【作者】 杨丽云；

【导师】 达万明； 朱平；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2010， 博士

【摘要】 [研究背景]穿孔素(Perforin PRF1)由T细胞,NK细胞等活性杀伤细胞分泌。其基因突变导致细胞毒介导的杀伤作用减弱,免疫系统清除病变细胞能力减弱,淋巴系统异常增生甚至发生肿瘤。目前发现高达60%家族性嗜血细胞综合征(familial hemaphagocytic lymphohistiocytosis,FHLH)与PRF1突变相关。Clementi等发现PRF1基因敲除小鼠易患淋巴系统肿瘤,Smyth等也再次证明了这一点。近年来人们发现再生障碍性贫血、淋巴瘤、自身免疫性淋巴增殖综合征都与PRF1突变有关。Cannella等发现儿童间变大细胞淋巴瘤存在PRF1突变Clementi等在弥漫大B细胞淋巴瘤病人中发现PRF1突变另外Clementi曾报道一例自身免疫性淋巴系统增殖性疾病转变为淋巴瘤的病例同时有PRF1、FAS突变;并发现该患者兄长具备同样的基因突变,其兄发生了嗜血细胞综合征,预示着PRF1突变单独或协同其他基因突变或在环境因素作用下可能引发各种淋巴系统增殖性疾病。急性淋巴细胞性白血病(acute lymphoblastic leukemia ALL)／林巴瘤是一种淋巴细胞单克隆增殖性疾病,并存在多种染色体异常、基因突变等细胞遗传学改变,因此很可能PRF1基因突变与其有一定关系,Santoro曾研究一百例白种儿童急淋白血病,认为与穿孔素基因突变A91V有关,但是Mehta曾研究二千余例白种儿童急淋白血病,认为与穿孔素基因突变无关,仅PRF191V(在美国白种人中约占5%)可能与BCR-ABL阳性儿童急淋白血病有一定关系。中国人与外国人种族不同,成人急淋白血病／淋巴瘤预后与儿童明显不同,本文拟观察PRF1基因突变、SNP(single nucleotide polymorphism单核苷酸多态性)与中国人急性淋巴细胞白血病/淋巴瘤的关系。[目的]了解PRF1突变、SNP是否与中国人ALL／淋巴瘤有关。[研究对象与方法]1.研究对象2006年2月-2008年12月期间从北京市道培医院和北京大学第一附属医院收集的111例ALL患者,2005年10月至2010年2月武警总医院和北大医院住院和门诊随访确诊的77例淋巴瘤患者,北京大学第一附属医院2008年6-10月63例健康体检人员作对照。该研究已取得北京市道培医院、北京大学第一医院和武警总医院伦理委员会通过。2.研究方法(1)基因组DNA提取病例组和对照组研究对象的基因组DNA提取自外周血、指甲或头发。(2)PRF1基因外显子及两旁内含子的扩增和测序(3)Ph染色体及BCR/ABL融合基因检测(4)免疫表型分析(5)EBER-1原位杂交[结果一]1.染色体分析和融合基因检测的结果111例ALL中B-ALL93例,T-ALL18例。111例ALL中93例作了核型分析,32例(34.4%)有t(9；22)(q34；q11)易位,即Ph染色体阳性,均为B-ALL。111例ALL都作了融合基因的检查,36例融合基因阳性,均为B-ALL。Ph染色体阳性的32例均为bcr/abl+,未作核型分析者中还有4例bcr/abl+。2.在ALL中检测到4种新的杂合性PRF1错义突变,它们引起氨基酸的改变为：G198R、R225Q、D486G和R509K。它们都是B-ALL并伴有核型异常3.在ALL中检测到2种新的PRF1杂合性同义突变：S388S、Q540Q,它们也都是B-ALL并伴有核型异常或融合基因4.PRF1基因内SNP rs885821、rs885822、rs10999427和rs10999426的杂合率在正常人与ALL患者之间未发现明显差异[结果二]1.在77例淋巴瘤中检测到8种新的PRF1错义突变.在77例淋巴瘤中检出了8种PRF1基因的错义点突变,它们引起氨基酸的改变为:L11P、Q164L、I125R、P188L、R 385W、L6P、A109G、F169S,前三种为纯合性错义突变,后五种为杂合性错义突变。2.在77例淋巴瘤中检测到3种新的PRF1同义突变我们还在3例淋巴瘤患者中检出了3种PRF1同义突变,c.9 C>T(A3A)和c.216 C>A(T72T)为双等位基因突变,c.180 A>G (P60P)为单等位基因突变。3. PRF1基因内SNP rs885821、rs885822、rs10999427和rs10999426的杂合率在正常人与淋巴瘤患者之间未发现明显差异。4.对4例病人指甲、头发分别测序,与外周血相同。5.对8例发生突变的淋巴瘤病人病理切片进行EBER检测,仅一例为阳性。[结论]1.PRF1基因突变与B-ALL有关,主要发生在Ph+或有其它核型异常的B-ALL。2.PRF1基因突变与淋巴瘤有关,与EB病毒感染无关。3.ALL／淋巴瘤中发生的PRF1基因突变是种系突变。更多还原

【Abstract】 Backgroundperforin(PRF1) was secreted by T cells, NK cells and other active killer cells. Mutations in PRF1 result in the decrease or absence of the protein and its activity, so that immunological surveillance to virus-infected and transformed tumor cells is compromised. Mutations in PRF1 are found in about 60% cases of familial hemaphagocytic lymphohistocytisis (FHLH) due to severely impaired CTL and NK functions.Knock-out mice for perforin have a high incidence of lymphocytic tumors. In recent years, mutations and polymorphisms in PRF1 have been detected in aplastic anemia, lymphomas and autoimmune lymphoproliferative syndromes. For example, a total of 6 different perforin mutations were identified in 12 of 44 cases with childhood anaplastic large cell lymphoma. In a group of 29 lymphoma patients, biallelic mutations were found in 4 patients and monoallelic mutations presented in 4 other patients. In another report, one case with mutations in PRF1 and FAS genes developed T-cell lymphoblastic lymphoma, and his brother carried the same mutations but manifested hemophagocytic lymphohistiocytosis, suggesting that additional genetic or environmental factors may have impact on clinical symptoms of the patients with PRF1 mutations. Acute lymphoblastic leukemia (ALL) is a malignant monoclonal lymphoproliferative disease, often associated with chromosomal abnormalities or mutations in several genes. A91V mutation in PRF1 has been investigated in childhood ALL with paradoxical results. This mutation was a frequent predisposing factor for childhood ALL from screening A91V mutation in 100 patients. However, in another study on 2272 ALL children, A91V polymorphism was not found to be a risk factor for childhood ALL. Here we want to investigated mutations and SNPs in PRF1 in 111 children and adults with ALL and 77 with lymphoma.ObjectiveTo identify whether mutations and single nucleotide polymorphism (SNP) in perforin gene (PRF1) are correlated with acute lymphoblastic leukemia (ALL) and lymphoma.Patients and methods1. ALL/lymphoma patients and healthy candidate recruitment111 ALL patients, who treated in Beijing Daopei Hospital or Peking University First Hospital during the period from February 2006 to December 2008, were recruited; The patients were diagnosed as ALL and supposed for hematopoietic stem cell transplantation.77 lymphoma patients, who treated in Peking University First Hospital and the General Hospital of Armed Police during the period from October 2005 to February 2010, were recruited; In addition,63 healthy medical students were recruited as controls. The research project was approved by the Medical Ethics Committee of Peking University First Hospital and the General Hospital of Armed Police and the Institutional Review Board of Beijing Daopei Hospital.2.Methods(1)DNA preparationGenomic DNA was extracted from mononuclear cells in peripheral blood or nail and hair (2)Sequencing of PCR products amplified from coding exons and their flanking introns in PRF1(3)Karyotype analysis and BCR-ABL fusion gene examination(4)Immunophenotype(5)EBER-1 in situ hybridizationResults 11. Karyotype and BCR-ABL fusion gene analysis Among111 ALL cases,93 cases are B-ALL,18 cases are T-ALL.We performed karyotype analysis in 93 cases of the 111 ALL patients. t(9;22)(q34;q11) trnaslocation, i.e., positive Ph chromosome, was found in 32 (34.4%) cases, of whom all were B-ALL patients. We also performed fusion gene examinations in 111 ALL cases. BCR-ABL fusion transcript was found in 36 cases, of whom 32 cases were Ph chromosome positive, and 4 cases were not performed karyotype analysis.2.Four novel monoallelic missense point mutations in PRF1 were found in 4 B-ALL cases with abnormal karyotype.The resultant amino acid changes are G198R、R225Q、D486G and R509K.3. Two novel monoallelic synonymous point mutations S388S and Q540Q in PRF1 were detected in 5 B-ALL cases with abnormal karyotype or fusion gene.4. No significant differences in the heterozygosity ratio of reported SNPs in PRF1 between ALL patients and normal controls. Results 21. Eight novel missense point mutations in PRF1 were found in 77 lymphomas In the 77 lymphoma cases,8 point mutations were found in PRF1. L11P、Q164L、I125R are biallelic missense mutations, P188L、R385W、L6P、A109G、F169S are monoallelic missense mutations.2. Three novel synonymous point mutations in PRF1 were found in 77 lymphomas In the 77 lymphoma cases,3 point mutations were found in PRF1. c.9 C>T (A3A)and c.216 C> A (T72T) are biallelic mutations, c.180 A> G (P60P) is monoallelic mutation.3. No significant differences in the heterozygosity ratio of reported SNPs in PRF1 between lymphoma patients and normal controls. Genotype and allele frequencies of rs885821, rs885822, rs10999427, rs10999426 and rs12161733 conform to Hardy-Weinberg equilibrium, and have no significant differences between lymphoma patients and normal individuals (P> 0.05)4. The same mutations could also be detected in nail or hair follicles in 4 lymphoma cases.5. Only one is EBER positive among 8 lymphoma cases with PRF1 mutations.Conclusions1.Mutations in PRF1 may occur in B-ALL patients, frequently seen in those with Ph chromosome or other karyotype abnormalities.2.Mutations in PRF1 can also occur in lymphoma patients and has no correlationship with EB virus.3. Mutations in PRF1 in ALL/lymphoma are germline mutations.更多还原