Direct Cytogenetic Analysis Research Articles

Aneuploidy in Human Oocytes: Some Facts and Questions

Studies on the origin of aneuploid conceptions have indicated that many chromosomal abnormalities arise during maternal meiosis. Therefore, the direct cytogenetic analysis of female gametes is of interest to evaluate underlying mechanisms. Human oocytes failing to fertilize after the application of assisted reproductive technologies have predominantly been used for this purpose. In addition, indirect data can be obtained from the examination of the first polar body that reflects the chromosomal constitution of the oocyte. Both approaches have been conducted with different methods and provided substantial information on the incidence of aneuploidy originating from errors during first meiotic division. However, the results reported so far display great variability, most probably due to technical problems and heterogeneity of the study population. Notwithstanding these limitations, it was unequivocally shown that two aneuploidy-causing mechanisms exist: non-disjunction, resulting in the loss or gain of whole chromosomes, and predivision, resulting in the loss or gain of single chromatids in mature oocytes. All chromosome groups are affected by aneuploidy but abnormalities in groups D, E, and G exceed the expected values. Considering only abnormal complements with one extra or missing chromosome/chromatid reveals a slight increase in predivision compared with non-disjunction. Chromosome and chromatid abnormalities are positively correlated to maternal age. The simultaneous analysis of oocytes and first polar bodies suggests that some cases of aneuploidy are caused by gonadal mosaicism instead of meiotic errors. Keywords: Oocyte, polar body, aneuploidy, non-disjunction, predivision, maternal age

Expression of HOX11 in childhood T-lineage acute lymphoblastic leukaemia can occur in the absence of cytogenetic aberration at 10q24: a study from the Children's Cancer Group (CCG).

Clonal genetic aberrations in tumour cells provide critical information for the development of new diagnostic and therapeutic strategies for patients. In paediatric T-cell acute lymphoblastic leukaemia (T-ALL) chromosomal translocations are present in 30-35% of cases. HOX11 and the closely related HOX11L2 genes play a key role in T-ALL. HOX11 is aberrantly activated by either of the two chromosomal translocations, t(7;10) and t(10;14). In this study, HOX11 expression levels were measured by real-time quantitative reverse-transcriptase polymerase chain reaction. We show that leukaemic blasts from 15/76 (19.7%) paediatric T-ALL patients expressed the HOX11 gene at high level and 22/76 (28.9%) at low level, yet the reported frequency for chromosomal rearrangement of 10q24 is 4-7%. Direct cytogenetic analysis revealed that only 2/16 specimens that showed HOX11 expression exhibited abnor-malities at 10q24. These results confirm and extend our previously published findings, and implicate mechanisms other than gross chromosomal translocations for the deregulation of HOX11. Analysis of clinical outcome for the whole study group showed a trend for better outcome for patients with leukaemic blasts expressing HOX11 at high level. A statistically significant difference in clinical outcome was found in a subgroup of 20 patients treated for high-risk disease on CCG-1901 from the Children's Cancer Group, where HOX11 expression in leukaemic blasts conferred a prognostic advantage (P=0.01).

Aneusomy and tumor heterogeneity in metastatic breast cancer detected by fluorescence in situ hybridization

Direct interphase cytogenetic analysis was performed on nuclei of metastatic effusions from breast cancer using fluorescence ill situ hybridization (FISH). DNA probes specific for repetitive pericentromeric regions on chromosomes 6, 8, 17, and human midi-satellite probe specific for one locus on the short arm of chromosome 1 were used to determine chromosome copy numbers in interphase tumor cells. The analysis showed cytogenetic heterogeneity in most nuclei examined with multiple subpopulations and a range of 0-6 chromosome signals per cell. The presence of subclones in these fluids, their correlation with highly malignant phenotypes and the diagnostic and prognostic applications of this method is discussed.

THE OUTCOME OF PREGNANCIES WITH CONFINED PLACENTAL CHROMOSOME MOSAICISM IN CYTOTROPHOBLAST CELLS

Cytogenetic findings and outcome of pregnancy are reported in 108 cases in which confined placental mosaicism (CPM, n=101) or generalized mosaicism (n=7) was found at or after first-trimester chorionic villus sampling. In all samples, a (semi)direct cytogenetic analysis of cytotrophoblast cells was performed. Two pregnancies with CPM ended in a spontaneous abortion before 28 weeks (1·9 per cent). In 15 cases the pregnancy was terminated: eight cases were shown to be examples of CPM; seven cases can be considered as examples of generalized mosaicism. A normal cytogenetic result was obtained after follow-up amniocentesis in 88 of the remaining 91 cases. In three cases, no amniocentesis was performed but confirmation of a normal karyotype was obtained in other cells. One of the 91 pregnancies was nevertheless terminated for psychosocial reasons. One child died perinatally and another on the seventh day after birth. The birth weight is known for 89 children; the curve shows a normal distribution. In 11 of these children (12·3 per cent), the birth weight was found to be below the tenth centile. The outcome in a subgroup of eight pregnancies with CPM and involvement of chromosome 13, 16, or 22, however, revealed two fetal losses and four children with a birth weight below the tenth centile (75 per cent).

THE OUTCOME OF PREGNANCIES WITH CONFINED PLACENTAL CHROMOSOME MOSAICISM IN CYTOTROPHOBLAST CELLS

Cytogenetic findings and outcome of pregnancy are reported in 108 cases in which confined placental mosaicism (CPM, n = 101) or generalized mosaicism (n = 7) was found at or after first-trimester chorionic villus sampling. In all samples, a (semi)direct cytogenetic analysis of cytotrophoblast cells was performed. Two pregnancies with CPM ended in a spontaneous abortion before 28 weeks (1.9 per cent). In 15 cases the pregnancy was terminated: eight cases were shown to be examples of CPM; seven cases can be considered as examples of generalized mosaicism. A normal cytogenetic result was obtained after follow-up amniocentesis in 88 of the remaining 91 cases. In three cases, no amniocentesis was performed but confirmation of a normal karyotype was obtained in other cells. One of the 91 pregnancies was nevertheless terminated for psychosocial reasons. One child died perinatally and another on the seventh day after birth. The birth weight is known for 89 children; the curve shows a normal distribution. In 11 of these children (12.3 per cent), the birth weight was found to be below the tenth centile. The outcome in a subgroup of eight pregnancies with CPM and involvement of chromosome 13, 16, or 22, however, revealed two fetal losses and four children with a birth weight below the tenth centile (75 per cent).

Identification of Philadelphia-negative granulocyte-macrophage colony- forming units generated by stroma-adherent cells from chronic myelogenous leukemia patients

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean (+/- SEM) percentage of Ph- metaphases was 3% +/- 1%. Mononuclear marrow cells from CML patients (n = 18) were incubated with mafosfamide (100 micrograms/mL) or control medium, seeded onto marrow stromal layers and allowed to adhere (2 hours, 37 degrees C). After a short-term (3-day) liquid culture, the cells were harvested, incorporated in methyl-cellulose, and individual colonies were analyzed by single colony karyotyping. The mean (+/- SEM) percentage of Ph- colonies generated from the stroma- adherent fraction was 35% +/- 6%. As compared with marrow colony- forming unit granulocyte-macrophage plated before any manipulation, the mean (+/- SEM) percentage of Ph- clones was significantly increased by stroma adherence (35% +/- 6% v 15% +/- 4%, P < or = .005) and mafosfamide (100 micrograms/mL) incubation of marrow cells before stroma adherence (58% +/- 9% v 35% +/- 6%, P < or = .005). An additive effect was observed by combining mafosfamide treatment and stroma adherence. Single-colony transfer experiments showed that 50% +/- 4% stroma-adherent and 70% +/- 4% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. To further characterize the stroma-adherent fraction, experiments were performed in which CD34+ marrow cells were used. The mean (+/- SEM) output of progenitors generated by 10,000 CD34+, stroma-adherent cells was 888 +/- 188 and 570 +/- 258 for untreated and mafosfamide-treated cells, respectively. Individual colonies were analyzed by single-colony karyotyping and fluorescent in situ hybridization using a biotinylated cosmid DNA probe that hybridize to abl oncogene. The CD34+, stroma-adherent fraction contained 38% +/- 14% (untreated) and 56% +/- 18% (mafosfamide-treated) (P < or = .025) Ph- progenitors. In conclusion, the present data show the possibility to select Ph- clones that (1) have a maintained capability of stroma adherence, (2) are mafosfamide resistant, (3) are derived from the CD34+ fraction, and (4) have high-replating potential.

Identification of Philadelphia-Negative Granulocyte-Macrophage Colony-Forming Units Generated by Stroma-Adherent Cells From Chronic Myelogenous Leukemia Patients

AbstractChronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the coexistence of Philadelphia-negative (Ph-) with Ph+ progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clono-genic cells generated from the stroma-adherent cell fraction. On direct cytogenetic analysis, the overall mean (± SEM) percentage of Ph- metaphases was 3% ± 1 %. Mononuclear marrow cells from CML patients (n = 18) were incubated with mafosfamide (100 μg/mL) or control medium, seeded onto marrow stromal layers and allowed to adhere (2 hours, 37°C). After a short-term (3-day) liquid culture, the cells were harvested, incorporated in methyl-cellulose, and individual colonies were analyzed by single colony karyotyping. The mean (± SEM) percentage of Ph-colonies generated from the stroma-adherent fraction was 35% ± 6%. As compared with marrow colony-forming unit granulocyte-macrophage plated before any manipulation, the mean (± SEM) percentage of Ph- clones was significantly increased by stroma adherence (35% ± 6% v 15% ± 4%, P ≤ .005) and mafosfamide (100 μg/mL) incubation of marrow cells before stroma adherence (58% ± 9% v 35% ± 6%, P ≤ .005). An additive effect was observed by combining mafosfamide treatment and stroma adherence. Single-colony transfer experiments showed that 50% ± 4% stroma-adherent and 70% ± 4% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. To further characterize the stroma-adherent fraction, experiments were performed in which CD34+ marrow cells were used. The mean (± SEM) output of progenitors generated by 10,000 CD34+, stroma-adherent cells was 888 ± 188 and 570 ± 258 for untreated and maf-osfamide-treated cells, respectively. Individual colonies were analyzed by single-colony karyotyping and fluorescent in situ hybridization using a biotinylated cosmid DNA probe that hybridize to abl oncogene. The CD34+, stroma-adherent fraction contained 38% ± 14% (untreated) and 56% ± 18% (mafosfamide-treated) (P ≤ .025) Ph- progenitors. In conclusion, the present data show the possibility to select Ph- clones that (1) have a maintained capability of stroma adherence, (2) are mafosfamide resistant, (3) are derived from the CD34+ fraction, and (4) have high-replating potential.

Variable growth in vitro of chromosomally different stem cells in myelodysplastic syndromes

Ten patients with myelodysplastic syndromes were investigated using in vitro colony assay of bone marrow cells and chromosome analysis of single colonies. The result was compared with conventional cytogenetic analysis of bone marrow cells. The chromosome abnormalities included were 5q−, +8, −7, 11q−, −Y and one complex karyotype. Erythroid colony formation was reduced in eight patients, while the number of granulocyte-macrophage colony-forming units was normal or increased. Cytogenetic examination of single colonies showed that both chromosomally normal and abnormal stem cells had colony forming ability. The proportion of cytogenetically abnormal colonies varied between 25 and 100% of analysed colonies. No further clones than those found in direct cytogenetic analysis were revealed after in vitro growth. One patient with a 5q− abnormality and one patient with a −7 abnormality showed a significantly lower proportion of cytogenetically abnormal colonies than the proportion of abnormal cells in the direct bone marrow chromosome preparation. One patient with a +8 abnormality showed a growth advantage of the +8 clone in comparison with the chromosomally normal clones, but this was not statistically significant. In two patients a chromosomally changed stem cell gave rise to both erythroid and myeloid colonies. The FAB-class did not seem to influence the growth of either chromosomally normal or abnormal colonies.

Biological and chemical selection of ph‐negative clones

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematopoietic stem cell characterized by the co-existence of Philadelphia-negative with Ph-positive progenitors. CML progenitor cells have been shown to be defective in adherence to marrow stroma. The present study investigated at the cytogenetic level marrow-derived CML clonogenic cells generated from the stroma-adherent cell fraction. Mononuclear marrow cells from CML patients (n = 20) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers, and allowed to adhere (2 h, 37 degrees C). Following a short-term (3 days) liquid culture, the cells were harvested, incorporated in methylcellulose, and individual colonies were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 9 +/- 20%. The mean (+/- SD) percentages of Ph-negative colonies grown from the stroma-adherent and the stroma-adherent mafosfamide-treated fraction were 41 +/- 32% and 62 +/- 40% (p < or = .005), respectively. Single colony transfer experiments revealed that 50 +/- 13% stroma-adherent and 70 +/- 24% stroma-adherent mafosfamide-treated progenitors gave rise to secondary colonies. In conclusion, the present data demonstrate the possibility to select Ph-negative clones that: 1) have a maintained capability of stroma adherence; 2) are mafosfamide resistant; and 3) have high-replating potential.

Fractionation of chronic myelogenous leukemia marrow cells by stroma adherence: implications for marrow purging.

Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their ability to adhere to marrow stroma. It was the aim of the present study to investigate at the cytogenetic level marrow-derived CML clonogenic cells fractionated on the basis of their ability to adhere to preformed, allogeneic, normal marrow-derived stromal layers. Mononuclear marrow cells from CML patients (n = 15) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers and allowed to adhere (3 hrs, 37 degrees C). Following a short-term liquid culture, the different cell fractions were harvested and incorporated in methylcellulose cultures. CFU-GM grown from these cultures were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 7 +/- 20%. Following stroma adherence and shortterm suspension culture, the mean (+/- SD) percentages of Ph-negative clones were as follows: 33 +/- 25% for adherent CFU-GM, 59 +/- 40% for adherent, mafosfamide-treated CFU-GM, 12 +/- 16% for non-adherent CFU-GM, and 32 +/- 26% for non-adherent-mafosfamide-treated CFU-GM. If only the patients showing a percentage of Ph-negative clones > or = 20% were included in this analysis, the mean (+/- SD) percentages of Ph-negative clones were 47 +/- 19% for adherent CFU-GM, and 81 +/- 21% for adherent-Mafosfamide-treated CFU-GM. In contrast, the majority of pH-positive CFU-GM were detected within the stroma non-adherent cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytogenetic analysis of primary neuroblastoma with del(1), del(14), hsr, and dmin chromosomes

The results of a direct cytogenetic tumor analysis in a 2 1 2 -year-old girl with metastatic neuroblastoma are presented in this paper. Investigations established a modal karyotype of 46 chromosomes with several marker chromosomes. Besides del(1)(p31), hsr, and dmin chromosomes in all the cells analyzed, terminal deletion of the long arm of chromosome 14 was also identified. Malignant cells were thus monosomic for segment 14q24→qter.

Mosaicism in Pallister i(12p) syndrome

The clinical diagnosis of Pallister syndrome in a 1-day-old white boy was confirmed by the presence of i(12p) in 100% of cells on direct bone marrow analysis. This is the second Pallister syndrome case in which cytogenetic diagnosis was made in bone marrow cells during the neonatal period. Other tissues analyzed in our patient included peripheral blood PHA-stimulated cultured lymphocytes and postmortem skin and lung cultured fibroblasts with 3%, 98.5%, and 97.5% of cells containing the i(12p), respectively. Serial skin fibroblast cultures re-established from frozen cells were analyzed sequentially over time for the isochromosome. There was slight reduction in the proportion of i(12p) cells until passage 15 with plateauing of the proportion of i(12p) cells at about 80% until culture senescence. Our review of such cytogenetic analyses suggests that in vivo and perhaps also in vitro isochromosome loss best explains the intra- and inter-tissue specific chromosomal mosaicism in the i(12p) syndrome. In any event, our results indicate that confirmation of the diagnosis in the neonatal period is possible by direct cytogenetic analysis of bone marrow.

Direct cytogenetic analysis of primary neuroblastoma

In this paper the results of cytogenetic analysis of a metastatic neuroblastoma from a 14-month-old boy are described. Direct cytogenetic analysis was performed on tumor pieces obtained from surgery prior to therapy. Consistent numerical and structural chromosome aberrations were identified. The modal chromosome number was 48, with 9.4% of the cell population being in the near-tetraploid range. In all karyotyped cells, the Y chromosome was missing and additions of chromosomes 7 and 14 were identified. Two rearranged #1 were observed: del(1)(p22 or p31) and t(1;18)(p22 or p31;q11–12), resulting in monosomy of the distal segment of the short arm and trisomy of the long arm. In two cells, single minutes were found; this chromosomal aberration has been previously described in a case of metastatic neuroblastoma.

Two new cases of primary peripheral neuroepithelioma of soft tissue with translocation t(11;22)(q24;q12)

A direct cytogenetic analysis was performed on tumor samples obtained from two patients with clinical and histopathologic diagnosis of primary peripheral neuroepithelioma. Both tumors presented the translocation t(11;22)(q24;q12). These results confirm those previously obtained by other authors and suggest a common histogenetic origin for this tumor with Ewing's sarcoma and Askin's tumor, in which the same translocation has been described.

Admixture of maternal metaphases in first trimester direct chromosome preparations?

The autoradiographic labelling of different cell types in chorionic villi and decidual tissue was investigated after [3H]-thymidine incorporation in vitro. Although the extent of labelling was found to be lower in decidual than in villus tissue the possibility that direct chromosome preparations may contain maternal metaphases should be considered. The need for careful selection of villi for direct cytogenetic analysis was stressed.

Trypanosomes of subgenus trypanozoon are diploid for housekeeping genes

The ploidy of trypanosomes has until now remained undetermined, although isoenzyme studies and direct measurements of DNA content and complexity suggest diploidy. Direct cytogenetic analysis is not possible, because the chromosomes do not condense at any stage of the cell cycle. We now present evidence from analysis of restriction site polymorphisms in and around three glycolytic enzyme genes (phosphoglycerate kinase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase) and the tubulin gene cluster, that trypanosomes of subgenus Trypanozoon are diploid for these housekeeping genes. This result is still compatible with the single copy nature of variant surface glycoprotein (VSG) genes in Trypanozoon, if different VSG genes are present in corresponding positions on paired chromosomes. Using pulse field gradient gel electrophoresis, we show that the genes for the three glycolytic enzymes are all located in very large DNA molecules, but the gene for triosephosphate isomerase is in another fraction from the genes for the other two enzymes. Since all three enzymes are located in glycosomes, which are trypanosome microbodies, the genes for glycosomal enzymes are not all clustered in one chromosomal segment of the trypanosome genome.

Chorionic villi sampling: Clinical experience, immediate complications, and patient attitudes

To develop chorionic villi sampling as a procedure for prenatal diagnosis, a pilot study was undertaken to perfect the obstetric and laboratory techniques, to evaluate our success with the procedure in continuing pregnancies, and to assess the attitudes of potential users of the procedure. Women about to have elective first-trimester abortions for nongenetic reasons were enrolled in the first phase of the study. Of the patients with a positive pregnancy test, 12.4% were found to have a nonviable pregnancy on ultrasound examination. Samples adequate for cytogenetic analysis were obtained in 130 of the 155 remaining cases, and the success rate was 93% in the 100 most recent cases. Direct cytogenetic analysis was undertaken in those cases successfully sampled, and karyotypes could be prepared in 97%. Immediate complications occurred in 5% of the pregnancies. Eight women at risk of bearing a child with a genetic defect had diagnostic chorionic villi sampling. Cytogenetic analysis was performed successfully on all of them. One had an induced abortion following the procedure because of the fetal diagnosis (a male with a 50% risk of Duchenne's muscular dystrophy). The other pregnancies are continuing uneventfully at 22 to 35 weeks' gestation. Finally, from preliminary analysis of our survey of potential users it appears that women 35 years old or over would prefer chorionic villi sampling to amniocentesis if the risks of the sampling were known to be low.