Conventional Cytogenetic Methods Research Articles

The impact of comparative genomic hybridization/single-nucleotide polymorphism microarray in risk stratification of pediatric acute lymphoblastic leukemia.

The objective of this study is to assess the concordance and added value of combined comparative genomic hybridization plus single-nucleotide polymorphism microarray (CGH/SNP) analyses in pediatric acute lymphoblastic leukemia (ALL) risk stratification compared to conventional cytogenetic methods. This is a retrospective study that included patients aged 1-18years diagnosed with de novo ALL at Sainte-Justine Hospital between 2016 and 2021. Results from conventional cytogenetic and molecular analyses were collected and compared to those of CGH/SNP. A total of 135 ALL patients were included. Sample failures or non-diagnostic analyses occurred in 17.8% cases with G-banding karyotypes versus 1.5% cases with CGH/SNP. The mean turnaround time for results was significantly faster for CGH/SNP than karyotype with 5.8 versus 10.7days, respectively. The comparison of ploidy assessment by CGH/SNP and G-banding karyotype showed strong concordance (r=.82, p<.001, r2=.68). Furthermore, G-banding karyotype did not detect additional clinically relevant aberrations that were missed by the combined analysis of CGH/SNP and fluorescence in situ hybridization. The most common gene alterations detected by CGH/SNP were deletions involving CDKN2A (35.8%), ETV6 (31.3%), CDKN2B (28.4%), PAX5 (20.1%), IKZF1 (12.7%), and copy-neutral loss of heterozygosity (CN-LOH) of 9p (9.0%). Among these, only ETV6 deletion was found to have a significant prognostic impact with superior event-free survival in both univariate and multivariate analyses (adjusted hazard ratio 0.08, 95% confidence interval: 0.01-0.50, p=.02). CGH/SNP provided faster, reliable, and highly concordant results than those obtained by conventional cytogenetics. CGH/SNP identified recurrent gene deletions in pediatric ALL, of which ETV6 deletion conferred a favorable prognosis.

Karyotype stasis but species-specific repetitive DNA patterns in Anguis lizards (Squamata: Anguidae), in the evolutionary framework of Anguiformes

Abstract Karyotype divergence may strongly affect the degree of hybridization between species. Western Palearctic slow worms (Anguis) are legless lizards forming different types of secondary contact zones. To identify the level of chromosomal variation in slow worms, we examined karyotype in multiple populations of all species except one and Pseudopus apodus as an outgroup. We applied conventional and molecular cytogenetic methods and whole-chromosome painting using macrochromosome probes from Varanus komodoensis to interpret results within the evolutionary framework of the common clade Anguiformes. All Anguis species and P. apodus have conserved karyotype structures composed of 44 chromosomes. Despite the conserved chromosome morphology, the phylogenetically oldest Anguis cephallonica living in partial sympatry with Anguis graeca, and parapatric Anguis colchica vs. Anguis fragilis exhibit distinct patterns of constitutive heterochromatin distribution and telomeric repeat accumulation. In contrast, the sister species A. colchica and A. graeca living in allopatry display highly similar karyotype features. Our findings thus indicate karyotype stasis in Anguis and Pseudopus for &amp;gt; 20 Myr, with fixed species-specific differences present in sympatric and parapatric species. These differences in repetitive DNA patterns may play a role as intrinsic factors co-maintaining species divergence. They may also be used as cytotaxonomic markers to identify slow worm species in practice.

Enhancing Cytogenetic Diagnostics: Incorporating Optical Genome Mapping in the Laboratory Routine

Key findings Introduction Optical Genome Mapping (OGM) is a is a novel high-throughput diagnostic technique that overcomes the limitations of conventional cytogenetic methods. The objective of this study was to evaluate the application of OGM in the daily workflow of the cytogenetics laboratory and compare it with standard techniques. Methods A total of 180 adult or pediatric patients referred to the Cytogenetics Laboratory at Hospital La Fe during disease onset or relapse were included. The patients had the following diagnoses: aplastic anemia (AA) n = 6, B-cell acute lymphoblastic leukemia (B-ALL) n = 39, T-cell acute lymphoblastic leukemia (T-ALL) n = 9, acute myeloid leukemia (AML) n = 51, myelofibrosis (MF) n = 36, polycythemia vera (PV) n = 3, essential thrombocythemia (ET) n = 2, multiple myeloma (MM) n = 11, and myelodysplastic syndromes (MDS) n = 22. Chromosome banding analysis (CBA) was performed on bone marrow samples, and in selected cases, fluorescence in situ hybridization (FISH) was carried out using specific probes following diagnostic recommendations. For OGM analysis, high molecular weight DNA was extracted and labeled from peripheral blood (PB, n = 62) or bone marrow (BM, n = 118) following the manufacturer's protocol (Bionano, San Diego, CA, USA). Results CBA was performed on all patients except MM, where an initial FISH panel targeting IGH rearrangement, TP53 deletion, and 1q amplification was applied. Among the CBA, 61/169 (36%) patients showed a CBA without alterations, 62/169 (37%) were abnormal and 46/169 (27%) were unsuccessful. OGM successfully detected cryptic alterations in 60% (37/61) of cases previously classified as normal with CBA. Additionally, OGM identified further alterations in 56% (35/62) of cases with abnormal CBA findings. Notably, OGM demonstrated a remarkable 93% success rate in resolving previously uninformative CBA results. AA OGM did not detect clinically significant alterations or copy neutral loss of heterozygozity (CN-LOH) on chromosome 6 in this subgroup of patients. B-ALL: OGM detected alterations in all patients with a normal or uninformative karyotype. Regarding cases with a previous abnormal CBA, OGM discovered 2 cases with masked hypodiploidy. Copy Number Alterations (CNA) affecting IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, PAR1 region, and ERG were detected in 27/39 (69%) patients, 3 of whom harbored the IKZF1plus Moreover, several cryptic rearrangements were uncovered, such as MEF2D::BCL9, RUFY1::ETV6, PDGFRB::EBF1, and PAX5::PML. T-ALL: All patients showed an uninformative or normal CBA. However, OGM discovered rearrangements in 7/9 (78%) patients, including PICALM::MLLT10, GATA3::RUNX1, or STIL::TAL1. AML OGM enabled the detection of 4 cases with cryptic NUP98::NSD1 rearrangements and 2 cases with tandem duplication of KMT2A. Additionally, OGM successfully identified the partner involved in a previously uncharacterized KMT2A rearrangement ( KMT2A::ELL). MPN (MF, PV and ET) = The main utility of OGM was observed in MF, as it resolved all cases with uninformative CBA. MM Primary limitations in these cases were low plasma cell infiltration and difficulty in DNA extraction from total nucleated cells. Only one case showed an infiltration rate higher than 15%, as determined by flow cytometry. Consequently, 7 cases obtained a normal OGM result despite alterations detected by FISH. However, OGM successfully identified a cryptic rearrangement involving MYC and provided a detailed characterization of CNA in the remaining cases. MDS OGM resolved uninformative karyotypes in the subgroup of MDS with fibrosis. Additionally, the resolution of OGM allowed the detection of a t(3;8)(q26.2;q24.21) with a variant allele frequency of 6%, involving MECOM rearrangement, in a patient who later progressed to AML. In total, 8 cases were uninformative with OGM, mainly because of poor sample quality or low cellularity. Conclusion This study demonstrates the utility of integrating emerging technologies like OGM into the daily routine of the laboratory. DNA extraction should be performed through selected CD138 cells in multiple myeloma

Evaluation of Y Chromosome Microdeletion and Chromosome Analysis Results in Infertile Male Patients

Objective: Genetic testing for male infertility is rarely performed in our country. Male infertility is caused by chromosome number or structural problems, Y chromosome deletions and gene alterations. Infertility is a problem seen in 15% of couples. Genetic causes are responsible for the etiology of 3-10% of those diagnosed with male infertility due to oligozoospermia and azoospermia. In this retrospective study, we aimed to determine both the chromosomal structure and the microdeletion of the azoospermic factor (AZF) region on the Y chromosome in infertile men admitted to our center before the application of assisted reproductive techniques.&#x0D; Method: We studied 327 patients who applied to our laboratory for routine analysis. Chromosome analysis was performed from peripheral blood by conventional cytogenetic method. DNA was isolated from peripheral blood and Y chromosome microdeletion was analyzed by fragment analysis method with Y chromosome microdeletion detection kit.&#x0D; Results: Out of 327 patients, 32 had cytogenetic and 18 had molecular abnormalities and 4 had both cytogenetic and molecular abnormalities. Numerical and structural anomalies were detected in patients with anomalous karyotype. Among the patients with Y microdeletions, 1 patient had AZFa, 2 patient had AZFb, 6 patients had AZFc, 3 patients had AZFc+d, 2 patients had AZFb+c+d, 1 patient had AZFb+c+sY160, 1 patient had AZFa+b+d+c+sY90, and 2 patient had AZFb+d+c+sY90.&#x0D; Conclusion: Our study shows that chromosomal abnormalities and Y chromosome microdeletions are important causes of male infertility and that chromosome analysis and Y chromosome microdeletion tests should be performed to explain these abnormalities. It also emphasizes the importance of genetic counseling in explaining male infertility.

Evaluation of the Sensitivity of Conventional Cytogenetic and Fluorescence in Situ Hybridization Methods for the Detection of Cytogenetic Abnormalities in Multiple Myeloma Patients: A Retrospective Study

Background: Identification of genetic abnormalities in multiple myeloma (MM) patients is of particular importance in order to design their treatment and management. Therefore, it is necessary to use the diagnostic methods with high sensitivity to detect abnormalities. In this study, we investigated the sensitivity of conventional cytogenetic and FISH methods in the diagnosis of genetic abnormalities in MM patients. Methods: This retrospective study included 246 patients who referred to the Kariminejhad Center for the Diagnosis of Genetic Abnormalities between 2009-2019. All patients were diagnosed based on diagnostic tests, as well as the approval of the relevant physician. The diagnosis of cytogenetic abnormality was made based on the two methods of conventional cytogenetic and FISH. Result: The results showed that out of 246 patients examined by conventional cytogenetic, only 17.8% had abnormal karyotypes. While out of 67 patients examined by FISH, 64.1% had abnormal results. The results also showed that 31 out of 50 patients with normal karyotype had abnormal FISH result. In the present study, the results showed that 25% of the patients had hyperdiploidy (57-47), which was diagnosed by conventional cytogenetic. Also, 40.90% had diploid abnormalities (pseudodiploid or structural abnormalities). FISH detected del 13q in 27.9% and t(11;14) IGH-CCND1 in 18.6% of patients, which were the most frequent compared to other abnormalities. Conclusion: Considering that the variety of mutations and translocations is high in different parts of the world and every day new mutations are detected, using both methods together can help to identify genetic disorders.

Analysis of chromosome aberrations in peripheral blood lymphocyte of medical radiation workers in a tertiary hospital

Objective: To analyze the level of chromosome aberration in lymphocytes of medical radiation workers and its influencing factors. Methods: From July to September 2020, 252 medical workers in a tertiary hospital were selected as the study subjects and 107 preserviceworkers were selected as the control group. The Chromosomal aberrations of peripheral blood lymphocytes were measured using conventional cytogenetic analysis method, and the differences were analyzed. Results: The frequencies of dicentric puls centric ring, total chromosome-type aberrations, and abnormal detection rate in the radiation group were significantly higher than those in the control group (Z=2.59, 3.74, 9.99, P<0.05). There was significant difference in the frequencies of dicentric plus centric ring and total chromosome-type aberrations among different types of work (χ(2)=8.59, 8.17, 11.39, P<0.05), and the frequencies of dicentric plus centric ring were significantly higher in the interventional radiology group than those in diagnostic radiology (χ(2)=2.90, P<0.05), While the rates of acentric fragment and total chromosome-type aberrations were significantly higher in the nuclear medicine group than those in diagnostic radiology (χ(2)=2.81, 3.19, P<0.05). The difference in the abnormal detection rate of chromosome aberrations between different types of work was statistically significant (P<0.05), and the rate in the interventional radiology group was significantly higher than that in the diagnostic radiology group (χ(2)=7.66, P<0.05). There was no significant difference in chromosome aberration level and abnormal detection rate among different working ages (P>0.05). Poisson regression analysis indicated that the type of work is a risk factor for chromosomal aberration [IRR=2.31 (nuclear medicine group), 1.66 (Radiation therapy), and 1.78 (interventional group) ; P<0.05]. Conclusion: Ionizing radiation causes certain radiation damage to medical radiology workers, and the frequencies of chromosome aberration in the radiation workers of nuclear medicine and interventional radiology groups are relatively high, so radiation protection should be strengthened to ensure the health of relevant workers.

Prenatally detected six duplications at Xp22.33-p11.22: a case report

BackgroundThe discrepancy between the results of cytogenetics and the results of chromosome microarray analysis (CMA) has often led to confusion over genetic counselling for prenatal diagnosis.Case presentationThe prenatal ultrasound results of a congenital heart defect (CHD) foetus displayed an apartial endocardial pad defect and permanently dilated coronary sinus and left superior vena cava at 21 weeks of gestation. Cytogenetic analysis, CMA, fluorescent in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) with foetal cord blood samples were used to detect the genetic aetiology. Routine G-binding cytogenetic analysis showed normal karyotypes in both the foetus’ and parents’ blood samples. CMA results demonstrated that there were 53.973-Mb recurrent CNVs at Xp22.33-p11.22, as confirmed by MLPA assay.ConclusionsHerein, we described the CNV of six duplications at Xp22.33-p11.22 and the 53.973 Mb duplication CNV that was not found in foetal cord blood samples by conventional cytogenetic methods, and it was confirmed by CMA and MLPA. Our novel findings will provide helpful information for prenatal diagnosis and genetic counselling for foetal CHDs.

Small Body, Large Chromosomes: Centric Fusions Shaped the Karyotype of the Amazonian Miniature Fish Nannostomus anduzei (Characiformes, Lebiasinidae).

Miniature refers to species with extraordinarily small adult body size when adult and can be found within all major metazoan groups. It is considered that miniature species have experienced severe alteration of numerous morphological traits during evolution. For a variety of reasons, including severe labor concerns during collecting, chromosomal acquisition, and taxonomic issues, miniature fishes are neglected and understudied. Since some available studies indicate possible relationship between diploid chromosome number (2n) and body size in fishes, we aimed to study one of the smallest Neotropical fish Nannostomus anduzei (Teleostei, Characiformes, Lebiasinidae), using both conventional (Giemsa staining, C-banding) and molecular cytogenetic methods (FISH mapping of rDNAs, microsatellites, and telomeric sequences). Our research revealed that N. anduzei possesses one of the lowest diploid chromosome numbers (2n = 22) among teleost fishes, and its karyotype is entirely composed of large metacentric chromosomes. All chromosomes, except for pair number 11, showed an 18S rDNA signal in the pericentromeric region. 5S rDNA signals were detected in the pericentromeric regions of chromosome pair number 1 and 6, displaying synteny to 18S rDNA signals. Interstitial telomeric sites (ITS) were identified in the centromeric region of pairs 6 and 8, indicating that centric fusions played a significant role in karyotype evolution of studied species. Our study provides further evidence supporting the trend of diploid chromosome number reduction along with miniaturization of adult body size in fishes.

Recurrent DNMT3B Alterations Are Associated with Unfavorable Outcome in Dicentric (9;20)-Positive Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

The dicentric chromosome dic(9;20)(p11∼13;q11), occurs in 2% of the cases with pediatric acute lymphoblastic leukemia (ALL), almost exclusively of the B-cell lineage. Although previous cytogenetic studies have provided first insights into the genetic alterations in cases with dic(9;20), they were limited by the lack of sensitive genome-wide and next generation sequencing-based methods. Furthermore, the prognostic relevance of the dic(9;20) remains unclear, as previous studies have reported variable outcomes among these cases. Therefore, a better understanding of the (epi)genomic landscape and prognostic relevance of dic(9;20) ALL is essential to further optimize treatment of children with these alterations. The aim of this study was to unravel the demographic, clinical, prognostic and molecular characteristics of dic(9;20) in pediatric ALL. The present study investigates a cohort of 57 children with B-ALL, enrolled in the German ALL-BFM 2000, 2009, or 2017 trials, which underwent cytogenetic diagnostics in our center. Detection of dic(9;20) by conventional cytogenetic and molecular methods was the prerequisite for inclusion into the study. All 57 dic(9;20) cases, and 56 cases for which RNA samples were available, were subjected to genome-wide CGH+SNP array and targeted RNA-sequencing analysis. In addition, we performed whole transcriptome, whole genome and whole genome bisulfite sequencing for a selected number of cases. Event-free survival (EFS) was estimated according to Kaplan-Meier, and the curves were compared by log-rank test. Overall, the cohort consists of 22 male and 35 female children, with a median age at diagnosis of 3 years (range: 1-17 years). Patients were treated according to the AIEOP-BFM treatment protocols and stratified in the standard risk (n=15), medium risk (n=37) or high risk (n=5) treatment arms. All 57 patients achieved remission, 11 experienced relapses, while two patients in remission were lost during follow-up. Integrative analysis of RNA-seq and CGH+SNP array revealed recurrent genomic rearrangements of the DNMT3B gene with PAX5 or adjacent ZCCHC7 gene (n=3, each). DNMT3B is a de novo DNA methyltransferase and deletions of this gene have been previously reported in various solid malignancies and AML. The rearrangement of DNMT3B and ZCCHC7 resulted in an in-frame chimeric fusion transcript, containing the 5’ end of the open reading frame of DNMT3B and the 3’ end of ZCCHC7. In contrast, due to opposing orientation of the DNMT3B and PAX5 genes, the resulting chimeric transcript contained the 5’ end of the open reading frame of DNMT3B or PAX5 and a run-through transcript of the antisense strand of the partner gene. All genomic rearrangements with their breakpoints and fusion transcripts were validated using whole genome and Sanger sequencing. In the total group of six cases with rearrangements in the DNMT3B and PAX5/ZCCHC7 genes, four experienced relapses, resulting in a 5-year EFS of 25% (SE=20.4), compared to 75.4% (SE=8.2) for the remaining cohort (p=0.015). All six patients were treated according to the medium risk arm of the respective AIEOP-BFM 2000 or 2009 protocols (n=1 and n=5, respectively), and all showed good initial response to prednisone. Three patients had non-quantifiable levels of minimal residual disease after induction, while one of each had measurable levels ranging from 10-3-10-5. The unfavorable prognostic marker IKZF1plus was detected in four patients (66%), two of which relapsed. Since the DNMT3B gene functions as de novo methyltransferase, we examined whether genomic rearrangements in these cases can lead to global changes in methylation, but we did not observe either global hypermethylation, or global hypomethylation in dic(9;20) compared to cases of other B-ALL subtypes. In this study we describe novel recurrent genomic rearrangements involving the DNMT3B and PAX5/ZCCHC7 genes in pediatric ALL with dic(9;20). Although our observations should be confirmed in an independent cohort and larger series of patients, cases with rearrangements involving DNMT3B and PAX5/ZCCHC7 genes appear to have very poor outcome compared to other cases with dic(9;20). Once confirmed in an independent cohort, dic(9;20)-positive cases with rearrangements involving DNMT3B and PAX5/ZCCHC7 genes should be considered as high risk for relapse and treated accordingly.

ACCURATE BREAKPOINT MAPPING USING OPTICAL GENOME MAPPING (OGM) HAS THE POTENTIAL TO INCREASE THE ACCURACY OF PREIMPLANTATION GENETIC TESTING FOR STRUCTURAL CHROMOSOMAL REARRANGEMENTS (PGT-SR)

PGT-SR is a genetic test performed on trophectoderm (TE) biopsy from IVF embryos to detect chromosomal structural rearrangements normally caused by balanced translocations and inversions. The feasibility and accuracy of PGT-SR is affected by the breakpoint defined by conventional cytogenetic methods. OGM enables direct high-resolution breakpoint mapping on balanced carriers of translocations. In this study, we applied OGM on the couple who underwent IVF with multiple embryos showing the loss or gain on the same segments, which didn’t match with partner’s previous karyotyping results.

Cytogenetic Analysis of the Members of the Snake Genera Cylindrophis, Eryx, Python, and Tropidophis.

The recent discovery of two independently evolved XX/XY sex determination systems in the snake genera Python and Boa sparked a new drive to study the evolution of sex chromosomes in poorly studied lineages of snakes, where female heterogamety was previously assumed. Therefore, we examined seven species from the genera Eryx, Cylindrophis, Python, and Tropidophis by conventional and molecular cytogenetic methods. Despite the fact that these species have similar karyotypes in terms of chromosome number and morphology, we detected variability in the distribution of heterochromatin, telomeric repeats, and rDNA loci. Heterochromatic blocks were mainly detected in the centromeric regions in all species, although accumulations were detected in pericentromeric and telomeric regions in a few macrochromosomes in several of the studied species. All species show the expected topology of telomeric repeats at the edge of all chromosomes, with the exception of Eryx muelleri, where additional accumulations were detected in the centromeres of three pairs of macrochromosomes. The rDNA loci accumulate in one pair of microchromosomes in all Eryx species and in Cylindrophis ruffus, in one macrochromosome pair in Tropidophis melanurus and in two pairs of microchromosomes in Python regius. Sex-specific differences were not detected, suggesting that these species likely have homomorphic, poorly differentiated sex chromosomes.

Molecular Cytogenetics Reveals Mosaicism in Human Umbilical Vein Endothelial Cells

Primary human umbilical vein endothelial cells (HUVECs) are consistently the most reliable in vitro model system for studying the inner lining of blood and lymphatic vessels or the endothelium. Primary human cells originate from freshly isolated tissues without genetic manipulation and generally show a modal number of 46 chromosomes with no structural alterations, at least during early passages. We investigated the cytogenetic integrity of HUVECs with conventional (G-banding) and molecular cytogenetic methods (spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH)). Our G-band data shows two X-chromosomes, confirming these HUVECs originate from a female donor. Notably, some cells consistently exhibit an unfamiliar banding pattern on one X chromosome toward the distal end of the long arm (Xq). Our FISH analysis confirms that approximately 50% of these HUVECs have a deletion of the Xq terminal region. SKY analysis indicates that the deleted region is apparently not integrated into any other chromosome. Finally, we demonstrated the presence of a similar Xq deletion in the daughter cell line, EA.hy926, which was generated by fusing HUVECs with A549 (a thioguanine-resistant clone of adenocarcinomic human alveolar basal epithelial cells). These findings will advance comprehension of HUVECs biology and will augment future endothelial studies.

Optical Genome Mapping and Single Nucleotide Polymorphism Microarray: An Integrated Approach for Investigating Products of Conception.

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, as well as microbial and maternal cell contamination (MCC). Optical genome mapping (OGM) is an emerging technology that has the potential to replace conventional cytogenetic methods. The use of OGM precludes the requirement for culturing (and related microbial contamination). However, a high percentage of MCC impedes a definitive diagnosis, which can be addressed by an additional pre-analytical quality control step that includes histological assessment of H&E stained slides from formalin-fixed paraffin embedded (FFPE) tissue with macro-dissection for chorionic villi to enrich fetal tissue component for single nucleotide polymorphism microarray (SNPM) analysis. To improve the diagnostic yield, an integrated workflow was devised that included MCC characterization of POC tissue, followed by OGM for MCC-negative cases or SNPM with histological assessment for MCC-positive cases. A result was obtained in 93% (29/31) of cases with a diagnostic yield of 45.1% (14/31) with the proposed workflow, compared to 9.6% (3/31) and 6.4% (2/31) with routine workflow, respectively. The integrated workflow with these technologies demonstrates the clinical utility and higher diagnostic yield in evaluating POC specimens.

EP406: Germline 16p13.1 microdeletion identified during routine hematologic testing

Inversion 16 or translocation 16;16, resulting in CBFB::MYH11 fusion, accounts for approximately 5–8% of acute myeloid leukemia (AML) and is recognized by the World Health Organization (WHO) as a distinct entity within the category of AML with recurrent genetic abnormalities. It is critical to correctly characterize AML patients with CBFB::MYH11 fusion for appropriate diagnosis and the association with a favorable clinical prognosis (a high rate of complete remission and favorable overall survival). However, germline microdeletions spanning the MYH11 gene region are associated with a rare, but recurrent, 16p13.1microdeletion syndrome. We present this case series to highlight the risk of neoplastic versus germline mutational results involving the MYH11 locus and to initiate a discussion on standardization for reporting incidental germline findings in the setting of hematologic testing. We performed a retrospective review of the Mayo Clinic Cytogenetics Database and identified four cases that were tested using the Mayo homebrew CBFB::MYH11 dual-color, dual-fusion FISH (D-FISH) probe strategy and had a probable MYH11 germline microdeletion (Table 1). In 3 of 4 cases, loss of the MYH11 probe was observed in the absence of CBFB::MYH11 gene fusion. Since the MYH11 deletion was identified in 97-100% of nuclei in each of these cases, this result strongly suggested a germline 16p deletion. In addition, a MYH11 gene deletion is not associated with diagnostic or prognostic significance in the context of myeloid neoplasia. Germline 16p13.1 microdeletion syndrome is characterized by a broad spectrum of clinical phenotypes, with the most severely affected having intellectual disability, epilepsy and additional phenotypic features. However, the 16p13.1 microdeletion has also been identified in individuals with very mild clinical phenotypes and in apparently normal individuals, suggesting either incomplete penetrance and/or variable expressivity of the phenotype. In 3 of 4 patient cases identified in this study through an initial hematologic evaluation, additional studies were recommended to document the constitutional origin of the MYH11 gene deletion. To our knowledge, none of these cases pursued follow-up germline testing. However, in one patient (patient 2) FISH performed at diagnosis showed CBFB::MYH11 fusion in addition to the MYH11 deletion. A follow-up bone marrow specimen demonstrated no evidence of CBFB::MYH11 fusion, but the MYH11 deletion persisted, representing additional evidence for a constitutional deletion of the MYH11 gene region in this patient. These cases illustrate the possibility of discovering incidental constitutional abnormalities when performing testing on hematologic neoplasms. Currently there are no defined guidelines for the detection and reporting of possible germline alterations detected by conventional cytogenetic methods in hematologic disease. Consequently, patients may not be counselled appropriately, and testing consent forms in cancer settings often do not address the possibility of the discovery of germline abnormalities. These cases highlight a need of standardization for reporting incidental germline findings from hematologic testing by clinical laboratories as well as consenting and genetic counselling from the clinical perspective.Tabled 1Age (years)SexReason for referralSpecimennuc ish(MYH11x1,CBFBx2) %Additional clonal abnormalitiesPatient 122FemaleAcute myeloid leukemiaBone marrow100.0NoPatient 259FemaleInversion 16 at diagnosisBone marrow99.6UnknownPatient 339FemaleAcute myeloid leukemia relapseBlood97.4NoPatient 459FemalePancytopeniaBone marrow100.0No Open table in a new tab

52. Optical genome mapping and SNP microarray: integrated workflow for optimizing analysis of products of conception

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, microbial contamination, and maternal cell contamination (MCC). Optical genome mapping is an emerging alternative to conventional cytogenetic methods but is limited to the need for ultra-high molecular weight DNA. Single nucleotide polymorphism (SNP) microarray has demonstrated higher performance compared to routine karyotype, but a high incidence of MCC precludes a valid clinical diagnosis.

Array-Based Comparative Genomic Hybridization Analysis in Children with Developmental Delay/Intellectual Disability.

Developmental delay (DD) is a condition wherein developmental milestones and learning skills do not occur at the expected age range for patients under 5 years of age. Intellectual disability (ID) is characterized by limited or insufficient development of mental abilities, including intellectual functioning impairments, such as learning and cause–effect relationships. Isolated and syndromic DD/ID cases show extreme genetic heterogeneity. Array-based comparative genomic hybridization aCGH) can detect copy number variations (CNVs) on the whole genome at higher resolution than conventional cytogenetic methods. The diagnostic yield of aCGH was 15.0–20.0% in DD/ID cases. The aim of this study was to discuss the clinical findings and aCGH analysis results of isolated and syndromic DD/ID cases in the context of genotype-phenotype correlation. The study included 139 cases (77 females, 62 males). Data analysis revealed 38 different CNVs in 35 cases. In this study, 19 cases with pathogenic CNVs (13.6%) and five cases with likely pathogenic CNVs (3.5%) were found in a total of 139 cases diagnosed with DD/ID. When all pathogenic and likely pathogenic cases were evaluated, the diagnosis rate was 17.1%. The use of aCGH analysis as a first-tier test in DD/ID cases contributes significantly to the diagnosis rates and enables the detection of rare microdeletion/microduplication syndromes. The clear determination of genetic etiology contributes to the literature in terms of genotype-phenotype correlation.

Cytogenetically Elusive Sex Chromosomes in Scincoidean Lizards.

The lizards of the species-rich clade Scincoidea including cordylids, gerrhosaurids, skinks, and xantusiids, show an almost cosmopolitan geographical distribution and a remarkable ecological and morphological divergence. However, previous studies revealed limited variability in cytogenetic traits. The sex determination mode was revealed only in a handful of gerrhosaurid, skink, and xantusiid species, which demonstrated either ZZ/ZW or XX/XY sex chromosomes. In this study, we explored the karyotypes of six species of skinks, two species of cordylids, and one gerrhosaurid. We applied conventional and molecular cytogenetic methods, including C-banding, fluorescence in situ hybridization with probes specific for telomeric motifs and rDNA loci, and comparative genomic hybridization. The diploid chromosome numbers are rather conserved among these species, but the chromosome morphology, the presence of interstitial telomeric sequences, and the topology of rDNA loci vary significantly. Notably, XX/XY sex chromosomes were identified only in Tiliqua scincoides, where, in contrast to the X chromosome, the Y chromosome lacks accumulations of rDNA loci. We confirm that within the lizards of the scincoidean clade, sex chromosomes remained in a generally poor stage of differentiation.

Cytogenetic Evidence for Sex Chromosomes and Karyotype Evolution in Anguimorphan Lizards

Anguimorphan lizards are a morphologically variable group of squamate reptiles with a wide geographical distribution. In spite of their importance, they have been cytogenetically understudied. Here, we present the results of the cytogenetic examination of 23 species from five anguimorphan families (Anguidae, Helodermatidae, Shinisauridae, Varanidae and Xenosauridae). We applied both conventional (Giemsa staining and C-banding) and molecular cytogenetic methods (fluorescence in situ hybridization with probes for the telomeric motifs and rDNA loci, comparative genome hybridization), intending to describe the karyotypes of previously unstudied species, to uncover the sex determination mode, and to reveal the distribution of variability in cytogenetic characteristics among anguimorphan lizards. We documented that karyotypes are generally quite variable across anguimorphan lineages, with anguids being the most varying. However, the derived chromosome number of 2n = 40 exhibits a notable long-term evolutionary stasis in monitors. Differentiated ZZ/ZW sex chromosomes were documented in monitors and helodermatids, as well as in the anguids Abronia lythrochila, and preliminary also in Celestus warreni and Gerrhonotus liocephalus. Several other anguimorphan species have likely poorly differentiated sex chromosomes, which cannot be detected by the applied cytogenetic methods, although the presence of environmental sex determination cannot be excluded. In addition, we uncovered a rare case of spontaneous triploidy in a fully grown Varanus primordius.

Cytogenetic analysis in couples with recurrent pregnancy loss

Background/Aim: Recurrent pregnancy loss (RPL), described as the loss of two or more pregnancies before 24 weeks of pregnancy, remains a concern for both the couples and the clinicians. Genetic factors tend to be strongly linked to reproductive failure among different etiologies. Our goal was to determine the rates and kinds of chromosomal defects in couples who had repeated pregnancy losses and a history of miscarriage in the first trimester. Methods: This cross sectional study was conducted at a single tertiary center over a 3-year period. Couples who visited the outpatient clinic due to recurrent pregnancy loss and underwent tests to investigate the etiology were included in the study. Ages, number of abortions and genetic results of the patients were recorded. A total of 253 pairs had been tested for karyotype. Conventional cytogenetic method was used to identify chromosomal aberrations. Results: Of 506 cases, chromosomal abnormalities were present in 15 (2.9%). Women were more frequently affected than men, with prevalences of 1.9% and 0.98%, respectively. Eight of the 15 cases (53.3%) showed structural deviations and 2 (13.3%) had numerical abnormalities. Additionally, 5 (33.3%) individuals were found to have chromosome variants. Conclusion: Pregnancy loss is a major adverse life event, and the recurring nature of RPL can intensify the grief experienced. Aside from routine analyses of couples on anatomical, endocrine, and infection factors, these findings suggest that cytogenetic testing is required for an accurate approach to determine the cause of recurrent miscarriages.

A PCR-RFLP method for genotyping of inversion 2Rc in Anopheles coluzzii

BackgroundGenotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity.MethodsUsing a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries.ResultsOf four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate.ConclusionsWe have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control.Graphical