Molecular Cytogenetic Methods Research Articles

Structural Variants in the SMC1A Gene Associated With Near-Haploidy in Undifferentiated Pleomorphic Sarcomas.

Near-haploidization, that is, loss of one copy of most chromosomes, is a relatively rare phenomenon in most tumors, but is enriched among certain soft tissue sarcomas, including undifferentiated pleomorphic sarcoma (UPS). Presumably, near-haploidization can arise through many mechanisms. This study aimed to identify gene rearrangements that could cause near-haploidization. We here present two UPS in which near-haploidization was an early event, identified through single nucleotide polymorphism (SNP) array analysis. One of the cases was studied further using whole genome and transcriptome sequencing, as well as cytogenetic and molecular cytogenetic methods. Both tumors had chromosomal rearrangements in the form of copy number shifts/structural variants affecting the SMC1A gene. These findings suggest that cohesin defects could contribute to mitotic errors resulting in massive loss of chromosomes. SMC1A encodes one of the components of the cohesin multiprotein complex, which is critical for proper alignment of the sister chromatids during S-phase and separation to opposite spindle poles. Further studies should explore the role of cohesin defects in near-haploidization in other sarcomas and to clarify its role in tumor development.

Classic and molecular cytogenetic findings in leukemia patients from the Western part of Romania.

Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in childhood and rare in adults, while acute myeloid leukemia (AML) is less common in children and more common in older adults. The aim of the study was to present our experience for the diagnostic of leukemia by using the classic and molecular cytogenetic methods. The study was conducted between 2009 and 2019 within the Classic and Molecular Genetic Laboratory of the Oncohematology Department from the Louis Ţurcanu Emergency Hospital for Children, Timişoara, Romania. The study group included 337 children and adults, evaluated between 2009 and 2019. By using the conventional and molecular cytogenetic technique, the cytogenetic anomalies found were 35 numerical chromosomal abnormalities, 10 (9;22)(q34;q11) [four ALL, one AML, five chronic myeloid leukemia (CML)] translocations, nine (15;17)(q24;q21) translocations, three (14;14)(q11;q32) translocations, two (4;11)(q21;q23) translocations, one (1;14)(p32;q11) translocation, one (7;14)(qter;q11) translocation, one (8;21)(q22;q22) translocation, one (9;14)(p12;q32) translocation, seven rearrangements of the MLL gene and two rearrangements of the core-binding factor subunit beta∕myosin heavy chain 11 (CBFB∕MYH11) gene. The use of conventional and molecular cytogenetic analysis is one of the most important prognostic indicators in acute leukemia patients, allowing the identification of biologically distinct subtypes of disease and selection of appropriate treatment approaches.

X chromosome rearrangement associated with premature ovarian insufficiency as diagnosed by molecular cytogenetic methods: a case report and review of the literature

BackgroundPremature ovarian insufficiency (POI) is a clinical condition characterized by ovarian dysfunction in women under 40. The etiology of most POI cases remains unidentified and is believed to be multifactorial, including factors such as autoimmunity, metabolism, infection, and genetics. POI exhibits significant genetic heterogeneity, and it can result from chromosomal abnormalities and monogenic defects.Case presentationThe study participant, a 33-year-old woman, presented with a history of irregular menstruation that commenced two years ago, progressing to prolonged menstrual episodes and eventual cessation. The participant exhibits a rearrangement of the X chromosome, characterized by heterozygosity duplication on the long arm and heterozygosity deletion on the short arm by whole exome sequencing(WES) combined with cell chromosome detection.ConclusionsThis study expands the spectrum of mutations associated with POI resulting from X chromosomal abnormalities. WES-Copy number variation analysis, in conjunction with chromosome karyotype analysis and other detection techniques, can provide a more comprehensive understanding of the genetic landscape underlying complex single or multi-system diseases.

Appraisal of current technologies for the study of genetic alterations in hematologic malignancies with a focus on chromosome analysis and structural variants.

During the last five decades, chromosome analysis identified recurring translocations and inversions in leukemias and lymphomas, which led to cloning of genes at the breakpoints that contribute to oncogenesis. Such molecular cytogenetic methods as fluorescence in situ hybridization (FISH), copy number (CN) arrays or optical genome mapping (OGM) have augmented standard chromosome analysis. The use of both cytogenetic and molecular methods, such as reverse transcription-polymerase chain reaction (RT-PCR) and next generation sequencing (NGS), including whole-genome sequencing (WGS), discloses alterations that not only delineate separate WHO disease entities but also constitute independent prognostic factors, whose use in the clinic improves management of patients with hematologic neoplasms.

Молекулярно-цитогенетическая характеристика малых сверхчисленных маркерных хромосом 15 и 22 у асимптоматических носителей

Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be identified unambiguously by standard cytogenetic methods. A comprehensive approach involving the use of molecular cytogenetic methods is required for the more thorough morphological assessment of such chromosomes, as well as for the development of strategy for genetic counseling of the patients being the sSMC carriers. It is widely accepted that the development of abnormal phenotype by the patients having sSMC in their karyotype is associated with the presence of euchromatic region material in the marker chromosome. Therefore, it results from the presence of relatively large DNA copy number variations (CNVs) in the form of duplication, triplication, and more increased copy numbers; which are localized in the pericentromeric region of the appropriate chromosome. Pericentromeric CNVs can be involved in the chromosome imbalance in asymptomatic carriers of sSMC as well, however, the boundaries of such imbalance have not been clearly identified. The study was aimed to acquire additional information about the genomic topology of the DNA regions insensitive to the genes copy number increase. FISH analysis with commercial and homemade DNA probes was performed in 18 carriers of sSMC 15 and 22 having no clinically significant phenotypic abnormalities. The molecular cytogenetic testing showed that pericentromeric euchromatic regions sized 1.2 Mb and 714 kb, respectively, were found in 33% of cases (6 out of 18). We assume that these regions comprise no potentially dosage-sensitive genes.

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 > 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.

Comparative cytogenetic study of Plantago species

Plantago is a large genus with a worldwide distribution characterized by three basic sets, x = 6 is the most common and is considered the ancestral set from which x = 5 and x = 4 derived following mechanisms of descending dysploidy. Advanced molecular cytogenetic methods are important for understanding evolutionary relationships but in Plantago their use has been rather limited. In this study the karyotypes of Plantago lanceolata L. (2n = 12), Plantago major L. (2n = 12) and Plantago coronopus L. (2n = 10) were characterized by using rDNA FISH, GISH, fluorochrome banding and chromosome morphology to obtain markers useful for evaluating their genomic affinity. The rDNA FISH mapping produced three distinct patterns but with evident similarities such as the chromosome position of 45S rDNA sites and the colocalization of 45S and 5S rDNA. Reciprocal GISH showed a low level of genome affinity between P. lanceolata and P. major and a total absence of homology between them and P. coronopus. This result supports the exclusion of the 2n = 12 species as possible ancestors of P. coronopus. This study confirms that a systematic use of in situ hybridization techniques, and in particular GISH, provide valid information for understanding the origin of species and can resolve many taxonomic and phylogenetic problematics.

Prenatal Screening of Cytogenetic Anomalies -A Ten Year Retrospective Study on 1510 Cases

Introduction: Prenatal diagnostic is a diagnostic method which is used to prove the presence of chromosome changes, a large number of metabolic disorders and other morphological fetus abnormalities. Prenatal genetic testing mostly refers to the molecular genetic and cytogenetic methods used during pregnancy to diagnose genetic fetal conditions. Aim: To investigate the existence and incidence of cytogenetics abnormalities in fetuses. Material and Methods: The retrospective research is based on cytogenetic analysis of the 1510 amniotic fluid samples collected from pregnant women sent to the cytogenetic laboratory from January, 2012 to December, 2022. Results: The karyotype without visible structural and numerical changes was detected in 96.8% (1462/1510) cases. The fetal karyotype was abnormal in 3.2 % (48/1510) of the cases. Trisomy 21 was the most frequent chromosome aberration detected in 1.12% (17/1510) cases followed by pericentric inversion 9 (10/1510; 0.66%) and trisomy 18 (4/1510; 0.26%). Mosaics were detected in five cases (5/1510; 0.33%). Comparing the prevalence of chromosome abnormalities according to maternal age, we come to know the prevalence of chromosome aberrations in the group of females above age 35 (26/790; 17.2/1000) is higher than in the group of females under age 25 (7/95; 4.63/1000), but not significantly different (P= 0.09). Conclusion: Conventional cytogenetics maintains its role as a powerful diagnostic tool in detecting chromosomal changes during prenatal screening.

Microdeletion 12p12.1 involving SRY-related HMG-box 5 gene with developmental delay and autistic behavior

The estimated prevalence of intellectual disability (ID) is 10.37/1000 population. One of the major causes of ID is a chromosomal abnormality. SRY-related HMG-box 5 (SOX5) gene encodes a member of the SOX family of transcription factors, which may act as a transcriptional regulator involved in the regulation of chondrogenesis and the development of the nervous system by regulation of the production of subcortical projection neurons. Array-comparative genomic hybridization (array-CGH) is a molecular-cytogenetic method used to detect submicroscopic copy number variants within the genome, which is not visible by conventional karyotyping. We describe the extensive application of array-CGH in a 4-year-old male child who presented with global developmental delay (DD), behavioral abnormality, microcephaly, mild dysmorphic facial features, and constriction ring around prepuce with 12p12.1 microdeletion (166 kb deletion) involving SOX5 gene. The patient is under follow-up with a pediatrician, geneticist, speech therapist, and physiotherapist. A high index of suspicion for cytogenetic abnormalities should be present in a pediatric patient presenting with DD, genitourinary abnormalities, and associated with or without facial dysmorphism.

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.

A genomic DNA-based NGS method for the simultaneous detection of multiple fusion genes in pediatric leukemia.

Fusion genes are products of chromosomal translocations that generate either a dysregulated partner gene or a chimeric fusion protein with new properties, and contribute significantly to leukemia development and clinical risk stratification. However, simultaneous detection of several hundreds of fusion genes has always been a challenge in a clinical laboratory setting. In the present study, a total of 182 pediatric patients with leukemia were screened for fusion genes by employing a novel genomic DNA-, instead of RNA-, based next-generation sequencing (NGS) method. This involved the comparison of the multiply targeted capture sequencing method with a detection panel of 270 fusion genes (MTCS-270) with an RNA-based multiplex reverse transcription-PCR technique with a detection panel of 57 fusion genes (MRTP-57). MRTP-57 has been well established in the clinical lab at Beijing Hightrust Diagnostics, Co. (Beijing, China) for an up-front leukemia diagnosis and served as the control technique in the present study. In the series, MTCS-270 and MRTP-57 yielded a positive fusion gene detection rate of 50.0% (91/182) and 41.8% (76/182), respectively, indicating an advantage of MTCS-270 over MRTP-57 in overall detection sensitivity. Specifically, all the fusion genes detected by MRTP-57 were also identified by MTCS-270, clearly signifying the respectable detection accuracy of MTCS-270. Notably, across the patients screened, MTCS-270 identified more samples with fusion genes than MRTP-57, illustrating a broader fusion gene detection coverage by MTCS-270. The present study provides solid evidence that this DNA-based NGS approach can be used as a potential detection tool together with other well-established molecular cytogenetic methods for leukemia management, and to the best of our knowledge, represents the largest leukemia fusion gene identification analysis by genomic NGS.

Fluorescence In Situ Hybridization of DNA Probes on Mitotic Chromosomes of the Mexican Axolotl.

Fluorescence in situ hybridization (FISH) is used extensively for visual localization of specific DNA fragments (and RNA fragments) in broad applications on chromosomes or nuclei at any stage of the cell cycle: metaphase, anaphase, or interphase. The cytogenetic slides that serve as a target for the labeled DNA probe might be prepared using any approach suitable for obtaining cells with appropriate morphology for imaging and analysis. In this chapter, we focus on the application of molecular cytogenetic methods such as DNA labeling, slide preparation, and in situ hybridization related to cells from Mexican axolotl.

NOVEL CHROMOSOMAL ALTERATION DER(2)T(2;15)(Q37;Q21) IN A PEDIATRIC PATIENT WITH MYELODYSPLASTIC SYNDROME: MOLECULAR CYTOGENETIC STUDIES

Pediatric Myelodysplastic Syndrome (p-MDS) comprises a heterogeneous group of clonal hematopoietic stem cell diseases with an increased risk of evolution to Acute Myeloid Leukemia (AML). Cytogenetic is one of the most important prognostic factors for MDS and it is present into score prognostic systems as the Revised International Prognostic Scoring System (IPSS-R). Chromosomal translocations are rare cytogenetic abnormalities in de novo MDS. Therefore, their reporting is essential for the identification of new cytogenetic prognostic risk groups and possible alterations associated with the MDS pathogenesis.The aim of this study was describe a yet unreported der(2)t(2;15)(q37;q21) in a de novo p-MDS associated with evolution to AML. Cytogenetic analysis was performed in Bone Marrow (BM) cells by G-banding. Fluorescence In Situ Hybridization (FISH) was performed to investigate the possible rearrangement involving chromosomes 2 and 15, using a Whole Chromosome Painting (WCP) probe for chromosome 2 and LSI PML/RARA for chromosome 15 (PML gene). A 12-year-old girl was admitted to the Pediatrics Institute of Federal University of Rio de Janeiro in January 2022. Peripheral blood count showed Hemoglobin (Hb) level of 6.5 g/dL, 794.000 platelets/mm 3 , and 2.600 white blood cell (WBC)/mm 3 . The myelogram showed dysplasias and 16% of myeloid blasts being classified with MDS-EB-t. The conventional cytogenetic analysis showed the karyotype: 46,XX,add(2)(q37?)[25]. Using molecular cytogenetic methods, we characterized the chromosomal alteration as an unbalanced translocation involving chromosomes 2 and 15. This translocation was observed in 71.6% (190/265) of the interphase nuclei and metaphases. The final karyotype was: 46,XX,der(2)t(2;15)(q37;q21)[25]. Five months later, the patient showed in the myelogram 50.7% of blast cells compatible with AML. The patient was indicated for allogeneic Hematopoietic Stem Cell Transplantation (HSCT) and initiated chemotherapy. In p-MDS, chromosomal translocations as sole chromosomal abnormality are rare and their real prognostic impact is unknown. The patients with this type of cytogenetic alteration are classified as intermediate-risk group according to the IPSS-R. Our report describes the clinical outcome in a novo p-MDS showing a der(2)t(2;15)(q37;q21). A broad scientific review showed that the der(2)t(2;15)(q37;q21) was not yet reported in p-MDS. It is interesting to note that this chromosomal translocation was associated with a poor clinical outcome, with progression from MDS to AML. The cytogenetic and clinical features were important tools for the indication to allogeneic HSCT. This study and literature review highlight the importance of cytogenetics and clinical follow-up of de novo p-MDS patients for the identification for HSCT. This is the first study that describe the der(2)t(2;15)(q37;q21) in a p-MDS associated with AML evolution.

Prenatal Diagnosis of Chromosome 22q11.21 Microduplication Syndrome in a Fetus with Congenital Malformation of the Cardiovascular System: Case Report

A clinical case of microduplication of chromosome 22q11.21 in a fetus with a malformation of the cardiovascular system (vascular ring) is presented. This anatomical variant of the vascular ring is extremely rare and is formed by the pathological circumflex retroesophageal left-sided cervical aortic arch with the left arterial ductus arteriosus. The anatomical features of the circumflex cervical aortic arch are the left-sided pathological curved aortic arch in combination with the right-sided descending aorta. This vascular pathology can cause compression of the trachea and esophagus. This variant of the vascular ring is extremely unfavorable for surgical correction. Chromosome 22q11.21 microduplication syndrome is associated with the clinical presentation of “cat eye syndrome”. The clinical case description shows unique data from instrumental and genetic studies. All stages of examination of a pregnant woman from primary to tertiary medical care are described. The features and importance of a multidisciplinary approach are also presented.
 Every case of congenital heart defect in fetus, confirmed by echocardiographic method in a specialized institution, requires medical and genetic counseling. Using of the highly sensitive specific molecular cytogenetic method, comparative genomic hybridization, in prenatal diagnosis allows for timely diagnosis of microstructural disorders of chromosomes in fetus. Follow-up of a pregnant woman with congenital heart defect in the fetus requires a multidisciplinary approach not only to improve diagnosis and treatment, but also to calculate genetic risk.

Fine mapping of powdery mildew and stripe rust resistance genes Pm5V/Yr5V transferred from Dasypyrum villosum into wheat without yield penalty.

The novel wheat powdery mildew and stripe rust resistance genes Pm5V/Yr5V are introgressed from Dasypyrum villosum and fine mapped to a narrowed region in 5VS, and their effects on yield-related traits were characterized. The powdery mildew and stripe rust seriously threaten wheat production worldwide. Dasypyrum villosum (2n = 2x = 14, VV), a relative of wheat, is a valuable resource of resistance genes for wheat improvement. Here, we describe a platform for rapid introgression of the resistance genes from D. villosum into the wheat D genome. A complete set of new wheat-D. villosum V (D) disomic substitution lines and 11 D/V Robertsonian translocation lines are developed and characterized by molecular cytogenetic method. A new T5DL·5V#5S line NAU1908 shows resistance to both powdery mildew and stripe rust, and the resistances associated with 5VS are confirmed to be conferred by seedling resistance gene Pm5V and adult-plant resistance gene Yr5V, respectively. We flow-sort chromosome arm 5VS and sequence it using the Illumina NovaSeq 6000 system that allows us to generate 5VS-specific markers for genetic mapping of Pm5V/Yr5V. Fine mapping shows that Pm5V and Yr5V are closely linked and the location is narrowed to an approximately 0.9Mb region referencing the sequence of Chinese Spring 5DS. In this region, a NLR gene in scaffold 24,874 of 5VS orthologous to TraesCS5D02G044300 is the most likely candidate gene for Pm5V. Soft- and hard-grained T5DL·5V#5S introgressions confer resistance to both powdery mildew and stripe rust in diverse wheat genetic backgrounds without yield penalty. Meanwhile, significant decrease in plant height and increase in yield were observed in NIL-5DL·5V#5S compared with that in NIL-5DL·5DS. These results indicate that Pm5V/Yr5V lines might have the potential value to facilitate wheat breeding for disease resistance.

Potentials and challenges of chromosomal microarray analysis in prenatal diagnosis.

Introduction: For decades, conventional karyotyping analysis has been the gold standard for detecting chromosomal abnormalities during prenatal diagnosis. With the development of molecular cytogenetic methods, this situation has dramatically changed. Chromosomal microarray analysis (CMA), a method of genome-wide detection with high resolution, has been recommended as a first-tier test for prenatal diagnosis, especially for fetuses with structural abnormalities. Methods: Based on the primary literature, this review provides an updated summary of the application of CMA for prenatal diagnosis. In addition, this review addresses the challenges that CMA faces with the emergence of genome sequencing techniques, such as copy number variation sequencing, genome-wide cell-free DNA testing, and whole exome sequencing. Conclusion: The CMA platform is still suggested as priority testing methodology in the prenatal setting currently. However, pregnant women may benefit from genome sequencing, which enables the simultaneous detection of copy number variations, regions of homozygosity and single-nucleotide variations, in near future.

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.

Establishment of a set of wheat-rye addition lines with resistance to stem rust.

Complete new wheat-rye disomic, telosomic addition lines and various chromosomal aberrations were developed and characterized by molecular cytogenetic method as novel chromosome engineering materials. A new stem rust resistance (Ug99) gene was located on 3RL. Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating fungal disease worldwide. A recently emerged great threat to global wheat production is Pgt strain Ug99 and its derivatives, which have overcome most of the commonly used resistance genes. Rye (Secale cereale L.), closely related to wheat (Triticum aestivum L.), is a significant and valuable resource of resistance genes for wheat germplasm improvement. It is of great importance and urgency to identify new resistance gene sources of rye and transfer them into wheat. In this study, two complete sets of wheat-rye addition lines were established through wide hybridization, chromosome doubling and backcrossing. A wheat-rye 3RL telosomic addition line was identified with high resistance to stem rust strain Ug99. PCR-based markers specific for the rye chromosome were developed. Furthermore, abundant chromosomal aberrations such as minichromosomes, ring chromosomes as well as centromere reduction and expansion were identified in the progeny of wheat-rye addition lines by multicolor GISH and FISH. The line carrying a novel resistance gene to stem rust can be utilized as a bridge material for wheat disease resistance breeding. The chromosomal and centromeric variation within the wheat-rye hybrids can further contribute to genetic diversity of their offspring.

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.

Structure and prognostic signifi cance of 13q14 deletion in chronic lymphocytic leukemia

Introduction. 13q14 deletion is the most common chromosomal abnormality in chronic lymphocytic leukemia (CLL), and as the sole abnormality determines the most favorable prognosis of the disease. Using molecular genetic methods two subtypes of 13q14 deletion were identifi ed based on the size of the lost chromosomal material: small (type I) with the involvement of the D13S319 segment containing MIR15A/MIR16-1 and DLEU1 genes and large (type II) containing centromeric region of 13q14 involving RB1 gene. Data on the impact of type I and II deletions on the course of CLL are controversial.Aim — to evaluate the prognostic signifi cance of different variants of 13q14 deletion in CLL.Patients and methods. The study enrolled two cohorts of CLL patients. Cohort 1: 256 patients who were studied by FISH with DNA probes for detection of 13q14/D13S319, 11q23/ATM, 17p13/TP53 deletions, and trisomy 12 before immunochemotherapy. 101 patients with identifi ed 13q14/D13S319 deletion were analyzed with a DNA probe for RB1 locus for determination of deletion size (type I or type II). Cohort 2: 28 patients at different stages of the disease with deletion 13q14 detected by FISH were studied by using combination of standard and molecular cytogenetic methods (mFISH, mBAND, arrayCGH) to clarify the structure of 13q abnormalities.Results. In Cohort 1 chromosomal aberrations were detected in 75 % of patients: 13q deletion — 52 % (isolated — 36 % of all cases and 48 % of cases with deletion), 11q deletion — 19 %, +12 — 13 %, 17p deletion — 6 %. 13q14 deletion type I was detected in 56 %, type II — in 44 % of patients. Type II deletion correlated with the presence of 11q deletion (p = 0.05). Isolated deletions of type I and II were found in 61 and 39 %, respectively. Biallelic deletion was identifi ed in 12.7 % of patients with 13q deletion. Statistically signifi cant differences in OS were obtained in type I and II groups of patients with isolated 13q14 deletions: median OS was not reached and made 67.5 months, respectively, p = 0.05. In Cohort 2 structural abnormalities of chromosome 13 by conventional cytogenetic analysis (CCA) were identifi ed in 50 % of cases: 13q deletion — 11 cases; translocations involving 13q14 — 6 cases. In 5 cases with biallelic deletion identifi ed by FISH, 13q14 deletion by CCA was detected in two patients, and only in one allele.Conclusion. In general, 13q14 deletion is a cytogenetic factor of favorable prognosis for CLL but its structure is heterogeneous. Loss of tumor suppressor RB1 (type II deletion) negatively affects OS in patients treated with immunochemotherapy