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.     

中国人遗传性非息肉病性结直肠癌MSH6基因胚系突变的测序研究

目的探讨中国人遗传性非息肉病性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系中MSH6基因胚系突变。方法采用PCR-直接测序的方法检测39个无胚系MSH2及MLH1基因突变、符合不同临床标准的中国人HNPCC家系先证者MSH6基因各外显子胚系突变;对137名正常人胚系基因组DNA进行错义突变相应外显子的测序分析。应用Envision二步法检测有突变的先证者肿瘤组织MSH6蛋白表达。结果在39个HNPCC先证者中共发现6个MSH6基因的胚系突变,分别位于第4、6、9和第10外显子;突变类型为4个错义突变、1个无义突变、1个剪接区的插入突变;对4个错义突变的相应外显子的测序分析显示:137名正常人胚系基因组DNA5例具有第6外显子1163密码子处的c.3488A>T的错义突变,约占3.65%(5/137),为单核苷酸多态性(single nucleotide polymorphism,SNP);其余错义突变在正常人群中均未发现。在6例有MSH6基因胚系突变家系的肿瘤组织中免疫组化染色除1例为SNP的肿瘤组织MSH6蛋白阳性表达外,其余均为阴性表达。经过查询国际HNPCC突变数据库及SNP数据库证实上述突变中5个为国际上尚未报道的病理性突变,1个为新发现的SNP。结论MSH6基因胚系突变在符合不同临床标准的中国人HNPCC中均起一定作用,对无MSH2及MLH1基因胚系突变的先证者行MSH6基因胚系突变的测序分析对确诊HNPCC家系是必要的。     

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。     

Partial duplications of the MSH2 and MLH1 genes in hereditary nonpolyposis colorectal cancer

Numerous reports have highlighted the contribution of MSH2 and MLH1 genomic deletions to hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch's syndrome, but genomic duplications of these genes have been rarely reported. Using quantitative multiplex PCR of short fluorescent fragments (QMPSF), 962 and 611 index cases were, respectively, screened for MSH2 and MLH1 genomic rearrangements. This allowed us to detect, in 11 families, seven MSH2 duplications affecting exons 1-2-3, exons 4-5-6, exon 7, exons 7-8, exons 9-10, exon 11, and exon 15, and three MLH1 duplications affecting exons 2-3, exon 4 and exons 6-7-8. All duplications were confirmed by an independent method. The contribution of genomic duplications of MSH2 and MLH1 to HNPCC can therefore be estimated approximately to 1% of the HNPCC cases. Although this frequency is much lower than that of genomic deletions, the presence of MSH2 or MLH1 genomic duplications should be considered in HNPCC families without detectable point mutations.     

Novel germline mutation (300-305delAGTTGA) in the human MSH2 gene in hereditary non-polyposis colorectal cancer (HNPCC)

Hereditary non-polyposis colorectal cancer (HNPCC) is a common hereditary syndrome characterized by the high incidence and early onset of colorectal cancer. The majority of the HNPCC families carry germline mutations in either the MSH2 or the MLH1 mismatch repair gene. A 46 year-old female patient whose family history fulfilled the Amsterdam criteria for HNPCC was diagnosed with undifferentiated adenocarcinoma of the transverse colon. Recognizing the Lynch 2 syndrome (the existance of multiple HNPCC related cancers in a pedigree), we used polymerase chain reaction followed by direct sequencing to screen the coding regions of both the MSH2 and the MLH1 genes for germline mutations in DNA from the patient. We detected a novel germline mutation (300-305delAGTTGA) in exon 2 of human MSH2. We noted microsatellite instability in four microsatellite loci. Immunohistochemistry showed a lack of expression of the MSH2 gene product in the tumor, suggesting that the mutation is a disease-causing mutation.     

Germline MSH2 and MLH1 mutational spectrum including large rearrangements in HNPCC families from Poland (update study)

Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 account for a significant proportion of hereditary non-polyposis colorectal cancer (HNPCC) families. One approach by which development of an efficient DNA-testing procedure can be implemented is to describe the nature and frequency of common mutations in particular ethnic groups. Two hundred and twenty-six patients from families matching the Amsterdam II diagnostic criteria or suspected HNPCC criteria were screened for MSH2 and MLH1 germline mutations. Fifty different pathogenic mutations were found, 25 in MSH2 and 25 in MLH1. Twenty-four of these had not previously been described in other populations. Among our 78 families with MSH2 or MLH1 mutations, 54 (69.2%) were affected by recurrent mutations including 38 found at least twice in our own series. Two of the most frequent alterations were a substitution of A to T at the splice donor site of intron 5 of MSH2 and a missense change (A681T) of MLH1 found in 10 and eight families, respectively. Among large deletions detected by the multiplex ligation-dependent probe amplification assay, exon 9 deletions in the MSH2 gene were found in two families. Our results indicate that a screening protocol specific for the Polish population that is limited to the detection of all reported mutations will result in the identification of the majority of changes present in MLH1 and MSH2 genes in Polish HNPCC kindreds.     

中国人家族性结直肠癌错配修复基因大片段变异分析

目的分析中国人家庭性结直肠癌错配修复基因大片段变异的特点。方法 采用多重连接探针扩增技术，分析32例具有家庭史结直肠癌、20例散发性结直肠癌患者错配修复基因MSH2的16个外显子、MLH1的19个外显子及7个其它基因外显子的拷贝数。研究工作包括：（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）分析结直肠癌患者外周血细胞DNA，筛查MSH2和MLH1基因大片段变异。结果 多重连接探针扩增技术分析系统稳定检出阳性对照样本的DNA大片段缺失；在3/32（9.4%）具有家庭聚集性结直肠癌患者中检出遗传性MSH2基因DNA大片段缺失。而在20例散发性结直肠癌患者未检出这类突变。结论 中国人家族性结直肠癌患者中错配修复基因的大片段变异是频发事件，对此类患者的遗传检测应包含错配修复基因大片段变异的筛查。     

Evaluation of enzymatic mutation detectiontrade mark in hereditary nonpolyposis colorectal cancer

In hereditary nonpolyposis colorectal cancer (HNPCC), the majority of reported mutations are dispersed throughout the 35 exons of the two principal susceptibility genes, MLH1 and MSH2, and because of this complexity, rapid mutation screening methods are required. The aim of this study was to evaluate the sensitivity of the Enzymatic Mutation Detection (EMD) assay in HNPCC using genomic DNA samples with known gene alterations in MLH1 and MSH2. The EMD assay relies upon the enzyme T4 Endonuclease VII recognizing and cleaving DNA mismatches, created when a PCR product containing a sequence alteration is hybridized with a wild type probe. A total of 68 different sequence variants from 30 exons were analyzed. The EMD assay was able to detect 62 of the 68 sequence variants (91%) with the majority showing strong cleavage products. One of the advantages of the EMD assay over other mutation screening techniques is that larger fragments can be analyzed in a single assay. No specialized equipment is required and one set of primers is sufficient for radioactive detection of the cleavage products. This method can be adapted to use fluorescent dye-labelled primers and may be automated to detect mutations accurately and rapidly in a large number of samples. One new MLH1 mutation (418delA) and two novel MSH2 mutations (1A>C; 227-228delAG) were also detected in HNPCC patients screened using this method.     

Identification and characterization of genomic rearrangements of MSH2 and MLH1 in Lynch syndrome (HNPCC) by novel techniques

It has recently been suggested that large genomic rearrangements account for 10-20% of all MSH2 mutations, and a lower proportion of all MLH1 mutations, among individuals with Lynch syndrome (hereditary non-polyposis colorectal cancer, HNPCC). These rearrangements are notoriously difficult to detect; moreover, for clinical purposes, simple tests must be devised to screen family members at risk. Here we used the multiplex ligation-dependent probe amplification (MLPA) method to screen for MSH2 and MLH1 deletions in 70 patients whose colorectal or endometrial tumors were MSI positive, yet no mutation had been found by genomic exon-by-exon sequencing of MSH2, MLH1, and MSH6. We identified five candidates with four different MSH2 deletions (exons 1-2, exons 1-6, exons 1-7 and exon 8) and one candidate with an MLH1 deletion (exons 3-6). To confirm the screening results and to characterize the breakpoints of these genomic deletions precisely, we used diploid-to-haploid conversion and inverse PCR as well as long-range PCR. In each case, we were able to pinpoint the breakpoint and design a simple diagnostic PCR. The procedures we used appear to be sensitive, specific, and simple enough for clinical use.     

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.     

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。     

Atypical HNPCC owing to MSH6 germline mutations: analysis of a large Dutch pedigree

Hereditary non-polyposis colorectal cancer (HNPCC) is the most common genetic susceptibility syndrome for colorectal cancer. HNPCC is most frequently caused by germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1. Recently, mutations in another MMR gene, MSH6 (also known as GTBP), have also been shown to result in HNPCC. Preliminary data indicate that the phenotype related to MSH6 mutations may differ from the classical HNPCC caused by defects in MSH2 and MLH1. Here, we describe an extended Dutch HNPCC family not fulfilling the Amsterdam criteria II and resulting from a MSH6 mutation. Overall, the penetrance of colorectal cancer appears to be significantly decreased (p<0.001) among the MSH6 mutation carriers in this family when compared with MSH2 and MLH1 carriers (32% by the age of 80 v >80%). Endometrial cancer is a frequent manifestation among female carriers (six out of 13 malignant tumours). Transitional cell carcinoma of the urinary tract is also relatively common in both male and female carriers (10% of the carriers). Moreover, the mean age of onset of both colorectal cancer (MSH6 v MSH2/MLH1 = 55 years v 44/41 years) and endometrial carcinomas (MSH6 v MSH2/MLH1 = 55 years v 49/48 years) is delayed. As previously reported, we confirm that the pattern of microsatellite instability, in combination with immunohistochemical analysis, can predict the presence of a MSH6 germline defect. The detailed characterisation of the clinical phenotype of this kindred contributes to the establishment of genotype-phenotype correlations in HNPCC owing to mutations in specific mismatch repair genes.     

Novel genomic insertion-- deletion in MLH1: possible mechanistic role for non-homologous end-joining DNA repair

Hereditary non-polyposis colorectal cancer (HNPCC) is an inherited cancer syndrome caused by a defect in the mismatch repair pathway. The majority of HNPCC mutations have been detected in MLH1 and MSH2. Most reported mutations are substitutions, small insertions and deletions, but standard methods of mutation analysis do not detect large rearrangements. It is now established that large deletions, insertions and rearrangements account for a significant proportion of MLH1 and MSH2 mutations. We report an unusual rearrangement resulting in the deletion of exons 6, 7 and 8 of MLH1, with the retention of part of intron 6 and insertions of two nucleotides each flanking the retained sequence. The 349-bp-retained sequence is made up of two closely spaced Alu sequences. The mutation was initially detected by protein truncation test and cDNA sequencing. Multiplex ligation-dependent probe amplification confirmed the deletion of three exons. PCR and sequencing were used to characterize the breakpoint. Despite the high density of Alu elements in MLH1, there is no homology at the deletion breakpoints or insertion junctions in this case to suggest that homologous recombination has occurred. We propose a mechanism involving non-homologous end joining to explain the occurrence of this complex deletion.     

Hereditary nonpolyposis colorectal cancer: pitfalls in deletion screening in MSH2 and MLH1 genes

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by a deficiency in DNA mismatch repair in consequence of germline mutations mainly in the genes MSH2 and MLH1. Around 10% of patients suspected of HNPCC are identified with large genomic deletions that cannot be detected by conventional methods of mutation screening. The recently developed multiplex ligation-dependent probe amplification (MLPA) proved to be an easy to perform method for deletion detection and is reliable when more than one exon is deleted. We show that, in some cases, apparent deletions of single exons may actually result from single base substitutions or small insertions/deletions in the hybridisation sequence of MLPA probes. We conclude that single exon deletions, detected by MLPA or multiplex PCR, should be validated with additional methods.     

A novel missense MSH2 gene mutation in a patient of a Korean family with hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant cancer-susceptible syndrome that predisposes to the early development of colorectal cancer. Germline mutations in DNA mismatch repair genes, particularly MLH1 and MSH2, are associated with the clinical phenotype of HNPCC. A previously unreported, novel missense mutation in exon 3 of the MSH2 gene (c.380A>T) was identified in the proband and a different missense mutation in exon 3 of MSH2 gene (c.505A>G) was noted in the mother, with a mutual splice mutation in intron 12 of the MSH2 gene in the proband, mother, and younger brother. Here, we report the clinical implications of a novel mutation in a patient with early-onset colorectal cancer and the significance of a common underlying splice site mutation occurring within a family with HNPCC.     

Eight novel germline MLH1 and MSH2 mutations in hereditary non‐polyposis colorectal cancer families from Spain

Germline mutations in the MLH1 and MSH2 genes, account for the majority of HNPCC families. We have screened such families from Spain by using DGGE analysis and subsequent direct sequencing techniques. In eight families we identified six novel MLH1 and two novel MSH2 mutations comprising one frame shift mutation (c.1420 del C), two missense mutations (L622H and R687W), two splice site mutations (c.1990‐1 G>A and c.453+2 T>C and one nonsense mutation (K329X) in the MLH1 gene as well as two frame shift mutations (c.1979‐1980 del AT and c.1704‐1705 del AG) in the MSH2 gene. Our analysis contributes to the further characterization of the mutational spectrum of MLH1 and MSH2 genes in HNPCC families. © 2001 Wiley‐Liss, Inc.