Conventional Banding Research Articles

Karyotype and Idiogram of Indian Hog Deer (<i>Hyelaphus porcinus</i>) by Conventional Staining, GTG-, High-Resolution and Ag-NOR Banding Techniques

Karyotype and Idiogram of Indian Hog Deer (<i>Hyelaphus porcinus</i>) by Conventional Staining, GTG-, High-Resolution and Ag-NOR Banding Techniques

CpG-ODN Stimulation in Population Screening Monoclonal B-Cell Lymphocytosis

Synthetic CpG-oligodeoxynucleotides (CpG-ODN) induce proliferation in normal and malignant lymphocytes B (LyB). This effect is widely exploited in CLL conventional chromosome banding analysis (CBA), which has become reliable only after the cultivation of CLL LyB using CpG-ODN stimulation in combination with IL-2. Monoclonal B-cell lymphocytosis (MBL) differs from CLL mainly in clone size. Only cytogenetic data on recurrent chromosomal aberrations analyzed by fluorescence in situ hybridization (FISH) are available for population screening MBL (sMBL). In sMBL the clone of malignant LyB is typically below 10/µL. We compared CpGODN stimulation in healthy donors and in individuals with sMBL. LyB and MBL LyB count were determined by flow cytometry in 15 samples from healthy subjects and 12 MBL cases. Mitotic indices were determined and CBA was done after cultivation of samples by CpG-ODN + IL2. In MBL samples, FISH analysis was performed on isolated LyB. MBL LyB clones in sMBL cases presented less than 1% of WBC and up to 33% of LyB. The MBL group was therefore compared to the group of healthy donors. Although normal and MBL group did not differ in WBC, overall LyB, and normal LyB count, a significantly higher mitotic index was observed in MBL samples (p = 0.0139). We were able to accomplish CBA in all samples which revealed a normal karyotype in all but one case. In this particular sMBL case FISH performed on isolated LyB showed 5% trisomy 12 which was later confirmed by CBA on CpG stimulated blood sample in 15% of metaphases. Our study, which was done on MBL cases obtained by population screening, confirmed that CpGODN preferentially induced proliferation in MBL LyB over normal LyB. Therefore, CBA can also be successfully accomplished in sMBL and can be used to additionally confirm clonality as well as to improve sensitivity of FISH analysis. Due to coexistence of comparable size of normal and malignant LyB, MBL can serve as a model for exvivo studying of LyB stimulation by CpG-ODN.

Cytogenetic place in managing myelodysplastic syndromes: an update by the Groupe francophone de cytogénétique hématologique (GFCH).

The myelodysplastic syndromes (MDS) are preleukemic diseases of elderly patients characterized by defective maturation of clonal hematopoietic progenitor cells resulting in peripheral blood cytopenias. Clonal chromosomal abnormalities are heterogeneous and can be detected in less than 50% of patients with de novo MDS and more frequently in secondary MDS (up to 80%). The karyotype plays an important role in the pathogenesis, diagnosis, and prognosis to evaluate the risk of leukemic transformation and, more recently, in treatment allocation. The gold standard for cytogenetic diagnosis in MDS is conventional chromosome banding analyses of bone marrow metaphases. The most frequent abnormalities are deletions and losses of chromosomes 5 (-5/5q-) and 7 (-7/7q-) and various isolated or combined abnormalities. Fluorescent in situ hybridization and array comparative genomic hybridization can reveal cryptic genetic abnormalities but are not recommended in routine diagnosis. New techniques including next generation sequencing revealed somatic driver mutations especially those affecting genes involved in RNA splicing or those harboring important prognostic value (TP53, ASXL1…) with potential applications in clinical practice in the future.

Chromosome Banding in Amphibia. XXXIII. Demonstration of 5-Methylcytosine-Rich Heterochromatin in Anura

An experimental approach using monoclonal anti-5-methylcytosine (5-MeC) antibodies and indirect immunofluorescence was elaborated for detecting 5-MeC-rich chromosome regions in anuran chromosomes. This technique was applied to mitotic metaphases of 6 neotropical frog species belonging to 6 genera and 4 families. The hypermethylation patterns were compared with a variety of banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and located exclusively in constitutive heterochromatin. They are found in centromeric, pericentromeric, telomeric, and interstitial positions of the chromosomes and adjacent to nucleolus organizer regions. 5-MeC-rich DNA sequences can be embedded both in AT- and GC-rich repetitive DNA. The experimental parameters that have major influence on the reproducibility and quality of the anti-5-MeC antibody labeling are discussed.

RETRACTED ARTICLE: Karyotype analysis and identification of extra chromosomes in primary aneuploid stocks of grass pea (Lathyrus sativus L.) by fluorescence chromosome banding

AbstractProminent primary aneuploid stocks namely seven primary trisomics, seven primary tetrasomics, and six double trisomic types were earlier developed in grass pea (Lathyrus sativus L.), a hardy legume. Despite distinct morphological features, identity and nature of their extra chromosome(s) were elusive, hampering assignment of desirable breeding traits into specific linkage groups. The present study aims to analyze the banding pattern and to reveal the identity of extra chromosome(s) involved in these 20 aneuploid types in grass pea. Conventional orcein banding was first done using the root-tip squash technique in all aneuploids along with disomic (2n = 2x = 14) parent, and karyomorphological features were noted. Chromosomes were classified following the total length of individual chromosomes and arranged in order of decreasing sizes, keeping their centromeres in a straight line. DNA-base specific chromomycin A3 (CMA) and 4,6ʹdiamidino-2-phenylindole (DAPI) banding pattern were finally employed to c...

Multicolor Spectral Analyses of Mitotic and Meiotic Mouse Chromosomes Involved in Multiple Robertsonian Translocations. I. The CD/Cremona Hybrid Strain

Multicolor spectral analysis (spectral karyotyping) was applied to mitotic and male diakinetic chromosomes of hybrid mice carrying a unique system of 18 autosomal Robertsonian translocation chromosomes with alternating arm homologies. Only the autosomes 19 and the XY sex chromosomes are excluded from these Robertsonian translocations. The translocations, previously identified by conventional banding analyses, could be verified by spectral karyotyping. Besides the Robertsonian translocations, no other interchromosomal rearrangements were detected. In diakineses of male meiosis, the 18 metacentric Robertsonian translocation chromosomes form a very large meiotic ‘superring'. The predictable, specific order of the chromosomes along this ‘superring' was completely confirmed by multicolor spectral analysis. In the majority of diakineses analyzed, the free autosomal bivalent 19 and the XY sex bivalent form a conspicuous complex which tightly associates with the 12;14 Robertsonian translocation chromosome in the ‘superring'.

Chromosomal analysis and nors polymorphism of bagarius suchus (Siluriformes: Sisoridae) by conventional banding and fish techniques

Chromosomal analysis and nors polymorphism of bagarius suchus (Siluriformes: Sisoridae) by conventional banding and fish techniques

Chromosomal analysis and nors polymorphism of bagarius suchus (Siluriformes: Sisoridae) by conventional banding and fish techniques

In the present study, conventional staining and NORs banding as well as Fluorescence in situ hybridizat ion (FISH) using the 18S rDNA and telomeric (TTA GGG)n probes were applied to stain the chromosomes of crocodile catfish, Bagariussuchus (Siluriformes, Sisoridae) fro m the Chao Praya River, Thailand. Kidney cells of 6 male and 6 female crocodile catfishes were used as a sample. The mitotic chro mosome preparations were done directly fro m kidney cells. The results showed that the diploid chro mosome number of B. suchus was 2n=56, the fundamental numbers (NF) were 102 in both male and female. The karyotype comprises 17m+17sm+12a+10t. The nucleo lar organizer reg ions (NORs) were detected by Ag -NORs banding and 18S rDNA probe mapping. The 18S rDNA are terminally located on the short arm adjacent to the telomere of the single pair of the 1st chromosome pair whereas NOR-bearing chro mosome is only one chromosome of the 1st chromosome pair (1a 1b, poly morphic characteristic) at the subtelomeric region of the short arm. Moreover, FISH with telomeric probe showed hybridizat ion signals on each telomere of all chro mosomes and interstitial telomeric sites were not detected. There were variations in signals of FISH and their position in the karyotype along with variat ion in DNA sequences. These markers are useful for future discrimination of population of closely related species and their poly morphis m.

Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level.

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR-, C- and fluorescent chromosome banding, and fluorescent insitu hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the 'Paranense Sea', probably forced the ancestors of these species to find refuge in unflooded areas for about 2Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.

New record on karyological analysis and first study of NOR localization of parthenogenetic brahminy blind snake, Ramphotyphlops braminus (Squamata, Typhlopidae) in Thailand

This is chromosomal analysis of the brahminy blind snake, Ramphotyphlops braminus (Daudin, 1803), a newly recorded species in northeastern Thailand. Mitotic chromosomes were prepared directly from intestinal epithelia cells of five brahminy blind snakes. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results showed that the triploid chromosome number was 3n = 42, while the fundamental number (NF) was 78. The types of chromosomes were 24 macrochromosomes (19 m + 5sm) and 18 microchromosomes (9 m + 3sm + 6 t). The region adjacent to the short arm near the telomere of one in macrochromosome pair 8 showed a clearly observable nucleolar organizer region/NOR. There were no irregularly sized chromosomes related to sex. The karyotype formula for R. braminus is as follows: 3n (42) = Lm 12 + Sm 7 + Ssm 5 + 18 microchromosomes.

Mid-term outcomes of patients undergoing adjustable pulmonary artery banding

ObjectiveThe adjustable pulmonary artery band (APAB) has been demonstrated by us earlier to be superior to the conventional pulmonary artery banding (CPAB), in terms of reduced early morbidity and mortality. In this study, we assessed the adequacy of the band and its complications over the mid-term. MethodsBetween 2002 and 2012, 73 patients underwent adjustable PAB, and their operative and follow-up data were collected and analyzed. ResultsThere was one early death following the APAB. Follow-up data were available for 57 patients of which 44 patients (61.7%) underwent definitive repair, 10 were awaiting definitive repair, and 3 patients were kept on medical follow-up because of inadequate fall in pulmonary artery (PA) pressures. 14 patients (19%) were lost to follow-up. Major PA distortion or stenosis was absent in the majority. 1 patient had pseudoaneurysm of the main pulmonary artery (MPA) with sternal sinus infection and required surgical reconstruction. 1 patient had infective endocarditis of the pulmonary valve managed medically. Band migration was not encountered. There were two deaths after definitive repair and one after APAB. ConclusionsPatients undergoing APAB fulfilled the desired objectives of the pulmonary artery banding (PAB) with minimum PA complications in the mid-term. This added to the early postoperative benefits, makes the APAB an attractive alternative to the CPAB.

Precision in chromosome identification with leads in molecular cytogenetics: An illustrated review.

Chromosomal aberrations are a major cause of human genetic diseases. Conventional cytogenetic banding techniques are the method of identification for both numerical and structural chromosomal abnormalities but with limited resolution. However, precise identification and characterization of the chromosomal abnormalities can only be achieved by advanced molecular cytogenetic techniques. These techniques are based mainly on fluorescence in situ hybridization, which have become an invaluable tool in the field of diagnostics. The advent of these molecular cytogenetic techniques has helped in the identification of chromosomal abnormalities to its minutest level. Apparently, the leads in molecular cytogenetic techniques have paved way for advanced molecular diagnosis, which now plays a significant role in both diagnostics and clinical research. These advances have led to the increased knowledge of the possible molecular mechanism involved in the chromosomal rearrangements and the genotype-phenotype correlation thus helping the patients towards better diagnosis and genetic counseling. This article highlights the advances in molecular cytogenetic techniques emphasizing the precision in identification of chromosomal rearrangements, and also illustrates few chromosomal abnormalities pediatric cases identified using these molecular cytogenetic techniques.

Karyotype homology between Calotes versicolor and C. mystaceus (Squamata, Agamidae) from northeastern Thailand

Karyotypes of Calotes versicolor and Calotes mystaceus from Khon Kaen Province, northeastern Thailand were investigated. Chromosome preparations were conducted by squash technique from bone marrow. Conventional Giemsa staining and Ag-NOR banding technique were applied to stain the chromosomes. The results showed that the diploid number of C. versicolor and C. mystaceus were 34 in both the species, while the fundamental number (NF) was 46 in both sexes of these species. The types of chromosomes were: four large metacentric, two large submetacentric, four medium metacentric, two small metacentric and 22 microchromosomes in the genus Calotes. NORs of these species were located at the secondary constriction to the telomere on the long arm of the large submetacentric chromosome pair no. 2. No difference was found in karyotypes of chromosomes prepared from the male and female agamid lizards used in the present study. The karyotype formulae of C. versicolor and C. mystaceus were found to be the same and were presented as 2n (34) = Lm4 + Lsm2 + Mm4 + Sm2 + 22 microchromosomes.

Fat-suppressed alternating-SSFP for whole-brain fMRI using breath-hold and visual stimulus paradigms

To achieve artifact-suppressed whole-brain pass-band-balanced steady-state free precession functional MRI from a single functional magnetic resonance imaging (fMRI) scan. A complete and practical data acquisition sequence for alt-SSFP fMRI was developed. First, multishot flyback-echo-planar imaging (EPI) and echo-time shifting were used to achieve data acquisition that was robust against eddy currents, gradient delays, and ghosting artifacts. Second, a steady-state catalyzation scheme was implemented to reduce oscillations in the transient signal when catalyzing in and out of alternate steady states. Next, a short spatial-spectral radiofrequency (RF) pulse was designed to achieve excellent fat-suppression while maintaining a repetition time <15 ms to sensitize functional activation toward smaller vessels and capillaries. Lastly, parallel imaging was used to achieve whole-brain coverage and sufficiently high temporal resolution. Breath-hold experiments showed excellent fat-suppression and alt-SSFP's capability to recover functional sensitivity from signal dropout regions of conventional gradient-echo and banding artifacts from conventional pass-band-balanced steady-state free precession. Applying fat-suppression resulted in improved activation maps and increased temporal SNR. Visual stimulus functional studies verify the proposed method's excellent functional sensitivity to neuronal activation. Artifact-suppressed images are demonstrated, showing a practical pass-band-balanced steady-state free precession fMRI method that permits whole-brain imaging with excellent blood oxygen level-dependent sensitivity and fat suppression.

Cytogenetic variations in a series of cases of Down Syndrome

IntroductionDown Syndrome (DS) is generally associated with mental retardation and developmental delay. The occurrence of DS is associated with multiple factors like maternal age, consanguineous marriage, early induced abortion etc. Chances of recurrences of DS in next pregnancy depend on the genetic constitution of the affected individual and the parents. So this study was done to find out the different types of cytogenetic abnormalities in DS patients and also the association of parental age to DS in a population in West Bengal. It is hoped that the present study will emphasize the need for genetic counseling of prospective parents as well as parents of individuals affected with DS, together with cytogenetic screening of pregnancies which are at high risk for DS. MethodsA cytogenetic analysis was performed using conventional GTG banding on 120 patients with clinical features of DS, referred to the Department of Genetics, Vivekananda Institute of Medical Sciences, Kolkata during the period from October, 2008 to September, 2014. ResultsCytogenetic analysis confirmed the diagnosis of DS in 117 cases, among them regular trisomy constituted 83.76%, mosaicism recorded in 11.11% and Robertsonian translocation in 5.12% of cases. The mean maternal age was higher in regular trisomy 21 (25.08 yrs) than in translocation (22.50 yrs). No significant difference was noted in mean paternal age among different categories of DS cases. DiscussionThis study documents the types of cytogenetic abnormalities in DS children of West Bengal population of India and thus emphasizes the need for genetic counseling in these cases.

Microstructural evolution of Cu–Al alloys subjected to multi-axial compression

Cu–Al alloys with Al contents of 1, 2, 4, and 6wt.% were deformed using a multi-axial compression (MAC) technique to obtain ultrafine-grained (UFG) and nanocrystalline (NC) structures. The effects of stacking fault energy (SFE) on the microstructural evolution and mechanical properties were investigated. After multiple MAC passes, homogeneous and equilibrium microstructures were achieved. The grain refinement mechanism mainly consists of conventional dislocation subdivisions, twinning and shear banding. Reducing SFE promotes deformation twinning and shear banding and thus can reduce the final equilibrium grain size. After obtaining UFG/NG Cu–Al alloys (<200nm), further deformation results in different microstructures for four Cu–Al alloys, indicating that a transition from de-twinning dominant mechanism to twinning dominant mechanism exists in UFG/NG Cu–Al alloys, occurring at a critical SFE in the range of 25mJm−2 (2wt.% Al) and 13mJm−2 (4wt.% Al). During deformation, the migration mechanism of dissociated incoherent twin boundaries (ITBs) was analyzed and the motion of ITBs was believed to be a way for twinning or de-twinning. In addition, hardness tests indicate that with increasing the strain the micro-Vickers hardness for the four alloys increased dramatically initially and then reached saturation at a relative stable value with further increasing the strain. The study suggests that gentle-MAC processing (at a low strain rate and room temperature) is a relative effective method to produce nano-grained materials (<100nm) with ultra-low-SFE (<10mJm−2).

Small supernumerary marker chromosomes and their correlation with specific syndromes.

A small supernumerary marker chromosome (sSMC) is a structurally abnormal chromosome. It is an additional chromosome smaller than one chromosome most often lacking a distinct banding pattern and is rarely identifiable by conventional banding cytogenetic analysis. The origin and composition of an sSMC is recognizable by molecular cytogenetic analysis. These sSMCs are seen in different shapes, including the ring, centric minute, and inverted duplication shapes. The effects of sSMCs on the phenotype depend on factors such as size, genetic content, and the level of the mosaicism. The presence of an sSMC causes partial tris- or tetrasomy, and 70% of the sSMC carriers are clinically normal, while 30% are abnormal in some way. In 70% of the cases the sSMC is de novo, in 20% it is inherited from the mother, and in 10% it is inherited from the father. An sSMC can be causative for specific syndromes such as Emanuel, Pallister-Killian, or cat eye syndromes. There may be more specific sSMC-related syndromes, which may be identified by further investigation. These 10 syndromes can be useful for genetic counseling after further study.

Comprehensive Genetic Characterization of Pediatric T-Cell Acute Lymphoblastic Leukemia

A comprehensive genetic characterization comprising conventional chromosome banding, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism (SNP) array analyses as well as large-scale sequencing of 75 genes were performed on a consecutive series of 47 pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. An abnormal karyotype was identified in 46% of the cases. Recurrent cytogenetic aberrations comprised T-cell receptor (TCR) translocations and deletions of 6q and 9p. FISH analyses of TCR rearrangements were positive in 26% of the investigated cases. The vast majority (37/39; 95%) of cases analyzed by SNP arrays displayed aberrations, with a median of 3 changes (range 0-11) per case. The genes recurrently deleted were CDKN2A, CDKN2B, LEF1, PTEN, RBI, and STIL. One case displayed chromothripsis involving 6q. No case had a whole chromosome uniparental isodisomy (wUPID); in fact, only one T-ALL of 123 informative cases in the literature has had a wUPID. However, segmental UPIDs (sUPIDs) were seen in 44% of the present cases, with most being sUPID9p. CDKN2A was homozygously deleted in all cases with sUPID9p, with a heterozygous deletion occurring prior to the sUPID9p in all instances. There was no evidence for chromosomal instability when comparing diagnostic and relapse samples. Among the genes sequenced, 14 were mutated in 28 cases. The genes targeted are involved in signaling transduction, epigenetic regulation, and transcription. In some cases, NOTCH1 mutations were seen in minor subclones and lost at relapse, showing that such mutations also can be secondary events. These findings support a multistep leukemogenic process. DisclosuresNo relevant conflicts of interest to declare.

강도다리(Platichthys stellatus)에 대한 세포유전학적 연구

【Cytogenetic analysis was conducted to obtain basic information for chromosome manipulation of starry flounder Platichthys stellatus. Nuclear surface area and volume of erythrocyte were $7.60{\pm}0.93{\mu}m^2$ and $12.80{\pm}1.75{\mu}m^3$ , respectively. The haploid DNA content of the species was 0.66 pg/haploid cell which correspond to 93% of olive flounder Paralichthys olivaceus. A karyotype analysis was also carried out with the species using conventional staining and Ag-NOR banding techniques. It was consisted of 48 acrocentric chromosomes and inter-sex or intra-individual polymorphism was not detected in all specimens analyzed. The NOR regions, appearing a terminal position of the short arm of the smallest acrocentric pairs.】

Cytogenetic and molecular genetic study of fetal supernumerary marker chromosomes and derivative chromosomes

Objective To investigate the prenatal diagnosis and phenotypic assessment strategies for fetal supernumerary marker chromosomes and derivative chromosomes. Methods Five cases of fetal supernumerary marker chromosomes and one case of fetal derivative chromosomes were diagnosed in the First Affiliated Hospital of Sun Yat-Sen University from March 12, 2010 to November 9, 2012 by conventional chromosome banding, fluorescence in situ hybridization （FISH） and spectral karyotyping （SKY）. These cases were retrospectively reviewed. Combined with the results of ultrasonography, abnormal phenotypes and pregnancy outcomes were evaluated in these cases. Results All of the five supernumerary marker chromosomes were de novo, in which two were mosaic and the remaining three cases were non-mosaic. Of these five cases, two were type 47, XX＋mar and ultrasound indicated abnormal phenotypes. FISH and SKY confirmed that they were＆amp;nbsp;derived from chromosome 4 and 22, respectively. The other three cases were marker chromosome with Turner syndrome karyotype （abnormal phenotypes were not found by ultrasound）, in which two cases were derived from chromosome Y （by FISH） and one case was identified as ring chromosome X （by FISH and SKY）. One de novo derivative chromosome was verified as a product of reciprocal translocation between chromosome 2 and 6 （by FISH and SKY）. Induced abortion was performed in all cases between 25 and 32 gestational weeks. Conclusions By combining conventional chromosome banding, FISH and SKY, the origin and content of supernumerary marker chromosomes and derivative chromosomes can be identified. On this basis, clinical phenotype evaluation and genetic counseling may be offered with the ultrasonographic result. Key words: Chromosome aberrations; Prenatal diagnosis; Genetic markers; Chromosome banding; In situ hybridization,fluorescence; Spectral karyotyping