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

PNH是一种获得性克隆干细胞疾病，其特征在于PIG-A基因的体细胞突变。虽然血管内溶血的分子机制已经很清楚，但PIG-A基因突变的病因和受影响细胞的选择性扩增机制仍然未知。基于PNH患者存在多个PIG-A突变、多个PIG-A突变克隆以及部分患者与MDS或白血病克隆共存，我们假设PNH患者的整个基因组存在遗传不稳定性。为了验证这一点，我们使用了三种不同的遗传不稳定性标记，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转移酶（HPRT）突变率我们首先检测了20例PNH患者的MSI，发现MSI罕见，（Eur J Haematol，64：430-2，2000）。为了评估染色体不稳定性，使用G显带和荧光原位杂交（FISH）分析了13名患者的7号和8号染色体的非整倍体。5例（38%）为7号染色体单体（-7），无1例为8号染色体三体，提示7号染色体不仅在PNH中是遗传不稳定的，而且-7号染色体是监测PNH向MDS或白血病进展的有用标记。12例患者中8例（67%）和17例健康志愿者中3例（18%）发现突变菌落（P<0.02）。患者中突变菌落的发生率（平均84，x10^<-6>）比健康供体（平均1.3，x10^<-6>）高得多。因此，HPRT基因突变在PNH患者中比在健康对照中更频繁。综上所述，我们得出结论，在PNH患者中，存在有利于血细胞发生多种体细胞突变的条件。

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.