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.

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.

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.

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.