Duplication of intrachromosomal insertion segments 4q32-->q35 confirmed by comparative genomic hybridization and fluorescent in situ hybridization / 대한생식의학회지

A 35-year-old man with infertility was referred for chromosomal analysis. In routine cytogenetic analysis, the patient was seen to have additional material of unknown origin on the terminal region of the short arm of chromosome 4. To determine the origin of the unknown material, we carried out high-resolution banding, comparative genomic hybridization (CGH), and FISH. CGH showed a gain of signal on the region of 4q32-->q35. FISH using whole chromosome painting and subtelomeric region probes for chromosome 4 confirmed the aberrant chromosome as an intrachromosomal insertion duplication of 4q32-->q35. Duplication often leads to some phenotypic abnormalities; however, our patient showed an almost normal phenotype except for congenital dysfunction in spermatogenesis.

Laboratory study on a rare case of chronic myeloid leukemia with ins(22;9)t(9;13) and Ph-negative / 中国实验血液学杂志

The study aimed to examine a rare case of Philadelphia (Ph)-negative chronic myeloid leukemia (CML) with t(9;13). Chromosome samples were prepared after culture of bone marrow cells for 24 hours, the karyotypes were analyzed by G banding technique. Chromosome painting analysis was performed by using whole chromosome paints for chromosomes 9 and 22. Fluorescence in situ hybridization (FISH) was done with dual color dual fusion LSI bcr/abl probe. Bcr/abl transcripts were detected by real time fluorescence quantitative polymerase chain reaction (RQ-PCR). As a result, G banding analysis showed a karyotype of 45, XX, der(9)t(9;13)(q34;q10), -13[20]. FISH assay using LSI bcr/abl DNA probe showed a red abl signal inserted into der(22) and a fusion signal of bcr/abl rearrangement was discovered. RQ-PCR detected high copies of bcr/abl transcripts. In conclusion, insertion of bcr/abl rearrangement was a rare variant t(9;22) and could be well detected by molecular techniques, however, regular cytogenetic banding technique and whole chromosome paintings may probably lead a misdiagnosis to such cases.

An analysis on chromosome X, Y and 18 in the spermatozoa of asthenospermia patients by triple-color fluorescence in situ hybridization / 中华男科学杂志

<p><b>OBJECTIVE</b>To analyze the numerical aberration of chromosome X, Y and 18 in the spermatozoa of asthenospermia patients by triple-color fluorescence in situ hybridization.</p><p><b>METHODS</b>The experiment included 10 asthenospermia patients and 5 healthy men with normal semen quality as controls. Fluorescence in situ hybridization (FISH) and probes for chromosomes including X, Y and 18 were used to determine the frequency of the aneuploid of the chromosomes in spermatozoa.</p><p><b>RESULTS</b>Of the 45,547 spermatozoa counted from the semen samples, the hybridization rate was 99.18%. The frequencies of the chromosome disomies including XX18, XY18, YY18, X1818 and Y1818 were (0.124 +/- -0.086)%, (0.360 +/- 0.380)%, (0.109 +/- 0.195)%, (0.342 +/- 0.746)% and (0.299 +/- 0.564)% in the case group and (0.014 +/- 0.019)%, (0.090 +/- 0.080)%, (0.030 +/- 0.031)%, (0.068 +/- 0.103)% and (0.075 +/- 0.083)% in the control. The sperm aneuploid rate was 9.25% in the former and 2.70% in the latter, with significant difference in between (P< 0.01).</p><p><b>CONCLUSION</b>Asthenospermia patients have a higher aneuploid rate of sperm chromosome than normal fertile men. However, larger samples are yet to be studied to obtain more scientific evidence.</p>

Analysis of complex chromosomal aberrations in patients with myelodysplastic syndromes using multiplex fluorescence in situ hybridization combined with whole chromosome painting / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the value of multiplex fluorescence in situ hybridization (M-FISH) in combination with whole chromosome painting (WCP) in the detection of complex chromosomal aberrations (CCAs) in myelodysplastic syndromes (MDS).</p><p><b>METHODS</b>M-FISH was used in seven MDS patients with R-banding CCAs to refine the complex chromosomal rearrangements, 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.</p><p><b>RESULTS</b>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 conventional cytogenetics (CC) were defined after M-FISH analysis; four 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 and -5/5q- were the two most frequent aberrations. Some misclassified and missed chromosomal aberrations by M-FISH were corrected by WCP.</p><p><b>CONCLUSION</b>M-FISH is a powerful molecular cytogenetic tool in clarification of CCAs. Complementary WCP helps us to identify misclassified and missed chromosomal aberrations by M-FISH. CC in combination with molecular cytogenetic techniques, such as M-FISH and WCP, can unravel complex chromosomal aberrations more precisely.</p>

Establishment of lymphocyte cell lines with abnormal chromosome karyotypes and its application in external quality assesment for chromosome karyotype analysis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop chromosome abnormal karyotype quality control cell and to explore the external quality assessment (EQA) method for chromosome karyotype analysis.</p><p><b>METHODS</b>The chromosome abnormal karyotype quality control cells were prepared by EB virus (EBV) transfection of human B lymphocyte strain establishment and were distributed to participating labs for EQA test of chromosome karyotype analysis project at appointed time. The evaluation results were obtained through 4 grades scoring.</p><p><b>RESULTS</b>Six kinds of chromosome abnormal karyotype quality control cells were initially developed, the karyotypes of which were 46,X, t(Y;5)(q12;q21), 46, XY, 15p +, 46, XX, t(13;18)(q12;q21), 46, X, r(Xp), 46,X,t(Y;Y), 46,XX,t(9;20)(p13;p13) respectively. In the external quality assessment, feedbacks from the participating labs on the sequencing results of the six kinds of quality control cells showed that the wholly overlapping rate were 82.1%, 92.0%, 84.6%, 80.8%, 86.2%, 74.1% and the wholly deviation rate were 10.7%, 8.0%, 11.5%, 19.2%, 13.8%, 18.5%. The overall wholly overlapping rate, partial overlapping rate, partial deviation rate and wholly deviation rate turned out to be 83.2%, 0.6%, 2.5% and 13.7% respectively.</p><p><b>CONCLUSION</b>The misdiagnose rate of chromosome karyotype analysis is rather high and regular external quality assessment is necessary to achieve dynamic information and improve diagnosis quality.</p>

The clinical application of whole chromosome painting probes in preimplantation genetic diagnosis for translocation carriers / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To make preimplantation genetic diagnosis (PGD) for female translocation carriers by analyzing first polar bodies (1PBs) with whole chromosome painting probe (WCP).</p><p><b>METHODS</b>WCP was used in fluorescence in situ hybridization (FISH) analysis of 1PBs for four female Robertsonian carriers presented for PGD with 45 XX, der(13;14)(q10;q10) karyotype. All the patients underwent ovarian stimulation and during 6 h after oocyte retrieval 1PBs were biopsied and WCP were used in FISH. On day 3 after fertilization embryos diagnosed as normal or balanced were transferred.</p><p><b>RESULTS</b>A total of 61 oocytes were collected in 4 PGD cycles. Of the 54 matured oocytes, 50 were biopsied and 45 were fixed successfully. Results were obtained in 40 1PBs. Overall, 74.1% (40/54) oocytes were diagnosed. The fertilization rate and good embryo rate were 64.8% (35/54) and 65.7% (23/35) respectively. Two clinical pregnancies were obtained. One patient delivered a normal female baby with karyotype 46, XX in June 2006. For another patient, the fetus spontaneously aborted at 9th week of pregnancy with karyotype of 45, X confirmed by amniotic villus diagnosis.</p><p><b>CONCLUSION</b>WCP can differentiate normal, balanced and unbalanced oocytes accurately and can be used as an efficient PGD method for female carriers of translocation.</p>

Cytogenetic and molecular genetic studies on a variant of t(15;17), ins(17;15)(q21;q14q22), in an acute promyelocytic leukemia patient / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To report a rare variant of t(15;17), ins(17;15)(q21;q14q22) in an acute promyelocytic leukemia (APL) patient and the results of cytogenetic and molecular genetic studies.</p><p><b>METHODS</b>Chromosomes were prepared after 24 hours culture of bone marrow cells and peripheral blood cells. R-banding technique was used to analyze karyotypes. Chromosome painting analysis was performed using whole chromosome paints for chromosomes 15 and 17. PML-RAR alpha and RAR alpha-PML fusion transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Karyotypic analysis using both specimens from bone marrow and peripheral blood leukemic cells revealed 15q- and 17q+. Chromosome painting analysis confirmed that the karyotypic abnormality was ins(17;15). PML-RAR alpha fusion transcript (S type) was detected by RT-PCR, while RAR alpha-PML fusion transcript was not detected.</p><p><b>CONCLUSION</b>Chromosome painting and RT-PCR are reliable methods for characterization of the insertion involving chromosomes 15 and 17 in APL patients.</p>

Cytogenetics and fluorescence in-situ hybridization in detection of hematological malignancies.

BACKGROUND: The technique of Fluorescence In-Situ Hybridization (FISH), a hybrid of cytogenetics and molecular biology has increased the resolution and application of cytogenetics in various neoplastic processes. In various types of leukemias, primary investigation by conventional cytogenetic [CC] technique followed by FISH has increased our understanding of the abnormal clonal formation involving different gene region. AIMS: Present study is aimed to use different kinds of in-house FISH probes in various hematological malignancies and its correlation with conventional cytogenetic finding. MATERIAL AND METHODS: Cytogenetic study was carried out in 360 patients either from peripheral blood or from bone marrow cells suspected for various types of leukemias. Four of 360 cases were further selected for FISH study by using different types of in-house probes, such as BAC [Bacterial Artificial Chromosome], PAC [Phague Artificial Chromosome], alphoid, PCP [Partial Chromosome Paint] and WCP [Whole Chromosome paint]. RESULTS: The results confirmed breakpoints of inversion 16 and del 16 in case 2 and 3 respectively. Whereas, case 1 did not confirm the cytogenetic findings of t(15;17) by PML/RARa fusion signals as multiple cell lines were involved in the patients. PCP and WCP were helpful in the identification of the marker chromosome in case 1. Telomeric and centromeric probes confirmed the cytogenetic findings of t(5;7) in case 4. CONCLUSION: We observe from this study that, in addition to the conventional cytogenetic study, FISH study provide further confirmation of chromosomal rearrangements. This facilitates our understanding of the neoplastic process more precisely for the better prognostication of the patient.

Chromosomal Alterations in Hepatocellular Carcinoma Cell Lines Detected by Comparative Genomic Hybridization / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: There have only been a few cytogenetic studies of hepatocellular carcinoma (HCC), and so far, no consistent specific chromosomal abnormalities have been described. Here, we have used comparative genomic hybridization (CGH), a powerful molecular cytogenetic technique for detecting changes of the copy number throughout the genome, to screen for genetic alterations in HCC cell lines. The CGH results were compared with those derived from G-banding and chromosome painting. MATERIALS AND METGODS: Conventional cytogenetic analyses were performed on five HCC cell lines, SNU-354, SNU-368, SNU-387, SNU-449 and SNU-475, using a G- banding staining technique. In CGH, equal amounts of differently labeled DNA from the cell lines, and normal reference DNA, were hybridized simultaneously to normal metaphase chromosomes. They were visualized by different fluorochromes, and the signal intensities quantified separately as gray levels along the single chromosomes. The over- and under-represented DNA segments were determined by computation of ratio images and average ratio profiles. To confirm the CGH results, florescence in situ hybridization (FISH), with chromosome specific painting, was performed using indirectly labeled chromosome specific paints. RESULTS: Complex unbalanced chromosomal aberrations, which could not be identified reliably by conventional cytogenetics in HCC cell lines, were successfully resolved by CGH analysis. CGH results were validated using FISH with chromosome specific probes. In HCC cell lines, gains in DNA copy number were more common than losses. The most prominent changes were gains of 1q12- qter (80% of cases), 1q41-qter (100%), 7 (80%), 8q12-qter (60%), 8q23-qter (80%) and 20q12-qter (60%). Recurrent losses were mapped on 4q13-qter (60%), 16q12-qter (60%), 16q21-qter (80%), 13q12-q14.2 (60%) and Yq11.2 (100%). All four male HCC cell lines showed loss or rearrangement of the Y chromosome. CONCLUSION: Conventional cytogenetics, CGH and FISH using painting probes, represent complementary approaches that, when employed in combination, could greatly facilitate the comprehensive analysis of chromosomal imbalances in HCC cell lines. Our results suggest the existence of an oncogene, or protooncogenes, on chromosome 1q41-qter, and the tumor suppressor genes on Yq11.2, that play a role in the development and/or progression of hepatocellular carcinogenesis.

Microsporogénesis y microgametogénesis de onoto (Bixa orellana L. ) / Microsporogenesis and microgametogenesis of annatto (Bixa orellana L. )

A series of buds of increasing maturity were individually sampled in order to examine cytological events of annatto (Bixa orellana L.), genotype Portuguesa. They were fixed in Carnoy II at 12:30 am, time of the highest rate of meiotic division. Three stain solutions were attempted. In the microspores mother cells, the use of acetic orcein 1% resulted in a good nucleus coloration and sharpness. In contrast, a well chromosome resolution was achieved with the application of propionic carmin 2%. The pollen grain mother cells (n = 8 chromosomes) at metaphase I were found in floral buds of 0.5 to 0.6 cm long; tetrad stage in buds of 0.6 to 0.7 cm long, uninucleate stage of microspores in buds of 0.7 to 0.8 cm long and the binucleate stage (pollen) in buds longer than 0.8 cm. Microphotographies showing the sequence of meiotic division (microsporogenesis) and subsequent mitosis to originate pollen grains were included

Sindrome de Pallister Killian ou tetrassomia do braco curto do cromossomo 12 em mosaico : relato de dois casos diagnosticados pela hibridizacao in situ por fluorescencia (FISH) / Pallister Killian syndrome or tetrasomy of the short arm of the chromosome 12 mosaicism : report on two cases diagnosed by fluorescense in situ hybridization

A sindrome de Pallister Killian (SPK) e uma doenca rara caracterizada por multiplas malformacoes, retardo mental profundo e presenca de um isocromossomo...

Chromosomal Aberrations in Ovarian Carcinoma Cell Line, SNU - S , Using Chromosome Painting / 대한암학회지

PURPOSE: The characterization of all recognizable chromosomal rearrangements was dis- turbed by technical limitation of conventional cytogenetic methods. Recently, the strong usefullness of generation of chromosome specific painting probes in identification of marker chromosomes has proven. This study was intended to analyze the chromosomal aberrations in human ovarian cancer cell line, SNU-8, by G-banding and multiple paintings. MATERIALS AND METHODS: Human ovarian cancer cell line, SNU-8 was cultured and harvested for cytogenetic analysis. Routine karyotyping was performed. For complete analysis of chromosomal aberrations, human chromosome-specific painting probes were constructed from somatic hybrid cells. The origins of the unidentified marker chromosomes were analyzed by fluorescent in situ hybridization (FISH) with these painting probes. RESULTS: All chromosome alterations were confirmed by the use of multiple chromosome paintings, which also demonstrated a number of additional alterations. SNU-8 had the karyotype 62-69,XXX, + der(1;10)(q10;p10),der(3;18) (q10;p10)X2,-4,+ 5,+ 7,del(9)(q21)X2,-11,-13,-15,-16,der(17;19)(q10;q10) X2, + 20,-22[cp51]. CONCLUSION: The chromosomal aberrations of SNU-8 cell line was effectively analyzed by FISH with these painting probes, and the approach methods of this study can be applied to cytogenetic analysis of chromosomal aberrations in the other cancers.

Chromosomal Alterations in Gastric Cancer Cell Lines Detected by Comparative Genomic Hybridization / 대한암학회지

PURPOSE: There are only a few cytogenetic studies in gastric cancer and so far no consistent specific chromosomal abnormalities have been described. In this study, we have used comparative genomic hybridization (CGH), a powerful molecular cytogenetic technique for detecting changes of the copy number throughout the genome, to screen for genetic alterations in gastric cancer cell lines. The CGH results were compared with those derived from G-banding and chromosome painting. MATERIALS AND METHODS: Conventional cytogenetic analysis was performed on five human gastric cancer cell lines, AGS, SNU-1, SNU-16, SNU-620, and SNU-719, by a G-banding staining technique. In CGH, equal amounts of differently labeled DNA from the cell lines and normal reference DNA were hybridized simultaneously to normal metaphase chromosomes. They were visualized by different fluorochromes, and the signal intensities were quantitated separately as gray levels along the single chromosomes. The over- and under- represented DNA segments were determined by computation of ratio images and average ratio profiles. To confirm the CGH results, fluorescence in situ hybridization (FISH) with chromosome specific painting was performed using indirectly labeled chromosome specific paints. RESULTS: Complex unbalanced chromosomal aberrations that could not be identified reliably by conventional cytogenetics in gastric cancer cell lines were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome specific probes. In gastric cancer cell lines, gains of DNA copy number were more common than losses. Gains were detected on 1p, 1q, 2p, 3q, 6p, 7q, 10q, 11p, and 19q, and losses were observed on 4p, 4q, 5q, 12p, 12q, and 18q. Interestingly, all the five gastric cancer cell lines tested showed gain of DNA copy number on the chromosome 20, suggesting an existence of oncogene. CONCLUSION: Conventional cytogenetics, CGH, and FISH using painting probes represent complementary approaches that, when employed in combination, could greatly facilitate the comprehensive analysis of chromosomal imbalances in gastric cancer cell lines. Our results suggest the existence of an oncogene or oncogenes on chromosome 20 that play a role in the development and/or the progression of gastric carcinogenesis.

Detection of the Pallister-Killian Syndrome by G-Banding and FISH in Cultured Skin Fibroblasts / 대한임상병리학회지

Pallister-Killian syndrome is a rare disorder characterized by multiple congenital anomalies, coarse face, profound mental retardation, and epilepsy. Chromosomes of peripheral lymphocytes are usually normal, however, tissue cultures show varying degree of mosaicism for an extra metacentric chromosome i(12)(p10). We report on a two-year-old boy with Pallister-Killian syndrome confirmed by FISH in cultured skin fibroblasts. The patient had myoclonic seizures beginning at 2 months and was delayed in physical and speech development. Craniofacial manifestations include sparsity of scalp hair, hypertelorism, sparse eyebrows, flat nasal bridge, and large ears. Cytogenetic analysis of peripheral lymphocytes done at another hospital was reported to be normal. Studies of his skin fibroblasts showed an extra small metacentric i(12p) chromosome in 100% of metaphases. FISH using of whole chromosome painting probe for chromosome 12 confirmed that the supernumerary chromosome was an isochromosome 12p.