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.     

PolyA deletions in hereditary nonpolyposis colorectal cancer: mutations before a gatekeeper

Microsatellite instability (MSI) secondary to loss of DNA mismatch repair (MMR) is present in adenomas and colorectal carcinomas from individuals with hereditary nonpolyposis colorectal cancer (HNPCC). To better characterize when MMR loss occurs during HNPCC progression, the extent of deletions in noncoding polyA sequences were compared between 6 adenomas (all < or = 1.0 cm in size) and 10 cancers. Numbers of deleted bases reflect time since loss of MMR because polyA deletions are stepwise. Adenoma deletions were nearly the same (85%) as the cancers with sum total deletions at four different polyA loci of -32.7 bases in adenomas and -38.4 bases in cancers. Intervals between negative clinical examinations and tumor removal (average of 2.1 years) were known for six tumors. There were no significant differences in the extent of deletions in tumors removed under clinical surveillance (-34.8 bases) versus tumors removed without prior negative examinations (-36.5 bases). These findings illustrate that MSI is extensive in both small adenomas, and tumors which appear after negative clinical examinations, consistent with an early loss of MMR in HNPCC, even before a gatekeeper mutation.     

A cost-effective algorithm for hereditary nonpolyposis colorectal cancer detection

Colorectal cancer with microsatellite instability (MSI) may occur sporadically or be inherited in cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. However, there is no consensus as to which patients must be tested and how to test MSI. In this study, MSI was tested by immunohistochemical analysis and by polymerase chain reaction in 148 cases of colorectal cancer, and methylation of the hMLH1 promoter was examined. MSI status was correlated with tumor phenotype. We found that localization, tumor infiltrating lymphocytes, and mucinous differentiation were predictive of high-frequency MSI (MSI-H) colorectal cancer and might be used to select cases for MSI analysis. Immunohistochemical analysis detected most MSI-H colorectal cancer and might constitute the first step in MSI detection. Absence of hMLH1 promoter methylation in MSI-H colorectal cancer could be predictive of hereditary colorectal cancer, and, hence, methylation analysis might constitute the second step in the identification of patients with HNPCC.     

遗传性非息肉性结直肠癌家系的MLH1基因两个胚系新突变

目的初步评价遗传性非息肉性结直肠癌（HNPCC）胚系MLH1基因突变中新突变的病理性。方法收集符合AmsterdamⅡ标准的12个不同家系的12例患者外周血,用特异引物和耐热性逆转录酶特异地逆转录MLH1的mRNA;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;利用PCR-Genescan技术和免疫组织化学染色分别检测有新突变患者肿瘤组织的5个微卫星位点（BAT26,BAT25,D5S346,D2S123和Mfd15）和MLH1蛋白的表达。结果在4例患者中检出4个MLH1突变,其中2个突变为第12外显子的第384密码子（1151bp处）GTT→GAT的突变,该突变是已报道的病理性突变;另外2个突变分别是第8外显子的第217密码子（649bp处）CGC→TGC突变和第16外显子的第581密码子（1742bp处）CC→CTG突变;后两者为尚未报道的新突变。2个新突变的患者肿瘤组织均呈高度微卫星不稳定性,两者的瘤组织MLH1蛋白均失表达。结论MLH1第8外显子的第217密码子突变和第16外显子的第581密码子的两个新突变很可能为病理性突变。     

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.     

一个遗传性非息肉性结直肠癌大家系调查及种系突变研究

目的探讨一个中国人遗传性非息肉性结直肠癌（heraditary nonpolyposis colorectal cancer，HNPCC）大家系的临床特点，报告基因突变筛查结果。方法调查一个HNPCC大家系，记录的数据包括患者性别，结直肠癌发生的部位，诊断年龄，是否具有同时和（或）异时结直肠癌及结肠外癌，肿瘤的组织病理特点等。抽取家族成员外周血，采用聚合酶链反应和扩增产物直接测序进行基因检测。结果该家系符合阿姆斯特丹Ⅰ标准，4代31人中17例患者共诊断21例次恶性肿瘤。12例（70．6％）患者患有直肠癌，且发病年龄早（平均42．9岁），右半结肠癌多见。基因检测发现一种国内外尚未见报道的MSH2基因的新突变。该突变位于MSH2基因的第7外显子中，由于4个核苷酸（CCGA）的重复导致移码突变，形成截短蛋白。结论HNPCC患者是恶性肿瘤（尤其是结直肠癌）的高发人群。新的MSH2基因突变（MSH2：C．1215-1218dupCCGA）导致该家系遗传性非息肉性结直肠癌的发生。     

中国汉族和韩国朝鲜族非患肉性结直肠癌家系临床及遗传学表型的比较分析

目的 分析和比较汉族和朝鲜族遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的临床及遗传学表型的异同点.方法 收集31个中国的汉族家系和63个韩国的朝鲜族家系先证者和家系成员的各项临床资料,对先证者外周血DNA进行相关基因hMLH和hMSH2的种系突变检测.应用聚合酶链反应-单链构象多态性分析或变性高效液相色谱法筛查突变,对结果异常的样本进行DNA测序.结果 31个汉族家系中共发生136例次恶性肿瘤,其中结直肠癌106例次,占所有肿瘤患者的77.9%,诊断年龄平均为(48.6±29.0)岁;其次为胃癌共14例.经突变检测,31例汉族先证者中有7例被检出含有hMLH1(3个)或hMSH2(4个)基因的病理性突变,总突变率为22.6%.其中错义突变2个、无义突变2个、移码突变2个、大片段缺失1个.63个朝鲜族家系中共发生293例次恶性肿瘤,其巾结直肠癌242例次,占所有肿瘤患者的82.6%,诊断年龄平均为(45.9±11.0)岁;胃癌同样也是第2大常见肿瘤类型,共发生21例.63例朝鲜族先证者中有19例被检测出含有hMLH1(17个)或hMSH2(2个)基因的突变,总突变率为30.2%.其中12个为移码突变,5个为错义突变,1个为无义突变,1个为剪接位点的碱基改变导致异常剪接.结论 (1)汉族与朝鲜族HNPCC家系在临床表现上相似,均有发病年龄轻、以远端结肠癌和直肠癌多见、多原发大肠癌发生率较两方国家低、肠外肿瘤以胃癌最多见等特点.(2)遗传表型方面,汉族与朝鲜族HNPCC家系的总突变率相似,但均低于西方国家的报道.两个种族的家系中的突变基因、突变类型和突变分布上存在差异及各自的特征.     

遗传性非息肉病性结直肠癌的分子生物学研究

目的：探索遗传性非息肉病性结直肠癌（HNPCC）基因突变规律。方法：用聚合酶链式反应和PCR-SSCP对9例HN-PCC患者及其家系成员4例政审对照的hMSH2、hHLH1基因进行检测。结果：9例来自不同家庭的患者中,7例出现电泳条带异常;4例来自上述家系的无症状成员,其中2例出现电泳条带异常。结论：聚合酶链式反应和PCR-SSCP联合应用,可用于 hMSH2、hMLH1基因突变的检测。     

Three new mutations in hereditary nonpolyposis colorectal cancer (Lynch syndrome II) in Uruguay

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common hereditary form of colorectal cancer (CRC). Our purpose is to describe three extended HNPCC families, each of which manifests novel germline mutations in Uruguay, a small country that is a study model for cancer investigation given its high cancer incidence and mortality rate. This is a study of three extended HNPCC families in which extensive genealogic information, medical history, and pathology findings are critically reviewed. DNA testing was performed for evidence of HNPCC mutations. The findings reveal three novel germline mutations, namely MLH1, with a deletion resulting in a frameshift and a premature stop codon (codon 228) in one of the families; in the second family, MSH2 exon 1, codon 61 at nucleotide 181, which results in immediate stop of translation; and in the third family, a mutation in MSH2 at exon 3: the amino acid at nucleotide 530, codon 117, causing a frameshift and a premature stop codon eight base pairs later. We conclude that it is important to study HNPCC mismatch repair genes because of emerging evidence for genotypic and phenotypic heterogeneity, which will harbor the potential to eventually translate this knowledge into specific screening and management protocols. Future projections for such mutations could even contribute to the emergence of molecular-based designer drugs developed through advances in genomics, proteomics, high-throughput screening, and bioinformatics, which would be effective therapeutically for these high-cancer risk patients.     

中国人遗传性非腺瘤病性结直肠癌家系hMSH2和hMLH1基因突变分析

目的 分析符合不同临床标准的中国遗传性非腺瘤病性结直肠癌(HNPCC)家系hMSH2和hMLH1基因种系突变状况,评价不同临床标准预示突变检测的敏感性。方法应用DNA直接测序对24个符合Amsterdam标准、15个符合日本标准家系先证者和19个符合Bethesda指导纲要患者(字系中仅1例患者)进行hMSH2和hMLH1基因种系突变检测。对检出突变的家系进行家庭成员的突变筛选。并对检出突变患者进行肿瘤组织突变的检测。结果在16例家系先证者中检测到6个hMSH2突变和11个hMLHl种系突变,其中12个突变是国际上尚未报道过的新突变。突变位于不同外显子中,其中6个突变位于hMLHl第14-16外显子。Amsterdam标准家系突变阳性率为50％(12／24),以日本标准所筛家系突变阳性率为3／15,以上两组家系以外的Bethesda指导纲要患者突变阳性率为1／19。突变类型包括移码突变、无义突变、剪接异常、框架内插入或缺失以及错义突变。基因突变与疾病共分离,检出突变家系先证者的肿瘤组织错配修复基因表现出3种不同基因型：(1)野生型等位基因丢失;(2)肿瘤组织基因型与生殖细胞一致;(3)突变型等位基因丢失。结论中国人HNPCC家系hMSH2和hMLHl突变谱广泛,突变类型多样,hMLHl突变较hMSH2突变多见,突变较为集中于hMLHl外显子14-16。不同临床标准预示突变的敏感性不同。突变基因型与疾病表现型共分离。家系成员中尚未发病的突变携带者应予密切监测。     

Germ line mutations of hMSH2 and hMLH1 genes in Japanese families with hereditary nonpolyposis colorectal cancer (HNPCC): usefulness of DNA analysis for screening and diagnosis of HNPCC patients

Mutations in hMSH2 and hMLH1 genes were analyzed in patients from 11 Japanese families that had been diagnosed as carrying hereditary nonpolyposis colorectal cancer (HNPCC) by clinical examination. Germ line mutations of hMSH2 gene were identified in 5 independent families in which colorectal (87% of patients), endometrial (30%), ovarian (17%), gastric (14%), and other cancers existed. Five mutations detected between codons 136 and 811 included single-base substitutions (CT and TG), a T deletion, and an A insertion, all of which produced stop codons resulting in truncated proteins, and an AT substitution at splice donor site of exon 5 which resulted in deletion of this exon. Moreover, one HNPCC family was presumed to have germ line mutation of hMSH2 gene because a somatic mutation of hMSH2 gene was detected in a cancer from a patient in this family. In addition to these 11 families already diagnosed with HNPCC, 3 new families with germ line mutations of hMSH2 gene and hMLH1 gene were found through analysis of DNA from patients who had multiple cancers with alteration in microsatellite DNA. These mutations included an AG deletion at codons 877–878 of hMSH2 gene, an AAG deletion at codons 616–618 of hMLH1 gene, and a CT single-base substitution at codon 217 of hMLH1 gene. Seven of eight germ line mutations found in this study are new mutations that have not been reported previously. In families in which germ line mutations were identified presymptomatic examination was then carried out using polymerase chain reaction single-strand conformation polymorphism analysis of DNA from peripheral blood, and the result was the detection of family members predisposed to HNPCC who did not yet show signs of cancer. These results indicate the value of DNA analysis in the screening and diagnosis of HNPCC patients and families.Abbreviations HNPCC Hereditary nonpolyposis colorectal cancer - PCR Polymerase chain reaction - SSCP Single-strand conformation polymorphism     

Evaluation of rapid microsatellite analysis of paraffin-embedded specimens in screening for hereditary nonpolyposis colorectal cancer.

Length alterations in short repetitive DNA sequences, termed microsatellite instability (MSI), are used as a diagnostic criterion of replication errors caused by various mutations in at least five mismatch repair genes. Therefore, MSI analysis is useful in clinical practice to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC). MSI can be detected by amplification of microsatellite loci in DNA extracted from paraffin-embedded tumor and corresponding peritumoral specimens after numerous time consuming steps limiting the clinical utilities. Rapid microsatellite analysis, a efficient and rapid DNA extraction technique based on Triton X-100 preincubation, was compared with the conventional DNA extraction for HNPCC screening in colorectal tumor specimens from 12 patients. Five complex and two noncomplex (CA)n microsatellite loci were tested, with use of multicolor fluorescent analysis. MSI and loss of heterozygosity in colorectal tumor samples could equally be assessed with the two DNA preparation methods, whereas the number of initially unsuccessful DNA extractions from paraffin-embedded tissue specimens and overall duration for MSI analysis were significantly reduced when rapid microsatellite analysis was used. A replication error-positive phenotype was detected in 2 of 10 patients with a positive family history for colorectal cancer, and diagnosis of HNPCC was finally confirmed by detection of a specific germline mutation. The described rapid microsatellite analysis is less time consuming and more efficient, and, in general, it reduces the risk of contamination by limiting the number of steps required. Therefore, it might replace current DNA extraction procedures. Furthermore, techniques using fluorescent polymerase chain reaction and semiautomated DNA sequencer allow for precise, observer-independent, and rapid scoring in MSI and loss of heterozygosity assessment. A combination of our rapid DNA extraction method and the use of a highly specific microsatellite marker might improve replication error analysis in HNPCC screening.     

Detection of Tn, sialosyl-Tn and T antigens in hereditary nonpolyposis colorectal cancer

The simple mucin-type carbohydrate antigens Tn, sialosyl-Tn, T and the cryptic sialylated variant of the last represent the mucin core oligosaccharide structures that are produced in the initial steps of the mucin biosynthetic pathway. Utilizing monoclonal antibodies anti-Tn antigen (HB-Tn1), anti-sialosyl-Tn antigen (HB-STn1), anti-T antigen (HB-T1) and the biotinylated Amaranthus caudatus agglutinin (ACA), we have investigated the expression of the simple mucin-type carbohydrate antigens in hereditary nonpolyposis colorectal cancer (HNPCC; 15 cases) compared with sporadic colorectal cancer (CRC; 60 cases) and normal colonic mucosa (30 cases). A variable positivity of Tn, sialosyl-Tn, T and the cryptic sialylated form of this latter antigen was encountered in both HNPCC and sporadic CRC cases; in addition, in normal colonic mucosa a constant reactivity was encountered only for Tn and the cryptic sialylated form of T, while negative results were always obtained for sialosyl-Tn and T antigens. Statistical analysis, performed using a Chi-square test, showed significantly lower (P=0.037) expression of sialosyl-Tn and higher (P=0.022) expression of T in HNPCC than in sporadic CRC, suggesting a greater presence of 1,3 galactosyl-transferase activity in HNPCC than in sporadic CRC. We were unable to identify a peculiar phenotype for HNPCC with simultaneous evaluation of reactivity for HB-Tn1, HB-STn1, HB-T1 and ACA; the biological significance of the preferential expression of T antigen in HNPCC remains to be investigated.     

Mean age of tumor onset in hereditary nonpolyposis colorectal cancer (HNPCC) families correlates with the presence of mutations in DNA mismatch repair genes

Fourteen Italian families affected with hereditary nonpolyposis colorectal cancer (HNPCC) were screened for germline mutations at three DNA mismatch repair (MMR) genes, MSH2, MLH1, and GTBP, by using a combination of different methods that included an in vitro synthesized protein assay, single-strand conformation polymorphism analysis, and direct sequencing. DNA alterations were observed in six instances, including a single base deletion in MSH2 exon 14, an A-to-G transition in the splice donor site of MLH1 exon 6, and two missense mutations in MLH1 exons 5 and 9. A previously reported common mutation affecting the splice donor site of MSH2 exon 5 was identified in two families. No mutations were detected in the GTBP gene. In total, eight of 16 Italian HNPCC families (50%), including two previously reported kindreds, were found to carry a mutation in MMR genes. We compared the mean age of colorectal cancer onset in the index cases (three patients for each family) between the two groups of kindreds, those with identified mutation vs. those without, and found that the first had a significantly lower value (43.0 vs. 53.7 years, P = 0.014). This finding suggests that HNPCC families with a more advanced age of tumor onset are less likely to be associated with known MMR genes. Genes Chromsom. Cancer 19:135–142, 1997. © 1997 Wiley-Liss Inc.     

An appendix carcinoid tumor in a patient with hereditary nonpolyposis colorectal cancer

Gastrointestinal carcinoid tumors are often associated with other tumors, particularly colon adenocarcinomas; but the association between carcinoid tumors and hereditary nonpolyposis colorectal cancer (HNPCC) syndrome has not yet been explored. We report an unusual case of a 28-year-old woman with HNPCC who underwent surgery for a transverse colon adenocarcinoma in whom an appendix carcinoid tumor was incidentally found. To assess whether the carcinoid tumor displayed the characteristic molecular features of HNPCC tumors, we investigated the expression of mismatch-repair (MMR) proteins and microsatellite instability (MSI) status in both tumors. Both tumors demonstrated normal expression of the MMR proteins hMLH1, hMSH2, hMSH6, and hPMS2. Interestingly, the adenocarcinoma exhibited an MSI phenotype but the carcinoid tumor did not, indicating that these 2 tumors arose through different molecular pathways.     

Unusual tumors associated with the hereditary nonpolyposis colorectal cancer syndrome.

The molecular pathogenesis of tumors outside the usual tumor spectrum for hereditary nonpolyposis colorectal cancer (HNPCC) is currently controversial. Specifically, it is not known whether these tumors are related to defects in DNA mismatch repair or arise independently of this defect in these patients. Here, we report two young patients, each with a known MSH2 mutation in the family, who developed rare tumors (adrenal cortical carcinoma and anaplastic carcinoma of the thyroid) that are not usually associated with HNPCC. Both of these patients were members of families that fulfilled modified Amsterdam (Amsterdam II) criteria for this familial cancer syndrome. Both the adrenal tumor and the thyroid tumor showed complete loss of immunohistochemical expression for MSH2 protein. Neither tumor was considered microsatellite instability-high following microsatellite instability analysis using the established National Cancer Institute panel of five microsatellite markers. To our knowledge, MSH2 defects in these types of tumors have not been previously reported in patients with the HNPCC syndrome. Our results suggest that microsatellite instability analysis using the National Cancer Institute panel of five microsatellite markers may not detect microsatellite instability in tumors that fall outside the usual tumor spectrum of this syndrome. Therefore, when analyzing unusual tumors in patients with known or suspected HNPCC syndrome, we advocate the performance of immunohistochemistry for mismatch repair gene products in addition to microsatellite instability analysis.