Methylation of the hMLH1 promoter but no hMLH1 mutations in sporadic gastric carcinomas with high-level microsatellite instability

Microsatellite instability (MSI) in tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, principally hMSH2 and hMLH1. In contrast, somatic mutations in MMR genes are relatively rare in sporadic MSI(+) colon cancers. Rather, the majority of mutation-negative, MSI(+) cases involve hypermethylation of the hMLH1 promoter and subsequent lack of expression of hMLH1. The details of the mechanisms of this epigenetic gene silencing remain to be elucidated. In some colon cancer cell lines, hMLH1 promoter methylation is accompanied by mutation of 1 of the 2 alleles, whereas in other cell lines and tumors, such combinations have not been reported. To contribute to the characterization of MSI in gastric cancer and to directly investigate whether hMLH1 promoter methylation is accompanied by gene mutation in these cancers, we have analyzed 42 gastric tumors and corresponding normal tissue for MSI, hypermethylation of the hMLH1 promoter, and mutations in hMLH1 as well as hMSH2. We found that 10 (23.8%) of 42 cases of sporadic gastric cancer were MSI(+) and that 8 had at least 2 of 12 altered microsatellite loci. All samples with at least 2 altered loci exhibited methylation of the hMLH1 promoter region, but none had detectable mutations in hMLH1 or hMSH2. Our results confirm the importance of methylation of the hMLH1 promoter region in MSI(+) gastric tumors and suggest that methylation takes place in the absence of hMLH1 mutations in these tumors.     

HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLalpha and hMLH1-hEXO1 complexes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common inherited form of neoplasia caused by germline mutations in DNA mismatch repair (MMR) genes. MMR proteins have been reported to associate with several proteins, including the human exonuclease 1 (hEXO1). We report here novel HNPCC-hMLH1 mutant proteins (T117M, Q426X and 1813insA) in Danish HNPCC patients. We demonstrate that these mutant HNPCC-hMLH1 proteins are unable to form complexes with hEXO1 and hPMS2 in vivo. The results indicate that mutations found in HNPCC gene carriers disrupt hMLH1-hEXO1 complex formation and hMutLalpha heterodimer assembly essential for MMR activity.     

Infrequent frameshift mutations in the simple repeat sequences of hMLH3 in hereditary nonpolyposis colorectal cancers

BACKGROUND: A recently identified mismatch repair gene, hMLH3, contains two simple repeat sequence regions, (A)9 and (A)8, in its coding region. To clarify the role of hMLH3 in hereditary nonpolyposis colorectal cancer (HNPCC), we searched for hMLH3 somatic and germline mutations, particularly in the repeat regions, in 41 HNPCC patient cells. METHODS: We analyzed the hMLH3 (A)9 and (A)8 repeats in 27 colorectal cancers with microsatellite instability (MSI) as well as in normal cells from 41 HNPCC patients by means of polymerase chain reaction-single-strand conformation polymorphism. hMSH3 (A)8 and hMSH6 (C)8 repeats were also examined in these cancers. RESULTS: Frameshift mutations in the hMLH3 (A)9 repeat were observed in 4/27 (14.8%) cancers with MSI, all of which showed the severe MSI phenotype. No mutations in the (A)8 repeat were found in any case. The mutation frequency of the hMLH3 (A)9 repeat was similar to that of the hMSH6 (C)8 repeat (5/26, 19.2%), but was significantly lower than that of the hMSH3 (A)8 repeat (16/27, 59.3%) (P < 0.001). All four cancers with hMLH3 mutations exhibited germline hMSH2 and/or somatic hMSH3 mutations. No germline mutation in the hMLH3 (A)9 or (A)8 repeat was detected in normal cells from the 41 HNPCC patients. CONCLUSION: hMLH3 mutations were infrequently observed in HNPCC cancers with MSI and they may be secondary to other mismatch repair gene mutations. Hence hMLH3 may only play a small role in HNPCC tumorigenesis.     

Application of a yeast assay to detect functional p53 mutations in archival prostate cancer tissue

Detection and functional evaluation of mutant p53 alleles using a yeast assay could yield significant information for predicting the prognosis of patients with prostate cancer (CaP). Since the current version of this yeast assay is not applicable to archival tissues, we developed a modified assay for use on formalin-fixed, paraffin-embedded tissue and have applied it to the study of patient samples. Using this modified assay, we examined archival CaP samples from 10 patients for mutations in exons 5-8 of p53 gene. Mutations were detected in four samples: three resulted in the formation of red yeast colonies indicating complete loss of function, while one gave pink yeast colonies, indicating that this mutant retained partial function. In parallel, we analyzed these samples for p53 abnormalities using a single-strand conformational polymorphism (SSCP) approach. Only three of the four yeast-positive samples gave abnormal SSCP bands. In each case where abnormal p53 was found by both methods, DNA sequencing revealed the identical base change. These results suggest that the modified yeast assay may be more sensitive than SSCP for detection of p53 mutations, and demonstrate that the modified method can be used to detect and evaluate the function of p53 mutants present in archival tissue.     

A human cell-based assay to evaluate the effects of alterations in the MLH1 mismatch repair gene

We describe a new approach to investigate alterations in the human MLH1 mismatch repair (MMR) gene. This is based on complementation of the phenotype of a MLH1-defective subclone of the ovarian carcinoma A2780 cells by transfection of vectors encoding altered MLH1 proteins. Measurements of resistance (tolerance) to methylating agents, mutation rate at HPRT, microsatellite instability (MSI), and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. The approach was validated by transfecting cDNA of wild-type (WT) MLH1, cDNAs bearing two previously identified polymorphisms (I219V and I219L) and two with confirmed hereditary nonpolyposis colorectal cancer (HNPCC) syndrome mutations (G224D and G67R). A low-level expression of two MLH1 polymorphisms partially reversed methylation tolerance and the mutator phenotype, including MSI. Higher levels of I219V resulted in full restoration of these properties to WT. Increased expression of I129L did not fully complement the MLH1 defect, because there was a simultaneous escalation in the level of oxidative DNA damage. The findings confirmed the important relationship between deficient MMR and increased levels of oxidative DNA damage. Mutations from Italian HNPCC families (G224D, G67R, N635S, and K618A) were all ineffective at reversing the phenotype of the MLH1-defective A2780 cells. One (K618A) was identified as a low penetrance mutation based on clinical and genetic observations.     

Contribution of BRCA1 germ-line mutations to breast cancer in Greece: a hospital-based study of 987 unselected breast cancer cases

Background:

In most Western populations, 5–10% of all breast cancer cases can be attributed to major genetic factors such as predisposing mutations in BRCA1 and BRCA2, with early-onset cases generally considered as an indicator of genetic susceptibility. Specific BRCA1 and BRCA2 mutations or different mutation frequencies have been identified in specific populations and ethnic groups. Previous studies in Greek breast and/or ovarian cancer patients with family history have shown that four specific BRCA1 mutations, c.5266dupC, G1738R, and two large genomic rearrangements involving deletions of exons 20 and 24, have a prominent function in the population''s BRCA1 and BRCA2 mutation spectrum.

Methods:

To estimate the frequency of the above mutations in unselected Greek breast cancer women, we screened 987 unselected cases independently of their family history, collected from major Greek hospitals.RESULTS: Of the 987 patients, 26 (2.6%) were found to carry one of the above mutations in the BRCA1 gene: 13 carried the c.5266dupC mutation (1.3%), 6 carried the exon 24 deletion (0.6%), 3 carried the exon 20 deletion (0.3%), and 4 carried the G1738R mutation (0.4%). Among 140 patients with early-onset breast cancer (<40 years), 14 carried one of the four mutations (10.0%).

Conclusion:

These results suggest that a low-cost genetic screening for only the four prominent BRCA1 mutations may be advisable to all early-onset breast cancer patients of Greek origin.     

Elimination of the chemotherapy resistant subpopulation of 4T1 mouse breast cancer by haploidentical NK cells cures the vast majority of mice

Metastatic breast cancer is currently incurable despite initial responsiveness, assumingly due to the presence of chemoresistant subpopulations that can be characterized as label retaining cells (LRC). In the 4T1 mouse breast cancer model, we previously achieved cure after Cyclophosphamide and Total Body Irradiation (CY + TBI) followed by haploidentical bone marrow and spleen transplantation (BMSPLT). CY + TBI without transplantation induced only transient impaired tumor growth indicating a critical role of donor immune cells. As it remained unknown if the 4T1 model resembles human disease with respect to the presence of subpopulations of chemoresistant LRC, we now demonstrate this is indeed the case. Chemoresistance of 4T1 LRC was demonstrated by in vitro co-incubation of fluorescently labeled 4T1 cells in limiting dilution with cyclophosphamide, doxorubicin or cisplatinum, after which only LRC containing colonies remained. LRC also remain in vivo after treatment with CY + TBI. Succeeding experiments set up to identify the haploidentical effector cell responsible for cure and, therefore, for the elimination of chemoresistant LRC designate donor NK cells crucial for the anti-tumor effect. NK cell depletion of the haploidentical graft fully abrogated the anti-tumor effect. Increased disease-free survival retained after transplantation of haploidentical bone marrow and NK cell-enriched spleen cell grafts, even in the absence of donor T-cells or of donor bone marrow. Tumor growth analysis indicates the anti-tumor effect being immediate (days). Based on these data, it is worthwhile to explore alloreactive adoptive NK cell therapy as consolidation for patients with metastasized breast cancer.     

Absence of PTEN germ-line mutations in men with a potential inherited predisposition to prostate cancer.

Epidemiological studies have demonstrated that men with a family history of prostate cancer are at an increased risk for this disease. This important observation has led a number of research teams, including our own, to collect DNA samples and clinical data from prostate cancer families, with the goal of localizing and characterizing prostate cancer susceptibility genes. The candidate tumor suppressor gene PTEN (also called MMAC1) has recently been shown to be somatically altered in several common malignancies, including cancers of the brain, kidney, skin, thyroid, endometrium, breast, and prostate. Germ-line mutations in this gene, which maps to chromosome 10q23, have been associated with Cowden disease, an autosomal dominant cancer predisposition syndrome that is characterized by multiple hamartomas. Although prostate cancer is not typically associated with Cowden disease, previous studies of sporadic prostate cancers demonstrate loss of heterozygosity at 10q23 loci in approximately 25% of cases. We, therefore, hypothesized that germ-line mutations in the PTEN gene may predispose to prostate cancer in a subset of families, particularly those in which cancers of the breast, kidney, and/or thyroid also segregate. To test this hypothesis, DNA was isolated from whole blood of 11 prostate cancer patients from 10 unrelated families. Four of the 10 families met the previously established clinical criteria for hereditary prostate cancer. Eight of the II men had at least one second primary malignancy, including cases of neuroendocrine cancer, glioblastoma multiforme, melanoma, kidney, and thyroid cancer. Although we identified some common as well as some unique polymorphisms, no nonsense or missense mutations were identified in any of the 11 samples. To further examine the possibility that PTEN mutations contribute to prostate cancer predisposition, we also studied the probands from each of 10 families with early-onset and/or multiple individuals with prostate cancer. Sequence analysis of the PTEN gene in these 10 men also revealed no mutations or novel polymorphisms. We conclude that germ-line mutations in the PTEN are unlikely to contribute in a significant way to the inherited predisposition to prostate cancer.     

食管癌相关基因1编码蛋白的相互作用蛋白筛选

目的　筛选与食管癌相关基因 1(ECRG 1)编码蛋白相互作用的蛋白 ,为其功能研究奠定基础。方法　将编码ECRG 1羧基端 378个氨基酸的DNA序列插入到pGBKT7 DNA BD载体 ,与编码Gal4DNA结合结构域的DNA序列拼接做融合基因 ,将此重组质粒与已克隆到pACT2载体上的人肝脏cDNA文库 (与编码Gal4激活结构域的DNA序列融合 )共转化酵母细胞AH10 9。ECRG 1与相应的人肝脏cDNA片段编码的蛋白发生相互作用后 ,可激活报告基因的表达。排除假阳性后 ,阳性克隆中的文库cDNA进行测序分析。同源性检索搜寻GenBank中与之相同或相似的序列。结果　共转化得到约 3× 10 6个转化子 ,2 3个克隆有报告基因的表达。排除假阳性后 ,得到 2个阳性克隆 ,它们分别编码Miz 1(myc interactingznfingerprotein 1)和FLNA(actin bindingprotein 2 80 )。结论　ECRG 1基因编码蛋白在酵母中可特异性的结合Miz 1和FLNA ,提示ECRG 1可能通过与MIZ 1、FLNA相互作用参与调控细胞周期的运行。     

Screening of the Men1 gene and discovery of germ-line and somatic mutations in apparently sporadic parathyroid tumors

Hyperparathyroidism is the first manifestation in a majority of multiple endocrine neoplasia (MEN1) patients. To discriminate between sporadic and hereditary parathyroid tumors and characterize MEN1 somatic mutations, we examined MEN1 gene mutations in patients who had undergone surgery for sporadic parathyroid tumors. DNA was extracted from fresh frozen parathyroid tumor specimens from 112 patients as well as from peripheral blood leukocytes from 64 of the 112 patients. Sequence analysis was performed to examine exons 2-10 of the MEN1 gene for mutations. Loss of heterozygosity (LOH) was also examined by an analysis of codon 418 and 541, which lie within a polymorphic region of MEN1. Somatic MEN1 mutations were found in 25 of the 112 patients (22%). Two patients had two point mutations (508del33 and Y341X and 363insT and 1767delT, respectively). A total of 27 mutations were characterized, 20 of which have not been reported previously. There were 7 nonsense mutations, 10 frameshift mutations, 2 splice site deletions, 5 missense mutations, and 3 in-frame mutations. Nineteen mutations (70%) predicted truncation of the menin protein. Germ-line MEN1 mutations were found in 3 of 64 patients (5%) who had no family history of endocrine tumors associated with MEN1, and these patients were identified as MEN1 gene probands. LOH at the MEN1 locus was detected in three parathyroid tumors showing germ-line mutation. LOH was significantly frequent in parathyroid tumors with somatic MEN1 mutations (15 of 22 tumors, 68%) but not in those without germ-line or somatic MEN1 mutations (14 of 51 tumors, 28%; P = 0.0011). Our findings suggest that alterations of both alleles of the MEN1 gene may be associated not only with endocrine tumors of affected MEN1 patients but also with sporadic parathyroid tumors. Germ-line MEN1 gene analysis can distinguish heritable from nonheritable parathyroid tumors, and MEN1 gene evaluation of patients with apparently sporadic parathyroid tumor is recommended before parathyroid surgery.     

A role for methylation of the hMLH1 promoter in loss of hMLH1 expression and drug resistance in ovarian cancer.

Experimental evidence from several sources has identified a link between mismatch repair deficiency and cytotoxic drug resistance. Selection for cisplatin resistance in the human ovarian cancer cell line A2780, results in loss of expression of the mismatch repair protein hMLH1 in most (90%) of the resultant cisplatin-resistant cell lines. Here we demonstrate that the cisplatin sensitive parental cell line displays methylation of the promoter of only one hMLH1 allele, but that the resistant cell lines all exhibit hyper-methylation of the promoters of both hMLH1 alleles. Full methylation of all sites tested was found to be invariably associated with loss of hMLH1 expression, whereas a partial increase in methylation appears compatible with either loss or maintenance of expression. In addition treatment of two of the resistant cell lines with 5-azacytidine, a known inhibitor of methylation, results in re-expression of hMLH1. Clonogenic assays demonstrate that the 5-azacytidine treated cells show increased sensitivity to cisplatin. Furthermore, 12.5% (3/ 24) of ovarian tumours show hypermethylation of the hMLH1 promoter. Expression of hMLH1 is absent in the tumours that are hypermethylated, while all the unmethylated tumours still express the protein. This analysis suggests that methylation of the hMLH1 promoter may be a common mechanism for loss of hMLH1 expression, and possibly for cisplatin-resistance, in ovarian cancer.     

Analysis for microsatellite instability and mutations of the DNA mismatch repair gene hMLH1 in familial gastric cancer

We examined 30 gastric-cancer patients with a varying degree of family history of stomach cancer and/or synchronous gastric tumors for microsatellite instability. We observed microsatellite instability at at least 1 of 8 loci tested in tumors of 14/30 patients; of these 14, 8 had single locus alterations and 6 had alterations at at least half of the 8 loci. Among the patients with microsatellite instability at >=4 loci, 3 patients showed a strong familial clustering of gastric cancer. Mutation analysis of the DNA mismatch repair gene hMLH1 on paired non-tumorous and tumor DNA from 10 patients 6 with microsatellite instability at 24 loci and 4 with an alteration at one locus, revealed a novel missense mutation, present in the normal and tumor DNA of one patient with microsatellite instability at multiple loci in his tumor. His family history of cancer included one second-degree relative affected with gastric cancer. These data suggest that germline mutations in the hMLH1 gene occur in some gastric-cancer patients and that in the majority of cases microsatellite instability in gastric tumors may be due to defects in other genes responsible for DNA replication fidelity than the hMLH1. © 1996 Wiley-Liss, Inc.     

IDH1 mutations are present in the majority of common adult gliomas but rare in primary glioblastomas

We screened exon 4 of the gene isocitrate dehydrogenase 1 (NADP+), soluble (IDH1) for mutations in 596 primary intracranial tumors of all major types. Codon 132 mutation was seen in 54% of astrocytomas and 65% of oligodendroglial tumors but in only 6% of glioblastomas (3% of primary and 50% of secondary glioblastomas). There were no mutations in any other type of tumor studied. While mutations in the tumor protein p53 gene (TP53) and total 1p/19q deletions were mutually exclusive, IDH1 mutations were strongly correlated with these genetic abnormalities. All four types of mutant IDH1 proteins showed decreased enzymatic activity. The data indicate that IDH1 mutation combined with either TP53 mutation or total 1p/19q loss is a frequent and early change in the majority of oligodendroglial tumors, diffuse astrocytomas, anaplastic astrocytomas, and secondary glioblastomas but not in primary glioblastomas.     

A new assay to evaluate cell growth and drug sensitivity in culture using a cell image processor

The use of a cell image processor for the in vitro assessment of drug effects on cell growth, cell kinetics and chromatin organization is described. We have studied the influence of two well documented cytotoxic drugs, i.e. BCNU and vincristine (VIN), on the above mentioned parameters of the P-388 mouse leukemia, MCF-7 human mammary and HBL human melanoma cell lines. The cells were cultured for 1 to 4 days on glass coverslips put in Petri dishes containing or not (control) 10, 1 or 0.1 microgram/ml medium of the drug, after which they were fixed for histology, Feulgen-stained and analyzed through a cell image processor, i.e. the System for Analytical Microscopic Biomedical Applications (SAMBA 200). Our results showed that both BCNU and VIN exerted a well-known antineoplastic effect that was assessed at three different but highly complementary levels on the same sample of cells in a very rapid and simple procedure.     

Functional characterization of BRCA1 sequence variants using a yeast small colony phenotype assay

Germline mutations that inactivate the tumor suppressor gene BRCA1 are associated with an increased risk of cancers of the breast and other tissues, but the functional consequence of many missense variants found in the human population is uncertain. Several predictive methods have been proposed to distinguish cancer-predisposing missense mutations from harmless polymorphisms, including a small colony phenotype (SCP) assay performed in the model organism, yeast (Saccharomyces cerevisiae). The goal of this study was to further evaluate this colony size assay. We constructed 28 missense mutations throughout the C-terminal 305 amino acid residues of BRCA1. Mutated proteins were expressed in yeast and evaluated using the SCP assay. We conclude there is as yet no evidence the assay can identify inactivating mutations upstream of the BRCT repeats. However, within and between the BRCT repeats, results of the assay are in general agreement with predictions based on structural modeling, other in vitro and in vivo assays, and cross-species sequence conservation. Thus, the yeast assay appears to provide confirmatory in vivo evidence to aid in characterizing some BRCA1 missense variants.     

Inherited predisposition to pancreatic adenocarcinoma: role of family history and germ-line p16, BRCA1, and BRCA2 mutations

Susceptibility to pancreatic adenocarcinoma appears to be linked to germ-line mutations in genes causing various familial cancer syndromes. The objectives of this study were to estimate the proportion of unselected pancreatic cancer patients belonging to hereditary cancer syndrome families and to determine the frequency ofp16, BRCA1, BRCA2, hMSH2, and hMLH1 germ-line mutations in patients with a personal or family history of cancer. The study population consisted of 102 patients with histologically verified pancreatic adenocarcinoma, unselected for age, sex, family history, or ethnic origin. Patients completed a family history questionnaire and provided blood for mutation analysis. Three-generation pedigrees were constructed and classified as high risk/familial, intermediate risk/ familial, intermediate risk/nonfamilial, or low risk according to defined criteria. High- and intermediate-risk cases were analyzed for germ-line mutations using a combination of methods. Thirty-eight of 102 (37%) patients were characterized as high or intermediate risk, and the remainder were classified as low risk. Germ-line mutations were identified in five (13%) of these cases [one in p16 (I49S); one in BRCA1 (5382 insC); and three in BRCA2 (6174delT)]. The BRCA1 and BRCA2 mutations were identified in Ashkenazi Jewish patients. Four of the mutation carriers had strong family histories of the syndromes associated with the mutated genes. No mutations were identified in patients in whom the sole risk factor was a family history of pancreatic cancer, and only one mutation was found among patients with early-onset disease. We conclude that known causes of genetic predisposition are an important risk factor in a small proportion of pancreatic cancer patients, especially if associated with a strong family history of syndromes associated with the disease. The lack of detectable germ-line mutations in most high- and intermediate-risk cases suggests that there are probably additional, as yet unidentified genes predisposing to this disease.     

Results of a population-based screening for hereditary breast cancer in a region of North-Central Italy: contribution of BRCA1/2 germ-line mutations

BRCA1/2 mutation status is of paramount importance to identify families at risk of Hereditary Breast and Ovarian Cancer (HBOC). Most HBOC and BRCA1/2 mutation studies have focused on highly selected sub-populations, and few data are available for large population cohorts. For this reason, as part of a regional cancer prevention strategy in North-Central Italy, we set up a population-based screening programme to identify all resident HBOC families, and to determine their BRCA1/2 mutation status. To date, 44 different BRCA1/2 variants have been identified in 55 HBOC families. Of the seven newly reported mutations, only BRCA1 Q284X is clearly deleterious. The analysis of clinical disease characteristics in relation to age of disease onset and family history showed a difference between BRCA1/2 wild type and mutation carrier families. Interestingly, BRCA1/2 mutations were significantly more common in women who developed breast cancer ≤40 years of age than in BRCA1/2 wild type women (50% vs. 29%, respectively, P = 0.005). The family history selection criteria most likely to indicate the presence of deleterious BRCA1/2 mutations are breast cancer ≤35 years (P = 0.012), two first-degree relatives with breast cancer ≤50 years (P = 0.022), and male breast cancer (P = 0.047). The penetrance of BRCA1/2 alterations in our cohort seems to be aligned with other published results. However, new data interpretations have emerged in relation to the clinical criteria and the presence of deleterious mutations. This information shows that a correct and accurate clinical selection could avoid unnecessary molecular tests and could better address genetic analysis and clinical management. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Establishment of a novel method to evaluate peritoneal microdissemination and therapeutic effect using luciferase assay

Peritoneal dissemination is a major cause of recurrence in patients with malignant tumors in the peritoneal cavity. Effective anticancer agents and treatment protocols are necessary to improve outcomes in these patients. However, previous studies using mouse models of peritoneal dissemination have not detected any drug effect against peritoneal micrometastasis. Here we used the luciferase assay to evaluate peritoneal micrometastasis in living animals and established an accurate mouse model of early peritoneal microdissemination to evaluate tumorigenesis and drug efficacy. There was a positive correlation between luminescence intensity in in vivo luciferase assay and the extent of tumor dissemination evaluated by ex vivo luciferase assay and mesenteric weight. This model has advantages over previous models because optimal luciferin concentration without cell damage was validated and peritoneal microdissemination could be quantitatively evaluated. Therefore, it is a useful model to validate peritoneal micrometastasis formation and to evaluate drug efficacy without killing mice.     

A novel germline mutation of hMLH1 in a patient with hereditary non-polyposis colorectal cancer

DNA mismatch repair genes, hMLH1 and hMSH2, assigned on chromosome 3p21-23 and 2p21-22 are involved in hereditary non-polyposis colorectal cancer (HNPCC). The heterozygous carrier of the mutated allele results in a mutator phenotype and accelerating tumorigenesis, which especially causes carcinomas in the gastrointestinal and genitourinary tracts. We screened germline mutations of mismatch repair genes hMLH1 and hMSH2 in a patient with multiple primary neoplasms (multiple stomach cancers, colon cancer and brain tumor) in a cancer clustered HNPCC family. Screening by long RT-PCR from the RNA extracted from puromycin-treated heparinized blood showed skipping of the exon 2 in hMLH1. The analysis of the genomic DNA showed a GT deletion in the splice-donor site of the exon 2, which is compatible with the splicing variant detected by long RT-PCR analysis. This is a novel germline mutation that has not been reported previously.