R-banding Technique Research Articles

The 450-Band Resolution G- and R-Banded Standard Karyotype of the Donkey (Equus asinus, 2n = 62)

Donkey chromosomes were earlier characterized separately by C-, G- and R-banding techniques. However, direct comparisons between G- and R-banding patterns have still not been carried out in this species. The present study reports this comparison at the 450-band level by using replication G- and R-banding patterns. Two sets of synchronized lymphocyte cultures were set up to obtain early (GBA+CBA-banding) and late (RBA-banding) BrdU incorporation. Slides were stained with acridine orange and observed under a fluorescence microscope. Reverse GBA+CBA- and RBA-banded karyotypes at the 450-band level were constructed. To verify G- and R-banding patterns in some acrocentric chromosomes, sequential GBA+CBA/Ag-NORs and RBA/Ag-NORs were also performed. The results of CBA-banding patterns obtained in 12 animals from 2 breeds showed a pronounced polymorphism of heterochromatin, especially in EAS1q-prox. Ideogrammatic representations of G- and R-banded karyotypes were constructed using only one common G- and R-banding nomenclature. In the present study both G- and R-banding patterns and relative ideograms are presented as standard karyotype for this species at the 450-band level.

Mechanisms of chromosomal evolution in three European species of the Sorex araneus-arcticus group (Insectivora: Soricidae)

Presented are the R-banding patterns of the karyotypes of three European species of the Sorex araneus-arcticus group (Insectivora: Soricidae). The eight species of this Holarctic complex are characterized by sharing a male chromosomal set of XY1Y2 elements. Robertsonian and tandem translocations are common in this complex, at the population level as well as at the species level. The rough morphology of the karyotypes looks similar between all the species presently described. An R-banding technique RHG, RBG) has allowed us to make a comparative analysis of the chromosomal similarities in three species, namely S. araneus, S. coronatus and S. granarius. The data provide evidence that Robertsonian and tandem translocations, accompaniei in some cases by centromeric shifts, are the main, if not the only, mechanisms of chromosomal evolution in this grou. It appears that S. granarius presents a karotype which is most similar to the hypotheticar ancestral type from which the chromosomar sets of two other European species might be derived.

Fluorescence in situ hybridization studies on a myeloid leukemia patient with ins(8;21)(q22;q22.1q22.3)

To report a case of acute myeloid leukemia (AML) with the insertion (8;21)(q22;q22.1q22.3). A 33-year-old Chinese woman was referred to our hospital. Hematologic data showed WBC 42.7 x 10(9)/L with monocytosis (monocyte counts 7.296 x 10(9)/L). Bone marrow aspirate was hypercellular with 4.5% monoblasts and 7.5% promonocytes. At first she was diagnosed with chronic myelomonocytic leukemia (CMML) according to the FAB criteria. Initially the patient received supportive care only, but her general condition rapidly became worse three months later. The monoblasts and promonocytes in the bone marrow rose to 20.5%. After two cycles of combined chemotherapy she obtained complete remission. Chromosome specimens were prepared by short-term culture of bone marrow cells. Karyotype analysis was carried out by R-banding technique. Three fluorescence in situ hybridization (FISH) analyses were performed using AML1-ETO dual color, dual fusion probe, whole chromosome painting 8 and 21 probes, and cen-8 and Tel 21qter probes, respectively. Reverse transcription polymerase chain reaction (RT-PCR) assay for detecting the AML1-ETO fusion transcript was also performed. Conventional cytogenetic analysis showed a karyotype of 46,XX,ins(8;21) (q22;q22.1q22.3)[7]/46,XX[3]. FISH tests confirmed the insertion. RT-PCR analysis detected the AML1-ETO fusion transcript. We consider that this patient should be rediagnosed as acute myeloid leukemia according to the criteria proposed by World Health Organization (WHO) and that FISH and RT-PCR play an important role in verification of the ins(8;21).

Karyotype Analysis of 58 cases myelodysplastie synarom

Objective To explore theralue of karyotype analysis in MDS for diagnosis, cure and progno-sis. Methods Chromosome analysis was performed in 58 cases of MDS using the short-term culture of bone marrow eell and R-banding technique, meanwhile,their clinical progress was followed up. Results Karyotype a-nalysis showed that 31 eases(53.4%) had elonal Karyotypie abnormalities. The nost common chromosomal ab-errations included +8 and high complex karyotypic, and then-7/7q-In the following up, 19 eases (30.5%) transformed into acute leukemia. Conclusion The Karyotype analysis is very important for diagnosis, cure and prognosis of MDS. Key words: Myelodysplastic syndrome; Chromosome; Karyotype

Familial interstitial deletion of the short arm of chromosome 4 (p15.33–p16.3) characterized by molecular cytogenetic analysis

This 15-month boy was expressed at the cytogenetic laboratory because of psychomotor development delay. He was tall and had plagiocephaly, micrognathia, high nasal bridge, anteverted nostrils and pectus excavatum. A 46,XY,del(4)(p16.1p16.3) karyotype was found using high-resolution R-banding technique. FISH studies using the LSI Wolf-Hirschhorn dual color 4p16.3 and the TelVysion 4p probes showed no deletion. Using BACs, the distal breakpoint was located in 4p16.3, between RP11-165K4 and RP11-717M10 and the proximal breakpoint in 4p15.33, between RP11-74M11 and RP11-1J7; therefore, approximately 7.96 Mb of the short arm were deleted. The maternal karyotype showed the same deletion, but in a mosaic status. Two distinct phenotypes have been recognized on the basis of the chromosomal bands involved in 4p deletion: the Wolf-Hirschhorn syndrome (WHS) and a proximal 4p deletion syndrome (4p15.2-p15.32). Our observation confirms that the basic WHS phenotype maps distally to this region.

Centromeric loss in translocation of centric fusion type in cattle and water buffalo.

Iannuzzi, L., Di Berardino, D., Gustavsson, I., Ferrara, L. and Di Meo, G. P. 1987. Centromeric loss in translocations of centric fusion type in cattle and water buffalo. - Hereditas 106: 73–81. Lund, Sweden. ISSN 0018–0661. Received March 15, 1986 Robertsonian translocations in Podolian and Romagna cattle (Bos taurus L.) and Asiatic river buffaloes (Bubulus bubalis L.) were studied with C, G + C, and R-banding techniques. The investigation demonstrated: (a) presence of one block of constitutive heterochromatin in the q-arm of the cattle translocation chromosome; (b) chromosomes identified as 1 and 25 according to the system used - but presumptively 1 and 29 as earlier described - being involved in the cattle centric fusions, with the centromere region of chromosome 25(29) being lost and that of chromosome 1 being retained; (c) no constitutive heterochromatin in the q-chromosome arm of the bi-armed chromosomes 1 and 5 in the water huffaloes (with retention of the constitutive heterochromatin in their p-arms) and partial losses of centromeric heterochromatin in both arms of chromosomes 2, 3 and 4; (d) presence of a C-positive segment in the telomere region of the p-arm of chromosome 4 in the water buffalo; (e) no bi-armed chromosome common for the two species. Karyotype evolution is discussed and the urgency of an appropriate nomenclature system for cattle and buffalo chromosomes is briefly stressed.

Der(20)idic(20)(p11)del(20)(q11q13) Is Seen Not Only in Myeloid Disorders but Also in Lymphoid Disorders: A Report of Further Seven Cases.

As a newly identified cytogenetic abnormality, idic(20q−) has been reported in thirteen cases of myelodysplastic syndrome (MDS), one case of chronic myelomonocytic leukemia, and one case of acute myeloid leukemia (AML) since 2004. Here we report additional seven cases consisting of 3 MDS, 2 AML and 2 acute lymphocytic leukemia (ALL). A karyotype analysis using R-banding technique revealed an i(20q−) as a sole anomaly in 5 of them and as a secondary change in other 2 cases: one was a B-cell ALL whose leukemic cells at presentation had a karyotype of 45, XY, dic(9;20)(p11;q11). However, an idic(20q−) was found coexistent with the dic(9;20) as relapse; another was a Ph positive ALL whose leukemic cells simultaneously contained t(9;22)(q34;q11) and idic(20q−). A series of FISH using multiple probes such as the centromeric probes specific for chromosomes 9 and 20, the subtelomeric probe for 20q, the locus-specific probe for 20q12, the whole chromosome paint probes for chromosomes 9 and 22 and the translocation probe for t(9;22) demonstrated the idic(20q−) to be actually a der(20)idic(20)(p11)del(20)(q11q13), confirmed the dic(9;20)(p11;q11) in case 6 and revealed a BCR/ABL fusion gene in case 7. Among them, 2 MDS patients survived and anemic except one lost to follow-up, 2 AML and 2 ALL patients did not obtain complete remission and died with short survivals. Our study brings the total number of cases with idic(20q−) to 22 and suggests that the disease spectrum of idic(20q−) is not restricted to myeloid disorders, and it also includes ALL.

The cytogenetic features of Chinese patients with myeloproliferative diseases

17533 Background: To investigate the cytogenetic features in Chinese patients with myeloproliferative diseases(MPD). Methods: The chromosomal abnormalities of 98 newly diagnosed essential thrombocytosis(ET) patients and 50 newly diagnosed polycythemia vera(PV) patients were studied by conventional cytogenetics (CC) and R-banding technique. Interphase fluorescence in situ hybridization (FISH) were used to detect trisomy 8 in 50 ET cases and detect trisomies 8 and 9 in 50 PV and 8 normal individuals. Results: CC showed 6.12% (6/98) of ET patients had chromosomal abnormalities including 1 case with 13q-, 2 with 5q-, 1 with 7q-, 1 with der(14)t(1;14) and 1 with Rob(13;14). FISH detected 1 case with trisomy 8 among 50 cases. CC showed 6% (3/50) of PV patients had chromosomal abnormalities including trisomy 8, deletion of chromosome Y and inversion of chromosome 11. FISH detected two cases of trisomy 8, including the confirmed one by CC technique, and one case of trisomy 9 neglected by CC technique. Conclusions: In our study, the incidence of chromosomal abnormalities in MPD by CC was rare. The frequency of trisomies 8 and 9was low in untreated MPD. No significant financial relationships to disclose.

Clinical and molecular cytogenetic characteristics of dic(9;20) in adult acute lymphoblastic leukemia: a case report of three patients

Dicentric (9;20) [dic(9;20)] is a rare recurring chromosome abnormality in leukemia patients. In this study, we report three adult patients with acute lymphoblastic leukemia (ALL) with dic(9;20) anomaly. All three patients were diagnosed as cluster of differentiation 10 (CD10) positive B cell ALL, achieved complete remission after induction chemotherapy, and died of leukemia relapse within 1 year after diagnosis. Specimens for chromosome analysis were prepared by direct method and/or short-time culture of bone marrow cells. Karyotyping was performed by R-banding technique. Dual-color fluorescence in situ hybridization (FISH) was performed using chromosome 9 and chromosome 20-specific centromeric probes. Karyotype analysis showed that all three patients had dic(9;20)(p11-13;q11), in which the dicentric nature of the derived chromosome was confirmed by interphase FISH. Dicentric (9;20)(p11-13;q11) may be specifically associated with ALL, and monosomy 20 may be a pointer to dic(9;20) in ALL. The prognostic significance of dic(9;20) in adult patients with ALL remains to be determined.

Whole Chromosome Painting and Multiplex Fluorescence In Situ Hybridization in Detecting Complex Chromosomal Aberrations in Myelodysplastic Syndromes.

Objective To explore the value of the technique of whole chromosome painting (WCP) and multiplex fluorescence in situ hybridization (M-FISH) in the detection of complex chromosomal aberrations (CCAs) of myelodysplastic syndromes (MDS).Methods M-FISH was used in seven MDS patients with CCAs detected by R-banding technique to refine CCAs, and to identify cryptic translocations and characterization of marker chromosomes. Dual-color WCP procedures were further performed in 7 cases to confirm some rearrangements detected by M-FISH.Results In all cases, M-FISH confirmed all results of R-banding.The composition and origin of 6 kinds of marker chromosomes, 9 kinds of chromosomes with additional material undetermined and 5 kinds of derivative chromosomes undefined by CC were defined after M-FISH analysis; 4 kinds of cryptic translocations overlooked by CC were found on derivative chromosomes and previously normal appearing chromosomes. In addition, M-FISH revealed some nonrandom aberrations: aberrations involving chromosome 17 (5/7) and -5/5q- (4/7) were the two most frequent aberrations. Fluorescence flaring is a main factor leading to misinterpretations. Some misclassified and missed chromosomal aberrations by M-FISH were corrected by WCP.Conclusions M-FISH is a powerful molecular cytogenetic tool in clarification of CCAs. Complementary WCP helped us identify misclassified and missed chromosomal aberrations by M-FISH. CC in combination with molecular cytogenetic techniques M-FISH and WCP can unravel complex chromosomal aberrations more precisely.

Karyotype analysis of 283 cases of myelodysplastic syndrome

To explore the implication of karyotype analysis in diagnosis and prognosis of myelodysplastic syndrome (MDS). The chromosomes were prepared with direct method, brief culture of cells and R-banding techniques, and then the karyotypic analysis was performed. Seventy-seven out of 283 patients (27.21%) had karyotypic abnormalities, including the numeral abnormalities of chromosomes and structural alterations. The most common chromosomal aberrations were +8, -20/20q-, -Y, translocation, -7/7q-, +9, -5/5q-. The rate of abnormal karyotype in refractory anemia with erythroblasts (RAEB) and refractory anemia erythroblasts-transformation (RAEB-t) was much higher than in refractory anemia (RA). Patients with abnormal karyotype or higher IPSS scores had a higher risk of transformation into acute leukemia than patients with normal karyotype or lower IPSS scores (P<0.05). MDS is a highly heterogenous disorder and karyotype analysis is helpful for its diagnosis and prognosis estimation.

Pentasomy 8q Resulting from Duplication of Isochromosome 8q in Chronic Myelomonocytic Leukemia

We report an interesting case of chronic myelomonocytic leukemia (CMML) with pentasomy 8q resulting from the duplication of isochromosome 8q in a 47-year-old male. His blood picture and myelogram showed CMML and the chromosome study, using R-banding and G-banding techniques, revealed a karyotype of 47,XY, m 8,+i(8)(q10) × 2. Dual-color fluorescence in situ hybridization (FISH) studies with a #8 centromeric probe and a locus-specific probe for C-myc gene completely confirmed the result of the conventional cytogenetic method. Reverse transcription polymerase reaction (RT-PCR) revealed no BCR/ABL fusion transcript. Hydroxyurea and 6-mercaptopurine therapy did not induce a complete remission and five months later he died of exacerbation of his disease. On reviewing another two cases with pentasomy 8q in the literature, we feel that pentasomy 8q, when present as a sole anomaly, may play a specific role in leukemogenesis and in determining the clinical characteristics such as monocytic involvement and poor prognosis.

T(8;21;8)(p23;q22;q22): A New Variant form of t(8;21) Translocation in Acute Myeloblastic Leukemia with Maturation

The complex variants of t(8;21) involving chromosomes 8 and 21 as well as a variable chromosome account for 1.1 ∼5% of acute myeloid leukemia (AML) patients. This paper reports a case of AML-M2 with t(8;21;8) translocation for the first time. The patient was a female, aged 47 years. Her myelogram was compatible with AML-M2. Chromosome study using R-banding technique revealed a karyotype 46, XX, t(8;8)(p23;q22). Dual-color FISH assay with two probes P1 164(green signal) and YAC 225B8 (red signal) both of which closely located on the 8q showed that one yellow signal consisting of a green signal and a red signal and one red signal appeared on the long and the short arm of the same der(8) chromosome, respectively, further confirming this translocation occurred between both homologous chromosomes 8. RT-PCR analysis detected the AML1/ETO fusion transcript in our patient, thus indicating that this chromosomal aberration was, in fact, a complex three-way rearrangement t(8;21;8)(p23;q22;q22). In conclusion, combining conventional karyotype, FISH or RT-PCR analyses is a rational strategy for identification of the complex variants of t(8;21) translocation.

ZOO-FISH and R-banding reveal extensive conservation of human chromosome regions in euchromatic regions of river buffalo chromosomes

Commercially available human chromosome (HSA) painting probes were hybridized on river buffalo (Bubalus bubalis, 2n = 50) chromosomes by using FISH and R-banding techniques. Clear hybridization FITC-signals revealed extensive conservation of human chromosome regions in this species and demonstrated that human chromosome probes primarily paint euchromatic regions (R-bands). The present results are discussed in the light of previous gene mapping data obtained in river buffalo and ZOO-FISH data in cattle, and in relation to the standard bovine chromosome nomenclatures. In particular, HSA 8, HSA 10, HSA 11, and HSA 16+7 paint, respectively, BBU 1p, BBU 4p, BBU 5p, and BBU 24, which are homoeologous, respectively, to cattle chromosomes 25, 28, 29 and 27. Thus, these river buffalo chromosome arms can serve as markers to resolve discrepancies in the nomenclature of cattle and related species.

Longitudinal differentiation of chromosomes of Asellus aquaticus (Crust. Isop.) by in situ nick translation using restriction enzymes and DNase I.

Asellus aquaticus is an isopod crustacean whose chromosomes cannot be differentiated by G- or R-banding techniques. In this work, we have obtained a longitudinal differentiation of these chromosomes by in situ nick translation using restriction enzymes (HaeIII, DraI and BamHI) and DNase I digestions. The four nucleases, with different efficiencies, have produced similar labelling patterns. Staining with DAPI, Giemsa and chromomycin A3 reveals that the DNA of the nick-translated regions is generally more resistant to extraction from the chromosome. The results obtained on the heteromorphic sex chromosome pair observed in about a quarter of the males of a natural population allow several hypotheses to be advanced on the nature and origin of chromosome dimorphism.

Chromosome painting: a method for testing chromosomal changes in lemur evolution

Chromosome painting using commercially available human chromosome-specific DNA libraries was performed to elucidate chromosomal rearrangements in lemur evolution. Human-specific probes for chromosomes 3, 14, 15, and 21 were used to paint chromosomes of six species: Eulemur fulvus mayottensis, Varecia variegata, Lemur catta, Hapalemur simus, H. griseus griseus, and H. aureus. All human chromosome libraries hybridized specifically to chromosome segments of varying length or to whole arms of Lemur chromosomes. The labeling was clearly visualized and permitted precise delineation of the hybridized Lemur chromosomal segments. The use of commercial probes of human chromosomes for chromosome painting appears efficient enough to investigate homology in different species of Lemur. In general, the results obtained by chromosome painting in this study confirm results previously obtained by the R-banding technique but modify the location of some chromosomal rearrangements on different branches of the evolutionary tree of the Lemuridae and reveal some new rearrangements that were not detectable with banding techniques. These results show that chromosome painting with human chromosome-specific DNA libraries can provide useful information in comparative studies on karyotypes of distantly related mammalian species, providing a powerful tool for evolutionary studies, especially in phylogeny.

Human cDNA mapping using a high-resolution R-banding technique and fluorescence in situ hybridization.

High-resolution fluorescence in situ hybridization (FISH) is now an essential element in both human gene mapping and clinical cytogenetics. To facilitate its application, a series of techniques have been developed using FISH to map DNA probes in the size range of 1-1,000 kb directly on R-banded human chromosomes. Distinctive reverse (R) banding is achieved by staining with chromomycin A3 and distamycin A following in situ hybridization. The use of such counterstains enables simultaneous viewing of both the fluorescent R-bands and in situ hybridization signals by either standard photomicroscopy or an automated image-acquisition system. This method is rapid and reproducibly reveals bands at the 350-700 stage. Further, specific methods for chromosome preparation, hybridization, and signal production have been developed and applied in combination with R-banding. These methods are used for precise chromosomal localization of DNA sequences in sizes ranging from that of cDNA (> 1 kb) through bacterial artificial chromosomes (100-150 kb) to yeast artificial chromosomes (> or = 1 Mb). These techniques provide high-resolution methods for rapid mapping of human genes, expanding the applications of FISH techniques in basic research and clinical analysis.

Nonrandom inactivation of the Y-bearing X chromosome in a 46, XX individual: evidence for the etiology of 46, XX true hermaphroditism

We previously reported a subject with 46,XX true hermaphroditism who had a 46,X,del(X) karyotype and Y-chromosomal sequences in genomic DNA. We hypothesized that the Y-chromosomal sequences were translocated to the deleted X chromosome and that the incomplete testis determination of this individual was the result of inactivation of the translocated X chromosome. In situ hybridization studies demonstrated that the Y-chromosomal sequences were located on the distal portion of the short arm of the deleted X chromosome. Investigation of the replication of the X chromosome, using a modified R-banding technique and localization of Y-chromosomal sequences by in situ hybridization, showed that the translocated X chromosome was late replicating in all 100 EBV-transformed lymphoblasts that were examined. By contrast, when cells from a subject with 46,XX maleness were studied, the translocated X chromosome was late replicating in only 21 of 47 cells. As the late-replicating X chromosome is presumed to be the inactive X chromosome, selection of cells in which the Y-bearing X chromosome has been inactivated may play a role in the incomplete testis determination in subjects with "Y-positive" 46,XX true hermaphroditism.

A genetic study of retinoblastoma

Eight patients with retinoblastoma (RB) were studied by high resolution chromosome R-banding technique, and the esterase D was quantitatively determined in red blood cells of the patients. Among these patients, 2 showed 13q14 deletion mosaicism, 1 showed a monosomy 13q14.1-q14.2, and the remaining 5 had normal karyotype. Our findings indicated that 1) the RB gene is located at 13q14.1. The result confirmed previous data; 2) 13q deletion is an important event in the development of RB; and 3) esterase D determination is an important diagnostic tool in the detection of 13q deletion, useful for prenatal diagnosis and genetic counselling.

Comparison of the 15q deletions in Prader‐Willi and Angelman syndromes: Specific regions, extent of deletions, parental origin, and clinical consequences

It has recently been shown that apparently similar deletions of chromosome 15q occur commonly in the Prader-Willi and Angelman syndromes. The distinctness of the syndromes suggests that the deletions are not identical. To address this possibility, the specific bands involved and the sizes of the deletions were compared in seven patients with Prader-Willi syndrome and 10 patients with Angelman syndrome using high-resolution G-, Q-, and fluorescent R-banding techniques. The parental origin of the nine cases of Angelman syndrome for which parents were available for study was determined. The same proximal band was deleted (q11.2) in both syndromes. In general, the deletion in patients with Angelman syndrome was larger, though variable, and included bands q12 and part of q13. All of the studied deletions in patients with Angelman syndrome were of maternal origin. This contrasts with the predominant paternal origin of the deletion in patients with Prader-Willi syndrome. Two possible reasons for these observations are postulated: 1) the deleted regions are different at the cytologic and/or molecular level because of different exchange points in meiosis in males and females or to different mechanisms of breakage in males and females, resulting in differing breakpoints; 2) the deleted regions are essentially the same, but differential expression of the genes in the homologous chromosome 15 has occurred (imprinting).