中国人遗传性非息肉病性结直肠癌错配修复基因大片段缺失研究

目的了解中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer．HNPCC)家系中MSH和MLH1基因大片段缺失情况及特点，以进一步完善中国人HNPCC家系遗传检测内容。方法取14个符合中国人HNPCC诊断标准的HNPCC家系肿瘤先证者外周血DNA，用荧光标记多重PCR技术结合GeneScan分析系统检测MSH2和MLH1基因大片段缺失。结果14例患者中有1例检测到MSH2基因第1～7外显子缺失，该家系另1例大肠癌患者和3个家系成员有同样的基因片段缺失。结论中国人HNPCC家系错配修复基因大片段缺失可能以MSH2比较常见。建议在中国人HNPCC家系遗传检测中常规包含错配修复基因大片段缺失检测。     

定量多重PCR-高效液相色谱分析错配修复基因大片段缺失

目的 建立一种以多重PCR-高效液相色谱(high performance liquid chromatography，HPLC)分析为基础的错配修复基因DNA大片段缺失检测技术。方法 设计合成35对引物，分8个多重PCR反应扩增MSH2和MLHl基因的35个外显子，PCR产物经高效液相色谱半定量分析，确定各外显子拷贝数。(1)双盲法分析阴性和阳性对照样本，完成方法学可靠性检验。(2)分析14例遗传性非息肉性大肠癌患者外周血细胞DNA和13例散发性大肠癌患者癌组织细胞DNA样本，筛查MSH2和．MLHl基因DNA大片段缺失。结果 (1)稳定检出阳性对照样本的DNA大片段缺失；(2)在筛查样本检出2例新的MSH2基因DNA大片段缺失，分别为MSH2基因外显子7遗传性缺失和MSH2基因外显子1～6体细胞性缺失。结论 多重PCR—HPLC分析系统可以作为突变基因分析系统的一个重要补充，在基因DNA大片段缺失检测中发挥作用。     

结直肠癌中错配修复蛋白缺陷与NTRK基因融合的相关性分析

目的探讨结直肠癌中错配修复蛋白缺陷(dMMR)与NTRK基因融合的相关性。方法收集南京大学医学院附属鼓楼医院病理科2015—2019年诊断为结直肠癌的组织蜡块830例, 分别运用免疫组织化学和荧光原位杂交(FISH)检测830例甲醛固定石蜡包埋(FFPE)肿瘤组织中错配修复蛋白表达情况以及NTRK1/2/3基因断裂情况。比较dMMR型和错配修复蛋白无缺陷(pMMR)结直肠癌中NTRK1/2/3基因融合的发生率, 进一步运用FFPE样本进行RNA Seq二代测序检测, 分析融合伴侣和融合方式。结果 830例原发性结直肠癌FFPE样本均成功进行免疫组织化学和FISH检测。dMMR病例82例(9.88%), pMMR病例748例(90.12%)。其中MLH1蛋白缺失比例为9.04%(75/830), PMS2蛋白缺失比例为9.04%(75/830), MSH2蛋白缺失比例为2.53%(21/830), MSH6蛋白缺失比例为4.10%(34/830), MLH1和PMS2共缺失比例为8.67%(72/830), MSH2和MSH6共缺失比例为2.17%(18/830)。伴有dMMR肿瘤与pM...     

PCR-RFLP法在脊髓性肌萎缩症基因检测中的局限性

目的 探讨聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)技术在脊髓性肌萎缩症(spinal muscular atrophy,SMA)基因诊断中的应用.方法 用PCR-RFLP分析935例临床疑似SMA患儿的运动神经元存活基因1(survival motor neuron,SMN1)第7和第8外显子的缺失,同时用多重连接探针扩增技术(multiplex ligationdependent probe amplification,MLPA)分析其中339例疑似病例的SMN1基因拷贝数改变.用Pearson卡方检验分析两种方法检测SMN1纯合和杂合性缺失的一致性.结果 共发现SMN1基因第7外显子纯合缺失590例,疑似患者的SMA基因诊断率为63.1％(590/935).用PCR-RFLP和MLPA技术联合分析疑似病例339例,PCR-RFLP共发现SMN1纯合缺失194例,MLPA发现196例,二者的一致性为98.9％,差异无统计学意义(x2=0.2,P=0.88).PCR-RFLP仅发现SMN1疑似杂合性缺失4例,而MLPA证实有17例,二者的一致性为23.5％,差异有统计学意义(x2=8.29,P＜0.01).结论 PCR-RFLP尽管简便、特异、实用,但对于5％～10％ SMN1杂合缺失合并点突变的病例则存在明显的局限性.     

染色体8q24区域内基因扩增在结直肠癌中的研究进展

恶性肿瘤常伴有基因组不稳定,而基因扩增是基因组不稳定常见表现形式.结直肠癌,常见消化系统恶性肿瘤,包含有多个区域出现扩增,chr6:42 008 700-42 937 90;chr8:125 620 117-128 955 220;chr12:24 175 625-27 444 930;chr13:27 392 825-27 439 502这4个区域在不同结直肠癌组织以及结直肠癌细胞系中存在扩增明显现象,其中,染色体8q24区域更是在4个不同的细胞系中均出现扩增.此外文献报道中称8q24区域的扩增影响肿瘤的转移.为了更好的了解该区域的基因对肿瘤的影响,本文就常见扩增区域8q24在结直肠恶性肿瘤中的研究进展作一综述.     

79例中国人结直肠癌 APC 基因突变分析

目的：研究中国人结直肠癌中 APC 基因的突变频率及分布特点。方法应用聚合酶链反应（ PCR ）产物直接测序法,对79例中国结直肠癌患者肿瘤标本中 APC 基因突变密集区（ MCR ）中的突变进行检测,并对该基因突变与相关临床病理参数之间的关系进行分析。结果在79例肿瘤标本中检出 APC 基因的总突变率为31.6％（25／79）。点突变频率为20.3％（16／79）,其中有12例无义突变及4例错义突变;另有11％（9／79）的突变是由缺失或插入导致的移码突变。12例无义突变及9例移码突变导致截短突变（27％）,占总突变率的84％。 APC 基因突变表现为区域密集,密码子1281-1352和1411-1465为突变频率较高的2个区段,其突变率分别为16.5％和15.2％。突变频率较高的位点为密码子1450、1421、1306和1286。统计分析结果显示,癌组织中 APC 基因突变与患者的年龄、性别、肿瘤位置、浸润深度、淋巴结转移情况、分期、大体分型和组织分化程度无关。结论中国人结直肠癌中存在较高频率的 APC 基因突变,截短突变为其主要突变类型。同时,本研究在 APC 基因的 MCR 中发现2个突变热点区及4个潜在突变热点。     

结直肠癌中错配修复基因表达与临床病理及Pgp、GSTπ、TopoⅡ、Ki-67表达的关系研究

目的：探讨结直肠癌错配修复基因(MLH1,PMS2,MSH2,MSH6)免疫组化表达特点及其与临床病理的关系,以及错配修复基因(MLH1,PMS2,MSH2,MSH6)与结直肠癌中P-糖蛋白(Pgp)、谷胱甘肽-S-转移酶(GSTπ)、DNA拓扑异构酶Ⅱ(TopoⅡ)、细胞增殖抗原Ki-67免疫组化表达关系。方法：回顾性分析2014年2月至2015年12月我院收治的93例结直肠癌患者的临床病理资料,采用免疫组织化学法,检测癌组织中错配修复基因(MLH1,PMS2,MSH2,MSH6)、Pgp、GSTπ、TopoⅡ、Ki-67的表达,分析其表达特点。采用多个样本率(或构成比)的比较(即：R×C表的χ2检验),分析结直肠癌错配修复基因(MLH1,PMS2,MSH2,MSH6)与临床病理的关系及其与Pgp、GSTπ、TopoⅡ、Ki-67免疫组化表达关系。利用Pearson相关分析法分析MLH1、PMS2、MSH2、MSH6与Ki-67表达相关性。结果：错配修复基因MLH1、PMS2、MSH2、MSH6表达缺失阳性率分别占14.0%、17.2%、10.8%、11.8%,Pgp(?)、Pgp (+)、Pgp (++)、Pgp (+++)表达率分别占3.2%、25.8%、51.6%、19.3%。GSTπ(?)、GSTπ(+)、GSTπ(++)、GSTπ(+++)表达率分别占3.3%、16.7%、56.7%、23.3%。TopoⅡ阴性表达及IV级表达未见,TopoⅡⅠ级、Ⅱ级、Ⅲ级表达率分别占19.3%、77.4%、3.2%。Ki-67表达<10%(+)、10-50%(+)、>50%(+)分别占1.3%、25%、50%。错配修复基因MLH1、PMS2、MSH2、MSH6缺失表达与患者的性别、年龄、病理分化、TNM分期无统计学差异(P>0.05)。MSH2缺失表达与发病部位无统计学差异(P>0.05)。MLH1、PMS2、MSH6基因缺失表达与发病部位有一定差异性(P<0.05),发生左半结肠的缺失表达阳性率分别为：10.7%、10.7%、7.1%,右半结肠缺失表达阳性率分别为28.6%、35.7%、25%,直肠缺失表达阳性率为5.4%、8.1%、5.4%。MLH1、PMS2、MSH2、MSH6缺失表达与Pgp、GSTπ、TopoⅡ的表达无统计学差异(P>0.05)。MLH1、PMS2、MSH2、MSH6缺失表达与Ki-67表达有一定差异性(P<0.05), Ki-67<10%(+)的MLH1、PMS2、MSH2、MSH6缺失表达率为62.5%,Ki-6710%~50%(+)的MLH1、PMS2、MSH2、MSH6缺失表达率分别为21.1%、0、10.5%、5.3%,Ki-67>50%(+)的MLH1、PMS2、MSH2、MSH6缺失表达率分别为6.1%、4.1%、8.2%、6.1%。MLH1、PMS2、MSH2、MSH6缺失表达与Ki-67表达呈负相关(相关系数分别为r=?0.969、r=?0.464、r=?0.143、r=?0.344, P<0.05)。结论：错配修复基因MLH1、PMS2、MSH6基因缺失表达发生右半结肠几率相对较高,其次依次是左半结肠、直肠。MLH1、PMS2、MSH2、MSH6缺失表达与Ki-67表达呈负相关。     

人错配修复基因hMLH1研究的新进展

人错配修复基因(mismatch repair,MMR)的主要功能是对DNA链中因某些原因造成的错误配对进行修复.目前已知MMR主要有hMLH1、hMSH2、hMSH6、hPMS2等.它们能够识别和修复在DNA复制过程中因插入、缺失或单核苷酸突变形成的错配,从而大大减低基因组微卫星不稳定性(MSI),维持基因组的稳定性.     

一例大片段重复变异导致血友病A的分析

目的:报告1例由罕见变异导致的重型血友病A(hemophilia A,HA),并探讨大片段重复变异的致病机制。方法:先后进行 F8基因第22内含子及第1内含子倒位检测、Sanger测序以及多重连接探针扩增(multiple ligation-dependent probe amplification,MLP...     

STK15基因单核苷酸多态性与结直肠癌风险的Meta分析

目的 综合评估STK15基因单核苷酸多态性与结直肠癌易感性的关系.方法 以"STK15"、"polymorphism"、"aurora-A"、"colorectal cancer"、"colorectal carcinoma"、"结直肠癌"、"STK15基因"和"基因多态性"等为主题词检索Pubmed、EMbase、S...     

A modified multiplex PCR assay for detection of large deletions in MSH2 and MLH1

A method for detection of large genomic deletions in the MSH2 and MLH1 genes based on multiplex PCR and quantitative evaluation of PCR products is presented. All 35 exons of MSH2 and MLH1 were screened simultaneously in seven PCR reactions, each of them including primers for both genes. The method is reliable for uncovering large genomic deletions in patients suspected of HNPCC. With this method, six novel deletions were identified, two in MSH2: EX1_10del and EX1_16del (representing deletion of the entire MSH2 gene); and four in MLH1: EX1_10del in two unrelated patients, EX3_5del, and EX4del. The deletions were detected in 18 unrelated patients in whom no germline mutation had been identified by SSCP and DHPLC. These results indicate that our modified multiplex PCR assay is suited for the detection of large deletions both in the MSH2 and MLH1 gene and therefore represents an additional valuable tool for mutation screening in HNPCC families.     

DNA错配修复基因MSH2和MLH1单核苷酸多态性与食管癌发生风险相关性研究

目的:探讨DNA错配修复基因MSH2和MLH1单核苷酸多态性对于食管癌易感性的潜在作用。方法:采用医院为基础的病例-对照研究方法,应用PCR-RFLP检测包括正常对照132例,食管癌患者169例MSH2c.2063TG和MLH1IVS14-19AG两个基因多态性位点的基因型。通过Logistic回归分析计算出比值比(OR)和95%置信区间(95%CI),估计不同基因型频率分布与食管癌发生风险的关系。结果:MSH2c.2063TG携带突变等位基因个体发生食管癌的风险是非携带者的3.24倍。MLH1IVS14-19AG突变等位基因携带者发生食管癌风险是非携带者的1.58倍。对MSH2和MLH1基因交互作用分析发现两突变基因型携带者发生食管癌风险大大增加并具有显著的统计学意义。结论:DNA错配修复基因MSH2c.2063G突变等位基因和MLH1IVS14-19G突变等位基因可能在促成食管癌发生过程起到一定作用。     

MLH1、MSH2基因 mRNA突变分析与遗传性非息肉性结直肠癌的基因诊断

目的检测胚系MLH1和MSH2基因mRNA突变，确立遗传性非息肉性结直肠癌（hereditary nonpolyposis colorectal cancer，HNPCC）家系。方法收集符合Amsterdam标准Ⅱ的12个家系14名家庭成员外周血，用特异引物和耐热性逆转录酶特异地逆转录MLH1和MSH2的RNA；利用长模板PCR扩增酶扩增逆转录产物（cDNA）；测序分析扩增产物。提取外周血的DNA，设计与利用上述方法检测出突变对应外显子的特异性引物，利用Taq DNA聚合酶扩增测序，以检测上述方法的有效性。结果利用基于外周血mRNA的方法，在6个家系中检出6个胚系突变，4个MLH1突变和2个MSH2突变，MLH1突变分别位于第8、12、16和第19外显子；MSH2突变分别位于第1和第2外显子。利用基于外周血DNA的方法，上述突变均在MLH1和MSH2相应的外显子中得到验证。突变类型为4个错义突变、1个同义突变和1个非编码区突变；其中5个突变国际上尚未报道；6个突变中有5个为病理性，分布于5个不同家系，该5个家系被确诊为HNPCC家系。结论基于外周血MLH1和MSH2 mRNA异常的检测能确诊HNPCC家系；该方法敏感、省时、节约成本。     

两个遗传性非患肉性结直肠癌家系中MLH1和MSH2基因的突变检测

目的 确定两个遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的致病基因,选择MLH1基因和MSH2基因进行突变检测.方法 采用聚合酶链反应结合DNA直接测序法,对两个遗传性非息肉性结直肠癌家系的患者进行MLH1基因和MSH2基因的突变检测;发现变异后,采用PCR-限制性片段长度多态性或直接测序法鉴定此变异是否属于突变.结果 在家系A的患者中发现了位于MLH1基因第3外显子内的新突变c.243_244 insA;在家系B的患者中发现了MSH2基因第7外显子内的c.1215_1218dupCCGA突变,这两个突变都导致了编码蛋白的提前终止.结论 MLH1基因的c.243_244insA突变和MSH2基因的c.1215_1218dupCCGA突变分别是导致家系A和家系B发生遗传性非息肉性结直肠癌的致病突变.     

Expression of DNA mismatch repair gene MSH2 in cytological material from lung cancer patients

Mismatch repair genes encode for proteins responsible for the correction of bases incorrectly paired in the DNA. Loss of DNA mismatch repair activity has been associated with various cancers including tumors of the lung. In the present study, we have analyzed by immunocytochemistry the expression of MSH2 DNA repair gene in cytological material obtained by fine needle aspiration from a panel of 42 primary lung cancer patients. Specimens included 13 adenocarcinomas, 11 small cell carcinomas and 18 squamous cell carcinomas. Loss of expression or low expression was detected in 6 out of 13 (46%) adenocarcinomas and in 7 out of 18 (39%) of squamous cell carcinomas, although all 11 small cell carcinomas expressed MSH2. Our results suggest that loss of MSH2 expression is frequent in nonsmall cell carcinomas of the lung (P < 0.01, chi2 test). Evaluation of MSH2 expression can be applied for the screening of cytological material from fine needle aspirations from the lung.     

Seven novel MLH1 and MSH2 germline mutations in hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent hereditary form of colorectal cancer and is caused by germline mutations in mismatch repair (MMR) genes. The majority of mutations occur in MLH1 and MSH2. We report hereby seven novel germline mutations in these two genes (five in MLH1 and two in MSH2). All mutations have been found in families fulfilling criteria of the Bethesda guidelines and four of which also fulfilled the Amsterdam criteria. We identified three insertions or deletions of 1 bp leading to premature stop codons (MLH1: c.341delC, c.1413‐1414insA; MSH2: c.1119delG) and three nonsense mutations (MLH1: c.67G>T [E23X], c.436C>T [Q146X]; MSH2: c.1857T>G [Y619X]). The corresponding tumors showed a high level of microsatellite instability (MSI‐H) and a complete loss of expression of the affected protein. In addition, a missense mutation in MLH1 was identified (c.1984A>C [T662P]). The respective tumor also showed a high level of microsatellite instability but a reduced, rather then lost, expression of the MLH1‐protein. This missense mutation was not found in 107 healthy control individuals and in 54 HNPCC patients. © 2001 Wiley‐Liss, Inc.     

Large genomic aberrations in MSH2 and MLH1 genes are frequent in Chinese colorectal cancer

Hereditary nonpolyposis colorectal cancer is caused by inactivating mutations in the genes of the DNA mismatch repair (MMR) system. Studies have shown that large-fragment aberrations in MMR genes are responsible for a considerable proportion of hereditary colorectal cancer (CRC), but it has been rarely reported in Chinese patients. Here we used multiplex ligation-dependent probe amplification to analyze the genomic rearrangements of 45 Chinese hereditary CRC families, 20 young-age CRC patients (onset of CRC at younger than 50 years and no family history), and 13 patients with sporadic CRC diagnosed at age 50 years or older. Overall, we found 9 (13.8%) large genomic deletions or duplications: 7 out of 45 CRC patients with family history and 2 out of 20 young CRC patients. In all alterations, five genomic deletions were uncovered in the MSH2 gene, as well as one deletion and three duplications in the MLH1 gene. Furthermore, two of the duplications unveiled in this study may have more than a four-copy increase of the exon showing duplication in MLH1. The results indicate that genomic aberrations, large-fragment deletions and duplications, in both MSH2 and MLH1 genes play a role in the pathogenesis of Chinese CRC patients with a family history, as reported in western populations. Moreover, the genomic aberrations in these genes might also be a frequent cause of CRC at a young age in China.     

Four new mutations in the DNA mismatch repair gene MLH1 in colorectal cancers with microsatellite instability

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with inherited mutation in one of four DNA mismatch repair genes. Somatic mutations in the same genes are also found in a subset of sporadic colorectal cancers. A defect in DNA mismatch repair results in an RER (replication error) tumor phenotype. We screened 110 archival and 11 prospectively acquired colorectal cancers for the RER phenotype. A total of 22 cancers were RER-positive. RER-positive tumors were investigated for mutations in the DNA mismatch repair gene MLH1 using single-strand-conformation-polymorphism (SSCP) analysis. We identified four previously undescribed mutations in four different samples. Three mutations were exonic: a point mutation at codon 69 (AGG→AAG [arg→lys]); a single base pair deletion at codon 42/43 (GCAAAATCC→GCAAATCC) leading to a new stop codon downstream; and a point mutation at codon 757 (TAA→TAT) [termination→tyr] which extend the MLH1 peptide by 36 amino acids. The fourth mutation was a 1 base pair insertion six base pairs 5′ to the start of exon 14 (tttgtttt→tttggtttt). The mutations were not seen in the patients' constitutional DNA. The somatic MLH1 mutations identified appear to be causally associated with the RER phenotype. Hum Mutat 12:73, 1998. © 1998 Wiley-Liss, Inc.     

MLH1 and MSH2 protein immunohistochemistry is useful for detection of hereditary non-polyposis colorectal cancer in young patients

AIMS: Hereditary non-polyposis colorectal cancer is related to germline mutations of DNA mismatch repair genes MLH1 and MSH2, which result in microsatellite instability and loss of protein expression of the corresponding mutated gene in the tumour tissue. METHODS AND RESULTS: MLH1 and MSH2 protein expression was studied by immunohistochemistry in paraffin-embedded surgical samples of 100 colorectal adenocarcinomas occurring before 50 years of age. Absence of tumour cell nuclear staining with positive internal control (normal mucosa, lymphoid follicles) was considered negative. Loss of MLH1 or MSH2 expression was found in 20 cases with microsatellite instability in 15 cases. Twelve of these patients had a family history of colorectal cancer. Compared with MLH1- and MSH2-positive cases, MLH1- or MSH2-deficient colorectal adenocarcinomas were significantly associated on multivariate analysis with a younger age (38 vs. 43 years, P;0.0224), a larger tumour size (60 +/- 6 vs. 46 +/- 2 mm, P=0.0291), an expanding margin (85% vs. 51%, P=0.0159), a higher number of tumour-infiltrating lymphocytes assessed by CD3 immunostaining (202 +/- 48 vs. 33 +/- 4 CD3+ lymphocytes/10 high-power fields, P=0.0039), and a grade 2 Crohn's like lymphoid reaction (70% vs. 9%, P=0.0037). The two groups were not different for tumour site, differentiation, pTNM stage, vascular and perineural invasion, peripheral adenomatous residue, and 5-year survival rates. CONCLUSIONS: MLH1- or MSH2-deficient colorectal carcinomas of young patients exhibit pathological and molecular features similar to hereditary non-polyposis colorectal cancer. This suggests that MLH1 and MSH2 immunohistochemistry is valuable for detecting hereditary non-polyposis colorectal cancer in young patients.