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

1. 我们报道了2例PNH患者，其PIGA突变细胞同时发生了12号染色体的获得性重排。在这两种情况下，der（12）在HMGA2的3个非翻译区有一个断裂，在许多良性间质肿瘤中，建筑转录因子基因失调控，导致HMGA2在骨髓中异位表达。这些观察结果表明，异常的HMGA2表达，与突变的PIGA一致，解释了克隆造血。为了研究HMGA2的异位表达是否也存在于其他无染色体异常的PNH患者中，我们建立了稳定和纯化患者及正常志愿者外周血或骨髓细胞mRNA的方法。我们可以通过Q-PCR检测正常志愿者和因染色体异常导致HMGA2异位表达的PNH患者血液中的mRNA，其在患者中的表达明显高于正常志愿者。我们还分析了4例无染色体异常的PNH患者的粒细胞HMGA2 3′utr的基因组序列，未发现基因组序列异常。刺激对GPI锚定蛋白表达的调节（Yoshiko Murakami）我们发现了一种以静脉血栓形成和癫痫发作为特征的新型疾病，其中GPI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，减少了PIGM的转录，导致部分GPI缺陷。我们制作了与患者具有相同突变的小鼠模型，以研究Pigm的表达在胚胎发生过程中是如何被调节的，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.