OBJECTIVETo explore the heterogeneous subclones in acute myeloid leukemia (AML) with t(8;21) by quantitative multicolor- fluorescence in situ hybridization (QM-FISH), and to figure out whether there is putative ancestral relationship among different subclones.

METHODSBacterial artificial chromosomes (BAC) clones that contain the targeted genes including AML1, ETO, WT1, p27 and c-kit were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP or dCTP to targeted genes by nick translation. Bone marrow mononuclear cells from t (8;21) AML patients are dropped on to the wet surface of glass slides after hypotonic treatment and fixation. After hybridization, the fluorescence signals were captured by Zeiss fluorescence microscope. The copy number of AML1, ETO, WT1, p27, c- kit and the AML1-ETO fusion gene in AML1-ETO positive cells was counted. The cells with same signals were defined as a subclone. Various subclones were recorded and their proportions were calculated, and their evolutionary relationship was deduced. The subclones in matched primary and relapsed samples were compared, the evolution of dominant clones were figured out and the genomic abnormality that is associated with relapse and drug resistance were speculated.

RESULTSIn this study, 36 primary AML with t(8;21) cases and 1 relapsed case paired with the primary case were detected. In these 36 primary cases, 4 cases (11.1%) acquired additional AML1-ETO fusion signal, 3(8.3%) had additional AML1 signal, 4(11.1%) had additional ETO signal, 20(55.6%) had additional WT1 signal, 15(41.7%) had additional p27 signal and 14(38.9%) had additional c-kit signal. In addition, 10(27.8%) displayed AML1 signal deletion, and such an aberration represents statistic significance in male patients. It seems that male patients usually accompany AML1 signal deletion. Of 36 cases, 28(77.8 %) harbored at least 2 subclones (ranged from 2 to 10). According to the genetic signature of subclones, we can assemble a putative ancestral tree, and the genetic architecture is linear or branching. In particular, the clonal architecture of the relapsed sample exhibited significant clonal evolution compared to its paired sample at diagnosis, including proportion changes in dominant clone, subclone disappearance and appearance of new dominant clones.

CONCLUSIONGenomic abnormality is very diverse in t(8;21) AML. Subclones have linear or complex branching evolutionary histories, and clonal architecture is dynamic.

Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Male ; Translocation, Genetic

OBJECTIVETo develop a rapid and reliable technique for the detection of Down's syndrome.

METHODSThe peripheral blood samples were collected from twenty-five Down's syndrome patients and fifty normal individuals. Four polymorphic loci on chromosomes 21, 1, 19 were amplified by real-time fluorescence quantitative PCR, and then four pairs of deltaCt values were analytically compared between the two groups.

RESULTSThe deltaCt values of Down's syndrome patients were significantly lower than those of normal individuals, and the reference ranges for clinical application were primarily established. The difference between the two groups was highly significant (P < 0.001), and the reference ranges between the two groups were not overlapped. Real-time quantitative PCR technique can effectively differentiates Down's syndrome samples from the normal fetuses; furthermore, the results were consistent with those of the karyotype analysis.

CONCLUSIONReal-time quantitative PCR is a fast and reliable method that may provide a new approach for rapid detection of Down's syndrome.

Chromosomes, Human, Pair 1 ; genetics ; Chromosomes, Human, Pair 19 ; genetics ; Chromosomes, Human, Pair 21 ; genetics ; Down Syndrome ; diagnosis ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; Reproducibility of Results

This study was purposed to investigate the acute myeloid leukemia with complex karyotype t(2;21;8)(p12;q22;q22) (AML-M(2)) by using morphologic, immunologic, cytogenetic and molecular biologic classification technique (MICM) and to analyze the MICM characteristics of AML-M(2) and their diagnostic significance. The FAB typing of bone marrow cells (BMCs) was performed by Wright-Giemsa staining and histochemical staining of BM smears; the immunophenotype of leukemic cells was detected by flow cytometry; the karyotypes of chromosome samples prepared by short-term (48 hours) conventional culture of fresh BMCs were analyzed by RHG banding technique; the FISH signaling in mitotic metaphase was determined by dual color and dual fusion AML/ETO probe and chromosome painting probe, and was compared with results of conventional cytogenetic assay; the AML/ETO fusion transcripts were detected by nested RT-PCR. The results indicated that the bone marrow smears of case 1 showed extremely hyperplasia with myeloblasts in which a ratio of eosinophilic granulocytes and monocytes increased. Case 2 accorded with AML-M(2b) in which abnormal increase of myelocytes mainly appeared. The complex karyotype t(2;21;8)(p12;q22;q22) was detected by cytogenetic analysis combined with FISH in both two cases and AML1/ETO fusion transcripts were found by RT-PCR as well. The immunophenotype assay showed high co-expression of CD34 and HLA-DR accompanied with CD19 and CD56 expressions. It is concluded that application of MICM has an important significance for correct diagnostic typing of AML-M2 with complex karyotype variant of t(8; 21)(p12;q22;q22).
Adult ; Chromosomes, Human, Pair 2 ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Female ; Humans ; Immunophenotyping ; Karyotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; immunology ; Male ; Middle Aged

OBJECTIVETo evaluate the feasibility of using fluorescence in situ hybridization(FISH) for the detection of a few common chromosome aneuploidies on interphase nuclei of uncultured amniotic fluid cells.

METHODSAmniotic fluid samples were taken from 55 women at 16-32 weeks of pregnancy; interphase FISH was performed for diagnosing Down syndrome and aneuploidies of other four chromosomes 13, 18, X and Y. Then the karyotypes from standard cytogenetic analysis after percutaneous umbilical blood sampling(PUBS) were compared to the FISH results.

RESULTSEach of the 55 uncultured amniotic fluid samples tested with FISH was enumerated 200 nuclei. Fifty-three samples were normal. Two samples were found to have trisomy 21(one is a case of standard trisomy 21 with three signals in all 200 nuclei, the other is a mosaic trisomy 21).

CONCLUSIONInterphase FISH analysis of uncultured amniotic fluid cells is a rapid, accurate and very sensitive method. It could be used in the prenatal cytogenetic laboratory.

Adult ; Amniocentesis ; Amniotic Fluid ; cytology ; Aneuploidy ; Chromosomes, Human, Pair 13 ; Chromosomes, Human, Pair 18 ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, X ; Chromosomes, Human, Y ; Down Syndrome ; diagnosis ; genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Pregnancy ; Prenatal Diagnosis ; methods ; Trisomy

The t(8;21)(q22;q22) is one of the most frequent structural chromosomal anomaly found in AML, occurring in about 5% of all AML and in 10% of AML with maturation (M2). And approximately 3.4% of AML with t(8;21)(q22;q22) occurs as a complex chromosomal abnormality and occasionally shows discrepancy between cytogenetic and molecular genetic analyses. We report a case of 42 yr old male patient that revealed morphological characteristics of AML-M2 and karyotypic abnormality of 45,X,-Y,t(8;17)(q22;p13) without visible involvement of chromosome 21 by conventional cytogenetic study with masked t(8;21) identified by FISH using RUNX1/RUNX1T1 probes. FISH confirmed nuc ish (RUNX1T1x3),(RUNX1x3), (RUNX1T1 con RUNX1x1). According to the results of conventional cytogenetic and FISH analyses, the karyotype was revised to 45,X,-Y,t(8;17;21)(q22;p13;q22).
Chromosome Aberrations ; Chromosomes, Human, Pair 21 ; Cytogenetics ; Humans ; Karyotype ; Leukemia, Myeloid, Acute ; Male ; Masks ; Molecular Biology

OBJECTIVE: To investigate the clinical characteristics of myelodysplastic syndrome (MDS) patients with chromosome 21 karyotype abnormality. METHODS: The clinical data of 155 patients with MDS were retrospectively analyzed, the clinical characteristics, survival and factors affecting prognosis of chromosome 21 karyotype abnormality patients were analyzed. RESULTS: Among 155 MDS patients, 4 were 5q- syndrome, 41 were MDS-EB-I, 35 were MDS-EB-II, 27 were MDS-SLD, 46 were MDS-MLD, 1 was MDS-RS-SLD, and 1 was MDS-U. The median follow-up time was 11.0(0.1-120.9) months. Among 155 MDS patients, 13 (9.0%) showed chromosome 21 abnormalities. Among the 13 patients with chromosome 21 karyotype abnormalities, there were 5 cases with simple +21 karyotype, 1 case with del (21q12), 1 case with +8, +21, 1 case with i(21q), 1 case with 20q-, +21, and 4 cases with complex karyotype involving chromosome 21; including 2 cases of MDS-SLD, 4 cases of MDS-MLD, 5 cases of MDS-EB-I and 2 cases of MDS-EB-II. The median survival time of the patients was 3.1 (0.1-6.7) months. CONCLUSION Chromosome 21 karyotype abnormality is rare in MDS, and the prognosis is worse than the patients without chromosome 21 abnormalities.
Chromosomes, Human, Pair 21 ; Humans ; Karyotype ; Karyotyping ; Myelodysplastic Syndromes/genetics* ; Retrospective Studies

AIMTo analyze the relationship between autosomal aberrations and testicular dysgenesis or spermatogenic arrest in Chinese patients and to map the corresponding regions on each autosome in regard to the recorded aberrations accompanying these distubances.

METHODSOne hundred and nineteen cases of aberrant karyotypes with testicular dysgenesis, azoospermia or oligozoospermia reported in five Chinese journals and one monograph were analyzed. For each autosome, the type and frequency of chromosomal aberrations were counted and the regions corresponding to the disturbances were mapped out.

RESULTSChromosomes 13, 14, 9, 21 exhibited a high frequency of aberration and bands 14q11 and 13p11 were the two regions showing the highest linkage to testicular dysgenesis or infertility. The frequency of chromosomal aberrations was higher in bands 9p11 and 22q than in others.

CONCLUSIONAutosomes 13, 14, 9 and 21 in the order of importance play a critical role in testicular development and spermatogenesis and other autosomes may also contribute; the following regions, 14q11, 13p11,9p11, and 22q, are of high significance.

Asian Continental Ancestry Group ; Chromosome Aberrations ; Chromosomes, Human, Pair 13 ; Chromosomes, Human, Pair 14 ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 9 ; Gonadal Dysgenesis ; genetics ; Humans ; Infertility, Male ; genetics ; Karyotyping ; Male ; Oligospermia ; genetics ; Testis ; abnormalities

OBJECTIVETo investigate molecular genetic alterations associated with primary and corresponding recurrent glioblastoma multiforme(GBM) and to identify which chromosomal regions of the whole genome may be involved in the recurrence of primary GBM.

METHODSA high-resolution allelotyping study of one patient's primary GBM and corresponding recurrent GBM was performed by PCR-based loss of heterozygosity(LOH) analysis with the use of 382 fluorescent dye-labeled polymorphic microsatellite markers covering all 22 autosomes. The mean genetic distance between two flanking markers is 10 cM.

RESULTSLOH at locus D9S157 on 9p21 and at loci D10S537, D10S185, D10S192, D10S597, D10S587, D10S217 on 10q21.3-26.3 was observed in the primary GBM. As for corresponding recurrent tumor, LOH was observed not only in expanded regions on 9p21 and 10q21.3-26.3 but also on multiple other chromosomal arms, including 1q, 7p,7q, 21q, 20p, 20q, 10p, 19p, 19q.

CONCLUSIONChromosome 9p and 10q may be involved in the development of this GBM. Although histopathological diagnoses of the primary and corresponding recurrent tumor are identical, the recurrence of GBM is characterized by an increased involvement of molecular genetic abnormalities and may be accompanied by inactivation of more tumor suppressor genes.

Adult ; Alleles ; Chromosome Mapping ; methods ; Chromosomes, Human, Pair 1 ; genetics ; Chromosomes, Human, Pair 10 ; genetics ; Chromosomes, Human, Pair 19 ; genetics ; Chromosomes, Human, Pair 20 ; genetics ; Chromosomes, Human, Pair 21 ; genetics ; Chromosomes, Human, Pair 7 ; genetics ; Chromosomes, Human, Pair 9 ; genetics ; DNA ; genetics ; Female ; Glioblastoma ; genetics ; pathology ; surgery ; Humans ; Loss of Heterozygosity ; Microsatellite Repeats ; Neoplasm Recurrence, Local

Holoprosencephaly of unknown definite causes, has been associated with several chromosome abnormalities involving the autosomes and the sex chromosomes. The most commonly reported associations include dup(3p), del(7q), deletions of chromosome 13, trisomy 13, trisomy 18, and triploidy. In previously reported cases in Korea, none were associated with chromosome 21 anomalies. In conclusion, we reported the first case of holoprosencephaly(semilobar type) associated with pure monosomy 21. We experienced a semilobar type holoprosencephaly with monosomy 21 in a neonate who had multiple congenital anomalies, including an abnormal face, a small thorax with widely spaced hypoplastic nipples and nail hypoplasia, lung hypoplasia with severe scoliosis and cardiac abnormalities. Chromosomal analysis revealed a 45, XY, -21.
Chromosome Aberrations ; Chromosomes, Human, Pair 13 ; Chromosomes, Human, Pair 21 ; Holoprosencephaly ; Humans ; Infant, Newborn ; Korea ; Lung ; Monosomy ; Nipples ; Scoliosis ; Sex Chromosomes ; Thorax ; Triploidy ; Trisomy

OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (NRBCs) could be distinguished from maternal cells in peripheral blood using an erythroblast scoring system. Presumptive fetal NRBCs were further analyzed through the use of fluorescent PCR amplification with polymorphic STR markers to prove fetal origin. METHODS: NRBCs were isolated by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection from peripheral blood of seven women who had undergone termination of pregnancy because of fetal trisomy 21 (n=4), 18 (n=1), and 13 (n=2). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR amplification of chromosome 21 short tandem repeat (STR) markers (D21S1411, D21S11) and chromosome 18 STR markers (D18S535). RESULTS: In all cases candidate fetal NRBCs were accurately identified based on erythroblast scoring system and confirmed to be fetal in origin based on the presence of shared and non-shared polymorphic DNA alleles when compared to DNA isolated from maternal cells. Also in five cases aneuploid fetal cells in maternal blood were identified through the use of fluorescent PCR amplification with polymorphic STR markers. CONCLUSION: We were able to distinguish fetal NRBCs from maternal cells and prove fetal origin independent of gender. These results suggest that this novel combined approach to fetal cell isolation through using an erythroblast scoring system and genetic analysis by STR analysis is a promising method for noninvasive prenatal diagnostic applications.
Alleles ; Aneuploidy* ; Cell Separation ; Chromosomes, Human, Pair 18 ; Chromosomes, Human, Pair 21 ; DNA ; Down Syndrome ; Erythroblasts* ; Erythrocytes ; Female ; Humans ; Microsatellite Repeats* ; Polymerase Chain Reaction ; Pregnancy