Investigation of the mechanism for clonal expansion of GPI negative cells in paroxysmal nocturnal hemoglobinuria

1. GPI阴性细胞克隆扩增机制的研究（Taroh Kinoshita）我们报道了2例PNH患者，其PIGA突变细胞同时发生了获得性12号染色体重排。在这两个病例中，der（12）在HMGA 2的3个非翻译区内有一个断裂，HMGA 2是一种在许多良性间叶肿瘤中失调的结构转录因子基因，导致HMGA 2在骨髓中异位表达。这些观察结果表明，异常HMGA 2的表达，与突变PIGA，占克隆hematopoietis.To调查是否异位表达的HMGA 2也是与其他PNH患者没有染色体异常的情况下，我们已经建立了稳定和纯化的方法从外周血细胞或骨髓细胞的mRNA的患者和正常志愿者。我们用Q-PCR方法检测到HMGA 2在正常人和PNH患者外周血中的表达，HMGA 2在PNH患者外周血中的表达明显高于正常人。我们还对4例无染色体异常的PNH患者的粒细胞HMGA 2的3 'UTR基因组序列进行了分析，未发现基因组序列异常.通过刺激调节GPI锚定蛋白的表达（Yoshiko Murakami）我们已经鉴定了一种以静脉血栓形成和癫痫发作为特征的新疾病，其中GPI的缺乏以常染色体隐性方式遗传。在患者中，甘露糖基转移酶编码基因PIGM起始密码子-270位点的点突变（c-g）破坏了转录因子Sp1与其同源启动子基序的结合，降低了PIGM的转录，导致部分GPI缺陷。我们已经建立了与患者具有相同突变的小鼠模型，以研究Pigm的表达在胚胎发育过程中是如何调节的，以及这种下调是如何引起症状的。

1. Investigation of the mechanism for clonal expansion of GPI negative cells (Taroh Kinoshita)We reported 2 patients with PNH whose PIGA mutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3 untranslated region of HMGA2,the architectural transcription factor gene dereguated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression,in concert with mutant PIGA, accounts for clonal hematopoiesis.To investigate whether ectopic expression of HMGA2 is also the case with other PNH patients without chromosomal abnormalities, we have established the method for stabilization and purification of mRNA from peripheral blood cells or bone marrow cells of patients and normal volunteers. We could detect mRNA in blood both from normal volunteers and the PNH patient who has ectopic expression of HMGA2 because of chromosomal abnormalities by Q-PCR, and its expression in the patient is significantly higher than in normal volunteers. We also analyzed the genomic sequences of 3'UTR of HMGA2 of granulocytes from four PNH patients who have no chromosomal abnormalities, and found no abnormalities in the genomic sequences.2. Regulation of expression of GPI anchored proteins by the stimulations (Yoshiko Murakami)We have identified a novel disease characterized by venous thrombosis and seizures in which deficiency of GPI is inherited in an autosomal recessive manner. In patients, a point mutation (c-g) at position -270 from the start codon of PIGM, a mannosyltransferase-encoding gene, disrupts binding of the transcription factor Sp1 to its cognate promoter motif and reduces transcription of PIGM, causing partial GPI deficiency. We have made the mouse model which has the same mutation as patients to investigate how the expression of Pigm is regulated during embryogenesis and how this down regulation causes the symptoms.