 single-nucleotide polymorphisms (SNPs) into one of the centromeres. Analysis of the distribution of SNPs in the recombinant centromeres reveals that recombination was initiated with about equal frequency within the conserved centromere DNA elements CDEII and CDEIII of the two centromeres. The conversion tracts range from about 50 bp to the full length of the homology between the two centromeres (340 bp). Breakage and repair events within and between the centromeres can account for the efficiency and distribution of DNA repair products. We propose that in addition to providing a site for kinetochore assembly, the centromere may be a point of stress relief in the face of genomic perturbations.

Breakage in breakage-fusion-bridge cycle: an 80-year-old mystery.

2021 marked the 80th anniversary of Barbara McClintock's pioneering article on the breakage-fusion-bridge (BFB) cycle. Of the three steps of the BFB cycle, breakage remains the least understood despite its major contribution to mutagenesis. We discuss recent findings shedding light on how chromatin bridges break in yeast and animal cells.

International Comparison Exercise for Biological Dosimetry after Exposures with Neutrons Performed at Two Irradiation Facilities as Part of the BALANCE Project.

In the case of a radiological or nuclear event, biological dosimetry can be an important tool to support clinical decision-making. During a nuclear event, individuals might be exposed to a mixed field of neutrons and photons. The composition of the field and the neutron energy spectrum influence the degree of damage to the chromosomes. During the transatlantic BALANCE project, an exposure similar to a Hiroshima-like device at a distance of 1.5 km from the epicenter was simulated, and biological dosimetry based on dicentric chromosomes was performed to evaluate the participants ability to discover unknown doses and to test the influence of differences in neutron spectra. In a first step, calibration curves were established by irradiating blood samples with 5 doses in the range of 0-4 Gy at two different facilities in Germany (Physikalisch-Technische Bundesanstalt [PTB]) and the USA (the Columbia IND Neutron Facility [CINF]). The samples were sent to eight participating laboratories from the RENEB network and dicentric chromosomes were scored by each participant. Next, blood samples were irradiated with 4 blind doses in each of the two facilities and sent to the participants to provide dose estimates based on the established calibration curves. Manual and semiautomatic scoring of dicentric chromosomes were evaluated for their applicability to neutron exposures. Moreover, the biological effectiveness of the neutrons from the two irradiation facilities was compared. The calibration curves from samples irradiated at CINF showed a 1.4 times higher biological effectiveness compared to samples irradiated at PTB. For manual scoring of dicentric chromosomes, the doses of the test samples were mostly successfully resolved based on the calibration curves established during the project. For semiautomatic scoring, the dose estimation for the test samples was less successful. Doses >2 Gy in the calibration curves revealed nonlinear associations between dose and dispersion index of the dicentric counts, especially for manual scoring. The differences in the biological effectiveness between the irradiation facilities suggested that the neutron energy spectrum can have a strong impact on the dicentric counts.

Co-occurrence of acrocentric chromosome associations with dicentric chromosomes in irradiated human lymphocytes.

PURPOSE: The occurrence of acrocentric chromosome association (ACA) after radiation exposure is an interesting cytogenetic endpoint, known to show a dose-dependent increase in irradiated lymphocytes suggesting its potential use in radiation biodosimetry. Here, an attempt was made to study the complexity and correlation of the occurrence of ACA with dicentric chromosomes (DC) in lymphocytes exposed to gamma radiation. METHODS: Ninety metaphases each with DC and without DC were chosen randomly from lymphocytes irradiated with different doses (0, 1, 2, 3, 4 and 5 Gy) of gamma radiation. ACA along with chromosomal types of aberrations were scored and analyzed for complexity and co-occurrence, retrospectively. RESULTS: The number of associations between 2 and ≥ 3 acrocentric chromosomes showed an increase with each irradiation dose. Concomitantly, the total number of chromosomal type of aberrations showed an increase in number at each radiation dose studied. The number of DC showed an increase, however, metaphases containing 1DC decreased while ≥ 2DC increased as the radiation dose increased. The number of tricentric chromosomes increased at doses higher than 2 Gy. Importantly, the association of DC with an acrocentric chromosome was noticed at doses 2 Gy and above. A significant (p < 0.05) increase was noticed in ACA frequency in 1DC and ≥ 2DC metaphases at 1 and 2 Gy, in 1DC at 3 Gy, and in ≥ 2DC 4 and 5 Gy compared to the frequency in no DC metaphases. When average ACA frequency was plotted against DC frequency, a significant (p = 0.0009) correlation was observed, producing regression equation y = 0.9025x + 0.1283; R2 = 0.9522. CONCLUSION: The present analysis showed increasing ACA complexity with increasing radiation dose. Furthermore, a higher frequency of ACA in cells with 1DC or ≥ 2DC compared to the ACA in cells without DC from the same sample of irradiated lymphocytes demonstrated the co-occurrence of ACA and DC in the same cells.

Revision of cytogenetic dosimetry in the IAEA manual 2011 based on data about radio-sensitivity and dose-rate findings contributing.

In order to achieve the goal of rapid response, effective controland protection of life inlarge-scale radiation events, the IAEA Manual 2011 has been revised based on the data of radio-sensitivity, dose-rate findings. Analyze individual differences in radiation sensitivity using 60 Co radiation (0.27 Gy/min). Chromosomal aberrations with different irradiation dose rates were used to establish the biological dose curve and analyze the excess of the "dicentric + ring" caused by the dose rate at each dose point; DAPI-images and Metafer 4 were used to capture metaphase images and make further analysis. The data were collected in 2020, Dicentric + ring/100 Cells was 17.5-43.8, the average value was28.32 ± 6.98. The mean value of Dicentric + ring/100 Cells was 31.37 in males while 25.27 in females, there are significant differences (p < .01). The irradiation dose is dominant, At each dose point, the value of"(dicentric chromosome + centric rings)/cell" is proportional to "dose rate", that is, Y = kx + b, within the dose range of 1-5 Gy, "(dicentric chromosome + centric rings)/Cell" holds a quadratic linear relationship with dose rate, that is, y = ax2 + bx + c; The DAPI-images might give you more hints than those of conventional Giemsa-stain. The authors recommend that the IAEA Manual 2011 could be revised based on data of radio-sensitivity and dose-rate, which may contribute to the establishment of a unified dose-response calibration curve and stimulation of potential for automation in cytogenetic biodosimetry. (1) Individual differences of radiosensitivity are very large. (2) At each dose point, "(dicentric chromosome + centric rings)/cell" is proportional to "dose rate", that is, Y = kx + b. (3) "(dicentric chromosome + centric rings)/Cell" is a quadratic linear relationship with dose rate, that is, y = ax2 + bx + c. (4) We created a "Unity Standard Curve of Biological Dose Estimation". Creating a Unity Standard Curve of Biological Dose, under these circumstances, we can form a joint and rapid response to a nuclear and radiological accident.

Multicentric evaluation of conventional dosimetry vs bio-dosimetry over a period of two years for a three-point contact.

Background: Dental radiology represents the best model for evaluating the effects of low-dose ionizing radiation. Therefore, this study evaluated the awareness on radiation hygiene among dental ancillary personnel through a questionnaire and their absorbed doses by physical and biologic dosimetry. Methods: The multicentric study included two groups. Group I (N = 30) consisted of dental staff involved in dental radiology. An equal number of personnel who were not related to radiology formed the control group. Knowledge (K), attitude (A), and practice (P) of participants were assessed using a KAP questionnaire. Radiation exposure was evaluated by physical dosimetry at 3 time periods: at the beginning of the study (T1), after 10 months (T2), and at the end after 20 months (T3). Similarly, biologic dosimetry was also carried out at 3 time points by dicentric chromosome aberration assay. The data were compared using percentage analysis, analysis of variance (one-way analysis of variance), and Student's t- test. Results: The KAP survey demonstrated enhanced understanding of radiation protection measures and its sound practice by the participants. Physical dosimetry showed a significant increase in absorbed dose at 3 time points: T1, T2, and T3. However, no chromosomal aberrations were observed in blood lymphocytes for any of the participants in the optimized 4-day biodosimetry protocol. Conclusion: Good radiation protection protocols-safe distance from the radiation source and wear of lead aprons and thyroid collars-ensured low absorbed doses. The 4-day protocol is an important step toward developing biodosimetry laboratories in the Armed Forces Medical Services for clinical and national radiation countermeasure strategies.

Postzygotic Breakages of Dicentric Chromosomes: A Rare Mechanism of Terminal Deletions.

We report a patient presenting with neurodevelopmental disorder, cleft palate, micrognathia, relatively mild microcephaly (-2 SD), and ventricular septal defect for whom a 9p terminal deletion was identified by aCGH at birth. The analyses of the samples taken prenatally showed that this terminal deletion resulted from the recombination of a dicentric chromosome which was transmitted to the zygote. Indeed, an inverted duplication with terminal deletion of the short arm of chromosome 9 [invdupdel(9p)] was found in a mosaic state in the placenta. To our knowledge, it is the first reported patient with a terminal deletion present in all tested cells of the blood associated with an invdupdel of the same chromosome in the placenta. This case highlights the role of postzygotic breakages of dicentric chromosomes, a possible underestimated mechanism of formation of terminal deletions. It raises the question of genetic counseling in cases of prenatally detected invdupdels.

High-Accuracy Relative Biological Effectiveness Values Following Low-Dose Thermal Neutron Exposures Support Bimodal Quality Factor Response with Neutron Energy.

Theoretical evaluations indicate the radiation weighting factor for thermal neutrons differs from the current International Commission on Radiological Protection (ICRP) recommended value of 2.5, which has radiation protection implications for high-energy radiotherapy, inside spacecraft, on the lunar or Martian surface, and in nuclear reactor workplaces. We examined the relative biological effectiveness (RBE) of DNA damage generated by thermal neutrons compared to gamma radiation. Whole blood was irradiated by 64 meV thermal neutrons from the National Research Universal reactor. DNA damage and erroneous DNA double-strand break repair was evaluated by dicentric chromosome assay (DCA) and cytokinesis-block micronucleus (CBMN) assay with low doses ranging 6-85 mGy. Linear dose responses were observed. Significant DNA aberration clustering was found indicative of high ionizing density radiation. When the dose contribution of both the 14N(n,p)14C and 1H(n,γ)2H capture reactions were considered, the DCA and the CBMN assays generated similar maximum RBE values of 11.3 ± 1.6 and 9.0 ± 1.1, respectively. Consequently, thermal neutron RBE is approximately four times higher than the current ICRP radiation weighting factor value of 2.5. This lends support to bimodal peaks in the quality factor for RBE neutron energy response, underlining the importance of radiological protection against thermal neutron exposures.

A comparative validation of biodosimetry and physical dosimetry techniques for possible triage applications in emergency dosimetry.

Large-scale radiological accidents or nuclear terrorist incidents involving radiological or nuclear materials can potentially expose thousands, or hundreds of thousands, of people to unknown radiation doses, requiring prompt dose reconstruction for appropriate triage. Two types of dosimetry methods namely, biodosimetry and physical dosimetry are currently utilized for estimating absorbed radiation dose in humans. Both methods have been tested separately in several inter-laboratory comparison exercises, but a direct comparison of physical dosimetry with biological dosimetry has not been performed to evaluate their dose prediction accuracies. The current work describes the results of the direct comparison of absorbed doses estimated by physical (smartphone components) and biodosimetry (dicentric chromosome assay (DCA) performed in human peripheral blood lymphocytes) methods. For comparison, human peripheral blood samples (biodosimetry) and different components of smartphones, namely surface mount resistors (SMRs), inductors and protective glasses (physical dosimetry) were exposed to different doses of photons (0-4.4 Gy; values refer to dose to blood after correction) and the absorbed radiation doses were reconstructed by biodosimetry (DCA) and physical dosimetry (optically stimulated luminescence (OSL)) methods. Additionally, LiF:Mg,Ti (TLD-100) chips and Al2O3:C (Luxel) films were used as reference TL and OSL dosimeters, respectively. The best coincidence between biodosimetry and physical dosimetry was observed for samples of blood and SMRs exposed toγ-rays. Significant differences were observed in the reconstructed doses by the two dosimetry methods for samples exposed to x-ray photons with energy below 100 keV. The discrepancy is probably due to the energy dependence of mass energy-absorption coefficients of the samples extracted from the phones. Our results of comparative validation of the radiation doses reconstructed by luminescence dosimetry from smartphone components with biodosimetry using DCA from human blood suggest the potential use of smartphone components as an effective emergency triage tool for high photon energies.

Genetic and epigenetic effects on centromere establishment.

Endogenous chromosomes contain centromeres to direct equal chromosomal segregation in mitosis and meiosis. The location and function of existing centromeres is usually maintained through cell cycles and generations. Recent studies have investigated how the centromere-specific histone H3 variant CENP-A is assembled and replenished after DNA replication to epigenetically propagate the centromere identity. However, existing centromeres occasionally become inactivated, with or without change in underlying DNA sequences, or lost after chromosomal rearrangements, resulting in acentric chromosomes. New centromeres, known as neocentromeres, may form on ectopic, non-centromeric chromosomal regions to rescue acentric chromosomes from being lost, or form dicentric chromosomes if the original centromere is still active. In addition, de novo centromeres can form after chromatinization of purified DNA that is exogenously introduced into cells. Here, we review the phenomena of naturally occurring and experimentally induced new centromeres and summarize the genetic (DNA sequence) and epigenetic features of these new centromeres. We compare the characteristics of new and native centromeres to understand whether there are different requirements for centromere establishment and propagation. Based on our understanding of the mechanisms of new centromere formation, we discuss the perspectives of developing more stably segregating human artificial chromosomes to facilitate gene delivery in therapeutics and research.

Characterization of chromatin at structurally abnormal inactive X chromosomes reveals potential evidence of a rare hybrid active and inactive isodicentric X chromosome.

X chromosome structural abnormalities are relatively common in Turner syndrome patients, in particular X isochromosomes. Reports over the last five decades examining asynchronous DNA replication between the normal X and isochromosome have clearly established that the structurally abnormal chromosome is the inactive X chromosome (Xi). Here the organization of chromatin at a deleted X chromosome, an Xq isochromosome, and two isodicentric chromosomes were examined. Consistent with previous differential staining methods, at interphase, the X isochromosome and isodicentric X chromosomes frequently formed bipartite Barr bodies, observed by fluorescence microscopy using numerous independent bona fide markers of Xi heterochromatin. At metaphase, with the exception of the pseudoautosomal region and the duplicated locus of the macrosatellite DXZ4 (if present on the abnormal X chromosome based on break points), euchromatin markers were absent from the Xi, whereas histone variant macroH2A formed reproducible banded mirror-image chromosomes. Unexpectedly, the isodicentric chromosome in 46,X,idic(X)(q28) cells, which carry a near full-length q-arm-to-q-arm fused chromosome, showed at interphase very rare instances of Xi chromatin bodies that were separated by large distances in the nucleus. Further examination using immunofluorescence and FISH support the possibility that these rare cells may represent ones in which one half of the isodicentric chromosome is active and the other half is inactive.

Genomic and functional variation of human centromeres.

Centromeres are central to chromosome segregation and genome stability, and thus their molecular foundations are important for understanding their function and the ways in which they go awry. Human centromeres typically form at large megabase-sized arrays of alpha satellite DNA for which there is little genomic understanding due to its repetitive nature. Consequently, it has been difficult to achieve genome assemblies at centromeres using traditional next generation sequencing approaches, so that centromeres represent gaps in the current human genome assembly. The role of alpha satellite DNA has been debated since centromeres can form, albeit rarely, on non-alpha satellite DNA. Conversely, the simple presence of alpha satellite DNA is not sufficient for centromere function since chromosomes with multiple alpha satellite arrays only exhibit a single location of centromere assembly. Here, we discuss the organization of human centromeres as well as genomic and functional variation in human centromere location, and current understanding of the genomic and epigenetic mechanisms that underlie centromere flexibility in humans.

Novel Complex Unbalanced Dicentric X-Autosome Rearrangement in a Thoroughbred Mare with a Mild Effect on the Phenotype.

Complex structural X chromosome abnormalities are rare in humans and animals, and not recurrent. Yet, each case provides a fascinating opportunity to evaluate X chromosome content and functional status in relation to the effect on the phenotype. Here, we report the first equine case of a complex unbalanced X-autosome rearrangement in a healthy but short in stature Thoroughbred mare. Studies of about 200 cells by chromosome banding and FISH revealed an abnormal 2n = 63,X,der(X;16) karyotype with a large dicentric derivative chromosome (der). The der was comprised of normal Xp material, a palindromic duplication of Xq12q21, and a translocation of chromosome 16 to the inverted Xq12q21 segment by the centromere, whereas the distal Xq22q29 was deleted from the der. Microsatellite genotyping determined a paternal origin of the der. While there was no option to experimentally investigate the status of X chromosome inactivation (XCI), the observed mild phenotype of this case suggested the following scenario to retain an almost normal genetic balance: active normal X, inactivated X-portion of the der, but without XCI spreading into the translocated chromosome 16. Cases like this present unique resources to acquire information about species-specific features of X regulation and the role of X-linked genes in development, health, and disease.

Extension of lymphocyte viability for radiation biodosimetry: Potential implications for radiological/nuclear mass casualty incidents.

Dicentric chromosome assay (DCA) is routinely used for estimating the absorbed radiation dose in exposed humans. Optimal lymphocyte viability is crucial for reliable dose estimation and most cytogenetic laboratories prefer the receipt of blood samples within 24 to 36 hours after collection. Delays in the shipment/receipt of samples can occur sometimes under certain unforeseen circumstances: (1) Adverse weather conditions, (2) distant location of blood collection sites, and (3) shipping and handling of a large number of samples after radiological/nuclear mass casualty incident(s). To circumvent some of these limitations, we evaluated the suitability of ex vivo irradiated blood samples stored in the presence of phytohemagglutinin (PHA) for 7 days at ambient temperature (22-24°C) for radiation biodosimetry. Blood samples stored in the presence of PHA for up to 7 days showed a higher mitotic index than blood samples stored without PHA. To verify the use of stored blood samples for DCA, frequencies of X-rays induced dicentric chromosomes were analyzed in the blood samples that were cultured either 24 hours after exposure or 7 days later after storage. Our results indicate that storage of ex vivo irradiated blood samples in the presence of PHA at ambient temperature was found optimal for DCA and that the radiation doses estimated by dicentric chromosome frequencies were grossly similar between the fresh and stored blood samples. Our study suggests that reliable and accurate biodosimetry results can be obtained for triage using blood samples stored for up to a week at ambient temperature in the presence of PHA.

What is behind "centromere repositioning"?

An increasing number of observations suggest an evolutionary switch of centromere position on monocentric eukaryotic chromosomes which otherwise display a conserved sequence of genes and markers. Such observations are particularly frequent for primates and equidae (for review see Heredity 108:59-67, 2012) but occur also in marsupials (J Hered 96:217-224, 2005) and in plants (Chromosome Res 25:299-311, 2017 and references therein). The actual mechanism(s) behind remained unclear in many cases (Proc Natl Acad Sci USA 101:6542-6547, 2004; Trends Genet 30:66-74, 2014). The same is true for de novo centromere formation on chromosomes lacking an active centromere. This article focuses on recent reports on centromere repositioning and possible mechanisms behind and addresses open questions.

Centromere Destiny in Dicentric Chromosomes: New Insights from the Evolution of Human Chromosome 2 Ancestral Centromeric Region.

Dicentric chromosomes are products of genomic rearrangements that place two centromeres on the same chromosome. Due to the presence of two primary constrictions, they are inherently unstable and overcome their instability by epigenetically inactivating and/or deleting one of the two centromeres, thus resulting in functionally monocentric chromosomes that segregate normally during cell division. Our understanding to date of dicentric chromosome formation, behavior and fate has been largely inferred from observational studies in plants and humans as well as artificially produced de novo dicentrics in yeast and in human cells. We investigate the most recent product of a chromosome fusion event fixed in the human lineage, human chromosome 2, whose stability was acquired by the suppression of one centromere, resulting in a unique difference in chromosome number between humans (46 chromosomes) and our most closely related ape relatives (48 chromosomes). Using molecular cytogenetics, sequencing, and comparative sequence data, we deeply characterize the relicts of the chromosome 2q ancestral centromere and its flanking regions, gaining insight into the ancestral organization that can be easily broadened to all acrocentric chromosome centromeres. Moreover, our analyses offered the opportunity to trace the evolutionary history of rDNA and satellite III sequences among great apes, thus suggesting a new hypothesis for the preferential inactivation of some human centromeres, including IIq. Our results suggest two possible centromere inactivation models to explain the evolutionarily stabilization of human chromosome 2 over the last 5-6 million years. Our results strongly favor centromere excision through a one-step process.

Chromosome Rearrangements Caused by Double Monosomy in Wheat-Barley Group-7 Substitution Lines.

Interspecific or introgressive hybridization is one of the driving forces in plant speciation, producing allopolyploids or diploids with rearranged genomes. The process of karyotype reshaping following homoploid interspecific hybridization has not been studied experimentally. Interspecific hybridization is widely used in plant breeding to increase genetic diversity and introgress new traits. Numerous introgression stocks were developed for hexaploid wheat Triticum aestivum L. (2n = 6x = 42, genome AABBDD). Double monosomic lines, containing one alien chromosome from the tertiary gene pool of wheat and one homoeologous wheat chromosome, represent a simplified model for studying chromosome rearrangements caused by interspecific hybridization. The pairing of a chromosome from the tertiary gene pool with a wheat homoeologue is restricted by the activity of the wheat Ph1 gene, thus, rearrangements caused by chromosome breakage followed by the fusion of the broken arms can be expected. We analyzed chromosome aberrations in 4 sets of lines that originated from double monosomics of barley (Hordeum vulgare L.) chromosome 7H and wheat group-7 chromosomes with dicentric or ring chromosomes. The dynamics of wheat-barley dicentric chromosomes during plant development was followed and an increased diversity of rearrangements was observed. Besides the targeted group-7 chromosomes, other wheat chromosomes were involved in rearrangements, as chromosomes broken in the centromeric region fused with other broken chromosomes. In some cells, multi-centric chromosomes were observed. The structure and dosage of the introgressed barley chromatin was changed. The transmission of the rearrangements to the progenies was analyzed. The observed aberrations emphasize the importance of cytogenetic screening in gene introgression projects.

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry.

Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®X (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

Molecular characterisation of a case of dicentric Y presented as nonobstructive azoospermia with testicular early maturation arrest.

The dicentric Y chromosome is the most common cytogenetically visible structural abnormality of Y chromosome. The sites of break and fusion of dicentric Y are variable, but break and fusion at Yq12 (proximal to the pseudoautosomal region 2/PAR 2) is very rare. Dicentric Y chromosome is unstable during cell division and likely to generate chromosomal mosaicism. Here, we report a case of infertile male with nonmosaic 46,XY where chromosome Y was dicentric with break and fusion at Yq12 (proximal to PAR 2). Clinical presentation of the case was nonobstructive azoospermia due to early maturation arrest at the primary spermatocyte stage. Various molecular techniques such as FISH, STS-PCR and DNA microarray were carried out to characterise genetic defect leading to testicular maturation arrest in the patient. The break and fusion was found at Yq12 (proximal to PAR 2) and resulted in near total duplication of Y chromosome (excluding PAR 2). The reason for maturation arrest seems due to CNVs of PARs (gain in PAR 1 and loss of PAR 2) and azoospermia factors (gain).