中国人食管p16基因突变分析

为了解ｐ１６基因与中国人食管癌遗传易感性的关系，应用ＰＣＲ－ＳＳＣＰ银染法和ＤＮＡ序列测定法对４０例食管癌及相应手术端正常组织ＤＮＡ标本ｐ１６基因进行研究，结果在４０例食管癌标本ＤＮＡ中共发现ＤＮＡ变异１４例，其中９例存在有ｐ１６基因的错义突变和剪接位点突变，５例分别在密码子４１和１４０存在与白种人已发现的相同的正常多态现象，而相应的正常手术切端组织标本中未发现有错义突变和剪接位点突变。提示：（１     

DukesC期大肠癌原发灶与淋巴结转移灶中p53基因突变的比较

目的探讨大肠癌转移与ｐ５３基因突变的关系。方法用ＰＣＲ－ＳＳＣＰ检测７３例ＤｕｋｅｓＣ期大肠癌及４０例相应的淋巴结转移灶ｐ５３基因第５至第９外显子的突变。结果７３例原发灶有２７例（３７％），４０例淋巴结转移灶中有１９例（４７．５％）检测到ｐ５３基因突变，总突变率为４４％。在原发灶和转移灶同时检测的４０例中，有２１例（５２．５％）两者均未见ｐ５３突变；另１２例（３０％）两者均可见完全相同的突变条带；余下的７例（１７．５％）两者ＰＣＲ－ＳＳＣＰ的结果不一致。这７例经ＤＮＡ直接测序确定了ｐ５３基因突变位点。其中５例原发灶未见ｐ５３突变，而转移灶ｐ５３有明确突变；７例中２例转移灶可见ｐ５３有二个外显子突变，而原发灶仅一个或无突变点。结论多数ｐ５３基因突变发生在转移之前并持续存在于大肠癌进展至转移的整个时期，大肠癌的转移可伴有ｐ５３基因的新突变，或转移对ｐ５３基因突变有一定选择，提示ｐ５３基因突变可能与大肠癌转移相关。     

p53基因突变与食管癌生物学行为的关系

目的为了探讨ｐ５３基因突变与食管癌生物学行为及预后的关系。方法应用ＰＣＲ－ＳＳＣＰ结合ＤＮＡ直接银染测序，对３０例散发性食管癌组织ｐ５３基因第５～８外显子进行检测。结果检出１１例阳性，突变检出率为３６．７％。９例为点突变，其中错义突变４例、无义突变２例、同义突变３例，其余２例为碱基插入和缺失导致的移码突变。统计学分析显示：中低分化食管癌的ｐ５３突变率为５６．３％，高分化组为１４．３％，两组相比有非常显著性差异（Ｐ＝０．０２５）；癌组织浸润累及食管壁全层的ｐ５３突变率５２．６％显著高于未累及全层组９．１％（Ｐ＝０．０２４）；有淋巴结转移组与无淋巴结转移组相比，ｐ５３突变率分别为６１．５％和１７．６％也具有非常显著性差异（Ｐ＝０．０２４）。结论ｐ５３基因突变与食管癌多种生物学行为如组织分化程度、肿瘤浸润程度及淋巴结转移有明显相关性。因此检测食管癌组织中是否存在ｐ５３基因突变有助于判断食管癌的恶性程度和患者的预后。还讨论了ｐ５３基因的“显性负效应”及同义突变的遗传学效应。     

p16基因突变及蛋白表达异常与人胰腺癌关系的研究

目的：研究ｐ１６基因的突变及蛋白表达异常在人胰腺癌发生及侵润转移中的作用。方法：应用ＰＣＲ、ＰＣＲ－ＳＳＣＰ、ＤＮＡ测序及免疫组织化学技术,对３５例人胰腺癌的ｐ１６基因第２外显子进行了检测,研究ｐ１６基因的突变及蛋白表达情况。结果：１２例在ｐ１６基因第２外显子部分存在至少５２２ｂｐ的纯合缺失。７例存在２个点突变（突变位点均相同）：其一为第１２６密码子碱基ＧＴＣ→ＡＡＴ的错义突变,氨基酸由缬氨酸（Ｖａｌ）变成了天冬酰胺（Ａｓｎ）;另一为第１２７密码子ＧＣＡ→ＧＣＧ的同义突变。缺失及突变共占５４．３％（１９／３５）。Ｐ１６蛋白三维结构模拟提示,Ｖａｌ１２６Ａｓｎ对蛋白空间结构有一定影响,从而影响Ｐ１６蛋白的功能。１２例缺失标本呈现Ｐ１６蛋白表达阴性,９例标本（其中７例点突变）出现Ｐ１６蛋白表达下降,共占６０％（２１     

卵巢上皮性肿瘤p16抑癌基因突变与HPV感染的研究

采用聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）技术，对同一卵巢上皮性肿瘤石蜡包埋组织中ｐ１６基因（第二外显子）突变及人乳头状瘤病毒（ＨＰＶ）感染进行相关性研究。并与正常卵巢组织进行对照。结果，２８例卵巢上皮性肿瘤组织中ｐ１６基因突变１５例，突变率为５３．６％（１５／２８），其中７例伴有ＨＰＶ１６型或ＨＰＶ１８型感染，占突变率的４６．７％。在卵巢上皮性肿瘤组ＨＰＶ１６、１８ＤＮＡ阳性率为５３．６％（１５／２８），对照组ＨＰＶ１６、１８ＤＮＡ阳性率为５．６（１／１８），二者比较有显著性差异。提示：卵巢上皮性肿瘤中ｐ１６基因突变与ＨＰＶ１６、１８型感染有关。ＨＰＶ１６、１８型感染与卵巢上皮肿瘤密切相关     

头颈部癌组织CDKN2基因突变及甲基化状态的研究

目的研究头颈部癌组织中ＣＤＫＮ２基因的突变状况及其在肿瘤发生中的作用。方法用ＰＣＲ－ＳＳＣＰ及ＰＣＲ产物直接测序方法检测３６例头颈部癌组织中ＣＤＫＮ２基因的突变，并用内切酶结合Ｓｏｕｔｈｅｒｎ杂交的方法检测其中２０例癌组织中ＣＤＫＮ２基因的ＣｐＧ岛甲基化状况。结果３６例肿瘤组织中４例ＣＤＫＮ２基因发现突变，其中２例为错义突变，另２例为移码突变，突变位点均在ＣｐＧ岛处。经甲基化状态检测，２０例癌组织中８例ＣＤＫＮ２基因第１外显子表现异常甲基化。结论ＣＤＫＮ２基因的失活除基因突变外，可能部分与该基因的异常甲基化状态有关。     

一个遗传性凝血因子Ⅴ缺乏症家系中凝血因子Ⅴ基因两种新突变的鉴定

目的对一个遗传性凝血因子Ⅴ缺乏症家系进行凝血因子Ⅴ（factorⅤ，FⅤ）基因突变的检测，探讨先天性凝血因子Ⅴ缺乏症的分子发病机理。方法PCR结合直接测序方法对先证者的FⅤ基因全部25个外显子及其旁侧序列进行分析，鉴别其中可能存在的基因变异。应用PCR产物反向测序法或PCR限制性内切酶分析技术对突变位点进行确证。随机选择100名健康体检者作为正常对照。结果先证者FⅤ基因存在两种突变，分别是第11外显子区的A1763C错义突变和位于第16内含子3′端剪接位点的G-T突变；家系分析表明这两个突变是双杂合子型，前者遗传自父亲，后者遗传自母亲。100名健康对照者均未发现A1763C错义突变。结论第11外显子A1763C错义突变和第16内含子3′端的剪接位点突变使先证者呈现双重杂合子型，可能是先证者FⅤ先天性缺乏的原因。     

人肺癌组织中p53,Rb基因突变研究

为探讨肺癌发生的分子遗传学机理，采用聚合酶链反应及聚合酶链反应－单链构象多态性技术，对４１例人肺癌组织中ｐ５３基因外显子５～８及Ｒｂ基因外显子１４～１６、２２～２３进行了突变分析。结果显示：ｐ５３基因突变１６例（１６／４１，３９％），分布于外显子５～７；Ｒｂ基因异常４例（４／４１，９．８％），其中外显子１４～１６区域部分缺失和外显子２２～２３区域突变各２例；在９例小细胞肺癌标本中，７例发生ｐ５３及Ｒ５基因的突变，其中１例存在ｐ５３基因两个外显子突变，另１例同时存在ｐ５３及Ｒｂ基因的突变。对部分ｐ５３基因突变标本序列分析，均在１个或３个密码子上存在导致ｐ５３蛋白异常的单碱基置换或插入突变。以上结果表明：肺癌、特别是小细胞肺癌的发生可能与ｐ５３及Ｒｂ基因的突变有关。     

乳腺癌p16基因的纯合缺失、高甲基化、突变及其表达

目的 研究ｐ１６基因在乳腺癌中的纯合缺失,高甲基化和突变等结构变化及其与表达之间的关系,方法 应用聚合酶链反应（ＰＣＲ）和ＰＣＲ甲基化银染分析技术（ＰＣＲ－ＭＡＳＳ）对６０例乳腺癌和２４例癌旁组织进行了ｐ１６基因纯合缺失和ｐ１６基在第１外显子的甲基化检测,用ＰＣＲ－单链构象多态性（ＳＳＣＰ）和ＤＮＡ测序技术进行了ｐ１６基因突变分析,检测了ｐ１６蛋白和ｍＲＮＡ的表达。结果 ６０例乳腺癌中,检出７例（     

中国人,日本人和德国人中hMLH1基因Val 384Asp的检测及 …

探讨ｈＭＬＨ１基因Ｖａｌ３８４Ａｓｐ错义突变在中国人，日本人和德国人中的存在频率及其在大肠癌发病中的可能作用。方法 应用ＰＣＲ－ＳＳＣＰ和ＤＮＡ序列分析技术检测了２６例中国汉族人大肠癌患列健康中国汉族人，８０例健康日本，１０９例德国人大肠癌患者和１００例健康德国人的正常体细胞ＤＮＡ，分析ｈＭＬＨ１基因的第１２外显子。     

Correlation between DNA alterations and p53 and p16 protein expression in cancer cell lines

To investigate the interaction between DNA abnormalities, p53 and p16 gene mutations, and methylation and protein expression, 20 cancer cell lines were examined by Western blotting. A clear relation was found to exist between p53 accumulation and mutation status. Of 20 cell lines examined, 14 demonstrated p53 homozygous mutations in exons 3-10, including 12 missense mutations, one nonsense mutation, and one frameshift mutation. Overexpression of p53 was always linked to missense mutations in exons 6-8. Intermediate expression of p53 was noted in cells with missense mutations or polymorphism to proline at codon 72 in exons 4-5, whereas there was slight or no visible expression in wild type cells and in cells with nonsense and frameshift mutations. DNA aberration in the p16 promoter gene correlated significantly with protein expression of the p16 suppressor gene. Overexpression was noted in six cell lines, intermediate expression in two, and slight or no visible expression in 12. Methylation-caused disappearance of p16 protein was noted in 40% (8/20) of the cell lines. Of six cell lines overexpressing p16 protein, two could be amplified with primers for both unmethylated and methylated forms in a methylation-specific RCP analysis. One cell line with no visible expression could also be amplified with both primers. Overexpression or disappearance of p16 protein may readily occur when one of two alleles has been methylated.     

Absence of somatic ATM missense mutations in 58 mammary carcinomas

Accumulating evidence indicates that germline missense mutations in the ATM gene predispose to breast cancer. To investigate the potential role of somatic ATM mutations in the tumorigenesis of breast cancer, the ATM gene was scanned in 58 mammary carcinomas using DOVAM-S (detection of virtually all mutations-SSCP [single-strand conformation polymorphism]), a robotically enhanced, highly redundant form of SSCP that detects virtually all mutations. A total of 1.65 megabases of tumor DNA sequence was scanned and 16 structural variants were identified, including one novel nonsense mutation, four novel missense mutations, and a common missense change in African-Americans. Sequencing from microdissected normal cells reveals that all variants were present in the germline. Thus, the ATM gene may be similar to the BRCA1/BRCA2 genes in that germline mutations are important in cancer predisposition, but somatic mutations are seldom present in tumors. Loss of heterozygosity (LOH) is common in these tumors, but ATM missense mutations occur with similar frequencies when LOH is present or absent (P=0.73). If germline ATM missense mutations predispose to breast cancer, the unmasking of a recessive missense allele by LOH does not seem to be a critical step in breast neoplasia.     

非小细胞肺癌中p16 INK4／CDKN2基因的缺失和点突变

目的为了探讨周期素依赖性激酶４抑制因子基因（ｐ１６ＩＮＫ４）与肺癌发生的关系。方法采用双重ＰＣＲ－ＳＳＣＰ和序列分析方法，对３１例原发性非小细胞肺癌中ｐ１６ＩＮＫ４基因的存在状况进行了研究。结果３例存在ｐ１６基因缺失（３／３１），其中１例整个ｐ１６基因发生缺失，另两例则分别为外显子１和外显子２缺失；ｐ１６基因点突变２例（２／３１），外显子１和外显子２各一例；并发现在这五例基因异常样品中，４例（２例缺失和２例点突变）样品均为淋巴转移的非小细胞肺癌（４／２０）。结论提示ｐ１６基因失活和某些非小细胞肺癌（５／３１）的发生有关，并且可能发生在癌症的晚期。     

原发胃癌中p16和p15基因表达及甲基化异常

为检测胃癌组织中抑癌基因p16,p15及其启动子区甲基化状态和P16、P15蛋白表达情况。选择p16、p15基因及启动子区域，用PCR－SSCP、MSP（甲基化特异的PCR）和测序法对100例胃癌患者的癌组织、癌旁正常组织和5例正常组织进行检测，同时用免疫组化法检测了癌组织和正常对照组织的P16和P15的表达。结果发现癌组织p16和p15基因启动子区甲基化率显著高于癌旁正常组织和正常对照；胃癌组织中，71％的病例P16表达阴性，54％的病例具有p16基因启动子区的高甲基化，无突变和纯合缺失检出；11％的病例P15表达阴性，9％的病例具有p15基因启动子区的高甲基化，p15异常与低分化胃癌有关，p15基因内含子1和外显子1内各发现1例DNA序列改变；癌组织中p16和p15基因启动子区甲基化与其蛋白表达密切相关。结果显示p16基因启动子区域高甲基化是胃癌中p16基因失活的关键因素之一，并在胃癌的发生发展中发挥重要作用；p15基因启动子区域高甲基化在胃癌中起一定作用。     

Wilm瘤中p16基因缺失一例报告

为调查ｐ１６基因在Ｗｉｌｍ瘤中的改变，采用ＰＣＲ－ＳＳＣＰ法对手术治疗的２４例Ｗｉｌｍ瘤患者肿瘤组织标本，进行了ｐ１６基因第１，２，３外显子的突变筛查，结果未发现异常泳动带。用双重对照ＰＣＲ，发现一例ｐ１６基因的纯合缺失。这一结果支持Ｗｉｌｍ瘤的发生涉及多个基因改变的假说     

Detection of new PTEN/MMAC1 mutations in head and neck squamous cell carcinomas with loss of chromosome 10.

Alterations of the candidate tumor suppressor gene PTEN/MMAC1 and the cell cycle control gene p16((CDKN2/MTS-1/INK4a)) have been detected in many types of human cancer. Here, we wanted to study the role of PTEN/MMAC1 in head and neck squamous cell carcinomas (HNSCC) in correlation to mutation and methylation of p16 and to previous in situ hybridization results concerning loss of chromosomes 9 and 10. We screened for alterations of PTEN/MMAC1 and p16 in 52 HNSCC of different sites. Mutations of PTEN/MMAC1 were found in 23% of tumor samples (missense mutations in 7 carcinomas, 13%). A loss of chromosome 10 was detected in five carcinomas with missense PTEN/MMAC1 mutations (71%). The missense mutations of PTEN/MMAC1 occurred in exons 5 (five different mutations in the neighborhood of the protein tyrosine phosphatase domain), 6, 7, and 8. Only one of these mutations had been described before. In addition, in three laryngeal carcinomas (6%), missense mutations of p16 (in exon 2) were detected and 14% of carcinomas showed a methylation of p16. Our results focus on the essential but not solitary role of PTEN/MMAC1 in the tumorigenesis or progression of a subset of HNSCC.     

Human papillomavirus infection in Egyptian esophageal carcinoma: correlation with p53, p21, mdm2, C-erbB2 and impact on survival

The etiological role of human papillomavirus (HPV) in esophageal carcinoma (EC) in relation to p53, mdm2, p21(waf), c-erbB2 and the overall survival (OS) rate was investigated. Tumor and normal tissues from 50 EC were evaluated by polymerase chain reaction and InnoLiPA for HPV. Single strand conformation polymorphism/sequencing were used to detect p53 gene mutations. Immunohistochemistry was performed to determine p53, mdm2, p21(waf)and c-erbB2 expression. Human papillomavirus was detected in 54% of tumors and in 24% of normal tissues. p53, mdm2 and c-erbB2 overexpression was detected in 68%, 70% and 60% of tumors and in 14%, 16% and 10% of normal samples, whereas loss of p21(waf) was evident in 64% of tumors. p53 mutations were detected in 20% of cases. Exon 8 and 5 showed the highest mutation rate (40% each), followed by exons 6 and 7 (10% each). There was a significant correlation between HPV and p53, mdm2, c-erbB2 overexpression. The OS was significantly associated with overexpression of p53 and loss of p21(waf). Human papillomavirus infection is frequent in Egyptian EC. Both p53-dependent and p53-independent pathways seem to be involved in HPV-associated EC. mdm2 and c-erbB2 are possible targets for HPV in the p53-independent pathway. However, only advanced stage and aberrant expression of p53 and p21(waf) are independent prognostic markers.     

Relevance of the viral RAK alpha gene in diagnosis of malignant versus nonmalignant tumors of the ovary and uterus

Human immunodeficiency virus type 1 (HIV-1)-like antigens RAK (named after the inventor E. M. Rakowicz) p120, p42, and p25, as well as HIV-1-like segments of cancer DNA (RAK gene alpha), have been found before in breast and prostate cancers. The present study focused on determining the value of markers RAK in the diagnosis and prognosis of gynecological cancer. Expression of RAK antigens in ovarian, uterine, cervical, and vulvar cancer, in benign tumors, in tissues adjacent to cancer, and in normal tissues was tested by Western blot hybridization of the electrophoretically separated proteins with monoclonal antibody RAK BrI. The RAK alpha gene was PCR amplified with HIV-1-derived primers SK68 and SK69. RAK antigens p120, p42, and p25 were found in 95% of ovarian, uterine, and cervical cancer cases and in 75% of vulvar cancer cases. The RAK alpha gene was expressed in 100% of cancer cases, in approximately 25% of benign ovarian tumors, and in 40% of benign tumors of the uterus. DNA sequences amplified in all cancer cases exhibited more than 90% homology to HIV-1 gp41 and were encoded for the functional peptide. DNA sequences found in benign tumors contained frameshift mutations and encoded truncated or nonfunctional peptides. Such sequences have not been amplified in normal tissues. RAK antigens and the RAK alpha gene seem to belong to a lentivirus type that is highly related to HIV-1. Beyond the diagnostic value of RAK markers, future cloning of the full viral genome would lead to a better understanding of the etiology of malignant and nonmalignant tumors of reproductive organs and to the development of novel therapeutic approaches.