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.     

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.     

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。     

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.     

Tumours from MSH2 mutation carriers show loss of MSH2 expression but many tumours from MLH1 mutation carriers exhibit weak positive MLH1 staining

Microsatellite analysis (MSA) in tumour tissue is useful for pre-selection of hereditary non-polyposis colorectal cancer (HNPCC) patients for mutation screening, but is time-consuming and cost-intensive. Immunohistochemistry (IHC) for expression of MLH1 and MSH2 proteins is simple, fast, and indicates the affected gene. IHC has therefore been proposed as an alternative pre-screening method. However, some authors report a lower sensitivity of IHC compared with MSA. The present study reports IHC results for MSH2 and MLH1 performed in 82 tumours with high microsatellite instability (MSI-H) from 81 carriers of pathogenic mutations in MSH2 or MLH1. One hundred per cent (38/38) of the tumours from MSH2 mutation carriers showed loss of MSH2 staining; in all cases, the affected MSH2 gene was predicted correctly by IHC. Complete loss of MLH1 expression was observed in 66% (29/44) of MLH1 mutation carriers. Weak positive MLH1 staining was observed in 14 (32%) cases and, in one case, normal MLH1 staining was seen. The pathologist was aware of the weak staining pattern as an indicator of an MLH1 mutation; 98% of the MLH1 mutations were predicted correctly. To evaluate whether weak positive MLH1 staining is observed more often with in-frame or missense mutations, IHC data from 23 MSI-H tumours from carriers of unspecified variants were added and mutations were grouped into truncating mutations, large non-truncating deletions, and small non-truncating mutations. Weak MLH1 staining was observed in all three categories and it is postulated that other factors, such as mutation of the second allele, also influence protein expression. In conclusion, IHC can be regarded as a very useful method for selecting HNPCC patients for mutation analysis, as long as it is interpreted by an experienced pathologist. The high specificity of IHC in terms of indicating the affected gene is useful for evaluating unspecified variants. However, the staining pattern does not predict whether the underlying germ-line mutation is truncating or not.     

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.     

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.     

Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.

Hereditary nonpolyposis colon cancer (HNPCC) is associated with malfunction of postreplicative mismatch repair (MMR). While a majority of HNPCC-associated mutations in the MMR genes MLH1, MSH2, or MSH6 genes cause truncations-and thus loss of function--of the respective polypeptides, little is currently known about the biochemical defects associated with nontruncating mutations. We studied the interactions of six MLH1 variants, carrying either missense mutations or in-frame deletions, with normal PMS2 and tested the functionality of these heterodimers of MLH1 and PMS2 (MutL(alpha)) in an in vitro MMR assay. Three MLH1 carboxy-terminal mutations, consisting of internal deletions of exon 16 (amino acids 578-632) or exon 17 (amino acids 633-663), or a missense R659P mutation in exon 17, affected the formation of a functional MutL(alpha). Interestingly, mutations C77R and I107R in the amino-terminal part of MLH1 did not affect its heterodimerization with PMS2. The complexes MLH1(C77R)/PMS2 and MLH1(I107R)/PMS2, however, failed to complement a MMR-deficient extract lacking a functional MutL(alpha). As all these five mutations were identified in typical HNPCC families and produce nonfunctional proteins, they can be considered disease-causing. In contrast, the third amino-terminal mutation S93G did not affect the heterodimerization, and the MLH1(S93G)/PMS2 variant was functional in the in vitro MMR assay, given thus the nature of the HNPCC family in question. Although the missense mutation segregates with the disease, the mean age of onset in the family is unusually high (approximately 65 years).     

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。     

Mutation sharing, predominant involvement of the MLH1 gene and description of four novel mutations in hereditary nonpolyposis colorectal cancer. Mutations in brief no. 144. Online

Worldwide, the DNA mismatch repair genes MSH2 and MLH1 account for a major share and almost equal proportions of hereditary nonpolyposis colorectal cancer (HNPCC). Furthermore, the predisposing mutation usually varies from kindred to kindred. In this study, we screen 29 verified or putative HNPCC kindreds from Finland for mutations in these two genes and found 8 different mutations, 7 in MLH1 and 1 in MSH2, occurring in 13 families. Four of these mutations were novel. Altogether, we have to date studied 81 kindreds for mutations and 12 different mutations in 52 families have been identified, 10 in MLH1 and 2 in MSH2. These data show that Finnish HNPCC kindreds are characterized by the predominant involvement of MLH1 (49/52, 94% of the families) and a high rate of shared mutations (5/12, 42%) offering unique possibilities for mutation screening for both research and diagnostic purposes.     

Functional analysis of MSH6 mutations linked to kindreds with putative hereditary non-polyposis colorectal cancer syndrome

To date, five mismatch-repair (MMR) genes, MLH1, MSH2, MSH6, MSH3 and PMS2, are known to be involved in human MMR function. Two of those, MLH1 and MSH2, are further the most common susceptibility genes for hereditary non-polyposis colorectal cancer (HNPCC), while MSH3 and PMS2 are seldom (PMS2) or not at all (MSH3 ) reported to be involved in HNPCC. Despite the increasing number of MSH6 germline mutations, their pathogenicity remains questionable, because the mutations are mainly linked to putative HNPCC families lacking the typical clinical and molecular characteristics of the syndrome, such as early age at onset and high microsatellite instability (MSI). High MSI is a consequence of MMR defect, and the pathogenicity of germline mutations in HNPCC is thus linked to malfunction of MMR. To address the question of whether and how MSH6 mutations cause susceptibility to HNPCC, we studied heterodimerization of four MSH6 variants with MSH2, and the functionality of these MutSalpha complexes in an in vitro MMR assay. All mutations occurred in putative HNPCC patients. Irrespective of the type or the site of the amino acid substitutions, all the variants repaired G.T mismatches to A.T as wild-type MSH6 protein. However, the MSH6 protein carrying a mutation in the MSH2/MSH6 interaction region was poorly expressed, suggesting problems in its stability. Our results are clinically relevant, since they demonstrate that under the stable in vitro conditions, when the amounts of the proteins are adequate for repair, the tested MSH6 mutations do not affect repair function. Consequently, while the typical HNPCC syndrome is associated with problems in repair reaction, the pathogenicity of mutations in putative HNPCC families may be linked to other biochemical events.     

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.     

Mutation sharing,predominant involvement of the MLH1 gene and description of four novel mutations in hereditary nonpolyposis colorectal cancer

Worldwide, the DNA mismatch repair genes MSH2 and MLH1 account for a major share and almost equal proportions of hereditary nonpolyposis colorectal cancer (HNPCC). Furthermore, the predisposing mutation usually varies from kindred to kindred. In this study, we screened 29 verified or putative HNPCC kindreds from Finland for mutations in these two genes and found 8 different mutations, 7 in MLH1 and 1 in MSH2, occurring in 13 families. Four of these mutations were novel. Altogether, we have to date studied 81 kindreds for mutations and 12 different mutations in 52 families have been identified, 10 in MLH1 and 2 in MSH2. These data show that Finnish HNPCC kindreds are characterized by the predominant involvement of MLH1 (49/52, 94% of the families) and a high rate of shared mutations (5/12, 42%) offering unique possibilities for mutation screening for both research and diagnostic purposes. Hum Mutat 11:482–483, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Four novel MSH2 and MLH1 frameshift mutations and occurrence of a breast cancer phenocopy in hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations of genes encoding for proteins of the mismatch repair (MMR) machinery. The majority of mutations occur in the MLH1 and MSH2 genes, and consist of splice-site, frameshift and nonsense changes, leading to loss of protein function. In this study, we screened 7 HNPCC families for MLH1/MSH2 mutations. Sequence changes were identified in 5 families. Four alterations were novel 1- or 2-bp deletions or insertions causing a frameshift and appearance of premature stop codons (MLH1: c.597-598delGA, c.1520-1521insT; MSH2: c.1444delA, c.119delG). The four small insertions/ deletions were located within stretches of simple repeated sequences. By reviewing the HNPCC mutation database, we found that the majority of 1-2 bp frameshift mutations similarly affects simple repetitive stretches, pointing to DNA polymerase slippage during replication as the most likely source of such errors. We also evaluated microsatellite instability (MSI) in a breast carcinoma (BC) from an MLH1 mutation carrier. While a colon cancer from the same individual showed MSI, the BC specimen was MSI-negative, indicating that development of the latter tumor was unrelated to MMR impairment, despite presence of a constitutional MLH1 mutation. Hum Mutat 17:521, 2001.