Cytogenetic Investigations Research Articles

O-236 Why one is not enough when studying pregnancy loss - a European perspective

Abstract Pregnancy loss is a distressing but still poorly understood condition, ending one in four pregnancies. With more than 30 million losses worldwide yearly, it is posing significant challenges to individuals, families, and healthcare systems. This presentation delves into the imperative for a multifaceted approach to studying pregnancy loss. By examining pregnancy loss through multiple lenses including fetal and parental genetics, male and female physiology, and psychosocial, lifestyle and political dimensions, we can achieve an improved understanding of the causes, impact, prevention, and treatment of pregnancy loss. Historically, pregnancy loss has been considered a part of the reproductive journey – a course of nature. And until a couple has suffered from recurrent pregnancy loss, the physiological and mental impact has been neglected. This is an outdated approach that prevents a full understanding of the consequences and underlying etiologies hindering the potential of prevention and treatment, and thereby contributes to maintaining the women’s health gap. Moreover, current practice ignores the association of euploid pregnancy loss with an increased risk of future losses and later maternal health consequences such as diabetes and cardiovascular disease. In Copenhagen, we are currently running a large-scale pregnancy loss study (Copenhagen Pregnancy Loss, COPL) involving 3000 pregnancy losses. By including the full trio (fetus, female, and male) we are gaining new insight to the mechanisms causing pregnancy loss and the consequences hereof, both in men and women. We know from previous cytogenetic investigation of lost pregnancies, that about 60% are caused by fetal aneuploidy. But the remaining part is still not well understood and leaves the opportunity of treatments and preventions if we knew the underlying mechanism. That we aim to cover by building the largest database and biobank in the field. Moreover, collected data from the COPL study will be linked to nationwide Danish registries including comprehensive information on medical history, social economic status, educational status, medication, and ancestry. We have validated the use of cell-free fetal DNA in maternal blood to separate the losses caused by fetal genetics or not. This differentiation is essential to address the heterogeneous nature of pregnancy loss, but also to provide an individual answer to the couples. In 1000 consecutive pregnancy losses between gestational age 5 and 22 weeks, we were able to provide a cffDNA based result of the fetal ploidy status in 89% of cases. When comparing the method to direct sequencing of the pregnancy tissue, the cffDNA-based testing had a higher success-rate and identified 85% of the aneuplodies (sensitivity) with a 93% specificity. These results show the potential and feasibility of the method to distinguish euploid and aneuploid pregnancy loss for improved clinical management and benefit of future research. From our close collaboration with couples that are experiencing a devastating event as pregnancy loss, we have seen the huge unmet need for better handling, but also a deep commitment and motivation from the men and women involved. We have investigated the conflicting feelings emerging in a next pregnancy and a need for closer support and care. We belief, that it takes a trio to improve the understanding and handling of pregnancy loss.

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.

Amplification of different satellite-DNAs in prostate cancer

In various solid tumors and corresponding cell lines, prior research has identified acquired copy number variations (CNVs) encompassing centromeric satellite-DNA sequences. This observation emerged from the application of centromeric probes (satellite-DNA) as controls in molecular cytogenetic investigations and diagnostics, although these accounts were largely anecdotal. In this study, we conducted a systematic screening for satellite-DNA sequence amplification in 31 prostate cancer (PCa) samples, a prevalent malignancy in men characterized by discernible molecular cytogenetic aberrations. Notably, PCa-typical genetic aberrations, such as TMPRSS2-ERG gene rearrangements and PTEN deletion, were identified in 12 and 6 out of the 31 PCa samples, respectively. Overall, PCa exhibited genomic instability marked by chromosomal gain or loss of signals across nearly all tested satellite-DNA regions, with particular emphasis on the Y-chromosome (18/31 cases). Remarkably, 5/12 PCa samples representing more advanced metastatic cancer displayed amplification of one or two satellite DNA stretches each, being detectable as blocks analogous to homogenously staining regions. Notably, these stretches included α-satellite DNA derived from chromosomes 2, 3, 4, 15, and 20, as well as satellite-III DNAs (D1Z1 and DYZ1). These findings align with recent discoveries indicating that α-satellite DNAs are expressed as long-non-coding RNAs in advanced cancer, particularly in the context of PCa.

Cytogenetics investigation in 151 Brazilian infertile male patients and genomic analysis in selected cases: experience of 14 years in a public genetic service

ObjectivesMale infertility accounts for approximately 30% of cases of reproductive failure. The characterization of genetic variants using cytogenomic techniques is essential for the adequate clinical management of these patients. We aimed to conduct a cytogenetic investigation of numerical and structural rearrangements and a genomic study of Y chromosome microdeletions/microduplications in infertile men derived from a single centre with over 14 years of experience.ResultsWe evaluated 151 infertile men in a transversal study using peripheral blood karyotypes and 15 patients with normal karyotypes through genomic investigation by multiplex ligation-dependent probe amplification (MLPA) or polymerase chain reaction of sequence-tagged sites (PCR-STS) techniques. Out of the 151 patients evaluated by karyotype, 13 presented chromosomal abnormalities: two had numerical alterations, and 11 had structural chromosomal rearrangements. PCR-STS detected a BPY2 gene region and RBMY2DP pseudogene region microdeletion in one patient. MLPA analysis allowed the identification of one patient with CDY2B_1 and CDY2B_2 probe duplications (CDY2B and NLGN4Y genes) and one patient with BPY2_1, BPY2_2, and BPY2_4 probe duplications (PRY and RBMY1J genes).

Defining autopolyploidy: Cytology, genetics, and taxonomy.

Autopolyploidy is taxonomically defined as the presence of more than two copies of each genome within an organism or species, where the genomes present must all originate within the same species. Alternatively, "genetic" or "cytological" autopolyploidy is defined by polysomic inheritance: random pairing and segregation of the four (or more) homologous chromosomes present, with no preferential pairing partners. In this review, we provide an overview of methods used to categorize species as taxonomic and cytological autopolyploids, including both modern and obsolete cytological methods, marker-segregation-based and genomics methods. Subsequently, we also investigated how frequently polysomic inheritance has been reliably documented in autopolyploids. Pure or predominantly polysomic inheritance was documented in 39 of 43 putative autopolyploid species where inheritance data was available (91%) and in seven of eight synthetic autopolyploids, with several cases of more mixed inheritance within species. We found no clear cases of autopolyploids with disomic inheritance, which was likely a function of our search methodology. Interestingly, we found seven species with purely polysomic inheritance and another five species with partial or predominant polysomic inheritance that appear to be taxonomic allopolyploids. Our results suggest that observations of polysomic inheritance can lead to relabeling of taxonomically allopolyploid species as autopolyploid and highlight the need for further cytogenetic and genomic investigation into polyploid origins and inheritance types.

Injection-based hairy root induction and plant regeneration techniques in Brassicaceae

BackgroundHairy roots constitute a valuable tissue culture system for species that are difficult to propagate through conventional seed-based methods. Moreover, the generation of transgenic plants derived from hairy roots can be facilitated by employing carefully designed hormone-containing media.ResultsWe initiated hairy root formation in the rare crucifer species Asperuginoides axillaris via an injection-based protocol using the Agrobacterium strain C58C1 harboring a hairy root-inducing (Ri) plasmid and successfully regenerated plants from established hairy root lines. Our study confirms the genetic stability of both hairy roots and their derived regenerants and highlights their utility as a permanent source of mitotic chromosomes for cytogenetic investigations. Additionally, we have developed an effective embryo rescue protocol to circumvent seed dormancy issues in A. axillaris seeds. By using inflorescence primary stems of Arabidopsis thaliana and Cardamine hirsuta as starting material, we also established hairy root lines that were subsequently used for regeneration studies.ConclusionWe developed efficient hairy root transformation and regeneration protocols for various crucifers, namely A. axillaris, A. thaliana, and C. hirsuta. Hairy roots and derived regenerants can serve as a continuous source of plant material for molecular and cytogenetic analyses.

Cytogenetic Investigation of Some Ranunculus L. Species Distributed in Bitlis and its Surroundings

Ranunculus L. genus has 84 species in Turkey, when subspecies and varieties are included in this number, it is represented by a total of 102 taxa. 22 of which are endemic, the endemism rate is 21.5%. Since many members of the genus Ranunculus cannot be distinguished from each other with definite differences, they have been tried to be defined and expressed as species complexes taxonomically. Most of the systematic studies are in the direction of regulating such complexes. It is known that karyological characters are of great importance in the phylogenetic classification of the Ranunculaceae family.
 
 In this study, it is aimed to reveal the karyological characteristics of three species which are very similar to each other, in Group A of Ranunculus subgenus of Ranunculus L. in Volume 1 of Flora of Turkey morphologically by using cytogenetic methods. In this context, the karyological characteristics of R. diversifolius Boiss. & Kotschy, R. poluninii Davis and R. crateris Davis species have been determined. R. poluninii and R. crateris species are endemic to our country and are Iran-Turanian element. R. diversifolius species, on the other hand, is distributed in the East of Turkey.
 
 It has been determined that all three species whose chromosomal characteristics were examined are diploid and their chromosome numbers are x=8, 2n=16. It has also been determined that these three species, which are morphologically similar to each other, are the same in terms of chromosome number but different in terms of chromosome morphology and each species has its own characteristics. With this study, the chromosomal characteristics of these three species have been determined for the first time by us and have been brought to the literature.

Lived experience of recurrent miscarriage: women and their partners’ experience of subsequent pregnancy and support within an NHS specialist clinic – a qualitative study

ObjectiveThis study aims to describe the lived experiences of couples with a history of recurrent miscarriage in subsequent pregnancies and their perception of clinic support and cytogenetic investigations.DesignA qualitative interview...

A rare case of EBV-positive gray zone lymphoma

Abstract Introduction/Objective Gray zone lymphoma (GZL) is a rare and novel category of lymphoid neoplasms, first described in 2005. It is derived from B-cells and exhibits characteristics that fall between diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma(cHL). Its association with EBV is extremely rare. Here we present one such case Methods/Case Report 80-year-old female presented with progressive neck swelling and significant weight loss for over a month. It was not associated with fever, pain, or night sweats. Her past medical history was significant for hypertension, cerebrovascular accident, breast carcinoma status post-mastectomy, and non-specific B-cell lymphoma on complete remission for 9 years (post rituximab therapy). Complete blood count and metabolic profile were within normal limits. A PET scan revealed hypermetabolic cervical, supraclavicular, right axillary, and mediastinal lymph nodes, suspicious for lymphoma recurrence. The patient underwent an excisional biopsy of the left cervical lymph node which on microscopy showed lymphoid tissue in a nodular pattern with fibrosis and large areas of necrosis. The well-preserved areas showed lymphohistiocytic proliferation composed of large atypical lymphoid cells with prominent nucleoli. An area with increased eosinophils was also noted. Immunohistochemical staining showed strong positivity for EBER, CD30, MUM1, and OCT2 with dim PAX5 positivity. The cells were negative for CD20 and BOB1. Cytogenetics and molecular studies did not reveal any rearrangements or mutations. Flow cytometric study was inconclusive due to insufficient cells. A diagnosis of EBV-positive GZL was rendered. She has received brentuximab and her symptoms are improving with regressing lymph node size. Results (if a Case Study enter NA) NA Conclusion The presence of EBV positivity in GZL further complicates the diagnostic process, as it adds another layer of complexity in determining the appropriate classification and treatment. Our case highlights the importance of comprehensive morphologic phenotypic and cytogenetic investigation to diagnose GZL.

The Types and Frequencies of X Chromosome Abnormalities in Women with Reproductive Problems

Introduction: X chromosome architecture and integrity are essential for normal ovarian function. Both numerical and structural X chromosome abnormalities play an important role in female infertility. This study aimed to determine the types and frequency of X chromosome aberrations detected in women referred for cytogenetic investigation due to reproductive problems. Methods: 2,936 women (average age: 37.5 years) were enrolled in the present study. Peripheral blood karyotyping was performed by conventional cytogenetic techniques. For each woman, 20 G-banded metaphases were studied and in case of suspected mosaicism, analysis was extended to 100 metaphases. Results: 2,588/2,936 (88.15%) of women had a normal karyotype (46,XX), while 348/2,936 (11.85%) had an abnormal one. Thirty-two women (1.09%) carried autosomal chromosome abnormalities and 316 (10.76%) had X chromosome rearrangements. In 311/2,936 women (10.59%), X chromosome numerical aberrations were detected (low-level mosaicism), and in 5/2,936 cases (0.17%), X structural abnormalities (two with pericentric inversion, one with Xq deletion and two 45,X mosaics, one with an Xp deletion cell line and the other with isochromosome Xq cell line). Low-level X mosaicism was a common finding in women >35 years as compared to younger ones (92.93% vs. 7.07%), a finding consistent with loss of chromosome X with aging. Other X chromosome abnormalities were detected in younger women (32.3 ± 4.13 vs. 41.04 ± 4.5 years). The mean age of women with Turner-like phenotype was 28.75 ± 6.6 years. Conclusion: The study confirms that the incidence of X chromosome abnormalities is increased in women with fertility problems and that karyotype is the gold standard for their identification. Genetic counseling is recommended in these cases to provide information concerning available treatment and fertility options.

Integrating Genomic and Chromosomal Data: A Cytogenetic Study of Transancistrus santarosensis (Loricariidae: Hypostominae) with Characterization of a ZZ/ZW Sex Chromosome System.

The plecos (Loricariidae) fish represent a great model for cytogenetic investigations due to their variety of karyotypes, including diploid and polyploid genomes, and different types of sex chromosomes. In this study we investigate Transancistrus santarosensis a rare loricariid endemic to Ecuador, integrating cytogenetic methods with specimens' molecular identification by mtDNA, to describe the the species karyotype. We aim to verify whether sex chromosomes are cytologically identifiable and if they are associated with the accumulation of repetitive sequences present in other species of the family. The analysis of the karyotype (2n = 54 chromosomes) excludes recent centric fusion and pericentromeric inversion and suggests the presence of a ZZ/ZW sex chromosome system at an early stage of differentiation: the W chromosome is degenerated but is not characterized by the presence of differential sex-specific repetitive DNAs. Data indicate that although T. santarosensis has retained the ancestral diploid number of Loricariidae, it accumulated heterochromatin and shows non-syntenic ribosomal genes localization, chromosomal traits considered apomorphic in the family.

Diagnostic Accuracy of Flow Cytometric DNA Index in Saudi Children with B Cell Acute Lymphoblastic Leukemia.

B cell Acute Lymphoblastic Leukemia (B-ALL) is one of the most common types of cancer diagnosed in children in Saudi Arabia. Cytogenetic investigations, including karyotyping and FISH, are used to determine the incidence and prognostic significance of chromosomal abnormalities in B-ALL. However, in ALL, accurate identification of the morphology of chromosomes is sometimes not achieved. Flow cytometric DI may have the advantage of being technically fast, using either fresh or frozen samples to correctly stratify the patient into the appropriate risk group for treatment. In this study, we evaluated the reliability and validity of using fixed samples instead of fresh samples to determine aneuploidy in cancer cells using a DNA index. The results of the DNA index obtained by flow cytometry were compared with those of conventional cytogenetic analysis to validate the accuracy. Fixed samples (n = 72) from children diagnosed with B-ALL at King Fahad Medical City in Riyadh between 2017 and 2019 were investigated. The results showed strong and statistically significant positive correlation between DNAI-FCM and conventional cytogenetic analysis (p = 0.000 < 0.01). The DNA index value by flow cytometry was proportional to the cytogenetic study in 94.36% (67) of the cases, while discrepancy was observed in 5.6% (four) cases. Our findings highlight the ability of the DNA index method to provide complementary information for the accurate diagnosis of aneuploidy in patients with B-ALL.

Bone Marrow Karyotype, Flow Cytometry, and FISH Analysis: Essential Tests to Improve the Initial Diagnosis of Patients with Myeloid Malignancies

Background and Aims: Diagnosing hematologic malignancies requires implementing several tests. This study aims to evaluate the chromosomal changes in patients with myeloid disorders and compare the results of flow cytometry and cytogenetics with the initial diagnosis performed by the oncologist.&#x0D; Materials and Methods: 115 patients with myeloid disorders, 57.2% males and 42.8% females with a mean age of 50.3 years, previously diagnosed by an oncologist based on the clinical features, complete blood count, and peripheral blood smear interpretations, were considered. Moreover, flow cytometry and cytogenetic analysis were implemented on the bone marrow samples.&#x0D; Results: Cytogenetic results showed that 30% of patients with myeloid disorders had abnormal karyotypes. 77% of patients with myelodysplastic syndromes, 65% of acute myeloid leukemia, and 30.7% of chronic myeloid leukaemia indices showed normal karyotypes, and the others resulted in common and uncommon abnormalities, including the translocation (13;17), 92, XXYY, and del (4q). Considering the flow cytometry and karyotype results, the improved diagnoses were made for 41 patients who had not been diagnosed initially.&#x0D; Conclusion: This study showed that, in some cases, an initial diagnosis is inconsistent with the flow cytometry and karyotype analysis results. Also, the flow cytometry results may differ from the karyotype depending on the case. Therefore, combining the results obtained by the cytogenetic investigation, flow cytometry, fluorescence in situ hybridization, and molecular testing is preferable to provide a comprehensive report for the appropriate disease diagnosis and prognosis.

Case Report: Spontaneous pneumothorax revealing multiple myeloma: a case report

Various causes like trauma, infection, pulmonary disease or neoplasm can lead to spontaneous pneumothorax. We report a rare case of a spontaneous pneumothorax as first manifestation of multiple myeloma. A 58-year-old patient presented suffering from dyspnea and right-sided chest pain, with no history of trauma. On examination, the patient had bilateral rib tenderness. The respiratory rate was 30 breaths/min and oxygen saturation was 88%. The chest physical exam revealed unequal breath sounds, an hyperresonance with percussion and decreased wall movement on the right side. The analysis of arterial blood gas revealed hypoxemia (arterial oxygen tension: 7.59 kPa) and hypercapnia (arterial carbon dioxide tension: 5.99 kPa). Laboratory data showed a raised C reactive protein level (133.8 mg/L), hyper-calcemia (serum calcium: 12.18 mg/dL) and a decreased plasma albumin level (31.9 g/L). Chest radiography and thoracic computed tomography revealed multiple ribs and sternum fractures leading to a partial pneumothorax on the right side. Subsequent workup for multiple myeloma showed elevated levels of immunoglobulin. Results of initial laboratory tests revealed an IgG gamma paraprotein, a urine protein electrophoresis of 1450 mg/24 hours and a β-2 microglobulin rate of 3.35. The diagnosis of multiple myeloma was confirmed with a bone marrow infiltration of 20% of atypical plasmatic cells. Cytogenetic investigations did not show any chromosomal abnormalities, especially the t (4,14) translocation. The patient was diagnosed with multiple myeloma stage IIIA according to Durie–Salmon classification. Appropriate treatment with oxygen therapy and systemic analgesic was started, associated with a cure of zoledronic acid in order to decrease the calcium level. The evolution was characterized by the complete resolution of the pneumothorax in 7 days and the normalization of the calcium level. The autologous stem cell transplant was the treatment of choice for this patient.

Allium cytogenetics: a critical review on the Indian taxa.

The genus Allium Linnaeus, 1753 (tribe Allieae) contains about 800 species worldwide of which almost 38 species are reported in India, including the globally important crops (onion, garlic, leek, shallot) and many wild species. A satisfactory chromosomal catalogue of Allium species is missing which has been considered in the review for the species occurring in India. The most prominent base number is x=8, with few records of x=7, 10, 11. The genome size has sufficient clues for divergence, ranging from 7.8 pg/1C to 30.0 pg/1C in diploid and 15.16 pg/1C to 41.78 pg/1C in polyploid species. Although the karyotypes are seemingly dominated by metacentrics, substantial variation in nucleolus organizing regions (NORs) is noteworthy. The chromosomal rearrangement between A.cepa Linnaeus, 1753 and its allied species has paved way to appreciate genomic evolution within Allium. The presence of a unique telomere sequence and its conservation in Allium sets this genus apart from all other Amaryllids and supports monophyletic origin. Any cytogenetic investigation regarding NOR variability, telomere sequence and genome size in the Indian species becomes the most promising field to decipher chromosome evolution against the background of species diversity and evolution, especially in the Indian subcontinent.

A pilot investigation of low-pass genome sequencing identifying site-specific variation in chromosomal mosaicisms by a multiple site sampling approach in first-trimester miscarriages.

Can multiple-site low-pass genome sequencing (GS) of products of conception (POCs) improve the detection of genetic abnormalities, especially heterogeneously distributed mosaicism and homogeneously distributed mosaicism in first-trimester miscarriage? Multiple-site sampling combined with low-pass GS significantly increased genetic diagnostic yield (77.0%, 127/165) of first-trimester miscarriages, with mosaicisms accounting for 17.0% (28/165), especially heterogeneously distributed mosaicisms (75%, 21/28) that are currently underappreciated. Aneuploidies are well known to cause first-trimester miscarriage, which are detectable by conventional karyotyping and next-generation sequencing (NGS) on a single-site sampling basis. However, there are limited studies demonstrating the implications of mosaic genetic abnormalities in first-trimester miscarriages, especially when genetic heterogeneity is present in POCs. This is a cross-sectional cohort study carried out at a university-affiliated public hospital. One hundred seventy-four patients diagnosed with first-trimester miscarriage from December 2018 to November 2021 were offered ultrasound-guided manual vacuum aspiration (USG-MVA) treatment. Products of conception were subjected to multiple-site low-pass GS for the detection of chromosomal imbalances. For each POC, multiple sites of villi (three sites on average) were biopsied for low-pass GS. Samples with maternal cell contamination (MCC) and polyploidy were excluded based on the quantitative fluorescence polymerase chain reaction (QF-PCR) results. The spectrum of chromosomal abnormalities, including mosaicism (heterogeneously distributed and homogeneously distributed) and constitutional abnormalities was investigated. Chromosomal microarray analysis and additional DNA fingerprinting were used for validation and MCC exclusion. A cross-platform comparison between conventional karyotyping and our multiple-site approach was also performed. One hundred sixty-five POCs (corresponding to 490 DNA samples) were subjected to low-pass GS. Genetic abnormalities were detected in 77.0% (127/165) of POCs by our novel approach. Specifically, 17.0% (28/165) of cases had either heterogeneously distributed mosaicism (12.7%, 21/165) or homogeneously distributed mosaicism (6.1%, 10/165) (three cases had both types of mosaicism). The remaining 60.0% (99/165) of cases had constitutional abnormalities. In addition, in the 71 cases with karyotyping performed in parallel, 26.8% (19/71) of the results could be revised by our approach. Lack of a normal gestational week-matched cohort might hinder the establishment of a causative link between mosaicisms and first-trimester miscarriage. Low-pass GS with multiple-site sampling increased the detection of chromosomal mosaicisms in first-trimester miscarriage POCs. This innovative multiple-site low-pass GS approach enabled the novel discovery of heterogeneously distributed mosaicism, which was prevalent in first-trimester miscarriage POCs and frequently observed in preimplantation embryos, but is currently unappreciated by conventional single-site cytogenetic investigations. This work was supported partly by Research Grant Council Collaborative Research Fund (C4062-21GF to K.W.C), Science and Technology Projects in Guangzhou (202102010005 to K.W.C), Guangdong-Hong Kong Technology Cooperation Funding Scheme (TCFS), Innovation and Technology Fund (GHP/117/19GD to K.W.C), HKOG Direct Grant (2019.050 to J.P.W.C), and Hong Kong Health and Medical Research Fund (05160406 to J.P.W.C). The authors have no competing interests to declare. N/A.

Enhanced mitotic arrest and chromosome resolution for cytogenetic analysis in the eastern mosquitofish, Gambusia holbrooki

Maximising the number of cells arrested at metaphase and their resolution is fundamentally important for molecular cytogenetic investigations, particularly in fish, which typically yield low mitotic index and have highly condensed chromosomes. To overcome these limitations, fish were injected with a mitotic stimulator (the yeast, Saccharomyces cerevisiae) to improve the mitotic index, and the intercalating agent ethidium bromide to produce elongated chromosomes. Specifically, adults were injected with activated yeast and then Colcemid (0.025 µg/µl solution, 10 µl per 1 g of body weight) at 24–96 h post yeast injections, followed by chromosome preparations from multiple tissues. Results showed that gill tissue had the highest number of dividing cells at 72 h post yeast exposure with no significant (p > 0.05) differences between the sexes. Nonetheless, sex-specific differences in the mitotic index were observed in spleen, kidney, and liver, which may be attributed to sex-specific differences in immune responses. For elongation of mitotic chromosomes, individuals (both sexes) were first injected with activated yeast and after 48 h with ethidium bromide (2 or 4 µg/ml) and Colcemid (0.05 µg/µl solution, 10 µl per 1 g of body weight). Following which, animals were sampled at three time points (1, 4 and 8 h) for chromosome preparations. The results show that the optimum elongation of metaphase chromosomes of males and females was achieved by using 2 µg/ml and 4 µg/ml, respectively, for 1 h. Interestingly, the average mitotic chromosome length (μm) of males and females post-ethidium bromide exposure was significantly different (p < 0.05) for both concentrations, except at 1 h exposure for 2 µg/ml EtBr. Such differences can be attributed to overall chromosomal condensation differences between sexes. Regardless, the increased mitotic index and chromosome resolution could benefit cytogenetic studies in other fish species.

Cytogenetic and Biochemical Responses of Wheat Seeds to Proton Irradiation at the Bragg Peak

The present study aimed to evaluate the morphological, cytogenetic and biochemical changes in wheat seedlings as affected by seed exposure to a proton beam at the Bragg peak. The average energy of the proton beam was of 171 MeV at the entrance into the irradiator room while at the point of sample irradiation the beam energy was of 150 MeV, with the average value of the Linear Energy Transfer of 0.539 keV/μm and the dose rate of 0.55 Gy/min, the radiation doses being of the order of tens of Gy. Cytogenetic investigation has revealed the remarkable diminution of the mitotic index as linear dose-response curve as well as the spectacular linear increase of the aberration index. Analyzing some biometric parameters, it was found that neither dry matter nor water content of wheat seedlings was influenced by proton beam exposure. Studying the biochemical parameters related to the antioxidant defense system, we found that the irradiation caused the slight increasing tendency of peroxidase activity as well as the decreasing trend in the activity of superoxidedismutase in the seedlings grown from the irradiated seeds. The level of malonedialdehyde (MDA) and total polyphenols showed an increasing tendency in all seedling variants corresponding to irradiated seeds, compared to the control. We conclude that the irradiation clearly induced dose-response curves at the level of cytogenetic parameters together with relatively slight variation tendency of some biochemical parameters related to the antioxidant defense system while imperceptible changes could be noticed in the biometric parameters.

New insights on partial trisomy 3q syndrome: de novo 3q27.1-q29 duplication in a newborn with pre and postnatal overgrowth and assisted reproductive conception

BackgroundDuplications of the long arm of chromosome 3 are rare, and associated to a well-defined contiguous gene syndrome known as partial trisomy 3q syndrome. It has been first described in 1966 by Falek et al., and since then around 100 patients have been reported. Clinical manifestations include characteristic facial dysmorphic features, microcephaly, hirsutism, congenital heart disease, genitourinary anomalies, hand and feet abnormalities, growth disturbances and intellectual disability. Most of cases are due to unbalanced translocations, inherited from a parent carrying a balanced aberration (reciprocal translocation or inversion), and rarely the genomic anomaly arises de novo. Very few studies report on the prenatal identification of such rearrangements.Case presentationHereby, we report on a newborn with a rare pure duplication of the long arm of chromosome 3. Noninvasive prenatal test (cell free fetal DNA analysis on maternal blood), performed for advanced parental age and use of assisted reproductive technique, evidenced a partial 3q trisomy. Then, invasive cytogenetic (standard and molecular) investigations, carried out through amniocentesis, confirmed and defined a 3q27.1-q29 duplication spanning 10.9 Mb, and including about 80 genes. Our patient showed clinical findings (typical facial dysmorphic features, esotropia, short neck, atrial septal defect, hepatomegaly, mild motor delay) compatible with partial trisomy 3q syndrome diagnosis, in addition to pre- and postnatal overgrowth.ConclusionsAdvanced parental age increases the probability of chromosomal and/or genomic anomalies, while ART that of epigenomic defects. Both conditions, thus, deserve more careful prenatal monitoring and screening/diagnostic investigations. Among the latter, cell free fetal DNA testing can detect large segmental aneuploidies, along with chromosomal abnormalities. It identified in our patient a wide 3q rearrangement, then confirmed and defined through invasive molecular cytogenetic analysis. Neonatologists and pediatricians must be aware of the potential risks associated to duplication syndromes. Therefore, they should offer to affected subjects an adequate management and early and careful follow-up. These may be able to guarantee to patients satisfactory growth and development profiles, prevent and/or limit neurodevelopmental disorders, and timely recognition of complications.

The Use of Fluorescence In situ Hybridisation in the Diagnosis of Hidden Mosaicism in Egyptian Patients with Turner Syndrome.

Turner syndrome (TS) is the most common chromosomal abnormality in females. The diagnosis of TS is based on karyotyping of 30 blood lymphocytes. This technique does not rule out tissue mosaicism or low-grade mosaicism in the blood. Because of the associated risk of gonadoblastoma, mosaicism is especially important in case this involves a Y chromosome. This study was set to determine the value of additional genetic studies such as fluorescent in situ hybridisation and the inclusion of buccal cells in search for mosaicism in TS patients. This cross-sectional, descriptive study was performed in Human Genetics Department, Medical Research Institute, Alexandria University. Fluorescence in situ hybridisation technique was applied to lymphocyte cultures as well as buccal smears using centromeric probes for X and Y chromosomes. Genotype phenotype correlation was also evaluated. Descriptive study where categorical variables were described using number and percentage and continuous variables were described using mean and standard deviation. Fluorescence in situ hybridisation technique study detected hidden mosaicism in 60% of studied patients; 20% of patients had a cell line containing Y material, while 40% had variable degrees of X, XX mosaicism, and in the remaining 40% no second cell line was detected. Fluorescence in situ hybridisation study helped identify the origin of the marker to be Y in all patients. The introduction of an additional cell line helped in identifying mosaicism in patients with monosomy X. Virilisation signs were only observed among TS patients with Y cell line mosaicism. The clinical manifestations were more severe in patients with monosomy X than other mosaic cases. Molecular cytogenetic investigation for all suspected cases of TS should be considered for appropriate treatment plan and genetic counselling.