聚合酶链式反应-单链构象多态性分析法检测大肠腺瘤及大肠癌APC基因突变

目的对１５例散发性大肠腺瘤组织和４５例大肠癌组织及其各自的正常肠粘膜组织，进行家族性腺瘤息肉病基因（ａｄｅｎｏｍａｔｏｕｓｐｏｌｙｐｏｓｉｓｃｏｌｉ，ＡＰＣ）第１５外显子ＭＣＲ区域的基因突变检测。方法应用聚合酶链式反应单链构象多态性分析（ＰＣＲＳＳＣＰ）技术进行检测。结果在散发性大肠腺瘤组织中检出突变率为２０．０％（３／１５），在大肠癌组织中的突变率为２２．２％（１０／４５）。实验结果表明，ＡＰＣ基因在散发性大肠腺瘤与大肠癌中均有较高突变频率，两者差异无显著性；ＡＰＣ基因突变在大肠癌中与肿瘤的大小、位置、组织分化程度和有无淋巴结转移无关；ＡＰＣ基因第１５外显子的ＭＣＲ区域是该基因突变的集中区域，且以１３３９～１４３６密码子区域突变率最高，１２６０～１３５９密码子区域突变率最低。结论证实了ＡＰＣ基因突变发生于腺瘤形成阶段，属于大肠癌发生的早期事件。     

聚合酶链式反应—单链构象多态性分析法检测大肠腺瘤及大肠癌APC …

目的 对１５例散发性大肠腺瘤组织和４５例大肠癌组织及各自的正常肠粘膜组织,进行家族性腺瘤肉病基因（ａｄｅｎｏｍａｔｏｕｓｐｏｌｙｐｏｓｉｓ,ｃｏｌｉ,ＡＰＣ第１５外显子ＭＣＲ区域的基因突变检测。方法 应用聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）技术进行检测。结果 在散发性大肠腺瘤组织中检出突变率为２０．０％（３／１５）在大肠癌组织中的突变率为２２．２％（１０／４５）。实验结果表明,ＡＰ     

国人大肠肿瘤与DCC基因mRNA表达缺失的相关性研究

应用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）技术检测了３４例大肠肿瘤及其相应正常粘膜中ＤＣＣ基因ｍＲＮＡ表达情况。结果：５例大肠腺瘤和２９例大肠癌组织中分别有１例（２０％）、１５例（５１．７％）ＤＣＣ基因ｍＲ－ＮＡ表达发生缺失。其中高、中、低分化的大肠癌组织中该基因ｍＲＮＡ表达缺失率分别为２５．０％（２／８）、４４．４％（４／９）、７５．０％（９／１２）（Ｐ＜０．０５）。而有转移者为７６．９％（１０／１３），无转移者３１．２％（Ｐ＝０．０１８）。ＤＣＣ基因ｍＲＮＡ表达缺失与性别、肿瘤大小及部位无关。提示：对大肠癌原发病灶ＤＣＣ基因ｍＲＮＡ表达的检测将有助于识别高度恶性的大肠癌及判断大肠癌的转移潜能。     

大肠癌的p53基因点突变

作者以改良的ＰＣＲ－ＳＳＣＰ的ＰＣＲ直接测序方法，分析了２２例大肠癌原发灶和１例转移淋巴结组织中ｐ５３基因第七外显子的基因结构变化。发现２７％（６／２２）大肠癌原发灶和检测的１例转移淋巴结中存在ｐ５３基因第七外显子点突变，突变位点分别为２４５、２５１、２５９和２６０密码子。５０％点突变是２４５密码子中碱基Ｇ∶Ｃ向Ａ∶Ｔ转换，其它突变形式是碱基插入和碱基缺失。本研究ｐ５３点突变阳性标本均为结肠癌，其中低分化腺癌突变率高于高、中分化腺癌（Ｐ＝０．０１７８），ＤｕｋｅｓＣ１期突变率高于ＤｕｋｅｓＡ２、Ｂ期（Ｐ＝０．０３６１）。此结果提示，临床检测大肠癌原发灶和转移淋巴结中是否存在ｐ５３基因第七外显子突变，可有助于识别高度恶性的大肠癌和判断大肠癌病员的预后。     

hMLH1基因Val384Asp的病因学作用及对大肠癌发病年龄的影响

目的：探讨ｈＭＬＨ１基因错义突变Ｖａｌ３８４Ａｓｐ在大肠癌发病中的作用。方法：取１０１例大肠癌患者、１００例正常对照的血细胞或正常大肠组织样本,提取基因组ＤＮＡ,ＰＣＲ－ＳＳＣＰ和ＤＮＡ测序技术分析ｈＭＬＨ１基因第１２外显同龄对照组（ｐ〈０．０５）,其中３５～４４岁年龄组的大肠癌患者与正常对照比较,ｐ〈０．０１。结论：ｈＭＬＨ１基因Ｖａｌ３８４Ａｓｐ将影响大肠癌的发病年龄,它的存在可能是中国人大肠     

大肠癌组织中抑癌基因APC突变的研究

为了解肿瘤抑制基因ＡＰＣ基因在大肠癌中突变的规律。方法应用聚合酶链反应┐单链构象多态性技术（ＰＣＲ┐ＳＳＣＰ）对３０例人大肠癌组织ＡＰＣ基因１５号外显子的三个区１５┐６、１５┐７、１５┐８进行了检测。检测步骤主要包括人基因组ＤＮＡ提取、ＰＣＲ反应扩增及ＰＣＲ产物的ＳＳＣＰ分析。结果检查结果显示在第１５┐６及１５┐７片段上共１０例发生了突变，突变率为３３．３％。突变的１０例中有８例发生于１５┐７外显子片段，占突变总数８０％（８／１０），２例发生于１５┐６片段，占突变总数的２０％（２／１０）。结论证明我国大肠癌患者中存在着ＡＰＣ基因的突变，且１５┐７片段是突变集中区。     

散发性大肠癌APC基因突变研究

ＡＰＣ基因不仅引起结肠腺瘤性息肉病，而且还是参与散发性大肠肿瘤形成的一重要抑癌基因。由于ＡＰＣ突变多位于第１５个外显子的５‘端，且９５％的突变产生终止密码，故本研究采用体外转录翻译系统来检测散发性大肠癌ＡＰＣ外显子１５的体细胞突变。     

大肠肿瘤基因调控模式初探

大肠肿瘤是一个典型的肿瘤遗传学多步骤模式。多个基因的变异发生在大肠粘膜,粘膜组织按顺序发展为过度增生、腺瘤、原位癌、最后形成浸润癌。参与的基因包括ｐ５３、ｒａｓ、ＡＰＣ、ＭＣＣ、ＤＣＣ、ＲＥＲ等,它们在肿瘤发展的不同阶段起不同的作用。本项研究从ｃｙｃｌｉｎＤ１、ｂｃｌ－２、ｐ５３、ｐ２１表达入手,探讨大肠肿瘤的基因调控模式。１材料与方法１．１标本来源１９９４～１９９８年白求恩医科大学中日联谊医院外科大肠肿瘤手术切除标本５８例,置－８０℃保存。其中大肠良性疾病（包括炎性息肉、腺瘤）５例,恶变患者９…     

大肠癌组织及结肠癌细胞系中FHIT基因的异常表达

目的：探讨ＦＨＩＴ基因与大肠癌的关系。方法：采用ＲＴ－ＰＣＲ及ＰＣＲ产物直接测序法,对３０例大肠癌组织及其配对正常组织,４个结肠癌细胞系的ＦＨＩＴ基因进行检测。结果：在８例（２６．７％）大肠癌组织和２个（５０％）结肠癌细胞系中检测到异常ＦＨＩＴ转录本,配对正常组织均无异常ＦＨＩＴ转录本;测序证实异常转录本缺失１￣３个ＦＨＩＴ外显子。异常ＦＨＩＴ转录本与大肠癌患者的年龄,性别、血清ＣＥＡ水平、肿瘤部     

大肠癌中DCC基因201密码子点突变的研究

目的：探寻结直肠癌缺陷基因（ＤＣＣ基因）２０１密码子在大肠癌中的突变规律。方法：采用等位基因特异性ＰＣＲＡＳ－ＰＧＲ结合ＳａｌⅠ酶切方法检测３５例大肠癌组织及配对的癌旁粘膜ＤＣＣ基因２０１密友子突变情况。结果：ＤＣＣ基因２０１密码子纯合突变率大肠癌（４０％）显高于癌旁粘膜（２．８％）,（Ｐ〈０．０５）。且与肿瘤侵袭深度、Ｄｕｋｅｓ分期相关,至少有１７例（４９％）大肠癌与相应癌旁粘膜相比增获一个密     

Two Chinese pedigrees for adenomatous polyposis coli: new mutations at codon 1309 and predisposition to phenotypic variations

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease caused by a mutation in the adenomatous polyposis coli (APC) gene. Some studies have attempted to correlate mutations at codon 1309 with classic FAP (≥100 colorectal polyps). We report two Chinese FAP pedigrees with new frameshift mutations at codon 1309, in which affected individuals manifest phenotypic variations. Comprehensive physical examinations were performed for all living individuals and the medical data of deceased patients were collected. Screening of the APC and human mutY homolog (MUTYH) genes for germline mutations was conducted by direct polymerase chain reaction (PCR) sequencing. In two pedigrees, a heterozygous deletion in exon 16 of the APC gene was present in all FAP patients but absent in the unaffected individuals. There were no changes to the MUTYH gene. The first pedigree, with a new frameshift mutation at c.3926_3930 del AAAAG (p. Glu1309Aspfs X4), exhibited obvious differences in the polyp number such that the proband manifested only three colorectal polyps, whereas another patients showed the symptoms of classic FAP. The second pedigree, also traced a new mutation at c.3922_3925 del AAAG (p. Glu1309Argfs X11). Although all of the patients presented with classic polyposis, one of them exhibited a delayed onset of colorectal cancer in his 50s. Two novel mutations at codon 1309 in two Chinese families suffering from FAP could enrich the germline mutation spectrum of the APC gene. Families of individuals might manifest different phenotypes, even with an identical codon 1309 mutation, unlike in previous studies.     

Difference in the role of loss of heterozygosity at 10p15 (KLF6 locus) in colorectal carcinogenesis between sporadic and familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer patients

OBJECTIVES: To clarify the role of the KLF6 (Kruppel-like factor 6) locus in multistep colorectal carcinogenesis, we analyzed loss of heterozygosity (LOH) at 10p15 (KLF6 locus) and mutations of the KLF6gene in 298 colorectal tumors at various pathological stages of sporadic and familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) patients. METHODS: 10p15 LOH was analyzed using KLF6M1 and KLF6M2, and KLF6 gene mutation was analyzed using PCR-SSCP and sequencing. RESULTS: It was found that the frequencies of LOH (sum of M1 and M2) were 4% in adenomas, 0% in intramucosal carcinomas, 35% in invasive carcinomas, and 33% in liver metastases in sporadic cases. Invasive carcinomas from FAP patients showed only 6% LOH, and invasive carcinomas from HNPCC patients exhibited 0% LOH. Mutation analysis of the KLF6 gene in 298 colorectal tumors detected no somatic mutations. CONCLUSIONS: The present data suggest that LOH of the KLF6 locus at chromosome 10p15 contributes to the invasion step from an intramucosal carcinoma to an invasive carcinoma specifically in sporadic colorectal carcinogenesis, but is rarely involved in the carcinogenesis of FAP and HNPCC cases. Moreover, the absence of somatic mutations suggests the uncertainty of the KLF6 gene as a classical tumor suppressor gene at the lost 10p15 region in colorectal carcinogenesis.     

Cell and molecular biology of gastrointestinal tract cancer.

The gene for familial adenomatous polyposis coli (APC or FAP), which has previously been linked to chromosome 5q21 has been identified. The APC gene has been found to be altered by point mutations in the germ line of both adenomatous polyposis coli and Gardner's syndrome patients and somatically in tumors from sporadic colorectal cancer patients. During the hunt for the APC gene, the closely linked MCC (mutated in colorectal cancer) gene was identified and found to be altered somatically in tumors from sporadic cancer patients. These data suggest that more than one gene on chromosome 5q21 may contribute to colorectal carcinogenesis and that mutations at the APC gene can cause both adenomatous polyposis coli and Gardner's syndrome. The identification of these genes should aid in the counseling of patients with genetic predispositions to colorectal cancer. Progress has also been made in identifying specific genetic changes that occur in other gastrointestinal cancers. A mutational "hotspot" in the p53 gene in human hepatocellular carcinomas has been identified that could reflect exposure to a specific carcinogen, one candidate being aflatoxin B1.     

Difference in characteristics of APC mutations between colonic and extracolonic tumors of FAP patients: variations with phenotype

To clarify the differences in characteristics of adenomatous polyposis coli (APC) mutations between colorectal tumors from various phenotypes of familial adenomatous polyposis (FAP) and between colorectal and extracolonic tumors, we analyzed APC mutations in 86 colorectal tumors from FAP patients including profuse, sparse and attenuated types, 23 sporadic colorectal tumors and 40 FAP extracolonic tumors including duodenal, gastric and desmoid tumors. In all tumors, 1 allele of the truncated APC gene retained armadillo repeats, 15-amino-acid (aa) repeats and various numbers of 20-aa repeats, but lacked SAMP repeats, as a result of germline and somatic mutations. Regarding 20-aa repeats, the truncated APC gene retained 1 repeat due to allele loss in 96% (27/28) of colorectal tumors from profuse-type FAP, 69% (36/52) of sparse-type retained 2 repeats due to somatic mutation, and 100% (6/6) of attenuated-type retained 2 or 3 repeats due to the third or second hit. In sporadic colorectal tumors 74% (17/23) retained 1 or 2 repeats. The truncated APC gene retained 3 repeats in 88% (7/8) of FAP duodenal tumors, 100% (26/26) of gastric tumors retained 2 or 3 repeats and 83% (5/6) of desmoid tumors retained 2 repeats. These data suggest that the number of beta-catenin downregulating 20-aa repeats in truncated APC gene associated with colorectal tumorigenesis is different in profuse, sparse and attenuated types of FAP, and that the association with tumorigenesis is also different between colorectal and extracolonic tumors.     

The relationship between frequencies of extracolonic manifestations and the position of APC germline mutation in patients with familial adenomatous polyposis

BACKGROUND: Familial adenomatous polyposis (FAP) patients develop various extracolonic lesions; however, the relationship between germline mutation of the APC gene and extracolonic manifestations is mostly unknown. To examine the genotype-phenotype relationship, we compared the APC mutation and clinical data. METHODS: Germline mutations from codon 157 to 1465 of the APC gene were identified in 39 families of FAP and clinical data were collected from 80 patients of these families. Germline mutations were classified into two groups: mutations from exon 4 to 9 (codon 157 to 416, Group 1) and those from exon 10 to 15H (codon 564 to 1465, Group 2). The complication rates of extracolonic manifestations were compared between these two groups. RESULTS: Frequencies of duodenal polyps and gastric adenomas in Group 2 were higher than those in Group 1 (p < 0.0001 and p < 0.0004, respectively) and development of osteoma was more frequent in Group 2 (p = 0.01). The number of colorectal polyps and retinal pigments also correlated with the germline mutation, which was consistent with previous reports. However, such correlations were less obvious with regard to gastric fundic polyps, desmoid tumors, soft tissue tumors and colorectal cancer. CONCLUSION: There are two types with regard to extracolonic manifestations of FAP: one is more severely affected according to the position of germline mutation of the APC gene and the other is not affected.     

Mutations of the PIK3CA gene in hereditary colorectal cancers

Somatic mutations of the PIK3CA gene have recently been detected in various human cancers, including sporadic colorectal cancer. However, mutations of the PIK3CA gene in hereditary colorectal cancers have not been clarified. To elucidate the mutation status in familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), which are the most common hereditary colorectal cancers, we investigated PIK3CA mutations in 163 colorectal tumors, including adenomas, intramucosal carcinomas and invasive carcinomas. For comparison, we also analyzed mutations of the same gene in 160 sporadic colorectal tumors at various histopathological stages. Analysis at exons 1, 7, 9 and 20 of the PIK3CA gene revealed somatic mutations in 21% (8 of 39) of FAP invasive carcinomas, 21% (7 of 34) of HNPCC invasive carcinomas, 15% (8 of 52) of sporadic invasive carcinomas, and 14% (7 of 50) of sporadic colorectal metastases in the liver. Mutations in FAP and HNPCC carcinomas predominantly occurred in the kinase domain (exon 20), while the majority of mutations in sporadic cases occurred in the helical domain (exon 9). Adenomas and intramucosal carcinomas from all patients exhibited no mutations (0 of 148). Our data suggest that PIK3CA mutations contribute to the invasion step from intramucosal carcinoma to invasive carcinoma in colorectal carcinogenesis in FAP and HNPCC patients at a similar extent to that seen in sporadic patients.     

Refining the relation between 'first hits' and 'second hits' at the APC locus: the 'loose fit' model and evidence for differences in somatic mutation spectra among patients

The site of the 'first hit' in the APC tumour suppressor gene determines the type of the 'second hit', both in familial adenomatous polyposis (FAP) and sporadic colorectal tumours. Mutations near codon 1300 are associated with loss of heterozygosity (LOH) of the wild-type allele; other tumours tend to have two protein-truncating mutations. In this study, we have confirmed and refined the LOH-associated region in colorectal FAP: allelic loss in adenomatous polyps tended to occur when the germline mutation lay in the region of the APC gene between the first and second beta-catenin degradation repeats (codons 1285-1378). LOH generally occurred by mitotic recombination, leaving two identical alleles, each encoding a protein with one remaining beta-catenin degradation repeat. For patients with germline mutations that truncated the protein before the first repeat (codon 1264), LOH was very rare and tumours generally acquired a somatic mutation which left two, or less often one, repeats remaining in the protein. In our sample set, patients with germline mutations after the second beta-catenin degradation repeat tended to have undetectable, presumably cryptic, somatic mutations in their polyps. Exceptions to these rules were, however, not uncommon. Although the site of the germline mutation was the strongest determinant of the somatic mutation in FAP tumours and most patients showed no clear tendency to acquire specific types of truncating 'second hit', a minority of patients did have unusual somatic mutation spectra in their polyps. Thus, some individuals may be predisposed to particular types of 'second hit' (for example, frameshift rather than nonsense changes). Overall, disease severity (polyp number) did not vary with individuals' spectrum of somatic APC mutations, providing no clear evidence for modifier genes that influence disease severity in this fashion. Our data are consistent with the hypothesis that there exists an optimal level of beta-catenin signalling in colorectal tumours and that the APC mutation spectrum principally reflects this fact. The association between 'first hits' and 'second hits' at APC is not, however, so strong as to suggest that tumorigenesis only occurs if the genotype is optimum; we suggest 'relaxed' terminology, the 'loose fit' model, to describe this situation.     

Correlation between the location of germ-line mutations in the APC gene and the number of colorectal polyps in familial adenomatous polyposis patients.

Recently we have isolated the adenomatous polyposis coli (APC) gene which causes familial adenomatous polyposis (FAP), and its germ-line mutations in a substantial number of FAP patients have been identified. On the basis of this information, we compared the location of germ-line mutations in the APC gene in 22 unrelated patients (12 of whom have been reported previously) with the number of colorectal polyps developed in FAP patients; 17 were sparse types and five were profuse types. All but one of the mutations were considered to cause truncation of the gene product by frame-shift due to deletion (14 cases) or nonsense mutation (seven cases). The location of the germ-line mutations seems to correlate with the two clinical types; germ-line mutations in five FAP patients with profuse polyps were observed between codon 1250 and codon 1464, whereas mutations in 17 FAP patients with fewer polyps were observed in the other regions of the APC gene. The result suggests that the number of colorectal polyps in FAP patients may be associated with a difference in the stability or biological function of the truncated APC protein.