Hereditary Nonpolyposis Colorectal Cancer Patients Research Articles

TCF-3, 4 protein expression correlates with β-catenin expression in MSS and MSI-H colorectal cancer from HNPCC patients but not in sporadic colorectal cancers

The beta-catenin-T-cell factor-4 (TCF-4) complex is the main control switch of cell proliferation and differentiation of normal and malignant intestinal cells. The aim of our study was to analyze the protein expression of components of the Wnt pathway in microsatellite stable (MSS) and highly unstable (MSI-H) sporadic and hereditary nonpolyposis colorectal cancer (HNPCC) in human colorectal cancers. Sixty seven colorectal tumors comprising of 15 sporadic MSS, 12 sporadic microsatellite instability colorectal tumors and 40 tumors from HNPCC patients, of which 20 were MSS and 20 MSI-H, were analyzed for the expression of APC, beta-catenin, and TCF-3, 4 proteins by immunohistochemistry. We found a significant difference in cytoplasmic APC expression frequency between sporadic MSS (52%) and HNPCC tumors (78%), whereas no difference was detected between MSI-H and MSS or HNPCC tumors. All tumor groups showed a similar pattern of decreased membranous staining and increased cytoplasmic and nuclear staining for beta-catenin compared to normal cells. Moreover, the TCF-3, 4 protein expression was higher (43%) in HNPCC-associated MSS tumors compared to sporadic tumors (14%; analysis of variance (ANOVA) p < 0.05). For HNPCC tumors, the subcellular beta-catenin expression (membranous, cytoplasmic, and nuclear) correlated with the nuclear TCF-3, 4 signal in MSS tumors (Spearman correlation p < 0.0007) and MSI-H tumors (Spearman correlation p < 0.0001). We have shown a previously unknown difference in TCF-3, 4 protein expression between sporadic and HNPCC MSS tumors. In addition, we found no difference in nuclear beta-catenin signal intensity, which may be caused by an alteration in Wnt pathway in MSS sporadic tumors by unknown mechanisms leading to lower TCF-3, 4 protein expression. This hypothesis has to be tested in future investigations.

Mucinous adenocarcinoma showing different clinicopathological and molecular characteristics in relation to different colorectal cancer subgroups

Mucinous adenocarcinoma (MAC) is frequently reported to be associated with patients of young-age sporadic colorectal cancer (YSCC) and hereditary nonpolyposis colorectal cancer (HNPCC). This study is aimed to investigate whether the clinicopathological characteristics of MAC of HNPCC patients are distinct from those of YSCC patients. Eighty-two HNPCC and 68 YSCC patients recorded in the colorectal cancer registry of Chang Gung Memorial Hospital at Linkou, Taiwan, between January 1, 1995 and December 31, 2001 were included in this study. Clinicopathological and molecular variables of MAC and non-MAC of HNPCC and YSCC patients were compared accordingly. Compared to non-MAC, MAC significantly showed higher frequencies of poor differentiation (32% vs. 8.2%, p = 0.001), advanced tumor stage (76% vs. 47%, p = 0.002), loss of mismatch repair protein (MMR) expression (74% vs. 44%, p = 0.023), and increased MUC2 expression (98% vs. 61%, p < 0.001). MAC of HNPCC patients showed predominant right-sided colon involvement, whereas MAC of YSCC patients displayed predominance in the left colon (79% vs. 22%, p = 0.001). Among the non-MAC counterparts, more differences were detectable including tumor stage, loss of MMR expression, and increased MUC1 expression. Furthermore, both MAC and non-MAC of YSCC patients showed higher frequencies of advanced tumor stage (81% vs. 62%, p = 0.072). In contrast, the incidence of loss of MMR expression in MAC and non-MAC of HNPCC patients is not significantly different (86% vs. 70%, p = 0.323). Significantly different tumor localization was observed between mucinous YSCC (left colon predominance) and mucinous HNPCC (right colon predominance).

Specific Hypomethylated Cpgs at the IGF2 Locus Act as an Epigenetic Biomarker for Familial Adenomatous Polyposis Colorectal Cancer

The identification of specific biomarkers for colorectal cancer is of primary importance for early diagnosis. The aim of this study was to evaluate if methylation changes at the IGF2/H19 locus could be predictive for individuals at high risk for developing sporadic or hereditary colorectal cancer. Quantitative methylation analysis using pyrosequencing was performed on three differentially methylated regions (DMRs): IGF2 DMR0 and DMR2 and the H19 DMR in DNA samples from sporadic colorectal cancer (n = 26), familial adenomatous polyposis (n = 35) and hereditary nonpolyposis colorectal cancer (n = 19) patients. We report in this article for the first time, that in sporadic colorectal cancer tumor DNA both the IGF2 DMR0 and DMR2 are hypomethylated, while the H19 DMR retains its monoallelic methylation pattern. In lymphocyte DNA, a striking hypomethylation of nine contiguous correlated CpGs was found in the IGF2 DMR2 but only in familial adenomatous polyposis patients. Methylation alterations at the IGF2 locus are more extensive than previously reported and DMR2 hypomethylation in lymphocyte DNA might be a specific epigenetic biomarker for familial adenomatous polyposis patients.

Comparison of strategies for identifying colorectal cancer patients with Lynch syndrome: A comparative-effectiveness analysis.

e16508 Background: Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC) is the most common form of inherited colorectal cancer (CRC). Identifying HNPCC is important for management of patients and their mutation-positive relatives. Germline testing (GT) can definitively identify HNPCC but is costly. Various strategies have been proposed to better select who should undergo GT but, the optimal strategy is unclear. This study evaluates the comparative effectiveness of strategies for identifying CRC patients with HNPCC. Methods: A decision-analytic model was used to evaluate 31 strategies for detecting patients with HNPCC, including use of cancer history assessment tools (Amsterdam Criteria [AC], Revised Bethesda Guidelines [RBG], or PREMM diagnostic model) with or without molecular evaluation (immunohistochemistry [IHC], microsatellite instability [MSI] testing and BRAF testing] of CRC tumors prior to GT. For an estimated 150,000 patients newly diagnosed with CRC in a year (3% of whom are expected to have HNPCC), the model estimated the number of HNPCC patients each strategy would detect and associated costs. Results: Universal GT identified all HNPCC patients and cost $386 million. The next most costly strategy at $167 million, universal IHC and MSI testing then GT, detected > 97% HNPCC. Strategies employing RBG then MSI and/or IHC detected > 85% of HNPCC patients, with costs from $58-$166 million. Strategies relying on AC for further testing were the least costly ($17-$22 million) but detected < 50% of HNPCC patients. Regardless of the strategy, inclusion of BRAF testing (detects sporadic CRC) reduced costs by 0.5%-26.7%. Considering cost-effectiveness, strategies using RBG or the PREMM model followed by molecular evaluation then GT, were more effective and less costly than strategies of universal molecular testing. Conclusions: Costs and effectiveness of strategies for identifying HNPCC patients vary greatly and some may be prohibitively expensive. However, programs that employ the PREMM model or the RBG followed by molecular testing may be optimal. If cancer history assessment is difficult, “Universal IHC” may be the next most cost-effective option. No significant financial relationships to disclose.

Microsatellite instability in the peripheral blood leukocytes of HNPCC patients

Most hereditary nonpolyposis colorectal cancer (HNPCC) patients inherit a defective allele of a mismatch repair (MMR) gene, usually MLH1 or MSH2, resulting in high levels of microsatellite instability (MSI-H) in the tumors. Presence of MSI in the normal tissues of mutation carriers has been controversial. Here we directly compare MSI in the peripheral blood leukocyte (PBL) DNA of seven HNPCC patients carrying different types of pathogenic MMR mutations in MLH1 and MSH2 genes with the PBL DNA of normal age-matched controls and of patients with sporadic colorectal cancer (SCRC). Small pool PCR (SP-PCR) was used studying three microsatellite loci for at least 100 alleles each in most samples. The average frequencies of mutant microsatellite fragments in each HNPCC patient (0.04-0.24) were significantly higher (p<0.01) relative to their age-matched normal controls with mutant frequencies (MF) from 0.00 to 0.06, or SCRC patients (MF from 0.01-0.03). The data support the conclusions that higher MF in the PBL DNA of HNPCC patients is real and reproducible, may vary in extent according to the type of germline MMR mutation and the age of the individual, and provide a possible genetic explanation for anticipation in HNPCC families.

Three synchronous primary carcinomas in a patient with HNPCC associated with a novel germline mutation in MLH1: Case report

BackgroundMLH1 is one of six known genes responsible for DNA mismatch repair (MMR), whose inactivation leads to HNPCC. It is important to develop genotype-phenotype correlations for HNPCC, as is being done for other hereditary cancer syndromes, in order to guide surveillance and treatment strategies in the future.Case presentationWe report a 47 year-old male with hereditary nonpolyposis colorectal cancer (HNPCC) associated with a novel germline mutation in MLH1. This patient expressed a rare and severe phenotype characterized by three synchronous primary carcinomas: ascending and splenic flexure colon adenocarcinomas, and ureteral carcinoma. Ureteral neoplasms in HNPCC are most often associated with mutations in MSH2 and rarely with mutations in MLH1. The reported mutation is a two base pair insertion into exon 10 (c.866_867insCA), which results in a premature stop codon.ConclusionOur case demonstrates that HNPCC patients with MLH1 mutations are also at risk for ureteral neoplasms, and therefore urological surveillance is essential. This case adds to the growing list of disease-causing MMR mutations, and contributes to the development of genotype-phenotype correlations essential for assessing individual cancer risk and tailoring of optimal surveillance strategies. Additionally, our case draws attention to limitations of the Amsterdam Criteria and the need to maintain a high index of suspicion when newly diagnosed colorectal cancer meets the Bethesda Criteria. Establishment of the diagnosis is the crucial first step in initiating appropriate surveillance for colorectal cancer and other HNPCC-associated tumors in at-risk individuals.

Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers

BackgroundDepending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (MMR) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from MLH1/MSH2 deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts.MethodsThe main MMR genes responsible for HNPCC, MLH1, MSH2, MSH6, and PMS2, were analyzed by MLPA (multiplex ligation-dependent probe amplification) in a total of 37 unrelated HNPCC-suspected patients whose MLH1/MSH2 genes gave negative results in previous sequencing experiments. An LOH study was performed on six tumors from LGR carriers by combining MLPA to assess LOH at LGR regions and sequencing to examine LOH at 28 SNP markers from the MLH1 and MSH2 genes.ResultsWe found six rearrangements in the MSH2 gene (five deletions and dup5-6), and one aberration in the MLH1 gene (del5-6). The MSH2 deletions were of three types (del1, del1-3, del1-7). We detected LOH at the LGR region in the single MLH1 case, which was determined in a previous study to be LOH-negative in the intragenic D3S1611 marker. Three tumors displayed LOH of at least one SNP marker, including two cases that were LOH-negative at the LGR region.ConclusionLGRs accounted for 25% of germline MMR mutations identified in 28 Slovakian HNPCC families. A high frequency of LGRs among the MSH2 mutations provides a rationale for a MLPA screening of the Slovakian HNPCC families prior scanning by DNA sequencing. LOH at part of the informative loci confined to the MLH1 or MSH2 gene (heterozygous LGR region, SNP, or microsatellite) is a novel finding and can be regarded as a partial LOH. The conversion begins within the gene, and the details of conversion tracts are discussed for each case.

Prognostic relevance of MLH1 and MSH2 mutations in hereditary non-polyposis colorectal cancer patients.

Colorectal carcinoma patients from hereditary non-polyposis colorectal cancer families are suggested to have a better prognosis than sporadic colorectal carcinoma cases. Since the majority of hereditary non-polyposis colorectal cancer-related colorectal carcinomas are characterized by microsatellite instability due to germline mutations in DNA mismatch repair genes, this is consistent with the prolonged survival observed in sporadic microsatellite instability-positive colorectal carcinoma compared to microsatellite stable cases. However, a fraction of colorectal carcinoma cases belongs to families that, despite fulfilling the clinical criteria for hereditary non-polyposis colorectal cancer, do not carry mismatch repair gene mutations. Our aim was to verify to what extent the genotypic heterogeneity influences the prognosis of hereditary non-polyposis colorectal cancer patients. A survival analysis was performed on 526 colorectal carcinoma cases from 204 Amsterdam Criteria-positive hereditary non-polyposis colorectal cancer families. Enrolled cases were classified as MLH1-positive, MSH2-positive and mutation-negative, according to the results of genetic testing in each family. Five-year survival rates were 0.73 (95% CI, 0.66-0.80), 0.75 (95% CI, 0.66-0.84) and 0.62 (95% CI, 0.55-0.68) for MLH1-positive, MSH2-positive and mutation-negative groups, respectively (logrank test, P = 0.01). Hazard ratio, computed using Cox regression analysis and adjusted for age, sex, tumor site and stage, was 0.71 (95% CI, 0.51-0.98) for the mutation-positive compared to the mutation-negative group. Moreover, in the latter group, patients with microsatellite instability-positive colorectal carcinomas showed a better outcome than microsatellite stable cases (5-year survival rates, 0.81 and 0.60, respectively; logrank test, P = 0.006). Our results suggest that the prognosis of hereditary non-polyposis colorectal cancer-related colorectal carcinoma patients depends on the associated constitutional mismatch repair genotype.

Prognostic Relevance of MLH1 and MSH2 Mutations in Hereditary Non-Polyposis Colorectal Cancer Patients

Colorectal carcinoma patients from hereditary non-polyposis colorectal cancer families are suggested to have a better prognosis than sporadic colorectal carcinoma cases. Since the majority of hereditary non-polyposis colorectal cancer-related colorectal carcinomas are characterized by microsatellite instability due to germline mutations in DNA mismatch repair genes, this is consistent with the prolonged survival observed in sporadic microsatellite instability-positive colorectal carcinoma compared to microsatellite stable cases. However, a fraction of colorectal carcinoma cases belongs to families that, despite fulfilling the clinical criteria for hereditary non-polyposis colorectal cancer, do not carry mismatch repair gene mutations. Our aim was to verify to what extent the genotypic heterogeneity influences the prognosis of hereditary non-polyposis colorectal cancer patients. A survival analysis was performed on 526 colorectal carcinoma cases from 204 Amsterdam Criteria-positive hereditary non-polyposis colorectal cancer families. Enrolled cases were classified as MLH1-positive, MSH2-positive and mutation-negative, according to the results of genetic testing in each family. Five-year survival rates were 0.73 (95% CI, 0.66-0.80), 0.75 (95% CI, 0.66-0.84) and 0.62 (95% CI, 0.55-0.68) for MLH1-positive, MSH2-positive and mutation-negative groups, respectively (logrank test, P = 0.01). Hazard ratio, computed using Cox regression analysis and adjusted for age, sex, tumor site and stage, was 0.71 (95% CI, 0.51-0.98) for the mutation-positive compared to the mutation-negative group. Moreover, in the latter group, patients with microsatellite instability-positive colorectal carcinomas showed a better outcome than microsatellite stable cases (5-year survival rates, 0.81 and 0.60, respectively; logrank test, P = 0.006). Our results suggest that the prognosis of hereditary non-polyposis colorectal cancer-related colorectal carcinoma patients depends on the associated constitutional mismatch repair genotype.

Hereditary Nonpolyposis Colorectal Cancer (HNPCC): A Pilot Study Assessing Understanding and Attitudes Towards Prenatal Testing

Purpose: Hereditary Nonpolyposis Colorectal Cancer Syndrome (HNPCC) is the most common hereditary cancer syndrome accounting for 2-3% of colorectal cancer (CRC). Patients with HNPCC gene mutations have a 70-80% chance of developing CRC. Little is known about the attitudes of individuals with HNPCC towards prenatal diagnosis. We conducted a pilot study to evaluate HNPCC patients' understanding of their disease and assess their attitudes towards various modalities of prenatal testing. Methods: 12 individuals with HNPCC were educated on the currently available options for prenatal diagnosis of HNPCC including amniocentesis, chorionic villous sampling (CVS), and preimplantation genetic diagnosis (PGD). They were then given a 40-item survey on personal and family history related to HNPCC, demographics, and attitudes toward prenatal testing of their offspring. Results: Nine women and 3 men participated. Mean age at diagnosis was 42.8 years. 83% were Caucasian and 8% African American. 83% were married. 67% had children none of which were yet tested for HNPCC. 83% of the subjects knew there was a 50% risk of their offspring being affected with HNPCC. None were aware of prenatal testing options for HNPCC prior to the study. 100% were of the opinion that prenatal testing options should be discussed and offered to all with a HNPCC gene mutation. 58% would consider undergoing prenatal testing. Of these 100% would consider amniocentesis or PGD and 86% would consider CVS. More subjects were aware of amniocentesis (83%) than PGD or CVS (50%, 25% respectively). 83% felt that all modalities were ethically sound. 50% of the subjects felt that learning the fetus was unaffected with HNPCC was the greatest advantage of PGD. The possibility of complications (58%), or concerns regarding procedure success (33%), seemed to be the greatest disadvantage. 25% of the subjects felt that a HNPCC diagnosis influenced their reproductive decisions. The most important reason against prenatal testing was the difficulty in subsequent decisions making regarding termination. Religious or personal beliefs played little role in decision-making. Conclusion: The majority of HNPCC affected patients surveyed were aware of the autosomal dominant inheritance pattern of HNPCC. All patients felt prenatal testing options should be offered routinely. Though all patients in this study had seen genetic counselors in the past, none were aware of prenatal testing options for their disease prior to this study. This pilot study demonstrates the importance of addressing prenatal testing options in HNPCC patients. Future studies should be conducted to assess if prenatal counseling referral should be required as standard of care in all patients diagnosed with HNPCC.

Determination of splice-site mutations in Lynch syndrome (hereditary non-polyposis colorectal cancer) patients using functional splicing assay

Lynch syndrome (hereditary non-polyposis colorectal cancer) is an inherited disease caused by germ-line mutation in mismatch repair genes such as MLH1, MSH2, and MSH6. The mutations include missense and nonsense mutations, small insertions and deletions, and gross genetic alterations including large deletions and duplications. In addition to these genetic changes, mutations in introns are also involved in the pathogenesis. However, it is sometimes difficult to interpret correctly the pathogenicity of variants in exons as well as introns. To evaluate the effect of splice-site mutations in two Lynch syndrome patients, we carried out a functional splicing assay using minigenes. Consequently, this assay showed that the mutation of c.1731+5G>A in MLH1 led to exon15 skipping, and that the mutation of c.211+1G>C in MSH2 created an activated cryptic splice-site 17-nucleotides upstream in exon1. These aberrant splicing patterns were not observed when wild type sequence was used for the assay. We also obtained concordant results by RT-PCR experiments with transcripts from the patients. Furthermore, additional functional splicing assays using two different intronic mutations described in earlier studies revealed splicing alterations that were in complete agreement with the reports. Therefore, functional splicing assay is helpful for evaluating the effects of genetic variants on splicing.

Papillary serous carcinoma in situ in ovarian endometriosis in an MSH2 mutation carrier

Hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome, is an autosomal dominant cancer caused by a germline mutation of mismatch repair (MMR) genes [1]. It is characterized by an early onset of colorectal, endometrial, and ovarian cancer as well as cancers of the gastrointestinal and upper urinary tracts, brain, and skin. The risk of gynecologic cancer in women with HNPCC equals or even exceeds that of colorectal cancer [1]. Typically, HNPCC-associated ovarian cancers are of nonserous histologies, such as endometrioid, mucinous, and clear cell types [1]. We report a unique case of noninvasive serous carcinoma that developed in an endometriotic cyst of the ovary in an MSH2 mutation carrier. A 47-year-oldwoman (G3P3)with a strong family history of cancer underwent surgery and adjuvant chemotherapy at age 42 for a welldifferentiated adenocarcinoma of the colon (stage pT3 pN1). This was subsequently identified to be HNPCC with an MSH2 mutation. An endometrial biopsy was negative for cancer. Pelvic sonography suggested ovarian endometriosis, CA125 level was 86 U/mL (normal b35 U/mL), CEAwas 0.6 ng/mL (normal b3.0 ng/mL), and the patient's Pap test was normal. At pathological inspection of the hysterectomy and bilateral salpingo-oophorectomy specimens, the adnexae and uterus appeared normal. The right ovary (4×2×2 cm) contained a 2.3×2.0×1.8 cm cyst filled with bloody fluid. The left ovary (2×1×0.8 cm) contained a few minute cystic structures filled with blood. Microscopically, both ovaries contained several foci of endometriosis. The cystic structure in the right ovary was lined by tuboendometrial type epithelium and focally displayed a papillary growth pattern and severe cytonuclear and nucleolar atypia, in all respects similar to high grade serous papillary carcinoma of the ovary or endometrium (Fig. 1). There was no invasion of the underlying endometrial type cellular stroma. By immunohistochemistry, the serous carcinoma in situ cells stained for p53 (30% of cells), p16 (90% of cells), and Ki-67, but not for estrogen/progesterone (ER/PR) and Wilms tumor 1 (WT1). The fimbriated segment of both tubes contained p53-negative epithelial hyperplasia without atypia. To the authors' knowledge, this is the first reported case of occult serous carcinoma in situ arising in cystic ovarian endometriosis in an HNPCC patient who underwent risk-reducing robotic hysterectomy and salpingo-oophorectomy. Both the light microscopic and immunophenotypic characteristics supported a serous rather than an endometrioid histotype of the atypical cells. Furthermore, consistent with the histological origin of the lesion together with positive immunoreactivity for p53 and p16 but not for WT1, the malignant cells were presumably of endometrial rather than ovarian serous phenotype. According to current concept, WT1 is a more reliable immunomarker of ovarian serous differentiation than p16 [2]. Also, WT1 is generally negative in serous carcinoma of the endometrium. The association of serous carcinoma in situ within endometriosis presents a management challenge. On the one hand, similar lesions in the endometrium are treated with chemoradiation for they are known to carry a potential for disseminatedmalignant disease [3]. On the other hand, there is no information on the clinical behavior of a case like ours, and preoperative cytology was negative for malignant cells. Hence, we elected a tight surveillance scheme rather than postoperative chemotherapy. The patient is well, without disease clinically, radiologically,

Meta-Analysis of MSH6 Gene Mutation Frequency in Colorectal and Endometrial Cancers

Studies on mutations and mutation frequencies of the MSH6 gene, which mainly focus on new types of mutations in small samples, have been published ever since the first report of MSH6 mutation in two atypical hereditary non-polyposis colorectal cancer patients. However, the results remain inconsistent. Therefore, a systematic review was conducted and a meta-analysis was undertaken to determine the frequency of MSH6 mutation in colorectal and endometrial cancers. From 27 studies, 180 cases with MSH6 mutation in a total of 3196 cases were detected. In colorectal and endometrial cancers the MSH6 mutation frequency is 7.2 and 9.6%, respectively. MSH6 mutation frequency was 10.4% in hereditary non-polyposis colorectal cancer patients, 7.1% in atypical hereditary non-polyposis colorectal cancer patients, and 5.9% in sporadic patients. The frequency of MSH6 mutation in high microsatellite instability (MSI-H) was 11.6% and in low microsatellite instability (MSI-L) cases was (13.3%), which were higher than in microsatellite stability (MSS) cases (1.7%). The mean age of the earliest onset of colorectal and endometrial cancers in MSH6 mutation carriers was 51.2 and 56.5 yr, respectively. Data suggest that the frequency of MSH6 mutation is higher in hereditary non-polyposis colorectal cancer patients than in atypical hereditary non-polyposis colorectal cancer and sporadic patients. MSH6 mutation frequency was also higher in endometrial than colorectal cancers. The mean age of earliest onset of endometrial cancer (56.5 yr) is older than for colorectal cancer (51.2 yr) in carriers of MSH6 mutation. Our results provide evidence for clinical genetic testing and counseling.

Four novel germline mutations in the MLH1 and PMS2 mismatch repair genes in patients with hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common cause of early onset hereditary colorectal cancer. In the majority of HNPCC families, microsatellite instability (MSI) and germline mutation in one of the DNA mismatch repair (MMR) genes are found. The entire coding sequence of MMR genes (MLH1, MLH2, MLH6, and PMS2) was analyzed using direct sequencing. Also, tumor tests were done as MSI and immunohistochemistry testing. We were able to find three novel MLH1 and one novel PMS2 germline mutations in three Iranian HNPCC patients. The first was a transversion mutation c.346A>C (T116P) and happened in the highly conserved HATPase-c region of MLH1 protein. The second was a transversion mutation c.736A>T (I246L), which caused an amino acid change of isoleucine to leucine. The third mutation (c.2145,6 delTG) was frameshift and resulted in an immature stop codon in five codons downstream. All of these three mutations were detected in the MLH1 gene. The other mutation was a transition mutation, c.676G>A (G207E), which has been found in exon six of the PMS2 gene and caused an amino acid change of glycine to glutamic acid. MSI assay revealed high instability in microsatellite for two patients and microsatellite stable for one patient. In all patients, an abnormal expression of the MMR proteins in HNPCC was related to the above novel mutations.

CAT25 Is a Mononucleotide Marker to Identify HNPCC Patients

Mismatch repair mutations are the cause of generalized genomic instability and are particularly evident at microsatellite loci, which is known as microsatellite instability (MSI). MSI is present in 85% to 90% of colorectal cancers and occurs in hereditary non-polyposis colorectal cancer (HNPCC). The National Cancer Institute recommends the "Bethesda panel" for MSI screening. Recently, a novel T(25) mononucleotide marker was described, termed CAT25. This microsatellite marker displays a quasi-monomorphic pattern in normal tissues. The aim of our study was to evaluate the performance of CAT25 in HNPCC patients and to compare its reliability with the results of the Bethesda panel. We tested 55 tumor tissues from HNPCC patients using both the Bethesda panel and the CAT25 mononucleotide marker. One hundred healthy blood donors were used as controls. The CAT25 microsatellite was found to be altered in all 13 colorectal cancers classified as MSI-H using the standard Bethesda panel. Colorectal tumors that showed a stable Bethesda pattern did not show altered CAT25 repeats. Additionally, CAT25 showed a monomorphic allele pattern in all tissue samples. In our series, the concordance between the Bethesda panel and CAT25 in identifying colorectal cancers with high MSI reached 100%. Our results suggest that the CAT25 microsatellite represents a sensitive and specific marker for MSI and could be, at least, included in the panel of markers for the identification of HNPCC patients.

Hereditaer Nonpolyposis Colorectalis Carcinoma előfordulásával szerzett tapasztalatainkr

Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is the most frequent genetic disease leading to colorectal malignancies. 3-5 percent of all colorectal cancer cases are related to Hereditary Non-Polyposis Colorectal Cancer. Currently there is no reliable data on the incidence of the disease in Hungary. The family history of 809 patients with colorectal cancer was investigated between 1997 and 2006. Data collection was performed in a retrospective manner in the rst and in a prospective manner in the second phase of the study. Subsequent immunohistochemical and microsatellite instability analysis, and DNA sequencing was carried out to detect any underlying MMR gene mutation. Moreover, multiple ligation dependent probe amplication was applied for recognizing large deletions in MMR genes. During the investigation 10 pathogenic mutations and several polymorphisms were found. Seven mutations were detected in families fulfilling the Amsterdam Criteria, while the remaining three fulfilled the Bethesda Criteria. In the second phase of the study -- which better reflects the real population characteristics -- six percent of our patients fulfilled completely the Amsterdam Criteria and we managed to nd pathogenic MMR gene mutations in 1,7 percent of our patients. 8 out of 10 pathogenic mutations were novel and published rst time by ourselves. No repeatedly occurring mutations were found in our patients. It seems that these genetic alterations are located sporadically on different exons of the involved MMR genes. Our mutation detection rate was 77 percent in the Amsterdam positive patients who were completely examined, which appears to be better than the published data in the relevant literature. Importantly, 30 percent of the mutation carrier could be missed applying only this single criteria system. In order to detect the highest number of HNPCC patients, we suggest using both the Amsterdam as well as the Bethesda Criteria.

Molecular Analysis: Microsatellite Instability and Loss of Heterozygosity of Tumor Suppressor Gene in Hereditary Non-Polyposis Colorectal Cancer (HNPCC)

HNPCC (Hereditary non-polyposis colorectal cancers) development is caused by mutation of genes included in system of mismatch repair genes. The mutation exists at 60% of patients in hMSH2 gene, 30% in hMLH1 and 10% both in hPMS1and hPMS2 genes. RER+ exists in about 90% in hereditary non-polyposis colorectal cancer and about 15-28% in sporadic cancers. The purpose of the study was to determine highly sensitive microsatellite markers which can be fast and efficient way of microsatellite screening for detection of HNPCC patients. Moreover, we have analysed the loss of heterozygosity of tumour suppressor genes which could have the diagnostic value in detection of HPNCC patients.

An Interstitial Deletion at 3p21.3 Results in the Genetic Fusion ofMLH1andITGA9in a Lynch Syndrome Family

Germline mutations in DNA mismatch repair genes, mainly MLH1 or MSH2, have been shown to predispose with high penetrance for the development of the clinical phenotype of hereditary nonpolyposis colorectal cancer (Lynch syndrome). Here, we describe the discovery and first functional characterization of a novel germline MLH1 mutant allele. A large kindred including 54 potential carriers was investigated at the molecular level by using different types of PCR experiments, gene cloning, transfection studies, Western blot experiments, and mismatch repair assays to identify and characterize a novel MLH1 mutant allele. Twenty-two of 54 putative carriers developed colon cancer or other tumors, including breast cancer. The identified MLH1 mutant allele emerged from an interstitial deletion on chromosome 3p21.3, leading to an in-frame fusion of MLH1 (exons 1-11) with ITGA9 (integrin alpha 9; exons 17-28). The deleted area has a size of about 400 kb; codes for LRRFIP2 (leucine-rich repeat in flightless interaction protein 2), GOLGA4 (Golgi autoantigen, golgin subfamily a, 4), and C3orf35/APRG1 (chromosome 3 open reading frame 35/AP20 region protein 1); and partly disrupts the AP20 region implicated in major epithelial malignancies. Tumor cells lost their second MLH1 allele. The MLH1*ITGA9 fusion protein provides no capability for DNA mismatch repair. Murine fibroblasts, expressing a doxycycline-inducible MLH1*ITGA9 fusion gene, exhibit a loss-of-contact inhibition phenotype. This is the first description of a functional gene fusion of the human MLH1 gene, resulting in the loss of mismatch repair capabilities. The MLH1*ITGA9 fusion allele, together with deletions of the AP20 region, presumably defines a novel subclass of Lynch syndrome patients, which results in an extended tumor spectrum known from hereditary nonpolyposis colorectal cancer and Muir-Torre syndrome patients.

Mismatch repair gene mutations in Chinese HNPCC patients

To explore the characteristics of DNA mismatch repair gene mutations in Chinese patients with hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome, the MLH1 and MSH2 genes from probands of 76 HNPCC families were sequenced. By doing so, two frame-shift mutations, three splice-site mutations and fourteen missense mutations (thirteen missense mutations and one nonsense mutation) were identified in the MLH1 gene. In addition, one splice-site mutation and six missense mutations were detected in the MSH2 gene. None of these mutations were detected in 100 matched healthy controls. The remaining mutation-negative cases were subjected to large fragment deletion analysis using multiplex ligation-dependent probe amplification (MLPA). By doing so, five large fragment deletions were detected in the MSH2 gene. No large fragment deletions were detected in the MLH1 gene. We conclude that the MLH1 and MSH2 genes in Chinese HNPCC families exhibit broad mutation spectra.

Functional analysis of HNPCC-related missense mutations in MSH2

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein–protein interactions.