阵发性睡眠性血红蛋白尿症克隆增殖相关分子异常研究

Paroxysmal nocturnal hemoglobinuria (PNH) is a benign clonal disease of hematopoietic stem cells. Somatic PIG-A gene mutations lead to defective biosynthesis of the GPI-anchor and are responsible for the PNH phenotype. However, it’s difficult to explain the clonal expansion and evolution of PNH solely based on the presence of the PIG-A mutation. Recently, application of the new generation sequencing to study malignant clonal diseases revealed greater than originally suspected complexity of clonal architecture with stepwise acquisition of mutations, and expansion of most permissive subclones. In preliminary experiments we have found that PNH, in analogy of leukemia, have a complex clonal architecture and PIG-A mutation is not a sole genetic lesion. Based on these results, we hypothesize that evolution of PNH clone may be associated with additional mutational events in diverse or recurrently affected genes. We plan to detect PIG-A mutation status in a larger PNH population, identify novel somatic mutations, investigate the acquisition pattern and associations of genetic defects in PNH (PNH clonal architecture). Additional somatic mutations may help to further clarify the mechanism of clonal expansion, persistence of mutated PNH stem cells, explain clinical diversity of PNH and distinct behavior of the PNH clones. Most importantly, new molecular lesions may constitute therapeutic targets and unlike the currently available therapies aiming at alleviation of the consequences of PIG-A mutations, they may aim at elimination of the mutant clones and therefore cure of PNH.

阵发性睡眠性血红蛋白尿症（paroxysmal nocturnal hemoglobinuria, PNH）的发生与PIG-A 基因突变导致糖基磷脂酰肌醇（glycosylphosphatidylinositol，GPI）锚连膜蛋白合成缺陷引起PNH细胞对补体敏感性增高相关。但迄今为止，PNH细胞增殖、疾病演变的具体机制尚不清楚。新一代测序技术证实恶性疾病中克隆增殖、疾病演变与基因突变及其所支持的亚克隆增殖密切相关。申请人前期研究发现PNH作为良性克隆性疾病，具有一个复杂的克隆构型，PIG-A突变并不是唯一存在的分子病变。申请人拟通过PIG-A突变的检测、再现性基因突变的发现、基于多基因突变PNH克隆构型的研究等进一步明确PNH克隆增殖的机制，解释PNH临床多样性表现的根本原因。更重要的是，新的分子异常的发现可能成为PNH治疗新的靶点，最终达到PNH克隆的消除，为PNH治疗提供新的思路。

PNH是一种干细胞起源的良性克隆性疾病，已经证实PNH中PIG-A基因突变决定了PNH表型特征。但关于PNH疾病演变、克隆增殖等机制至今不明。本课题应用全外显子测序（whole exome sequencing, WES）技术分析了12例PNH患者体细胞突变事件；应用目的基因靶向测序技术（targeted deep sequencing，TDS）分析了36例PNH病例；应用Sanger PCR法测序分析了58例患者包括PIG-A、TET2、JAK2等基因突变；应用深度测序技术明确PNH体细胞突变基因对应的克隆大小；进一步应用单细胞集落培养测序的方法分析部分病例中体细胞基因突变事件和克隆演变的相关性。WES测序分析12例PNH中10例发现PIG-A突变在内的局限于PNH阳性克隆的体细胞突变事件共35个，累及23个不同基因。TDS分析36例PNH病例发现SUZ12、DHX29、MECOM、BCOR、U2AF1、KDM6A、TET2等基因突变。单细胞集落培养证实具有双位点PIG-A突变病例对应存在双PNH克隆。对9例患者克隆构型分析证实PIG-A既可以作为起源突变同样也可以以继发突变形式发生。以上说明PNH中存在与白血病、MDS等恶性血液病类似的克隆性突变事件，且与特征性的PIG-A突变共同参与决定PNH的表型特征，额外体细胞突变事件与PIG-A协同承担了克隆选择、疾病演变的作用。PNH中一些突变事件在恶性血液病中具有明确的致病和预后意义，提示我们具有此类突变的PNH病例可能为恶性血液病的一种特殊表现形式，尚需进一步分析其相关性。我们的研究尚未发现具有明确病理意义的再现性突变事件。.本课题基于骨髓衰竭性疾病包括再生障碍性贫血（AA）和骨髓增生异常综合征（MDS）与PNH可能存在一定的病理相关性，进一步分析了PNH表现的多样性与免疫抑制、PNH克隆大小及额外亚克隆的驱动等是否相关。本课题通过筛选检测AA和MDS中尤其是表现为溶血患者和骨髓衰竭表现患者中PNH克隆的存在，分析比较不同疾病中PNH克隆阳性患者临床特征、预后等的差异发现PNH可伴发于AA和MDS，PNH克隆大小直接决定了PNH临床表现；对重型再生障碍性贫血（SAA）患者免疫治疗前后PNH克隆演变的研究发现SAA患者ATG治疗后原有PNH可持续存在或增大；免疫治疗前后PNH克隆的存在不影响其疗效。

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