Cytogenetic Techniques Research Articles

A detailed karyological investigation of three endemic Cobitis L. 1758 species (Teleostei, Cobitidae) in Anatolia, Türkiye.

Comparative cytogenetics is a vital approach for diagnosing chromosome abnormalities and identifying species-specific patterns. In this study, chromosomal analysis of three Anatolian endemic Cobitis species was performed. Conventional cytogenetic techniques such as Giemsa staining, C-banding and Ag-NOR staining were applied, followed by measurements of chromosome arm lengths including analysis of the measured data. The diploid chromosome number, 2n = 50, was determined for all three species. The karyotype formulas were as follows: four pairs of metacentric, five pairs of submetacentric, and 16 pairs of subtelo-telocentric chromosomes in Cobitis bilseli; 11 pairs of metacentric, seven pairs of submetacentric, and seven pairs of subtelo-telocentric chromosomes in C. fahireae; and four pairs of metacentric, four pairs of submetacentric, and 17 pairs of subtelo-telocentric chromosomes in C. turcica. Dark C-bands were observed on the pericentromeres of nearly all chromosomes in C. bilseli and C turcica, whereas light C-bands appeared on the pericentromeres of some chromosomes in C. fahireae. Silver-stained metaphases revealed signals on the short arm of a submetacentric chromosome pair in C. fahireae (each homologous chromosome carries one signal), while in C. bilseli and C. turcica, Ag-NOR signals were detected on the long arm of a single metacentric chromosome (only one homologous chromosome carries the signal, and the signal-carrying chromosome is the largest chromosome in the karyotype). This study provides new cytogenetic data consistent with the phylogenetic distances between the studied species, indicating pericentric inversions and/or translocations that govern the formation of Cobitis karyotypes.

Complex chromosomal rearrangements in female carriers experiencing recurrent pregnancy loss or poor obstetric history and literature review.

Complex chromosomal rearrangements (CCRs) often remain unidentified as they are rarely observed in the general population. Females with CCRs are generally recognized on the identification of an affected child with multiple congenital anomalies (MCA) or having a history of repeated pregnancy loss/bad obstetric history (RPL/BOH). In contrast, males with CCRs are diagnosed primarily due to infertility. This study aimed to carry out a systematic epidemiological analysis of CCRs in one of the largest series from the Indian population. In addition, a review of the literature on female CCR carriers experiencing RPL/BOH has been compiled in an attempt to identify the genomic landscape of breakpoints, commonly involved chromosomes, and the breakpoint regions. A total of 8560 healthy individuals with normal physical and mental well-being and had no history of any obvious genetic disorder at the time of presentation were referred for chromosome analysis in view of RPL/BOH between 1994 and 2024. Of them, 8158 had a normal chromosome complement whereas, 402 (4.7%) showed chromosomal aberrations. CCRs were detected in seven individuals, i.e., one partner in each of seven couples with structural rearrangements, all of whom were females. Comprehensive characterization of CCR was carried out using various molecular cytogenetic techniques. Seven CCR carriers had a total of 25 pregnancies: 20 leading to miscarriages (80%), one leading to the birth of an abnormal child (4%), two medically terminated pregnancies (8%) due to abnormal antenatal findings, and the remaining two were healthy (8%). A total of 13 different chromosomes with 24 non-recurring breakpoints were identified in these cases. Chromosome (#) 2 showed four breaks (16.7%), followed by #1, #4, #6, and #13 with three breaks each (12.5% each), while one break each (4.2% each) was seen on the remaining eight chromosomes (#3, #5, #8, #11, #14, #15, #17, and #21). Type I and type IV CCRs were observed in five (71.4%) and one case (14.3%), respectively, along with a "not a true" CCR (14.3%) in the present study group. Overall, the prevalence of CCRs in couples with RPL/BOH was 0.16%. To the best of our knowledge, this is the first study on the epidemiology of CCRs in couples with RPL/BOH of Indian origin. The incidence of CCRs in couples experiencing RPL/BOH in the present cohort was found to be 0.16% with type I CCR being the most predominant of all types, which is congruent with observations from non-Hispanic white and South East Asian populations. The uniqueness and rarity of each CCR pose a challenge in genetic and reproductive counseling.

Aegilops crassa Cytotypes in Some Regions of Türkiye.

A new hexaploid cytotype of Aegilops crassa has been identified in Türkiye. To assess the ploidy levels of native populations, 50 samples from Adıyaman, Batman, Bitlis, Diyarbakır, Hakkari, Mardin, Siirt, Şanlıurfa, Şırnak, and Van were analyzed using flow cytometry and cytogenetic techniques. DNA content was determined by comparison with standard plants. Results confirmed two cytotypes in Türkiye: tetraploid populations from Batman, Bitlis, Diyarbakır, Hakkari, Mardin, Siirt, Şanlıurfa, and Şırnak, and hexaploid accessions from Adıyaman and Van. Ten metaphase plates were analyzed. The tetraploid cytotype exhibited chromosome lengths of 8.95 ± 0.27 to 13.96 ± 0.13 µm, a total genome length of 165.51 ± 0.34 µm, and nuclear DNA content of 18.53 ± 0.29 to 20.37 ± 0.49 pg. Most chromosomes were metacentric, except for chromosomes 7, 8, 10, and 12, which were submetacentric. Two satellite pairs were found on chromosomes 4 and 10. The hexaploid cytotype showed chromosome lengths of 8.90 ± 0.16 to 14.06 ± 0.06 µm, a total genome length of 230.47 ± 0.23 µm, and nuclear DNA content of 33.40 ± 0.52 to 35.01 ± 0.31 pg. Most chromosomes were also metacentric, with three satellite pairs on chromosomes 3, 6, and 10. In conclusion, both tetraploid (2n = 2x = 28) and hexaploid (2n = 6x = 42) cytotypes of Ae. crassa exist in Türkiye, with the hexaploid cytotype having potential for wheat breeding programs.

Impact of sex chromosome abnormalities in male chronic myeloid leukemia patients

IntroductionChronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the Philadelphia chromosome (Ph) resulting from the reciprocal translocation t(9;22)(q34;q11). Sex chromosomal abnormalities are found rarely in CML patients. Materials and methodsConventional and molecular cytogenetic analysis (Fluorescent In Situ Hybridization (FISH)) were performed to reveal the presence of Ph and additional chromosomal abnormalities (ACA). ResultsThree patients with abnormalities within sex chromosomes, one Klinefelter syndrome (KS) patient and two cases with LOY (Loss of Y) diagnosed with CML CP were included in the study. All three patients initially responded to the targeted therapy, became resistant to their treatment course and switched to 2nd line therapies. In the 15th month, the KS patient abruptly turned into BC and showed clonal evolution with four karyotypic patterns. According to Mitelman databases, this is the first KS-CML patient who showed three major route abnormalities, +8, i(17) and + der(22) in a single clone. Survival analysis showed one patient with LOY chromosome expired on the 48th month of diagnosis. ConclusionThe application of cytogenetic techniques in identifying the constitutional abnormality of KS, ACA at initial CML diagnosis, revealing of extra Ph during the treatment course and clonal evolution in BC (Blast Crisis) assist the proper monitoring of disease transformation and serves as a major diagnostic tool for CML patients thereby switching the treatment protocols. A systematic stratification of patients according to the chromosomal abnormalities is needed in CML patients.

Culture failure and suboptimal analysis in cytogenetics: A data review as a quality improvement metric from a resource-limited country

Abstract Introduction/Objective Conventional cytogenetic failures can affect hematological cancer diagnosis and prognosis. Resource-constrained regions without contemporary genetic testing facilities may experience this impact more. The effectiveness of this technique is contingent on cultural success, availability of metaphases, and morphological adequacy. The results can be influenced by the underlying diagnosis, inherent biological features of the leukemic clone, sample conditions, and transit time. Culture failure (CF) and suboptimal analysis (SOA) data can identify problems and set standards. Thus, we designed a cohort to find areas of improvement and reduce negative contributing factors, making this cytogenetic technique more accessible and cost-effective. Methods/Case Report A retrospective study was conducted at Cytogenetics laboratory at Indus hospital, Karachi. From January 2021 to March 2023, 1234 blood and bone marrow samples were processed. CF was defined as no growth, whereas suboptimal cases showed a low mitotic index or poor morphology. Data analysis included collection and processing time, sample volume, cell count, specimen type, in-house or out-reach sample, and diagnosis. Armonk, NY-based IBM SPSS 24.0 used to analyze the data. Chi-square used to find association of various factors with karyotyping. A p-value < 0.05 indicated significance. Results (if a Case Study enter NA) Of 1234 samples, 1110 (90%) were bone marrow and 124 (10%) were peripheral blood. There were 32/1234 (2.6%) CF cases. Each CF case was found in bone marrow samples, making the true incidence 32/1110 (2.9%). Constitutional blood tests found no CF in 124 samples. Additionally, chromosomal analysis quality was assessed. SOA occurred in 105/1234 (8.5%) instances, with 58% having poor morphology and 31% having a low mitotic index. Among 137 CF and SOA patients, 134/1110 (12%) were found in marrow and 3/124 (2.4%) in blood. Conclusion Lower CF rates suggest that appropriate standards and quality management may reduce cytogenetic failure rates despite resource constraints and newly established services.

Exploring Chromosomal Polymorphism and Evolutionary Implications in Rineloricaria lanceolata (Günther, 1868) (Siluriformes: Loricariidae): Insights from Meiotic Behavior and Phylogenetic Analysis.

Chromosomal polymorphism is a significant aspect of population genetics, influencing the adaptation and evolution of species. In Rineloricaria lanceolata, a Neotropical fish species, chromosomal polymorphism has been observed, yet the underlying mechanisms and evolutionary implications remain poorly understood. This article aims to investigate the chromosomal polymorphism in Rineloricaria lanceolata, focusing on elucidating the meiotic behavior of karyotypic variants and tracing the phylogenetic origins of this polymorphism within the genus. By employing molecular markers and cytogenetic techniques, we aim to uncover the mechanisms driving chromosomal rearrangements and their potential role in speciation and adaptation. Understanding the genetic basis of chromosomal polymorphism in R. lanceolata not only contributes to our knowledge of species evolution but also holds implications for the conservation of genetic diversity within this vulnerable group of Neotropical fishes.

Genomic structural variants analysis in leukemia by a novel cytogenetic technique: Optical genome mapping.

Genomic structural variants (SVs) play a pivotal role in driving the evolution of hematologic malignancies, particularly in leukemia, in which genetic abnormalities are crucial features. Detecting SVs is essential for achieving precise diagnosis and prognosis in these cases. Karyotyping, often complemented by fluorescence insitu hybridization and/or chromosomal microarray analysis, provides standard diagnostic outcomes for various types of SVs in front-line testing for leukemia. Recently, optical genome mapping (OGM) has emerged as a promising technique due to its ability to detect all SVs identified by other cytogenetic methods within one single assay. Furthermore, OGM has revealed additional clinically significant SVs in various clinical laboratories, underscoring its considerable potential for enhancing front-line testing in cases of leukemia. This review aims to elucidate the principles of conventional cytogenetic techniques and OGM, with a focus on the technical performance of OGM and its applications in diagnosing and prognosticating myelodysplastic syndromes, acute myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia.

Combined biological effects of CBCT and therapeutic X-ray dose on chromosomal aberrations of lymphocytes

Background and purposeCone beam computed tomography (CBCT) is routinely used in radiotherapy to localize target volume. The aim of our study was to determine the biological effects of CBCT dose compared to subsequent therapeutic dose by using in vitro chromosome dosimetry.Materials and methodsPeripheral blood samples from five healthy volunteers were irradiated in two phantoms (water filled in-house made cylindrical, and Pure Image CTDI phantoms) with 6 MV FFF X-ray photons, the dose rate was 800 MU/min and the absorbed doses ranged from 0.5 to 8 Gy. Irradiation was performed with a 6 MV linear accelerator (LINAC) to generate a dose–response calibration curve. In the first part of the investigation, 1–5 CBCT imaging was used, in the second, only 2 Gy doses were delivered with a LINAC, and then, in the third part, a combination of CBCT and 2 Gy irradiation was performed mimicking online adapted radiotherapy treatment. Metaphases were prepared from lymphocyte cultures, using standard cytogenetic techniques, and chromosomal aberrations were evaluated. Estimate doses were calculated from chromosome aberrations using dose–response curves.ResultsSamples exposed to X-ray from CBCT imaging prior to treatment exhibited higher chromosomal aberrations and Estimate dose than the 2 Gy therapeutic (real) dose, and the magnitude of the increase depended on the number of CBCTs: 1–5 CBCT corresponded to 0.04–0.92 Gy, 1 CBCT + 2 Gy to 2.32 Gy, and 5 CBCTs + 2 Gy to 3.5 Gy.ConclusionThe estimated dose based on chromosomal aberrations is 24.8% higher than the physical dose, for the combination of 3 CBCTs and the therapeutic 2 Gy dose, which should be taken into account when calculating the total therapeutic dose that could increase the risk of a second cancer. The clinical implications of the combined radiation effect may require further investigation.

Integrating Genetic and Cytogenetic Data: A Diversity Study of Astyanax and Psalidodon (Characidae) Species from the Paraná River Basin.

Astyanax is one of the most specious fish groups in the Neotropical region, with many cryptic species, which represents a challenge for correct identification through traditional taxonomic methods. Psalidodon is a recently resurrected genus group of species previously belonging to Astyanax, specifically those with extensive chromosomal variation of the A. scabripinnis and fasciatus complexes. In the present study, the mitochondrial genes cytochrome c oxidase subunit 1 (COI), mitochondrial ATP synthase 6 and 8 (ATPase 6/8), and NADH dehydrogenase subunit 2 (ND2) were used in conjunction with chromosomal data to characterize molecularly and cytogenetically populations of Astyanax and Psalidodon from rivers and streams of the Ivaí River Basin (Paraná Basin). The results demonstrated the effectiveness of the integrative use of molecular and cytogenetic techniques, with the confirmation of at least three species for the sampled sites: A. lacustris, P. paranae, and P. fasciatus, which showed inter- and intrapopulation karyotype variations. In addition, extensive haplotypic variation can be observed for these species within the Ivaí River Basin and throughout the Paraná River Basin. The data demonstrate a hidden diversity among the species analyzed, enrich the ichthyofaunistic knowledge of small rivers and streams, and contribute to future conservation projects in these areas.

Determination of aneuploidy/polyploidy in Holstein cows with a history of abortion

Objective. Determine chromosomal aberrations such as aneuploidies/polyploidies (ap/pp) of cows with a history of abortion from a municipality in Zacatecas, Mexico. Materials and methods. 107 blood samples were collected from cows with a history of abortion and ages between 3 and 10 years in the municipality of Enrique Estrada Zacatecas Mexico, a chromosomal analysis was performed with the conventional cytogenetic technique, to determine chromosomal aberrations such as aneuploidies/polyploidies. Results. The ap/pp cows range from those that did not present any aberrations (0%), a median of (2%) and a maximum of (16%) chromosomal aberrations, the recorded age of the cows was a minimum of 3.5 years, a median of 5.8 and a maximum of 7.4. With these results it can be predicted that there is the possibility that with each year a cow will abort and it increases by 31.7%. However, the p of this coefficient is 0.384—not significant. On the other hand, the possibility of a cow aborting increases by 16.5% with each unit percentage increase in the degree of polyploidy, and the p for this coefficient is 0.029, which is significant. Conclusions. Aneuploidies/polyploidies in correlation with the age of the cows are a possible probability of abortion.

G-Banding and Molecular Cytogenetics Detect Novel Translocations and Cryptic Aberrations in Human Immortal Endothelial Cells.

Endothelial cells (ECs) maintain vessel tone and barrier integrity, regulate blood homeostasis, and prevent the extravasation of leukocytes under normal physiological conditions. Because of the limited lifespans and batch-to-batch differences with respect to the genetic make-up of primary ECs, established immortal EC lines are extensively used for studying endothelial biology. To address this issue, the immortal endothelial cell line EA.hy926 was developed by fusing primary human umbilical vein endothelial cells (HUVECs) with human lung carcinoma A549 cells. EA.hy926 cells share a number of similar endothelial properties with HUVECs and are considered the immortal counterpart to primary HUVECs. However, the cytogenetic integrity of EA.hy926 cells is not fully elucidated. We characterized EA.hy926 cells with conventional G-banding and molecular cytogenetic techniques such as spectral karyotyping and subtelomeric fluorescence in situ hybridization. Cytogenetic analysis revealed an array of numerical and stable structural chromosomal rearrangements including one deletion, one duplication, one isochromosome, seven simple translocations, and five complex translocations in Ea.hy926 cells. These findings will advance comprehension of EA.hy926 cell biology and augment future endothelial studies, specifically in comparison studies between HUVECs and EA.hy926 cells.

Mapping of SINEs in the genome of Proechimys (Mammalia: Rodentia)

This study aimed to analyze the distribution of short interspersed elements (SINEs) in the chromosomes of five species of rodents of the genus Proechimys and in a variant karyotype of P. guyannensis. Molecular cytogenetic techniques were used to characterize the sequences of the B1, B4, MAR and THER SINEs, which were used as probes for hybridization in metaphase chromosomes. A wide distribution of SINEs was observed in the chromosomes of the Proechimys species examined, thus indicating differentiation of these retroelements. The signal of the B4 SINE was more evident than that of the B1 SINE, especially in P. echinothrix, P. longicaudatus, and P. cuvieri. Although the signal of the MAR SINE was more explosive than that of the THER SINE, in the species P. echinothrix, P. guyannensis (2n = 46) and P. longicaudatus, its distribution in the karyotypes was similar. The signals of these retroelements occurred at specific heterochromatic sites and were centromeric/pericentromeric and at the terminal regions in most chromosomes. This appears to be a typical distribution pattern of the SINEs and may indicate involvement with rearrangements during karyotypic diversification in Proechimys. The variation of the SINEs in the genome of Proechimys species demonstrates that these elements are distributed in a specific way in this genus and the preference for some sites, considered hotspots for chromosomal breakage, allows us to propose that these elements are related to the karyotypic evolution of Proechimys.

Chromosomal analysis of two Acanthodoras species (Doradidae, Siluriformes): Insights into the oldest thorny catfish clade and its karyotype evolution.

The Doradidae fishes constitute one of the most diverse groups of Neotropical freshwater environments. Acanthodoradinae is the oldest lineage and the sister group to all other thorny catfishes, and it includes only the genus Acanthodoras. The diversity of Acanthodoras remains underestimated, and the use of complementary approaches, including genetic studies, is an important step to better characterize this diversity and the relationships among the species within the genus. Therefore, we conducted a comprehensive analysis using conventional cytogenetic techniques and physical mapping of three multigene families (18S and 5S ribosomal DNA [rDNA], U2 small nuclear DNA [snDNA]) and four microsatellite motifs, namely (AC)n, (AT)n, (GA)n, and (GATA)n, in two sympatric species from the Negro River: Acanthodoras cataphractus and Acanthodoras cf. polygrammus. We found significant differences in constitutive heterochromatin (CH) content, distribution of the microsatellite (AT)n, and the number of 5S rDNA and U2 snDNA sites. These differences may result from chromosome rearrangements and repetitive DNA dispersal mechanisms. Furthermore, the characterization of the diploid number (2n) of these Acanthodoras species enables us to propose 2n = 58 chromosomes as the plesiomorphic 2n state in Doradidae based on ancestral state reconstruction. Acanthodoradinae is the oldest lineage of the thorny catfishes, and knowledge about its cytogenetic patterns is crucial for disentangling the karyotype evolution of the whole group. Thus, this study contributes to the understanding of the mechanisms behind chromosome diversification of Doradidae and highlights the importance of Acanthodoradinae in the evolutionary history of thorny catfishes.

P-538 Liquid biopsy (niPOC) as a new approach to genetic studies in early pregnancy loss. First clinical experience in Spain

Abstract Study question How accurate is the use of liquid biopsy as a new tool for aneuploidy testing in cases of pregnancy losses? Summary answer This study demonstrates that genome-wide cfDNA testing in the maternal bloodstream (niPOC) constitutes a simple, tissue-independent, straightforward and reliable complement/alternative to traditional POC analysis. What is known already Around 50-70% of clinical miscarriages are caused by aneuploidies, mostly trisomies. To date, conventional cytogenetic and advanced molecular techniques are used for the analysis of POC to identify the genetic cause of miscarriage, providing valuable information for genetic counseling. However, both approaches are based in the direct analysis of the abortive tissue, which entails several limitations due to a risk of culture failure and/or maternal cell contamination. To solve these drawbacks, maternal cell-free DNA (cfDNA) testing emerges as a promising alternative due to the accumulated evidence. Study design, size, duration Retrospective study and first clinical experience, both conducted in our facilities from January 2021 to December 2023. Before carrying out the fetal tissues collection for the POC analysis, a blood sample was drawn to evaluate possible aneuploidies by cfDNA testing. Using NGS+STR POC results as the gold standard, results derived from both studies were compared to assess the percentage of concordance and the cases of non-informativeness (fetal fraction (FF) <2%), false positives, and false negatives. Participants/materials, setting, methods Herein we present a clinical validation (n = 152) and the first clinical experience (n = 103). Maternal peripheral blood samples were extracted for aneuploidy evaluation using Illumina’s sequencing platform. Genetic testing on POCs was performed using NGS (Thermo Fisher Scientific) to detect aneuploidies, and short tandem repeats to detect cases with MCC and/or polyploidy. In both groups (niPOC and POC), the percentage of concordance and cases of non-informativeness (by fetal fraction <2%, or CCM) were evaluated. Main results and the role of chance In the validation set, no significant differences in non-informativity were observed between POC and niPOC [9.2% (14/152) vs 13.8% (21/152)]. Excluding triploids, the sensitivity of niPOC compared with the pregnancy tissue sequencing result was 90.1%, specificity of 97.1% and an accuracy of 88%. In the first clinical experience, non-informativity was similar to that found in the validation (17/103, 16.5%). The percentage of abnormal cases was 43%, very close to the 50% expected according to the scientific literature. Regarding turnaround time, reports in niPOC were released in an average of 3.8 working days. Limitations, reasons for caution Current cfDNA testing technology fails in polyploidy, uniparental disomy identification, which is a potential cause of pregnancy loss. Aneuploidy detection is only feasible if the blood collection was performed before the fetal remains evacuation. Wider implications of the findings CfDNA testing before curettage or pharmacological treatment could be an alternative to POC analysis. If optimized, cfDNA testing could be used contingently with the molecular POC analysis in cases where maternal cell contamination is present. As a result, the overall success rate in the POC program could be substantially improved. Trial registration number Not applicable

P-529 Optical Genomic Mapping (OGM) enable cryptic chromosomal rearrangements in a couple with recurrent miscarriages undergoing preimplantation genetic diagnosis

Abstract Study question Could Optical Genomic Mapping (OGM) detect chromosomal structural variations (SVs) below the detection limit of conventional cytogenetic diagnostics in couples with recurrent miscarriages? Summary answer The Next-generation cytogenetics of Optical Genomic Mapping confirmed a cryptic chromosomal translocation in a couple with recurrent miscarriages undergoing preimplantation genetic testing for aneuploidies (PGT-A). What is known already Chromosomal structural variants (SVs) significantly contribute to infertility, recurrent pregnancy losses and congenital defects... Approximately 2-4% of couples facing recurrent miscarriages carry chromosomal abnormalities. Currently, their detection is based on convencional cytogenetic methods (karyotyping, FISH or array-CGH), however cryptic chromosomal rearrangements are difficult to identify. Next-generation cytogenetics such as OGM is able to detect SVs with a resolution of < 500 kb, allowing a better understanding of the cause of patient’s infertility. In our study, we utilized OGM to confirm a cryptic translocation in a couple experiencing recurrent miscarriages, aiming to enhance our understanding of the underlying causes of their infertility. Study design, size, duration Our study focuses on a non-consanguineous couple diagnosed with recurrent pregnancy loss that attended to our private fertility clinic to undergo assisted reproductive treatment (ART) with their autologous semen, oocytes and PGT-A. Conventional cytogenetic technique was performed on both partners by G-banding karyotyping. PGT-A was performed by NGS (Veriseq, Illumina). Optical Genomic Mapping (OGM) served as a diagnostic tool, complemented by lymphocyte FISH analysis to confirm chromosomal structural variations in these infertile patients. Participants/materials, setting, methods Ultra High Molecular Weight genomic DNA (UHMW gDNA) was extracted from frozen human peripheral blood of both partners. UHMW gDNA was quantified using Qubit dsDNA assay BR kit and Qubit 4 Fluorometer (Thermo Fisher Scientific). We used Saphyr system (Bionano Genomics) for cytogenetic diagnosis. The consensus genomic map was constructed by de novo assembly in the Bionano Solve data analysis software. SVs were identified by aligning Optical Genomic Map with the reference genome GRCh38/hg38. Main results and the role of chance The couple had suffered four prior miscarriages and undergoing three PGT-A cycles. Both partners provided a normal karyotype. Among 13 biopsied embryos, four showed CNVs on chromosomes 2(p24.3è25.3, 12Mb) and 6(p24.1è25.3, 11Mb). Following this result we suspected that the couple might has a cryptic chromosomal structural abnormalities. To test our hypothesis, we performed OGM. We analysed UHMW gDNA samples from both partners. OGM generated an average of 823.67Gb. The average N50 molecule length (≥150kb) was 318kb. The average mapping rate was 90.75% and the average labeling rate was 15.30 labels per 100kb. The average effective coverage depth was 242X. OGM successfully identified a cryptic translocation in the male partner ogm[GRch38]t(2;6)(p24.3;24.1), while the female partner showed no abnormalities. Translocation breakpoints identified by OGM were Chr2:12.560.508 and Chr6:11.622.593. The translocated segments were near to the detection limit of conventional cytogenetic analysis and with similar pattern that could be the reason for missed detection by karyotyping. Importantly, we identified one gene disrupted by OGM rearrangement breakpoint: MIR3681HG. A previous study considered that CNV loci of 2p24.3, including MIR3681HG gene, could be associated with spermatogenesis. The lymphocyte FISH analysis finding corroborated the OGM result. Limitations, reasons for caution OGM is a very powerful method for detecting chromosomal structural variants in human genomes and is able to detect cryptic structural variants. However, this is a very new diagnostic application, so there is limited experience with OGM for correlating structural variations results to infertility diagnosis in couples with recurrent miscarriages. Wider implications of the findings A balanced cryptic translocation has been identified in a couple with recurrent miscarriages. Our case report demonstrates the high diagnostic capability of Optical Genomic Mapping for cryptic SVs. OGM technology allows a more accurate cytogenetic diagnosis by detecting cryptic structural variants with high resolution in infertile patients with recurrent miscarriages. Trial registration number not applicable

The likelihood of detecting abnormal karyotypes in fetuses with a single major anomaly or "soft" marker on ultrasonographic scanning.

Fetuses with abnormal karyotypes often exhibit distinctive ultrasonographic markers, including major anomalies and "soft" markers, indicating potential chromosomal issues. A crucial consideration arises when a single fetal anomaly is detected, raising the question of whether karyotyping is warranted, given the associated procedural risks. Our objective was to establish correlations between single fetal anomalies identified through ultrasound and chromosomal abnormalities. A cross-sectional study analyzed the karyotype of 1493 fetuses and detected a single ultrasonographic anomaly over a 16-year period. Karyotyping was performed using the standard karyotype technique. Moreover, data regarding the type of anomaly detected ultrasonographically, karyotype results, and outcomes following interventions were collected. Among other methods, the use of positive likelihood ratios (LR+) was used to evaluate the diagnostic accuracy of ultrasound compared to karyotyping. In total, an aberrant karyotype was identified in 99 fetuses (6.6%). This was most commonly observed in cases involving a "soft" marker, occurring in 27 out of 218 fetuses (12.4%). The most frequently detected aberrant karyotype resulted from aneuploidies (80.6% of cases), notably trisomy 21 (50.5%). "Soft" markers predicted chromosomal issues (LR+ = 1.9; OR = 2.4), and isolated polyhydramnios (LR+ = 1.54; OR = 1.6) showed significance in predicting fetal chromosomal aberrations. When assessing the necessity for karyotyping in fetuses with single major anomalies or "soft" markers, it is crucial to consider individual risks for chromosomopathies, including the LR+ of the detected marker. In cases where fetuses exhibit isolated anomalies with a normal karyotype, additional diagnostic measures, such as molecular cytogenetic and molecular genetics techniques, may become necessary.

Optical Genome Mapping for Applications in Repeat Expansion Disorders.

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow.

An alternative approach of TUNEL assay to specifically characterize DNA fragmentation in cell model systems

DNA damage is one of the most important effects induced by chemical agents. We report a comparative analysis of DNA fragmentation on three different cell lines using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, generally applied to detect apoptosis. Our approach combines cytogenetic techniques and investigation in detached cellular structures, recovered from the culture medium with the aim to compare the DNA fragmentation of three different cell line even beyond the cells adherent to substrate. Consequently, we detect any fragmentation points on single chromosomes, whole nuclei and other cellular structures. Cells were exposed to resveratrol (RSV) and doxorubicin (Doxo), in single and combined treatments. Control and treated astrocytes showed DNA damage in condensed nuclei and detached structures. Caco-2 cells showed fragmented DNA only after Doxo-treatment, while controls showed fragmented chromosomes, indicating DNA damage in replicating cells. MDA-MB-231 cells showed nuclear condensation and DNA fragmentation above all after RSV-treatment and related to detached structures. This model proved to perform a grading of genomic instability (GI). Astrocytes show a hybrid level of GI. Caco-2 cells showed fragmented metaphase chromosomes, proving that the DNA damage was transmitted to the daughter cells probably due to an absence of DNA repair mechanisms. Instead, MDA–MB-231 cells showed few or no fragmented metaphase, suggesting a probable activation of DNA repair mechanisms. By applying this alternative approach of TUNEL test, we obtained data that can more specifically characterize DNA fragmentation for a suitable application in various fields.

The impact of COVID-19 on microRNA and CD marker expression in AML patients

Acute myeloid leukaemia (AML) is an aggressive leukaemia characterised by uncontrolled blast cell proliferation. miRNAs and Clusters of Differentiation (CD) molecules play essential roles in AML progression. This study aims to investigate the effect of COVID-19 on the expression of circulating miRNA and CD molecules in AML. This cross-sectional study recruited 32 AML patients and 20 controls. Blood samples were collected and analysed using molecular cytogenetic, miRNA/mRNA expression, and flow cytometry techniques. The expression of miRNAs varied significantly between patients with AML and control individuals. The co-expression of these miRNAs was higher (P < 0.05), indicating that the presence of one miRNA led to increased expression of other miRNAs. A differential correlation was observed between miRNAs and CD markers. Additionally, miRNA 16, miRNA 21, and miRNA 221 showed significant downregulation (P < 0.05 and P < 0.01, respectively) in AML patients with COVID-19 infection compared to those without a disease. Interestingly, this study identified a higher expression level (P < 0.01) of miRNA 137 as a novel biomarker for AML patients. Moreover, the expression of miRNA 137 showed a high correlation (P < 0.05) with most of the CD markers examined in this study and FISH features data. Furthermore, a strong correlation (P < 0.01) was observed between CD markers and miRNA among AML patients with positive and negative COVID-19 infection. These data demonstrated that COVID-19 contributed to increased expression of microRNAs in AML patients. MicroRNA 137 was identified as a novel microRNA that exhibited significant differences between patients and healthy individuals, highlighting its role in AML pathogenesis.

Hematological cytomorphology: Where we are.

The manuscript discusses the historical evolution of observing blood cell morphology under an optical microscope, from the earliest microscopes in the 17th century to the modern digital era, highlighting key advancements and contributions in the field. Blood has historically held symbolic importance in various cultures, with early medical observations dating back to Hippocrates and Galeno. The discovery of cells and subsequent advancements in microscopy by scientists like Hooke and van Leeuwenhoek paved the way for understanding blood cell morphology. Influential figures such as Hewson, Donné, and Ehrlich followed. Diagnostic cytology evolved from manual cell counting to the development of automated hematological systems. Automated complete blood counting came to support microscopic examination in diagnosing hematological disorders. Morphology is crucial in predicting disease outcomes and guiding treatment decisions, particularly hematological neoplasms. The introduction of flow cytometry and its integration with traditional morphological analysis and the new cytogenetic and molecular techniques revolutionized the classification and prognostication of hematologic disorders. Digital microscopy has emerged as a powerful tool in recent years, offering rapid acquisition and sharing of blood cell images. Integrating Artificial Intelligence with digital microscopy has further enhanced morphological analysis, improving diagnostic efficiency. We also discuss the prospects of AI in pre-classifying blood cells in bone marrow aspirate samples, potentially revolutionizing diagnostic pathways for hematologic diseases. Overall, the manuscript provides a comprehensive overview of the historical development, clinical significance and technological advancements in observing blood cell morphology, underscoring its continued relevance in modern hematology practice.