Conventional G-banding Analysis Research Articles

Clinical Expression of an Inherited Unbalanced Translocation in Chromosome 6

Unbalanced chromosomal rearrangements are not common; however, they have a significant clinical expression. The parental balanced translocation produces unbalanced chromosome, which is transmitted to next generation through fertilization of gametes carrying the derivative chromosome. The carriers of balanced rearrangements mostly do not have recognizable phenotypic expression. We report a family comprising of healthy and non-consanguineous young parents and their preemie newborn severely affected with congenital anomalies and systemic disorders. Conventional Gbanding analysis of somatic chromosomes identified a balanced translocation, t(6;10)(p23;q24), in mother and an unbalanced rearrangement, der(6)t(6:10)(p23;q24)mat, in the child. The child has inherited a derivative chromosome 6 with partial deletion of 6(p23-pter) and partial trisomy 10(q24-qter), which has resulted in fusion of genes of two different chromosomes. The prominent phenotypic features of del(6p), including high forehead, flat nasal bridge, agenesis of left ear, atrial septal defect (ASD), craniosynostosis, and growth retardation, are overlapping with specific Axenfeld-Reiger-, Larsen-, and Ritscher-Sinzel/3-C syndromes, however, lacking in ocular anomalies, skeletal laxity, or cerebellar malformation. Therefore, this paper rules out the isolated effect of del(6p23) or trisomy 10(q24) on distinct previously reported syndromes and proposes the combined effect of unbalanced chromosomal alteration.

Polymicrogyria and infantile spasms in a patient with 1p36 deletion syndrome

A 3-months-old boy presented with partial seizures that soon evolved into infantile spasms. Magnetic resonance imaging revealed bilateral perisylvian polymicrogyria with right-sided predominance. ACTH therapy successfully controlled epilepsy and electroencephalograms were normalized. Conventional G-banded chromosomal analysis was performed due to his distinctive features and a derivative chromosome 1 derived from parental balanced translocation with a karyoptype of 46,XY,der(1)t(1;4)(p36.23;q35) was detected. Fluorescent in situ hybridization analysis confirmed the deleted region of 1p36 as large as 8.6 Mb. This is the first delineation of concurrent complications of infantile spasms and polymicrogyria in patient with 1p36 deletion. 1p36 deletion syndrome should be broadly recognized as a differential diagnosis of regional polymicrogyria and/or infantile spasms.

Prenatal Diagnosis of mos45,X/46,X,+mar in a Fetus With Normal Male External Genitalia and a Literature Review

Prenatal diagnosis of mos45,X/46,X,+mar is difficult in genetic counseling. Patients with the presence of a Y-derived marker may manifest male or female external genitalia. Here, we report a fetus with phenotypically male external genitalia of mos45,X/46,X,+mar. In addition, the cases with prenatally detected mos45,X/ 46,X,del(Y)(q11.2) and normal male external genitalia are reviewed. A 30-year-old, primigravid woman was referred for amniocentesis because of an abnormal Down syndrome screening result at 20 weeks' gestation. Cytogenetic analysis showed mos45,X/46,X,+mar without a normal Y chromosome. Prenatal ultrasound detected symmetric intrauterine growth restriction and normal male external genitalia. After termination of the pregnancy, a phenotypically normal male fetus was delivered smoothly without apparent structural defects. Based on conventional G-banded analysis, the marker chromosome appeared as a Y chromosome that originated with a deleted Yq, designated as del(Y)(q11.2). Based on a literature review, the addition of fluorescence in situ hybridization and molecular analysis to the conventional cytogenetic techniques can provide more accurate identification of a Y chromosome aberration in the prenatal detection of mos45,X/46,X,+mar, thus allowing more appropriate genetic counseling for the family.

Jumping translocation in acute monocytic leukemia (M5b) with alternative breakpoint sites in the long arm of donor chromosome 3

An 86-year-old man presented with acute hepatic failure, worsening thrombocytopenia, and anemia having been diagnosed and managed expectantly with cytogenetically normal RAEB-1. After 20 months a diagnosis of disease transformation to acute monocytic leukemia (M5b) was made. Conventional G-banded analysis of unstimulated bone marrow cultures demonstrated a jumping translocation (JT) involving proximal and distal breakpoints on donor chromosome 3 at bands 3q1?2 and 3q21, respectively. Recipient chromosomes included the long-arm telomeric regions of chromosomes 5, 10, 14, 16, and 19. A low-level trisomy 8 clone was also found in association with both proximal and distal JT clones. Conventional G-banded analysis of unstimulated peripheral blood cultures detected the proximal 3q1?2 JT clone involving recipient chromosome 10 several weeks after transformation to acute monocytic leukemia. Interestingly, JTs involving recipient chromosomes 5, 14, 16, and 19 were not detected in this peripheral blood sample. Palliative care was administered until his demise 2.2 months after disease transformation. There have been fewer than 70 cases of acquired JTs reported in the literature, including one myeloproliferative neoplasm and five acute myeloid leukemias involving a single breakpoint site on donor chromosome 3. Our case is unique as it is the first acquired case to demonstrate a JT involving alternative pericentromeric breakpoint sites on a single donor chromosome consisting of a proximal breakpoint at 3q1?2 and a more distal breakpoint at 3q21.

Cytogenetic and real-time quantitative reverse-transcriptase polymerase chain reaction analyses in pleomorphic rhabdomyosarcoma

Pleomorphic rhabdomyosarcoma (PRMS) is a rare variant of rhabdomyosarcoma that occurs mostly in adults. A few cytogenetic studies of PRMS have been reported, but no consistent specific chromosome aberrations were detected. We herein report a cytogenetic study of three cases of pleomorphic rhabdomyosarcoma using a conventional G-banded karyotyping analysis. The three cases appeared to exhibit an extremely complex karyotype with numeric and structural rearrangements. Although the three cases displayed several common aberrations, including –2, –4, –9, –13, –14, –15, –19, –21, add(X)(p11), add(1)(q11), add(7)(p11), and add(13)(p11), no recurrent characteristic chromosomal aberrations could be detected. In addition, among these cases and seven other cases of previously reported PRMS, the most frequent chromosomal alterations were –2, –13, –14, –15, –16, and –19. No obviously consistent structural alterations can be found in these 10 PRMS cases, however, thereby suggesting that it is difficult to confirm whether these complex karyotypes correlated with the diagnosis or clinical outcome in PRMS. In this study, we detected MyoD1 and myogenin gene transcripts at the mRNA level in four cases of PRMS together with other soft-tissue sarcomas, including seven cases of malignant fibrous hitiocytoma, five cases of liposacroma, and three cases of leiomyosacroma using a real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. High-level expressions of MyoD1 and myogenin gene transcripts were determined in all cases of PRMS. In contrast, the other non-PRMS sarcomas showed either no expression or extremely weak expressions for both genes. Our findings suggest that the detections of MyoD1 and myogenin transcripts using real-time quantitative RT-PCR, combined with immunohistochemical stains, are extremely sensitive and useful for the diagnosis of PRMS.

Limitations of G-banding Karyotype Analysis with Peripheral Lymphocytes in Diagnosing Mixed Gonadal Dysgenesis

Mixed gonadal dysgenesis (MGD) is an abnormal sexual differentiation syndrome usually presenting with ambiguous genitalia. Karyotype analysis is one of the essential components in the diagnosis of MGD and is conventionally done with peripheral lymphocytes by the G-banding technique. It is speculated that this conventional karyotype analysis has limitations since there are often difference in gonadal tissue analysis. Here we present four cases of MGD, in which karyotype analysis were performed by peripheral lymphocytes fluorescence in situ hybridization (FISH), gonad fibroblasts FISH and gonad fibroblasts G-banding technique, in addition to the conventional peripheral lymphocytes G-banding technique. In Case 1, the percentage of the 45,X cell line in lymphocytes decreased after birth and detection of mosaicism could only be done by karyotype of gonads at 7 mo of age. In Case 2, FISH analysis with peripheral lymphocytes was more useful for detecting low frequency mosaicism. In all cases, phenotype of gonads and external genitalia were more consistent with karyotype of gonads than that of the peripheral lymphocytes G-banding technique. In conclusion, conventional G-banding karyotype analysis with peripheral lymphocytes has limitations in the diagnosis and evaluation of MGD. Karyotype analysis by FISH or by using gonads is useful for diagnosing MGD and understanding of the phenotype of gonadal tissue.

Establishment of a new human epithelioid sarcoma cell line, FU-EPS-1: Molecular cytogenetic characterization by use of spectral karyotyping and comparative genomic hybridization

A small number of human epithelioid sarcoma cell lines have been reported, but their characterization at a molecular cytogenetic level is not well known. In this study, a new permanent human cell line, FU-EPS-1, derived from a metastatic epithelioid sarcoma developing in the axillary lymph node of a 21-year-old man is described. This cell line was characterized by use of immunocytochemistry, conventional G-banding analysis, spectral karyotyping (SKY) and comparative genomic hybridization (CGH). FU-EPS-1 cells were round, polygonal or spindle shaped with an abundant cytoplasm, and have been maintained continuously in vitro for over 100 passages during more than 15 months. Histologic features of the heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original tumor, revealing a multinodular proliferation of eosinophilic epithelioid and spindle cells. Both in vitro and in vivo, the cells were immunopositive for cytokeratins (AE1/AE3 and CAM5.2), epithelial membrane antigen, vimentin and CD34, but were negative for desmin, S-100 protein, CD31 or factor VIII-related antigen. By G-banding and SKY analyses, FU-EPS-1 revealed a hyperdiploid karyotype with the following chromosomal abnormalities: +i(5)(p10), -8, +13, der(13)t(8;13)(q?;p11), +der(19)t(9;19)(?;?) and del(22)(q13). In addition, CGH analysis identified gains of 5p, 9q, 19q and 22q and a loss of 8p. This study demonstrates the value of molecular cytogenetic techniques such as SKY and CGH in defining genomic alterations in greater detail. The FU-EPS-1 cell line will be exceedingly useful for biologic and molecular pathogenetic studies of human epithelioid sarcoma.

Increased frequency of multiradial chromosome structures in mouse embryonic fibroblasts lacking functional Werner syndrome protein and poly(ADP-ribose) polymerase-1

To determine whether the mouse Werner syndrome homologue (Wrn) and the poly (ADP-ribose) polymerase-1 (PARP-1) enzymes act in concert to prevent specific chromosomal rearrangements, mice with a mutation in the helicase domain of the Wrn gene ( Wrn Δhel/Δhel mice) were crossed to PARP-1 null mice. Spectral karyotyping of the mouse metaphases was used in correlation with conventional G-banded karyotype analysis to precisely define the chromosomal aberrations in cells. Although there was no recurrent clonal chromosome aberration, PARP-1 null/ Wrn Δhel/Δhel fibroblasts were distinguished by an increased frequency of chromatid breaks. Interestingly, multiradial structures were the only type of DNA rearrangement that was significantly higher in such PARP-1 null/ Wrn Δhel/Δhel cells. These results indicate that Wrn and PARP-1 enzymes may be part of a protein complex involved in the processing of DNA breaks that can ultimately lead to multiradial structures when both enzymes are nonfunctional. Finally, regions of chromosomes known to be fragile sites in the mouse genome are not more prone to DNA rearrangements in the absence of both PARP-1 and functional Wrn proteins. Moreover, the low number of recurrent rearranged chromosome at any given site suggest a random mutagenesis process in PARP-1 null/ Wrn Δhel/Δhel fibroblasts.

SKY detection of chromosome rearrangements in two cases of tMDS with a complex karyotype

In this study, we used spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) as complementary techniques for the analysis of two therapy-related secondary myelodysplastic syndrome (t-MDS) cases with complex karyotypes, previously analyzed by G-banding. Different types of SKY's cytogenetic contributions include confirmation of G-banding results, identification of partially characterized rearrangements, identification of marker chromosomes unidentified by G-banding, and detection of cryptic reciprocal translocations. In particular, the ability of SKY to clarify a number of markers led to the comprehension of clonal evolution. The common aberration found in these two t-MDS cases was the fragility of chromosome 5 and monosomy of chromosome 18. We clearly present that the use of SKY combined with conventional G-banding analysis and FISH has assisted in the identification of important chromosomal events that may play a key role in the development of t-MDS.

Cytogenetics of the Chronic Myeloid Leukemia-Derived Cell Line K562: Karyotype Clarification by Multicolor Fluorescence In Situ Hybridization, Comparative Genomic Hybridization, and Locus-Specific Fluorescence In Situ Hybridization

The transformation of chronic myeloid leukemia (CML) from a chronic phase to an acute phase is frequently accompanied by additional chromosome changes. Extensive chromosome G-banded studies have revealed the secondary changes are nonrandom and frequently include trisomy 8, isochromosome 17q, trisomy 19, or an extra copy of the Philadelphia chromosome. In addition to these secondary chromosome changes, complex structural rearrangements often occur to form marker structures that remain unidentified by conventional G-banded analysis. The CML-derived cell line, K562, has been widely used in research since it was originally established in 1975. The K562 karyotype however, has remained incomplete, and marker structures have never been fully described. Recent advances in fluorescence in situ hybridization (FISH) technology have introduced the possibility of chromosome classification based on 24-color chromosome painting (M-FISH). In this study, we report a clarified karyotype for K562 obtained by a combination of the following molecular cytogenetic techniques: comparative genomic hybridization (CGH), FISH mapping using locus-specific probes, and M-FISH. Multicolor FISH has identified the marker structures in this cell line. The characteristic marker chromosome in K562 has been confirmed by this study to be a der(18)t(1;18). Multicolor FISH confirmed the identity of marker structures partially identified by G-banding as der(6)t(6;6),der(17)t(9;17),der(21)t(1;21),der(5)t(5;6). In addition M-FISH has revealed a deleted 20q and a complex small metacentric marker comprised of material from chromosomes 1, 6, and 20. A cryptic rearrangement was revealed between chromosomes 12 and 21 that produced a structure that looks like a normal chromosome 12 homologue by G-banding analysis. Finally, M-FISH detected regions from chromosome 13 intercalated into two acrocentric markers.

Characterization of a Chromosomally Complex Lung Cancer Cell Line Using Multiwell Fluorescence In Situ Hybridization

The chromosomal characterization of a non-small cell lung cancer cell line (NCIH358) is described. This characterization was achieved using a simple, cheap and technically straightforward multiwell fluorescence in situ hybridization (FISH) method. The many and complex chromosome rearrangements identified by this method could not be defined using conventional G-banded chromosome analysis, and have not been previously described. For the detailed characterization of complex cell lines, multiwell FISH has many advantages over more technically demanding and expensive FISH techniques, and opens up the possibility of screening for consistent rearrangements, leading to the identification of unique fusion genes.

Interphase Fluorescence In Situ Hybridization Overcomes Pitfalls of G-Banding Analysis with Special Reference to Underestimation of Chromosomal Aberration Rates

Fluorescence in situ hybridization (FISH) is suitable for detecting different types of chromosome aberrations on interphase nuclei even in specimens with no or few chromosome metaphases. However, it is not known why FISH is superior to conventional G-banding analysis. The sensitivity of interphase FISH was compared to that of G-banding analysis in 288 leukemia/lymphoma patients for 10 different types of chromosome aberrations: t(9;22) (M- and m-BCR), t(8;21), 11q23 abnormalities, t(15;17), del(5)/ −5, del(13)/ −13, +8, −7, and +12. The results revealed that t(15;17) positive cells could not proliferate well in culture, leading to underestimation of abnormality by G-banding. Monosomy 7 in acute myelocytic leukemia (AML) and myelodysplastic syndrome (MDS) as well as trisomy 12 and deletion chromosome 13 in chronic lymphocytic leukemias (CLL) were also severely underestimated by G-banding. On the other hand, no discrepancies were observed in t(8;21), t(9;22), translations involving 11q23, or in trisomy 8. These findings indicate the superiority of interphase FISH over conventional cytogenetics for detecting chromosome abnormalities in small clones, especially for monosomy 7 or (15;17) translocations.

A case of recurrent congenital fetal anomalies associated with a familial subtelomeric translocation

The potential of a new fluorescent in situ hybridization technique is discussed, which uses a complete set of telomeric probes to reveal cryptic chromosome rearrangements that remain undetected by standard cytogenetic analysis. We report the obstetric history of a patient who had a termination of pregnancy at 20 weeks for a fetus with multiple congenital anomalies but a normal male karyotype using conventional G-banding analysis on a mid-trimester placental biopsy. In a subsequent pregnancy, a diaphragmatic hernia and intra-uterine growth restriction were detected at 34 weeks' gestation and a fetal blood sample showed a normal female karotype. However, her child was born with dysmorphic features and additional severe abnormalities including microcephaly, anophthalmos and left fixed talipes. The child has shown marked developmental delay. In view of a strong family history of congenital abnormalities and recurrent miscarriage suggestive of a familial translocation, a fluorescent in situ hybridization technique using specific telomeric probes was performed on blood from the affected child and her parents. An unbalanced subtelomeric translocation was detected involving the long arms of chromosomes 2 and 7 in the child and a balanced translocation was detected in her father. Accurate genetic counselling and the opportunity for early prenatal diagnosis can now be offered to this family.

Comparative Analysis of G-Banding, Chromosome Painting, Locus-Specific Fluorescence In Situ Hybridization, and Comparative Genomic Hybridization in Chronic Myeloid Leukemia Blast Crisis

The molecular basis for blast transformation of chronic myeloid leukemia (CML) remains poorly understood. Cytogenetic alterations associated with CML blast crisis have previously been extensively studied by conventional G-banding analysis. However the complexity of some chromosome abnormalities or poor chromosome morphology or both has exceeded the resolution of G-banding analysis in a significant proportion of CML cases, and complex chromosome rearrangements have remained unidentified. In this study, comparative genomic hybridization (CGH) was used to elucidate genome imbalances in chronic phase or blast crisis samples or both from 12 CML patients. CGH and G-banding results were compared, and discrepancies were further clarified by using multipaint chromosome analysis and locus-specific DNA probes. No imbalances were detected in the 4 early disease phase samples studied. Eleven blast crisis samples were analyzed by G-banding and CGH, and the commonest genomic abnormality detected was overrepresentation of the long arm of chromosome 8, which was detected in 5 patients. This overrepresentation was attributable to trisomy 8 in 4 patients, whereas amplification of the entire long arm of chromosome 8 was detected in 1 patient. The formation of isochromosomes of the long arm of chromosome 8 was observed as a mechanism for gene amplification in this patient. Additional material originating from chromosome 8 was also observed intercalated into three marker chromosomes in peripheral blood metaphase spreads from this patient. These markers may further define areas on chromosome 8 that harbor oncogenes implicated in transformation of chronic myeloid leukemia.

Screening for Trisomy 12 in Bone Marrow from Patients with CLL by the Application of Fluorescence In Situ Hybridization to Interphase Nuclei

Trisomy 12 is the most common chromosomal abnormality in chronic lymphocytic leukemia (CLL) of B-cell origin. We have studied 12 CLL patients with fluorescence in situ hybridization (FISH) to detect trisomy 12 and with conventional cytogenetic trypsin G-banding analysis to detect other chromosomal abnormalities. Fresh uncultured heparanized bone marrow from each patient was analyzed using a chromosome-12-specific a-satellite DNA probe for FISH and also by conventional trypsin G-banding analysis. Two patients (2 of 12) with trisomy 12 were detected by FISH. One of these was confirmed by conventional trypsin G-banding analysis, but the other was not. On conventional cytogenetic analysis, one patient had mosaic trisomy 12, six patients had normal karyotypes, three patient samples failed to produce metaphase spreads, and three patients had different types of chromosomal abnormalities. In conclusion, FISH to interphase nuclei is a sensitive and powerful tool for detecting trisomy 12 in a large number of patients without the need for metaphase preparations.

Molecular cytogenetic analysis of RB-1 deletions in chronic B-cell leukemias.

Deletions or translocations of 13q, most commonly involving band 13q14, belong to the most frequent structural chromosome abnormalities in B-cell chronic lymphocytic leukemia (B-CLL). In a combined metaphase and interphase cytogenetic study using conventional G-banding analysis and fluorescence in situ hybridization (ISH) we previously analysed the retinoblastoma susceptibility gene (RB-1) and its chromosomal locus 13q14 in 35 patients with chronic B-cell leukemias. We report here on the interphase cytogenetic analysis of 109 cases with chronic B-cell leukemias [B-CLL = 90; B-prolymphocytic leukemia (B-PLL) = 6, hairy cell leukemia (HCL) = 13]; a subset of 49 patients (B-CLL = 45; B-PLL = 4) was studied by conventional G-banding analysis. By G-banding, 5/45 (11%) patients with B-CLL had deletions or translocations affecting band 13q14; in contrast, ISH to interphase cells showed RB-1 deletion in 19/90 (21%) patients with B-CLL. No 13q14 abnormalities or RB-1 deletion were detected in patients with B-PLL and HCL. Our data confirm the high frequency of RB-1 deletions in B-CLL and further emphasize the possible pathogenetic role of this genomic region.