Comparison of Capillary Electrophoresis with Cellulose Acetate Electrophoresis for the Screening of Hemoglobinopathies / 대한진단검사의학회지

BACKGROUND: beta-thalassemia is primarily found in individuals of Mediterranean and Southeast Asian ancestry. With rapid growth in the Southeast Asian segments of the Korean population, the geographic distribution of hemoglobinopathies is expected to become significantly different from what it is today. In this study, Hb fractions were measured in patients with hypochromic microcytosis to detect thalassemia and Hb variants. To evaluate the feasibility of replacing cellulose acetate electrophoresis (CA) with capillary electrophoresis (CE) in a clinical laboratory, both techniques were performed and the outcomes were compared. METHODS: To evaluate hemoglobinopathies, complete blood cell counts (CBC), CA, and CE were carried out on samples from healthy and microcytic hypochromic groups. The microcytic hypochromic group consisted of 103 patients whose mean corpuscular volume (MCV) was less than 75 fL and mean corpuscular hemoglobin (MCH) was less than 24 pg. Quantitative analysis of Hb fractions was performed on 143 whole blood samples. RESULTS: There was a good correlation for measurements of HbA (r=0.9370, P<0.0001), HbA2 (r=0.8973 P<0.0001), and HbF (r= 0.8010, P=0.0304) between the two methods. In the microcytic hypochromic group, there were 29 cases (28.2%) with decreased HbA2, 2 cases (1.9%) with increased HbA2, 3 cases (2.9%) with increased HbF, and 2 cases (1.9%) with increased HbA2 and HbF. CONCLUSIONS: CE is comparable to CA for reliable measurement of Hb fractions. It is suitable for screening of hemoglobinopathies in many clinical laboratories.

A Rare Case of Acute Lymphoblastic Leukemia with t(12;17)(p13;q21) / 대한진단검사의학회지

Patients with ALL rarely present with t(12;17)(p13;q21) as the primary clonal abnormality; this abnormality is associated with the expression of myeloid antigens. In this study, we have reported presumably the first case of this chromosomal abnormality in Korea, thereby facilitating the delineation of a distinct subtype of ALL. A 57-yr-old woman was referred to our hospital because of pancytopenia. Peripheral blood examination showed 55% blasts. The bone marrow was markedly hypercellular, and about 82.4% of all nucleated cells were blasts. The results of immunophenotyping and cytochemical staining suggested early precursor B-ALL. Cytogenetic analysis of the bone marrow cells showed a complex karyotype, including a reciprocal translocation between the short arm of chromosome 12 and the long arm of chromosome 17, t(12;17)(p13;q21). Data from array comparative genomic hybridization were almost consistent with the cytogenetic findings.

Relationship between the Levels of Holotranscobalamin and Vitamin B12 / 대한진단검사의학회지

To date, the determination of serum vitamin B12 levels has been the most common laboratory test for the assessment of vitamin B12 status; however, the diagnostic accuracy of this test is low. To obtain a more sensitive marker, a new test to measure holotranscobalamin (holoTC) levels has been introduced. In this study, we assessed 45 patients for whom a vitamin B12 test had been requested and 139 anemic patients. We investigated the associations between the levels of homocysteine (Hcy) and those of holoTC, serum vitamin B12, and folate and assessed the diagnostic value of holoTC levels as a marker for vitamin B12 deficiency. We also determined the precision of the AxSYM holoTC assay by calculating the coefficient of variance (CV). The within-run and between-run precision values were excellent, as all CV values were less than 3.5%. The holoTC levels were low (12 micromol/L) indicated vitamin B12 deficiency. Thus, the holoTC levels were more sensitive than the serum vitamin B12 levels for indicating vitamin B12 status. If the serum vitamin B12 level is 151-300 pmol/L, the levels of holoTC alone or in combination with serum vitamin B12 levels are likely to be more useful markers than serum vitamin B12 levels alone.

Cytogenetic Characterizations of Central Nervous System Tumors: The First Comprehensive Report from a Single Institution in Korea

The World Health Organization (WHO) classification of central nervous system (CNS) tumors incorporates morphology, cytogenetics, molecular genetics, and immunologic markers. Despite the relatively large number of CNS tumors with clonal chromosome abnormalities, only few studies have investigated cytogenetic abnormalities for CNS tumors in Korea. Thus, we investigated 119 CNS tumors by conventional G-banded karyotypes to characterize patterns of chromosomal abnormalities involving various CNS tumors, and 92.4% of them were cultured and karyotyped successfully. Totally, 51.8% of karyotypable CNS tumors showed abnormal cytogenetic results, including neuroepithelial tumors (75.0%), meningeal tumors (71.1%), pituitary adenomas (4.2%), schwannomas (44.4%), and metastatic tumors (100.0%). Glioblastomas had hyperdiploid, complex karyotypes, mainly involving chromosomes Y, 1, 2, 6, 7, 10, 12, 13, and 14. Monosomy 22 was observed in 56.4% of meningiomas. There was a significant increase in the frequencies of karyotypic complexity according to the increase of WHO grade between grades I and II (P=0.0422) or IV (P=0.0101). Abnormal karyotypes were more complex at high-grade tumors, suggesting that the karyotype reflects the biologic nature of the tumor. More detailed cytogenetic and molecular characterizations of CNS tumors contribute to better diagnostic criteria and deeper insights of tumorigenesis, eventually resulting in development of novel therapeutic strategies.

Cytogenetic Characterizations of Central Nervous System Tumors: The First Comprehensive Report from a Single Institution in Korea

The World Health Organization (WHO) classification of central nervous system (CNS) tumors incorporates morphology, cytogenetics, molecular genetics, and immunologic markers. Despite the relatively large number of CNS tumors with clonal chromosome abnormalities, only few studies have investigated cytogenetic abnormalities for CNS tumors in Korea. Thus, we investigated 119 CNS tumors by conventional G-banded karyotypes to characterize patterns of chromosomal abnormalities involving various CNS tumors, and 92.4% of them were cultured and karyotyped successfully. Totally, 51.8% of karyotypable CNS tumors showed abnormal cytogenetic results, including neuroepithelial tumors (75.0%), meningeal tumors (71.1%), pituitary adenomas (4.2%), schwannomas (44.4%), and metastatic tumors (100.0%). Glioblastomas had hyperdiploid, complex karyotypes, mainly involving chromosomes Y, 1, 2, 6, 7, 10, 12, 13, and 14. Monosomy 22 was observed in 56.4% of meningiomas. There was a significant increase in the frequencies of karyotypic complexity according to the increase of WHO grade between grades I and II (P=0.0422) or IV (P=0.0101). Abnormal karyotypes were more complex at high-grade tumors, suggesting that the karyotype reflects the biologic nature of the tumor. More detailed cytogenetic and molecular characterizations of CNS tumors contribute to better diagnostic criteria and deeper insights of tumorigenesis, eventually resulting in development of novel therapeutic strategies.

Expression of dendritic cell markers on cultured neutrophils and its modulation by anti-apoptotic and pro-apoptotic compounds

Neutrophils are also known to acquire the characteristics of dendritic cells (DCs) under the appropriate conditions. In this study, neutrophils were cultivated in vitro in the presence or absence of compounds modulating their survival in an attempt to characterize the expression profile of the DC markers. Higher MHC-II, CD80, CD86, CD83, and CD40 expression levels were detected on the surface of the cultured neutrophils for 24 h than on the freshly isolated cells. The annexin V-positive cells showed a higher expression level of the DC markers than the annexin V-negative cells. The population of neutrophils double stained with annexin V and the DC markers increased after being incubated with agonistic anti-Fas Ab. LPS, the anti-apoptotic compound, decreased the CD86 and MHC-II expression levels but 50-60% of the DC marker-positive cells were detected in the annexin V-positive cells. In contrast, CD80, CD86, CD83, and HLA-DR mRNA levels increased in the GM-CSF-treated neutrophils but not in the anti-Fas Ab-treated neutrophils. T cell proliferation was inhibited by co-culturing them with anti-Fas Ab- or LPS-treated neutrophils at a high neutrophil:T cell ratio. However, the superantigen-mediated T cell proliferation was increased by the LPS-treated neutrophils but decreased by the anti-Fas Ab-treated neutrophils. There was a lower level of interferon-gamma production in the T cells co-cultured with anti-Fas Ab-treated neutrophils than with the LPS-treated neutrophils. This suggests that apoptotic neutrophils express DC markers on their surface and the differential expression of DC markers might have a detrimental effect on the immune reaction.

Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif

Checkpoint kinase 1 (Chk1) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chk1 and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1alpha were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1alpha were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.

Netrin Inhibits Regenerative Axon Growth of Adult Dorsal Root Ganglion Neurons in Vitro

Netrin is a neuronal guidance molecule implicated in the development of spinal commissural neurons and cortical neurons. The attractive function of netrin requires the receptor, Deleted in Colorectal Cancer (DCC), while the receptor Unc5h is involved in the repulsive action of netrin during embryonic development. Although the expression of netrin and its receptor has been demonstrated in the adult nervous system, the function of netrin in adult neurons has not yet been elucidated. Here, we show that netrin treatment inhibited neurite outgrowth of adult dorsal root ganglion (DRG) neurons in explant and dissociated cultures. In addition, unc5h1-3 mRNAs, but not the dcc mRNA, are abundantly expressed in the adult DRG. An in situ hybridization study demonstrated that unc5h mRNAs were expressed in DRG neurons. This finding indicates that netrin/Unc5h signaling may play a role in the neurite outgrowth of adult DRG neurons and that netrin may be involved in the regulation of peripheral nerve regeneration.

Detection of single nucleotide insertion of BCR/ABL region in imatinib-resistant human myelogenous leukemia SR-1 cells

Imatinib mesylate is a selective Bcr/Abl kinase inhibitor and an effective anticancer agent for Bcr/Abl-positive chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Mutations within the BCR/ABL kinase domain are the most commonly identified mechanism associated with relapse. To overcome the imatinib resistance in CML, many investigators have tried to clarify molecular mechanism for imatinib resistance in cells of patients who failed to respond to imatinib. Our aim was to invesitigate underlying mechanism for imatinib resistance in SR-1 cells, which were derived from a CML patient in blast crisis. We detected the new mutation of BCR/ABL, resulting in premature termination and loss of BCR/ABL fusion protein expression, which might be possible mechanism for the resistance to imatinib in SR-1 cells.

Involvement of mitogen-activated protein kinases and p21Waf1 in hydroxyurea-induced G1 arrest and senescence of McA-RH7777 rat hepatoma cell line

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21Waf1, p27Kip1 and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21Waf1 was increased, while p27Kip1 and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21Waf1 over-expression.

Subject The Effect of Apolipoprotein E Overexpression on Plasma Lipoprotein Profile in Mice Fed on Long-term High Cholesterol Diet

BACKGROUND AND OBJECTIVES: Apolipoprotein E (apoE), a 34-kD plasma glycoapolipoprotein, plays a key role in lipoprotein metabolism by facilitating cellular uptake of remnants of triglyceride-rich chylomicrons and VLDL and may have other important biological functions. Various studies using apoE-knockout mice have elucidated the role of apoE in lipolysis, remnant clearance, and atherogenesis. Despite the growing evidence of the protective role exerted by apoE against atherosclerosis, the direct in vivo effects of the apoE overexpression on lipoprotein metabolism in the presence of endogenous mouse apoE are not yet fully understood. In this study, the technique of adenovirus-mediated gene transfer was employed to investigate the in vivo effect of apoE overexpression on lipid level and lipoprotein profile in mice fed on normal chow or high cholesterol diet. MATERIALS AND METHODS: Recombinant adenovirus (rAd.mApoE) containing mouse apoE cDNA driven by a cytomegalovirus promoter was generated and infused via tail vein in mice fed on normal chow or high cholesterol diet. Recombinant adenoviruses have emerged as the most efficient vectors for transient delivery of functional genes to the mammalian liver. RESULTS: rAd.mApoE in the various mouse tissues one week after injection was expressed mainly in the liver. ApoE overexpression decreased the cholesterol and triglyceride concentration in mice fed on normal chow. In mice fed on high cholesterol diet, apoE overexpression resulted in decrease in triglyceride concentration and increase in cholesterol. VLDL and LDL fraction were decreased, HDL was increased by apoE overexpression in both mice fed on normal chow and high cholesterol diet. CONCLUSION: These data suggest that overexpression of mouse apoE in mice with endogenous apoE may exert antiatherogenic effect by inducing favorable change in the lipoprotein profile, regardless of diet and consequent plasma lipid level. In the future, the studies regarding the effect of human apoE overexpression on the lipid and lipoprotein profile in mice fed on normal chow and high cholesterol diet will be helpful to understand the species differences or similarities in apoE activity.

Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the nonadherent cells were harvested and recultured with erythropoietin in the second phase for another 3-5 days. We examined cellular morphology, and counted the number of benzidine- positive cells and the percentage of glycophorin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, and fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1x10(6)-1.0x10(6)/mL with a purity of 84.0-97.3%. Hb F stain showed total erythroid cells of approximately 0.4x10(4)-9.8x10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides enriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures.

Identification of Tumor Suppressor Gene in Renal Cell Carcinoma using Arbitrarily Primed-Polymease Chain Reaction (AP-PCR) / 대한비뇨기과학회지

No abstract available.

DNA-Protein Interaction of gamma-Globin Gene Promoter by Differentiation Inducers / 대한혈액학회지

BACKGROUND: The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic cells. There are numerous similarities between the erythroid or megakaryocytic lineages. In this study, we examined role of the region -269~-240 of gamma-globin gene promoter in fetal hemoglobin expression during either erythroid or megakaryocytic differentiation. METHODS: K562 cells were cultured and treated with differentiation inducers. Hemoglobin content was scored by benzidine staining, and hemoglobin F was stained by acid elution technique. To determine whether transcription factor binding to the gamma-globin gene promoter is critical to lineage determination, DNA-protein interaction of gamma-globin gene promoter was examined under both uninduced and induced conditions of K562 cells using gel mobility shift assay and southwestern blot analysis. RESULTS: Phorbol 12-myristate 13-acetate (PMA) induced a megakaryocytic differentiation, but suppressed erythroid differentiation. On the other hand, hydroxyurea (HU), hemin, n-butanol, and sodium butyrate (NaB) induced the expression of erythroid phenotypes. Parallel to hemoglobinization, increase in gamma-globin mRNA was observed in HU- and hemin-treated K562 cells. Gel mobility shift assay and southwestern blot analysis revealed that binding of a erythroid-specific protein (p120) to the region -269~-240 of gamma-globin gene promoter occurred with treatment of erythroid differentiation inducers and did not occur with treatment of PMA. CONCLUSION: These results suggest that erythroid differentiation inducers may act via DNA- protein interaction at the gamma-globin gene promoter region to induce erythroid differentiation.

Isolation of Candidate Tumor Suppressor Gene in Renal Cell Carcinoma / 대한임상병리학회지

BACKGROUND: Although renal cell carcinoma (RCC) is the most common malignancy originated from kidney in adults, pathogenesis of RCC remains unknown. The purpose of this work is to find tumor suppressor gene in RCC. METHODS: The arbitrarily primed polymerase chain reaction (AP-PCR) has been used to detect somatic genetic alterations in RCC. DNA fingerprints generated by single arbitrary primers were compared between normal and tumor tissues of the same individuals. AP-PCR bands showing decreased intensities in tumor tissue DNA, relative to normal, have been cloned after reamplification with the same arbitrary primer. We have performed Southern blot hybridization and DNA sequencing. RESULTS: For a given primer, at least 5 differences in band patterns between normal and tumor tissues were observed and band C was deleted in tumor tissues of clear cell type RCC. We found this band was split into 3 bands. Because band C2 was consistantly deleted in tumor tissue, we decided to clone and characterize this fragment. Partial DNA sequences of this fragment showed no homology with other genes by BLAST search. Southern blot analysis showed this fragment was deleted in 2 cases of clear cell type and 1 case of mixed cell type RCC. CONCLUSIONS: These results suggest that fragment C2 might be a candidate for novel tumor suppressor gene and loss of this fragment might be necessary for malignant development to clear cell type RCC. Further characterization of this fragment is expected to give us useful informations about RCC tumorigenesis.

Changes in expression of cell cycle regulators and their hepatic lobular distribution in partial hepatectomy-induced regenerating rat liver

Partial hepatectomy (PH) endorses quiescent hepatocytes to reenter the cell cycle. The regenerating liver returns to its preresection weight after 7 days, following one or two cell division and maintains nearly its original volume after then. We focused on the inhibition of further hepatocyte proliferation, hypothesizing possible involvement of cell cycle upregulators and inhibitors. We studied protein levels in expression of cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs), and their in situ hepatic lobular distributions in partial hepatectomized rat liver. Cyclin E was expressed in the same levels in normal liver and after PH. Expression of cyclin A, not detected in normal liver, increased in following times after PH and reached a maximum at 7 day. CDK2 and 4 showed increased expression toward terminal period. Contradictory findings of cyclin A and these CDKs might play an important role in the inhibition of further cell division, although still unclear. Constitutively expressed CDK6 decreased after 1 day. p18 showed peak expression within 1 day, and p16, p21, p27 and p57 were stronger at terminal periods. During the expected period of their activity, intranuclear translocations were observed in cyclin E, p18 and p16. There was no evidence of regional distribution in hepatic lobular architecture, instead, diffuse in situ expression, corroborating synchronous event, was found.

Calyculin A modulates activation of the NADPH-oxidase in Me2SO-differentiated HL-60 cells

Human promyelocytic leukemia cells (HL-60) have been used as a model system in which to study the effects of protein phosphatase inhibitors on NADPH-oxidase activation. Since O2- is generated by NADPH-oxidase, we examined the effect of calyculin A pretreatment on oxidase activation in response to various agonists. When Me2SO-differentiated HL-60 cells were treated with calyculin A prior to the addition of phorbol 12-myristate 13-acetate (PMA), O2- production was inhibited; however, calyculin A enhanced O2- production by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The decreased O2- production seen with calyculin A pretreatment followed by PMA may be due to diminished translocation of the p47-phox and p67-phox, cytosolic components of the oxidase, and inhibition of arachidonic acid release. Interestingly calyculin A pretreatment followed by either agonist significantly enhanced mitogen-activated-protein kinase (MAPK) activity. The differential effects of pretreatment with calyculin A on subsequent oxidase stimulation elicited by FMLP or PMA provide further evidence for substantial heterogeneity in the activation of the respiratory burst.

Role of Gamma Globin Promoter Region -269~-240 in Hydroxyurea Treated Erythroid Progenitor Cells / 대한임상병리학회지

BACKGROUND: Recently, a great deal of interest has been focused on the use of hydroxyurea and hemin that may augment Hb F levels in patients with hemoglobinopathies and thalassemia, although the molecular mechanism of those chemicals remains unclear. In this study, we examined the effects of hydroxyurea and hemin on human adult peripheral and cord blood erythroid cells grown in a two-phase liquid culture system. METHODS: Four adult peripheral and four cord blood cells were cultured in two-phase liquid culture, and were treated with hydroxyurea or hemin. We counted isolated erythroid cells by acid benzidine and glycophorin A stains. To determine whether transcription factor binding to the promoter is critical, we also examined the promoter region of gamma globin gene both under uninduced and hydroxyurea or hemin induced conditions using gel mobility shift assay and southwestern blot analysis. RESULTS: When added together with erythropoietin, hydroxyurea led to significant increase in the percentage of erythroid cells in cord blood. In contrast, hemin greatly accelerated hemoglobin accumulation in adult erythroid progenitor cells. At -230 and -264 regions of gamma globin gene promoter, different protein binding patterns were observed in uninduced and hydroxyurea or hemin induced conditions between adult and cord blood. CONCLUSIONS: These results suggest that hydroxyurea and hemin may act via alteration in DNA-protein interactions to induce gamma globin gene expression. In addition, we can conclude that different transcription factors may be involved in the gamma globin induction process between the adult and cord blood erythroid cells.

Quantitative Analysis of Cultured Erythroid Progenitors in Liquid System / 대한혈액학회지

BACKGROUND: Hemopoiesis and erythropoiesis have been studied mainly in immortalized cell lines and semisolid medium. But cell lines do not represent normal erythropoiesis, besides, in semisolid medium the cells are immobilized that it is difficult to do additional immunologic, biochemical, and molecular biologic experiments. In the present study we used a two-phase liquid culture system to isolate and quantify erythroid progenitors from peripheral blood and cord blood. METHODS: Peripheral and cord blood were obtained from three healthy donors and three full-term deliveries, respectively. Mononuclear cells were separated by density gradient centrifugation and were cultured in the first phase at a density of 5x106/mL in alpha- minimal essential medium ( -MEM). After 5~7 days of incubation at 37degrees C in an atmosphere of 5% CO2 with extra humidity, the nonadherent cells were harvested and recultured in the original volume of -MEM containing 10ng/mL stem cell factor and 1U/mL erythropoietin (EPO). Cellular morphology was observed by preparing cytocentrifuge slides stained with Wright Giemsa. On days 8, 10, 12, and 16 of the second phase, hemoglobin (Hb)- containing cells were counted on hemocytometer after staining with acid benzidine and glycophorin A-positive erythroid cells were scored by a flow cytometer. RESULTS: Pronormoblasts first started to appear on days 4~5 in the secondary culture. On day 10 basophilic normoblasts could be seen and on days 12~14 orthochromatic normoblasts were present. Both from peripheral and cord blood the maximum number of benzidine and glycophorin A-positive cells were achieved after 10 days and the total erythroid cell yield was approximately 1x106/mL. CONCLUSION: Using two-phase liquid culture, erythroid cell yield reached 1x106/mL both from peripheral and cord blood. In addition, this culture system permits the study of the effect of various culture conditions and components without terminating the culture, therefore it might provide us a useful experimental tool for studying pathogenesis and therapeutic modalities in genetic erythroid disorders as well as erythroid cell development.

Identification of an i(21q) by Using Dinucleotide Repeat Polymorphisms / 대한임상병리학회지

BACKGROUND: Recent DNA polymorphism analysis using numerous DNA markers has been used to determine the parental origin of the extra chromosome 21 in Down syndrome. In this study we used seven dinucleotide repeat polymorphisms on chromosome 21 to characterize a case of rea(21q21q) and to know whether it is consistent with an isochromosome or a true Robertsonian translocation. METHODS: Cytogenetic investigation was done by conventional G banding DNA was extracted from whole blood of a proband and her parents and was amplified by PCR using seven sets of (GT)n repeat dinucleotide markers located on the long arm of chromosome 21 After electrophoresis of the PCR product in polyacrylamide gel and silver staining the parental origin and number of DNA copy were determined by visual comparison of the band intensities within and between individuals. RESULTS: Conventional cytogenetics showed that the proband had a 46.XX.re(21q21q) chromosome pattern. Parental chromosome studies were normal, therefore, the rearrangement was a de novo event. All seven DNA markers showed one or two alleles, demonstrating rea(21q21q) to be an isochromosome. For D21S215 and D21S156 markers both parents were heterozygous and the proband inherited one copy of paternal allele and two copies of maternal allele which both parents did not share. This finding was consistent with a maternally derided isochromosome. CONCLUSION: Use of dinucleotide repeat DNA polymorphisms after PCR amplification will be very useful to detect the parental origin of additional chromosome 21 or rearrangement of chromosome 21 in Down syndrome. Besides employing siltier staining of a PCR product we will be able to avoid using of radioisotopes and apply to clinical laboratory diagnosis.