THE ETIOLOGY OF SOMATIC MUTATIONS IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)

PNH是一种以猪- a基因体细胞突变为特征的获得性克隆干细胞疾病。虽然血管内溶血的分子机制已经被很好地理解，但猪- a基因突变的病因和受影响细胞选择性扩增的机制仍然未知。基于多个猪- a突变的存在，单个患者中存在多个猪- a突变克隆，并且在一些患者中存在MDS或白血病克隆，我们假设PNH的整体基因组存在遗传不稳定性。为了验证这一点，我们分析了PNH患者遗传不稳定性、微卫星不稳定性（MSI）、染色体异常和次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HPRT）基因突变率的三个不同标记。我们首先检查了20例患者的MSI，发现MSI的罕见性（Eur J Haematol, 64: 430- 2,2000）。为了评估染色体的不稳定性，使用g带和荧光原位杂交（FISH）分析了13例患者的7号和8号染色体的非整倍体。5例（38%）有7号单体（-7），但没有8号三体，这不仅表明7号染色体在PNH中遗传不稳定，而且-7是监测疾病进展为MDS或白血病的有用标记。利用HPRT基因突变试验结合T细胞集落形成，我们检测了T细胞集落突变的频率。12名患者中有8名（67%）和17名年龄匹配的健康志愿者中有3名（18%）存在突变菌落（p<0.02）。患者中突变菌落的发生率（平均84个，x10^<-6>）远高于健康供者（平均1.3个，x10^<-6>）。因此，PNH患者的HPRT基因突变比健康对照组更频繁。综上所述，我们得出结论，在PNH患者中，存在有利于血细胞中多种体细胞突变发生的条件。

PNH is an acquired clonal stem cell disorder characterized by a somatic mutation in the PIG-A gene. Although the molecular mechanism of intravascular hemolysis is well understood, the etiology of PIG-A gene mutation and the mechanism of selective expansion of affected cells still remain unknown. Based on the presence of multiple PIG-A mutations, more than one PIG-A mutant clones in a single patient and a coexistence with MDS or leukemia clones in some patients, we hypothesize the genetic instability in the overeall genome in PNH.To test this, three distinct markers of genetic instability, microsatellite instability (MSI), chromosomal abnormality and mutation rate of the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene were analyzed in PNH patients.We first examined MSI in 20 patients and found rarity of MSI as reported (Eur J Haematol, 64 : 430-2, 2000).To asses the chromosomal instability, aneuploidy for chromosome 7 and 8 was analyzed in 13 patients using G-banding and fluorescence in situ hybridization (FISH). Five (38%) had monosomy 7(-7) but none had trisomy 8, suggesting not only that chromosome 7 is genetically unstable in PNH but also that -7 is a useful marker for monitoring the disease progression to MDS or leukemia.Using HPRT gene mutation assay combined with T cell colony formation, we examined the frequency of mutations in T cell colonies. Mutant colonies were found in 8 of 12 (67%) patients and 3 of 17 (l8%) age-matched health volunteers (p<0.02). The incidence of mutant colonies was extremely higher in the patients (mean 84, x10^<-6>) than in the healthy donors (mean 1.3, x10^<-6>). Thus, the HPRT gene mutate more frequently in patients with PNH than in healthy controls.Taken together, we concluded that in PNH patients, conditions exist that favor the occurrence of diverse somatic mutations in blood cells.