”TAM”ing of the shrew-challenges in the diagnosis of Neonatal leukemia with Down's syndrome -A case report with literature review

The latest WHO (2017) classification describes the hematological abnormalities of Down's syndrome as a separate entity under 'Myeloid proliferations associated with Down's syndrome'. It includes Transient Abnormal Myelopoiesis and Myeloid leukemia of Down's syndrome. Here we report a case of a 3 days old neonate with Down's syndrome, presenting with a leukemic blood picture. The baby had icterus, fever and hepatosplenomagaly. Peripheral blood showed megakaryoblasts and giant platelets. A diagnosis of transient abnormal myelopoiesis was made by confirming with karyotyping and immunophenotyping. We attempt to address all the diagnostic challenges faced by a clinician and pathologist same, upon encountering such a case,by following an algorithmic approach. The mandatory need for follow up and cytogenetic studies in identifying high risk cases that will become myeloid leukemia of Down's syndrome are stressed. Our case also throws light upon the significance of identification of GATA1 mutation in diagnosing and prognostication of such cases.

Construction,Protein Expression,and Subcellular Localization of Various Kinase Mutant Plasmids of GATA1 / 中国医科大学学报

Objective The GATA1 mutant GATA1 S161A S187A (death type) and GATA1 S161D S187D (activated) eukaryotic expression vectors were constructed using the large primer method,and,to explore their biological function and potential tumor treatment targets,the expression and localization of the fusion protein in cells were confirmed. Methods S161A,S187A,S161D,and S187D mutants were amplified by GFP-GATA1 WT,which served as the template. The recombinant plasmid was cloned into a pEGFP-C1 expression vector and transfected into HEK293 cells by immunoblotting expression of the fusion protein. Results The eukaryotic expression vectors pEGFP-GATA1 S161A S187A and pEGFP-GATA1 S161D S187D were successfully constructed using the high primer PCR method,and expression of the fusion protein was verified. Confocal laser microscopy showed that the fusion protein was mainly located in the nuclei of HEK293 cells. Conclusion A eukaryotic expression vector of a GATA1 mutant was successfully constructed using the large primer method. This work lays the foundation for further studies on the structure and function of the mutant.

A Case of Transient Myeloproliferative Disorder Associated with Clonal Trisomy 21 in a Chromosomally Normal Newborn / 임상소아혈액종양

Transient myeloproliferative disorder (TMD) is similar to congenital leukemia, with leukocytosis, increased peripheral blast cells, and hepatomegaly in the neonatal period. Although TMD occurs only in patients with Down syndrome and trisomy 21 mosaicism, there have been reports of congenital leukemia with trisomy 21 limited to hematopoietic cells showing spontaneous resolution. We identified trisomy 21 in the leukemic cells in a patient with congenital leukemia. As there was no other gene abnormality, we stopped chemotherapy, and the disease resolved spontaneously. We reviewed the cases of clonal trisomy 21 TMD and found that their clinical features were similar to those of TDM associated with Down syndrome. In conclusion, in a phenotypically normal patient with suspected congenital leukemia, it is necessary to confirm the presence of 21 trisomy. If the neonate has only trisomy 21 without other gene abnormalities, intensive chemotherapy may not be required.

GATA1 Expression in BCR/ABL1-negative Myeloproliferative Neoplasms

BACKGROUND: This study aimed to determine GATA1 expression levels to better characterize subgroups in BCR/ABL1-negative myeloproliferative neoplasms (MPNs). METHODS: This study enrolled 49 patients diagnosed as having BCR/ABL1-negative MPN on the basis of the 2016 World Health Organization classification : nine polycythemia vera (PV), 17 essential thrombocythemia (ET), 12 prefibrotic primary myelofibrosis (prePMF), and 11 overt primary myelofibrosis (PMF). Relevant clinical and laboratory data were retrieved from the medical records. The molecular analysis of CALR and MPL mutations and quantification of JAK2 V617F allele burden were performed. GATA1 expression was assessed by an immunohistochemical assay on bone marrow biopsy. GATA1 expression was analyzed serially in 18 patients. RESULTS: GATA1 expression decreased significantly in PMF compared with that in other subtypes, while no statistical difference was identified between ET and prePMF. GATA1 expression did not differ according to the mutation profiles or the allele burden of JAK2 V617F, but it decreased significantly in patients with overt fibrosis or leukemic transformation. CONCLUSIONS: Our results suggest that GATA1 expression is significantly low in PMF and decreases with progressive fibrosis and possibly with leukemic transformation, although our attempt to accurately distinguish between subgroups using GATA1 immunohistochemical approach did not achieve statistical significance. A large patient cohort with long term follow-up is required to evaluate the prognostic value of GATA1 expression.

GATA1 mutations in a cohort of Malaysian children with Down syndrome-associated myeloid disorder

<p><b>INTRODUCTION</b>Children with Down syndrome (DS) are at increased risk of developing distinctive clonal myeloid disorders, including transient abnormal myelopoiesis (TAM) and myeloid leukaemia of DS (ML-DS). TAM connotes a spontaneously resolving congenital myeloproliferative state observed in 10%-20% of DS newborns. Following varying intervals of apparent remission, a proportion of children with TAM progress to develop ML-DS in early childhood. Therefore, TAM and ML-DS represent a biological continuum. Both disorders are characterised by recurring truncating somatic mutations of the GATA1 gene, which are considered key pathogenetic events.</p><p><b>METHODS</b>We herein report, to our knowledge, the first observation on the frequency and nature of GATA1 gene mutations in a cohort of Malaysian children with DS-associated TAM (n = 9) and ML-DS (n = 24) encountered successively over a period of five years at a national referral centre.</p><p><b>RESULTS</b>Of the 29 patients who underwent GATA1 analysis, GATA1 mutations were observed in 15 (51.7%) patients, including 6 (75.0%) out of 8 patients with TAM, and 9 (42.9%) of 21 patients with ML-DS. All identified mutations were located in exon 2 and the majority were sequence-terminating insertions or deletions (66.7%), including several hitherto unreported mutations (12 out of 15).</p><p><b>CONCLUSION</b>The low frequency of GATA1 mutations in ML-DS patients is unusual and potentially indicates distinctive genomic events in our patient cohort.</p>

Expression relevance of GATA-1 and GATA-2 in bone marrow CD71 +cells of high-altitude polycythemia rat model / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the expression relevance of transcription factors GATA-1 and GATA-2 in the bone marrow CD71 +cells of a high-altitude polycythemia (HAPC) rat model.METHODS:Male SD rats (n=48) were randomly divided into normal control group and HAPC model group .HAPC model was established at an altitude of 4 300 m in the natural environment and verified by the morphology and quantity of the bone marrow cells and hematologic parameters detection .A relative change in the trend of bone marrow CD 71 +cell numbers was detected by flow cytometry analysis .The expression of GATA-1 and GATA-2 at mRNA and protein levels in the CD 71 +cells was examined by RT-qPCR and West-ern blot .CD71 +cells were cultured under hypoxic condition and transfected with the optimal interference sequence of GA -TA-1shRNA1 for 96 h.The expression of GATA-1 and GATA-2 at mRNA and protein levels was detected by RT-qPCR and Western blot .RESULTS:The establishment of the animal model with HAPC was successful as the bone marrow smears and the hematologic parameters showed compared with the control .The quantity of the bone marrow CD 71 +cells of HAPC rats were significantly increased and the expression of GATA-1 at mRNA and protein levels in the CD 71 +cells were higher than those of the control .The expression of GATA-2 at mRNA and protein levels was similar to that of the control .The correla-tion analysis showed that the expression of GATA-1 was negatively correlated with that of GATA-2 in the control, while no obvious correlation between them was observed in the HAPC rats .The expression of GATA-1 at mRNA and protein levels in HAPC group was lower than that in control group after interfered by GATA-1 shRNA1 for 96 h, but no obvious diversity of GATA-2 expression between the 2 groups was observed .CONCLUSION:GATA-1 and GATA-2 are abnormally expressed and their negative correlation is destroyed in HAPC , which may be one of the pathogenesis of HAPC .

Effect of simulated microgravity on proliferation and differentiation of the human megakaryocyte cell / 实用医学杂志

Objective To investigate the effect of simulated microgravity on the proliferation and differentiation of the human megakaryocyte cells in vitro. Methods The fourth generation rotating cell culture system (RCCS-4) was used to generate the simulated microgravity environment. The cell viability was assessed by trypan blue staining method. The proliferation of cells was assessed by cell counting method and CCK8 method. The CD41+/CD61+ cells rate and the cells cycle were detected by flow cytometry. The expression levels of thrombopoietin receptor (c-mpl) and transcription factors were detected with RT-PCR. Results After 24, 48, 72 h, culture under simulated microgravity resulted in a significant decrease in the cell number , proliferative activity, cells in the G2/M phase and levels of c-mpl mRNA expression in comparison with that under the normal gravity (P < 0.05). After 48 h and 72 h culture, CD41+/CD61+ cells ratio decreased and RUNX-1 mRNA expression was down-regulated in cells of the group SMG compared with that of the group NG (P < 0.05). Conclusion Microgravity can inhibit the proliferation and differentiation of human megakaryocyte cells in vitro. The mechanism may be that TPO/c-mpl pathway was inhibited by down regulating the expression of c-mpl which transcriptional inhibition lead to.

Effect of resveratrol on proliferation and differentiation in K562 cells / 中国药理学通报

Aim To investigate the effect of resveratrol on proliferation and differentiation in K562 cells. Methods K562 cells were treated with different con-centrations of resveratrol for 6d. The colony number of K562 cells was detected with semi-solid culture assay. Expression of GATA-1 and PU. 1 in K562 cells was re-spectively measured with immunocytochemistry and Western blot. Expression of differentiation related anti-gen, CD11b, CD14 and CD42b, was measured with flowcytometry on K562 cells. Results Resveratrol could significantly decrease the colony number of K562 cells in a dose-dependent manner, and enhance the ex-pression of GATA-1,PU. 1,CD11b, CD14 and CD42b in K562 cells. Conclusion Resveratrol could inhibit the proliferation and induce differentiation of K562 cells via up-regulating the expression of GATA-1 and PU. 1 protein.

Effect of Bu-Shen Yi-Sui Sheng-Xue Method for Medicated Serum of Rats with Aplastic Anemia on Mechanism of Proliferation and Differentiation of Hematopoietic Progenitor Cells / 世界科学技术-中医药现代化

This study was aimed to investigate the effect of Bu-Shen Y i-Sui Sheng-Xue (BSYSSX) method on pro-liferation and differentiation mechanisms of hematopoietic progenitor cells. The rat models were established by 60Co-γrays and cyclophosphamide. Compound Chinese medicine was gavaged to rats of the normal control group, model group, stanozolol group, Yi-Sui Sheng-Xue (YSSX) group, Wen-Shen Sheng-Xue(WSSX) group and Zi-Shen Sheng-Xue (ZSSX) group. Then, serum of rat was prepared. Rat bone marrow cells were incubated with AA rats serum ac-counted for 20% and the number of hematopoietic progenitor cells colony-forming units (CFU) were counted. The level of GATA-1 and PU.1 mRNA in colony cells were detected with RT-PCR. The results showed that compared with the normal control group, the number of bone marrow cells, CFU-E, BFU-E, CFU-GM, as well as the expres-sion of GATA-1 and PU.1 mRNA in the model group decreased significantly (P< 0.01). Compared with the model group, the number of bone marrow cells, CFU-E, BFU-E, CFU-GM of each treatment group were significantly in-creased (P< 0.01). CFU-E and BFU-E of the ZSSX group were better than the YSSX group (P < 0.01). CFU-GM of the ZSSX group was better than the YSSX group and the WSSX group. The expression of GATA-1 and PU.1 mR-NA in each treatment group were significantly higher than the model group (P< 0.01). The expression of GATA-1 mRNA in the ZSSX group was better than the YSSZ group and WSSX group (P< 0.05). The expression of PU.1 mR-NA in the ZSSX group was higher than the YSSX group and WSSX group. It was concluded that BSYSSX method may increase the expression of GATA-1 and PU.1 mRNA in order to promote the proliferation and differentiation of bone marrow hematopoietic progenitor cells. The ZSSX method was better than the YSSX method and WSSX method.

Caracterización de mutaciones en el gen GATA-1 en pacientes con Síndrome de Down y diagnóstico de mielopoyesis anormal transitoria o leucemia megacarioblástica aguda / Mutation characterization in the GATA-1 gene in patients with Down's Syndrome diagnosed with transient abnormal myelopoiesis or acute megakaryoblastic leukemia

Los pacientes con síndrome de Down tienen un riesgo más elevado de presentar leucemia megacarioblástica aguda (LMCA). Un 10% de los recién nacidos con ese síndrome presentan un cuadro de mielopoyesis anormal transitoria (MAT), indistinguible de la LMCA, que en general remite espontáneamente. En ambos grupos de pacientes se describió una alta incidencia de mutaciones en el gen GATA-1. Se analizaron 14 muestras de ADN de médula ósea (10 MAT/4 LMCA) correspondientes a 13 pacientes con Síndrome de Down mediante PCR y secuenciación, para describir la frecuencia y las características de las mutaciones en el gen GATA-1 en la población estudiada y sus consecuencias a nivel proteico. Se detectaron mutaciones en 10 de 10 MAT y en 3 de 4 LMCA, que a nivel proteico originarían un codón de terminación prematuro (n= 5), alteraciones en el sitio de corte y empalme (splicing) (n= 6) o cambio de secuencia (n= 3). Se confrmó la alta frecuencia de mutaciones en el gen GATA-1 en recién nacidos con Síndrome de Down y MAT o LMCA.

Patients with Down's Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Down's Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Down's Syndrome and MAT or AML.

GATA1-positive Acute Megakaryoblastic Leukemia in a 6.9-year-old Patient with Down Syndrome: What is the Prognosis? / 임상소아혈액종양

We describe a very rare case of 6.9-year-old boy with Down syndrome (DS) and a prior history of transient myeloproliferative disorder. He was diagnosed with acute megakaryoblastic leukemia and found to have a novel GATA1 gene mutation, as well as a complex karyotype without recurrent acute myeloid leukemia (AML) aberrations. The patient achieved an early bone marrow response to chemotherapy. However, relapse occurred during treatment, 9 months after the initial diagnosis. Although GATA1 mutations are closely associated with DS-AML, we speculate that factors other than the presence of the GATA1 mutation can affect the overall outcome in older pediatric patients.

GATA1-positive Acute Megakaryoblastic Leukemia in a 6.9-year-old Patient with Down Syndrome: What is the Prognosis? / 임상소아혈액종양

We describe a very rare case of 6.9-year-old boy with Down syndrome (DS) and a prior history of transient myeloproliferative disorder. He was diagnosed with acute megakaryoblastic leukemia and found to have a novel GATA1 gene mutation, as well as a complex karyotype without recurrent acute myeloid leukemia (AML) aberrations. The patient achieved an early bone marrow response to chemotherapy. However, relapse occurred during treatment, 9 months after the initial diagnosis. Although GATA1 mutations are closely associated with DS-AML, we speculate that factors other than the presence of the GATA1 mutation can affect the overall outcome in older pediatric patients.

Eosinophil Development, Regulation of Eosinophil-Specific Genes, and Role of Eosinophils in the Pathogenesis of Asthma

Eosinophils arise from hematopoietic CD34+ stem cells in the bone marrow. They acquire IL-5Ralpha on their surface at a very early stage during eosinophilopoiesis, and differentiate under the strong influence of interleukin (IL)-5. They then exit to the bloodstream, and enter the lung upon exposure to airway inflammatory signals, including eotaxins. In inflamed tissues, eosinophils act as key mediators of terminal effector functions and innate immunity and in linking to adaptive immune responses. Transcription factors GATA-1, CCAAT/enhancer-binding protein, and PU.1 play instructive roles in eosinophil specification from multipotent stem cells through a network of cooperative and antagonistic interactions. Not surprisingly, the interplay of these transcription factors is instrumental in forming the regulatory circuit of expression of eosinophil-specific genes, encoding eosinophil major basic protein and neurotoxin, CC chemokine receptor 3 eotaxin receptor, and IL-5 receptor alpha. Interestingly, a common feature is that the critical cis-acting elements for these transcription factors are clustered in exon 1 and intron 1 of these genes rather than their promoters. Elucidation of the mechanism of eosinophil development and activation may lead to selective elimination of eosinophils in animals and human subjects. Furthermore, availability of a range of genetically modified mice lacking or overproducing eosinophil-specific genes will facilitate evaluation of the roles of eosinophils in the pathogenesis of asthma. This review summarizes eosinophil biology, focusing on development and regulation of eosinophil-specific genes, with a heavy emphasis on the causative link between eosinophils and pathological development of asthma using genetically modified mice as models of asthma.

CpG methylation at GATA elements in the regulatory region of CCR3 positively correlates with CCR3 transcription

DNA methylation may regulate gene expression by restricting the access of transcription factors. We have previously demonstrated that GATA-1 regulates the transcription of the CCR3 gene by dynamically interacting with both positively and negatively acting GATA elements of high affinity binding in the proximal promoter region including exon 1. Exon 1 has three CpG sites, two of which are positioned at the negatively acting GATA elements. We hypothesized that the methylation of these two CpGs sites might preclude GATA-1 binding to the negatively acting GATA elements and, as a result, increase the availability of GATA-1 to the positively acting GATA element, thereby contributing to an increase in GATA-1-mediated transcription of the gene. To this end, we determined the methylation of the three CpG sites by bisulfate pyrosequencing in peripheral blood eosinophils, cord blood (CB)-derived eosinophils, PBMCs, and cell lines that vary in CCR3 mRNA expression. Our results demonstrated that methylation of CpG sites at the negatively acting GATA elements severely reduced GATA-1 binding and augmented transcription activity in vitro. In agreement, methylation of these CpG sites positively correlated with CCR3 mRNA expression in the primary cells and cell lines examined. Interestingly, methylation patterns of these three CpG sites in CB-derived eosinophils mostly resembled those in peripheral blood eosinophils. These results suggest that methylation of CpG sites at the GATA elements in the regulatory regions fine-tunes CCR3 transcription.

NDRG2 Promotes GATA-1 Expression through Regulation of the JAK2/STAT Pathway in PMA-stimulated U937 Cells

BACKGROUND: N-myc downstream-regulated gene 2 (NDRG2), a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMA-stimulated U937 cells. METHODS: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression. RESULTS: NDRG2 overexpression in U937 cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1 were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells. CONCLUSION: The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.

GATA1 Mutation in Transient Myeloproliferative Disorder of Down Syndrome / 대한혈액학회지

Children with Down syndrome (DS) have a higher risk of developing leukemia than do healthy children, and they especially have a higher risk for developing transient myeloproliferative disorder (TMD) or acute megakaryocytic leukemia (AMKL). In recent studies, it has been reported that most of these patients have acquired mutation of the GATA1 gene, which encodes the erythroid/megakaryocytic transcription factor GATA1. GATA1 mutations have not been found in AMKL patients who did not have DS and other hematologic malignancies in DS. Most of the GATA1 mutations in DS-TMD/AMKL are nonsense mutations that are mainly located in exon 2. We observed a nonsense mutation in exon 2 of GATA1 [c.189_190delCA (Tyr63X)] in one case of DS-TMD. The GATA1 mutation has been thought to be an early event in the leukemogenesis of DS-TMD/AMKL and it could be used as a stable molecular marker to assess the treatment response or to monitor for the recurrence of DS-TMD/AMKL.

A Novel Mutation in the GATA1 Gene Associated with Acute Megakaryoblastic Leukemia in a Korean Down Syndrome Patient

Although acquired mutations in the GATA1 gene have been reported for Down syndrome-related acute megakaryoblastic leukemia (DS-AMKL) in Caucasians, this is the first report of a Korean Down syndrome patient with AMKL carrying a novel mutation of the GATA1 gene. A 3-yr-old Korean girl with Down syndrome was admitted to our hospital complaining of pallor and fever. The findings of a peripheral blood smear and bone marrow study were compatible with the presence of AMKL. A chromosome study showed 48,XX,-7,+21c,+21,+r[3]/47,XX,+21c[17]. Following GATA1 gene mutation analysis, a novel mutation, c.145dupG (p.Ala49GlyfsX18), was identified in the N-terminal activation domain of the GATA1 gene. This mutation caused a premature termination at codon 67 and expression of an abnormal GATA-1 protein with a defective N-terminal activation domain, and the absence of full-length GATA-1 protein. This case demonstrates that a leukemogenic mechanism for DS-AMKL is contributed by a unique collaboration between overexpressed genes from trisomy 21 and an acquired GATA1 mutation previously seen in Caucasians and now in a Korean patient.

Rastreamento de mutações no gene GATA1 em crianças com síndrome de Down

Crianças com síndrome de Down (SD) apresentam um risco 10 a 20 vezes maior de desenvolver leucemia do que crianças normais, particularmente a leucemia megacarioblástica aguda (LMA-M7) e uma forma reversível denominada doença mieloproliferativa transitória também conhecida como leucemia transitória (LT), devido ao fato de que geralmente há uma remissão espontânea dentro de 3 meses. A LT pode ser considerada uma pré-condição leucêmica, já que cerca de 20% dos pacientes podem desenvolver a LMA-M7 no prazo de 4 anos. Recentemente, foi relatado que mutações somáticas no GATA1, localizado no cromossomo X, estão presentes tanto em blastos de LT quanto em LMA-M7 de crianças com SD. O GATA1 é um fator de transcrição e está presente na diferenciação normal das linhagens eritróides e megacariocíticas. O modo pelo qual as alterações no GATA1 contribuem para a leucemia ainda é desconhecido. A partir disso, estabelecemos um programa nacional, a fim de determinar a incidência de mutações no GATA1 (éxons 2 e 3) em uma coorte de recém-nascidos com SD. Para isso, utilizamos a técnica de cromatografia líquida desnaturante de alta performance (dHPLC) e seqüenciamento automático. Esta técnica de dHPLC se baseia nas variações de heteroduplex e homoduplex dos fragmentos de DNA e apesar de o seqüenciamento automático ser o padrão ouro para a identificação de mutações, este método pode ser lento quanto à análise da mutação, ao passo que o dHPLC tem se mostrado eficaz e rápido para a análise das variações genéticas de diversos genes de interesse médico. Para este estudo utilizamos medula óssea e/ou sangue periférico de 111 crianças com SD (recém-nascidos e crianças sendo a grande maioria com menos de 4 anos de idade) obtidos entre janeiro de 2000 e dezembro de 2007, sem tratamento prévio. Um total de 127 amostras de crianças com SD foram analisadas, sendo 66 crianças com SD e doenças hematológicas identificadas clinicamente e 61 recémnascidos com SD e sem evidência clínica de doenças hematológicas. A análise através do dHPLC e seqüenciamento automático identificou dezenove mutações no éxon 2 exclusivamente em crianças com LT e LMA-M7 com SD e em uma criança com LT e SD foi detectada alteração no éxon 3.A freqüência de anomalias genéticas não foi estatisticamente significativa em relação ao sexo ou cor da pele e alterações no GATA1 não foram detectadas em nossa coorte de recém-nascidos sem sinal de distúrbios hematológicos. A concordância da detecção através da técnica de dHPLC foi de 100% com o seqüenciamento automático. Em conclusão, nossos resultados indicam que alterações no GATA1 são especificas do subtipo LMA-M7 e LT da SD e que a técnica de dHPLC é eficaz e uma valiosa ferramenta para análise mutacional no GATA1 e, além disso, podemos consolidar o GATA1 como um marcador molecular com o intuito de uniformizar os critérios diagnósticos precoces da criança com SD melhorando assim sua taxa de sobrevida.

Children with Down syndrome (DS) have a 10 to 20-fold elevated risk of developing leukaemia, particularly acute megakaryoblastic leukemia (AMKL) and a reversible form of myeloproliferative disorder, known as transient leukemia (TL), which usually spontaneous resolves within 3 months. TL can be considered a preleukemic condition, as approximately 20% of TL patients will develop AMKL within 4 years. Recently, it has been reported that somatic mutations in the X-linked GATA1 gene are present in TL and AMKL blasts of DS infants. GATA1 gene encodes a transcription factor that is critical for normal development of erythroid and megakaryocytic lineages. The precise pathway by which mutagenesis of GATA1 contributes to leukemia is still unknown. Then, we established a national program in order to determine the incidence of GATA1 mutations in a cohort of DS newborns and children with DS presenting hematological disorders, furthermore we have evaluated the efficacy of denaturing high-performance liquid chromatography (dHPLC) screening method for detecting mutations in GATA1 gene. Bone marrow and/or peripheral blood from 111 DS children (newborns and children with the vast majority less than 4 years old) obtained between January 2000 and December 2007 without previous treatment. They were screened for GATA1 mutations (exons 2 e 3) by the denaturing High-Performance Liquid Chromatographic (dHPLC) and direct sequencing in an automated sequencer. dHPLC has been developed to screen for DNA variations by separating heteroduplex and homoduplex DNA fragments by ion-pair reverse-phase liquid chromatography. Although the automatic sequencing is the gold standard technique for identifying mutations, this method can be time consuming for analysis, while the dHPLC was effective and fast for the analysis of genetic variations A total of 127 samples from DS children were analyzed, with 66 DS children with hematological disorders identified clinically and 61 newborns without clinical evidence of hematological disorders by dHPLC and direct sequencing methods. Nineteen mutations were detected exclusively in exon 2 of DS children with AMKL nd TL disorders and one was detected in exon 3 of DS child with TL. The frequency of genetic abnormalities was no statistically significant regarding to sex or ethnicityand GATA 1 mutation was not detected in our cohort of newborns without sign of hematological disorder. The overall detection rate of dHPLC screening was 100%. In conclusion, our results indicate that dHPLC is an efficient and valuable tool for GATA1 mutational analysis.

Capsaicin promotes the development of burst-forming nits-erythroid (BFU-E) from mouse bone marrow cells

Capsaicin, the pungent component of chilli peppers, is known to induce mediators of hematopoiesis. We investigated the effect of capsaicin on hematopoiesis in mouse progenitor cells. Treatment of mouse bone marrow cells with capsaicin induced the formation of colony of burst-forming units-erythroid (BFU-E). We also found that the number of erythropoietin receptor (EpoR)-positive cells was increased by capsaicin. To clarify the effect of capsaicin on erythroid lineage, BFU-E colonies were separated from non-BFU-E colonies by colony-picking after in vitro culture of mouse bone marrow cells. Quantitative RT-PCR analysis revealed that capsaicin stimulated the expression of the erythroid-specific genes encoding EpoR, glycophorin A (GPA), beta-globin (Hbb-b1), GATA-1, PU.1, nuclear factor erythroid-derived 2 (NF-E2), and Kruppel-like factor 1 (KLF1) in the BFU-E colonies. Furthermore, capsaicin could effectively stimulate the transfected GATA-1 promoter in K562 cells. GATA-1 is known as an essential transcription factor for the development of erythroid cells. Our results show that development of the erythroid lineage from bone marrow cells can be induced by treatment with capsaicin, and that GATA-1 seems to play a role in this induced erythroid maturation.

Effects of a new recombinant fusion protein rTMP-GH on proliferation of megakaryocytes in vitro / 第三军医大学学报

Objective To investigate the effects of rTMP-GH, a recombinant fusion protein of thrombopoietin mimetic peptide (TMP) and human growth hormone (GH), on the proliferation and thrombocytopoiesis of cultured megakaryocytes. Methods After being treated with 100 ng/ml of rTMP-GH for 7 d, Dami cells, a kind of megakaryocyte cell line, were analyzed by observing the numbers of colony forming unit. Meanwhile, Western blotting and RT-PCR were applied to detect the expression changes of globin transcription factor-1 (GATA-1), which is a main regulator of thrombocytopoiesis in megakaryocytes. Results The numbers of colony forming unit were markedly increased in cultured Dami cells incubated with rTMP-GH. Compared with normal control and GH treatment groups, both the mRNA and protein levels of GATA-1 were up-regulated significantly in Dami cells treated with rTMP-GH. Conclusion rTMP-GH has a strong ability to promote the proliferation and thrombocytopoiesis of megakaryocytes.