骨髓增生异常综合征与线粒体细胞色素氧化酶基因突变初探

Mutation of Mitochondria Cytochrome Oxidase Gene in Patients with Myelodysplastic Syndrome

骨髓增生异常综合征(MDS)与线粒体基因突变的关系是近年来血液肿瘤发病机制研究的热点。本研究旨在分析MDS患者线粒体细胞色素氧化酶基因COI和COII是否存在不同于正常组织的突变,这些突变是否为"热点突变"。18例MDS患者纳入本研究,患者年龄20-70岁。18例中RA2例,RCMD 3例,RAEB 7例,AML(MDS转化)5例,MPD/MDS 1例。分别提取MDS患者骨髓单个核细胞和颊粘膜细胞的总DNA,用PCR方法扩增线粒体细胞色素氧化酶基因COI和COII的高度突变区,以获得528 bp(7181-7709)基因片段,产物经纯化后双向测序,将结果与DNA文库标准序列和患者自身正常组织对应序列比较,对突变结果进行分析。结果表明:所检测的18例患者线粒体细胞色素氧化酶基因COI和COII中高度突变区528 bp的片段中共发现3个单核苷酸序列突变,分别为7674 T→C,7353 A→G和7702 Ins G。前2个突变存在于相同个体的颊黏膜细胞和骨髓细胞,导致编码的异亮氨酸改变为蛋氨酸,蛋氨酸改变为缬氨酸。7702 Ins G插入仅见于骨髓细胞,导致移码,这可能与MDS细胞突变有关。结论:本研究病例中没有发现文献报道的细胞色素氧化酶基因COI和COII"热点突变",但发现3个新突变,提示mtDNA突变在MDS发病中仍然是一个异质性很大的复杂现象,可能是疾病过程中的一个前期或伴随现象。

The relationship between mitochondria gene mutation and hematological malignancies has been focusing on as a key point in recent studies. This study was aimed to investigate whether in patients with myelodysplastic syndrome （MDS） exists mitochoudria cytochrome oxidase COl and COl/gene mutations different from normal tissues and to analyze whether these mutations are ＂hot spot＂ mutations. Eighteen MDS patients aged from 20 to 70 years old were brought into this study, including 2 of RA, 3 of RCMD, 7 of RAEB, 5 of AML（transformation from MDS）, and 1 of MDS/MPD. The total DNA was extracted both from bone marrow cells and buccal cells of the same patients. A pair of primers was designed to amplify a fragment with 528 base pair（7181 -7709 ）by PCR technique, which contained high frequency mutation area of cytochrome oxidase COⅠ and COⅡ gene based on the literature reports. The PCR products were purified and sequenced as bidirection to confirm if there is any mutation. The results of sequence of COⅠ and COⅡ gene from MDS patient bone marrow cells were compared with both the standard sequence from GenBank and the sequence from MDS patient buccal cells. The results showed that 3 single nucleotide changes in 528 bp cytochrome oxidase gene fragment from 18 MDS patients were confirmed. They were 7674 T→C, 7353 A→G, and an insert mutation of G at 7702. The former two mutations caused isoleucine→methionine, and methionine→viline. The 7702G ins was only confirmed with marrow cells in a patient, and caused a frame shift, which suggested that the mutation might be related to MDS cells. It is concluded that some of ＂hot spots＂ of mtDNA mutation in cytochrome oxidase（ COⅠ, COⅡ）gene from our MDS patients are failed to be confirmed, but 3 new mutations on this gene are found, which suggested that mitochondria DNA mutations in MDS patients still have much complexity and heterogeneity, mtDNA mutation may be a prophase or an accompany phenomenon of this disease.