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。     

Four novel MSH2 / MLH1 gene mutations in portuguese HNPCC families

Hereditary non-polyposis colorectal cancer (HNPCC) is considered to be determined by germline mutations in the mismatch repair (MMR) genes, especially MSH2 and MLH1. While screening for mutations in these two genes in HNPCC portuguese families, 3 previously unreported MSH2 and 1 MLH1 mutations have been identified in families meeting strict Amsterdam criteria. Hum Mutat 15:116, 2000.     

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.     

Clinical and molecular characteristics of hereditary non-polyposis colorectal cancer families in Southeast Asia

Lee S-C, Guo J-Y, Lim R, Soo R, Koay E, Salto-Tellez M, Leong A, Goh B-C. Clinical and molecular characteristics of hereditary non-polyposis colorectal cancer families in Southeast Asia.Hereditary non-polyposis colorectal cancer (HNPCC), predominantly due to germline MLH1/MSH2 mutations, is the commonest form of hereditary colorectal cancer (CRC), but data in Asians are sparse. We sequenced the MLH1/MSH2 coding and promoter core regions in CRC patients diagnosed below age 40, and/or with multiple primary cancers or familial cancer clustering suggestive of HNPCC, and correlated deleterious mutations with clinical and tumour features. Forty-six Chinese, Malay and Indian kindreds participated. Of the 153 cancers reported in the 46 kindreds, stomach (14%) and urogenital cancers (13%) were the most common extracolonic cancers, whereas endometrial cancer comprised only 7%. Eleven different MLH1 and 12 MSH2 mutations were identified, including nine novel and four recurring mutations in the Chinese. One Indian was a compound heterozygote for an MLH1 and MSH2 mutation. The MLH1/MSH2 mutation data in the Malays and the Indians represents the first in these ethnic groups. Factors strongly associated with deleterious mutations were the Amsterdam criteria, family history of stomach or multiple primary cancers, and MSI-high tumours, whereas family history of endometrial cancer and young cancer age alone correlated poorly. Distinct clinical and molecular characteristics were identified among Asian HNPCC kindreds and may have important clinical implications.     

Strategies for screening for hereditary non-polyposis colorectal cancer

Germline mutations in DNA mismatch repair genes (MLH1, MSH2, PMS1, PMS2, and MSH6) predispose to hereditary non-polyposis colorectal cancer (HNPCC). In the absence of pathognomonic clinical features, diagnosis of HNPCC is often based on family history. Microsatellite instability (MSI) analysis has successfully been used for screening colorectal cancer patients for HNPCC. The aim of this study was to evaluate the feasibility of a recently introduced logistical model based on family history data in detecting HNPCC patients with germline mutations. A series of 509 kindreds with a proband with colorectal cancer was studied. MSI analysis and subsequent germline mutation analysis (MLH1 and MSH2) in MSI positive patients had been performed previously. Of the 509 patients, 63 (12%) were MSI positive and 10 (2%) had a germline mutation in MLH1 or MSH2. The power of the logistical model was tested to determine its value in predicting the probability of a germline mutation. The model proposed a high probability in three out of 10 mutation positive cases when data on cancer in first degree relatives were considered (typically three generation pedigrees, consisting, on average, of eight people). Using extended pedigrees and family cancer data in the 10 mutation positive kindreds (an average of 38 family members available), the model suggested high probabilities in seven out of 10 mutation positive cases. We conclude that for the model to predict germline mutation cases, extensive pedigrees and family history data are required. When screening colorectal cancer patients for HNPCC, a model using a combination of family information and MSI has optimal specificity and sensitivity.     

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.     

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.     

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.     

Novel MLH1 and MSH2 germline mutations in the first HNPCC families identified in Slovakia

Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly-inherited cancer predisposition syndrome, in which the susceptibility to cancer of the colon, endometrium and ovary is linked to germline mutations in DNA mismatch repair (MMR) genes. We have recently initiated a cancer prevention program in suspected HNPCC families in the Slovak Republic. The first ten families fulfilling Amsterdam criteria or Bethesda guidelines were screened for germline mutations in MLH1 and MSH2, two MMR genes most frequently mutated in HNPCC families. Six mutations were identified, five of which have not been reported previously. Two of the three new mutations in MLH1 (c.380+2T>A; c.307-2A>C) were absent from 100 chromosomes of healthy controls and probably cause a splicing defect, while the third was a 1 bp deletion (c.1261delA). In the MSH2 gene, one new nonsense (c.1030C>T [p.Q344X]) and one missense (c.524T>C [p.L175P]) mutation were identified. This latter variant was not found in 104 alleles of healthy control individuals. Moreover, a previously-reported pathogenic mutation (c.677G>T [p.R226L]) was found in one kindred. The clinical data and the genotypic and phenotypic evaluation of the tumors indicate that all the new alterations are pathogenic HNPCC mutations.     

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。     

Putative common origin of two MLH1 mutations in Italian-Quebec hereditary non-polyposis colorectal cancer families

Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common inherited cancer syndromes, accounting for 3-5% of all cases of colorectal cancer. In most HNPCC families, the disease is caused by a germline mutation in MLH1 or MSH2. In some populations, founder mutations appear to explain a substantial fraction of HNPCC. We report here the identification and preliminary characterization of two putative MLH1 founder mutations. The mutation MLH1c.1831delAT was shown to segregate in two Quebec families of Italian origin who fulfilled the Amsterdam criteria for HNPCC. Haplotype analysis using five intragenic microsatellite/single nucleotide polymorphism markers spanning MLH1 on chromosome 3 showed that these two unrelated families share an identical haplotype. In addition, two other Italian kindred whose affected members carry MLH1g.IVS6 + 3A>G also share a common haplotype, suggesting that, similarly, the latter mutation has a common origin. These mutations are the first putative founder MLH1 mutations to be identified in HNPCC kindred of Italian origin.     

Identification of germline MSH2 gene mutations in endometrial cancer not fulfilling the new clinical criteria for hereditary nonpolyposis colorectal cancer

Endometrial cancer is the second most common malignancy in patients with hereditary nonpolyposis colorectal cancer (HNPCC). This cancer is caused by germline mutations in one of the DNA mismatch repair (MMR) genes. The present study was undertaken to analyze the relation between microsatellite instability (MSI) and germline mutations of MMR genes. We analyzed MSI in 38 cases of endometrial cancer. MSI was present in one or more (out of 5 examined) regions in 11 (29%) cases. Furthermore, alterations in MLH1 and MSH2, two culprit genes representative of HNPCC, were examined in the 11 MSI-positive patients using polymerase chain reaction-single-strand conformation polymorphism and sequencing. Germline mutations, namely, 1) a missense mutation at codon 688 (ATG-->ATA, Met-->Ile) and 2) a missense mutation at codon 390 (CTT-->TTT, Leu-->Phe) of the MSH2 gene, were found in 2 of the 11 patients (18%). Although these two cases do not fulfill the new Amsterdam criteria, they had strong family histories of colorectal and endometrial carcinoma. Our results show that genetic testing is important in cases of endometrial cancer with a history suggestive of HNPCC even if the new Amsterdam criteria are not fulfilled.     

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.     

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.     

Microsatellite instability in early onset and familial colorectal cancer.

Hereditary non-polyposis colorectal cancer syndrome (HNPCC) is often considered to be the most common form of inherited colorectal cancer, although its precise incidence is unknown. The clinical diagnosis of HNPCC relies on a combination of family history and young age of onset of colorectal cancer, but as many familial aggregations of colorectal cancer do not fulfil the strict diagnostic criteria, HNPCC might be underdiagnosed. The majority of HNPCC families have germline mutations in mismatch repair (MMR) genes, such as MSH2 or MLH1, so that HNPCC cancers characteristically exhibit DNA replication errors (RERs) at microsatellite loci. Although an RER positive phenotype in tumours can also result from somatic mutations in an MMR gene, the prevalence of RER + tumours should provide a maximum estimate of the incidence of germline MMR gene mutations in patients with early onset and familial colorectal cancer. We investigated colorectal cancers for RERs from (1) a population based study of 33 patients with colorectal cancer aged 45 years or less, (2) 65 kindreds with familial colorectal cancer which only partially fulfilled the criteria for the diagnosis of HNPCC, and (3) 18 cancers from 12 HNPCC kindreds. Seven of 33 patients (21%) with colorectal cancer aged 45 years or less had an RER + cancer, with only two of these having a clear family history of HNPCC. A greater proportion of RER + tumours (5/7) occurred proximal to the splenic flexure than RER - tumours (4/26; chi2 = 6.14, p < 0.025). RERs were detected in all 18 cancers from HNPCC patients but in only six of 65 non-HNPCC familial colorectal cancer kindreds (9%; chi2 = 52.2, p < 0.0005). These findings suggest that most cancers in patients diagnosed at 45 years of age or less and familial aggregations of colorectal cancer which do not fulfil HNPCC diagnostic criteria do not have germline mutations in MSH2 and MLH1. Hence population screening for germline mutations in these genes is unlikely to be an efficient strategy for identifying people at high risk of developing colorectal cancer.     

Germline mutations in MLH1, MSH2 and MSH6 in Korean hereditary non-polyposis colorectal cancer families

Hereditary non-polyposis colorectal cancer (HNPCC), the most common hereditary colon cancer syndrome, is a dominant disorder caused by germline defects in mismatch repair (MMR) genes. Identification of MMR gene mutations can have direct clinical implications in counseling and management of HNPCC families. We screened 44 HNPCC and 97 suspected HNPCC Korean families for germline mutations in three MMR genes: MLH1, MSH2 and MSH6. We identified twelve novel mutations: nine in MLH1(c.632_633insT, c.808_811delACTT, c.845C>G, c.1625A>C, c.1730+1delG, c.1907T>C, c.1918C>T, c.2104-2A>G and c.2170T>A), two in MSH2 (c.1886A>G, c.1316_1318delCCT) and one in MSH6 (c.3488A>T). In addition, two statically significant cSNPs in MLH1: c.1128T>C ( p=0.008 in HNPCC and p=0.037 in early-onset CRC) and c.2168C>A ( p<0.001 in HNPCC). Interestingly, the most frequent mutation, c.1757_1758insC in MLH1, was a founder mutation inherited from a common Korean ancestor.     

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。