We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer. Mlh1-Pms1 also binds to DNA, but independently of a mismatch. Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1-Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.

Using immunolocalization of the mismatch-repair protein MLH1 in oocytes and spermatocytes of the Japanese quail and the zebra finch, we estimated the average amount of recombination in each sex of both species. In each case the number of MLH1 foci is statistically equivalent in males and females and the resulting sex-averaged map lengths are 2800 cM in the Japanese quail and 2275 cM in the zebra finch. In the Japanese quail the MLH1 foci are regularly distributed along the macrobivalents and recombination rates per Mb pair are somewhat lower compared to the chicken. In the zebra finch the MLH1 foci on the macrobivalents are substantially reduced in number relative to the Japanese quail and they show remarkable localization in both sexes. It is proposed that the lack of sex-dependent differences in recombination might be an extended feature among birds and that the different recombination patterns observed here reflect different controls of crossing over in spite of similarities regarding karyotypic asymmetry and DNA content. We discussed possible causes of the differences between birds and mammals, which show sex-dependent recombination differences.

OBJECTIVE

The Lynch syndrome (LS) is an inherited cancer syndrome showing a preponderance of colorectal cancer (CRC) in context with endometrial cancer and several other extracolonic cancers, which is due to pathogenic mutations in the mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2. Some families were found to show a LS phenotype without an identified MMR mutation, although there was microsatellite instability and absence of MSH2 expression by immunohistochemistry. Studies of a subset of these families found a deletion at the 3' end of the epithelial cell adhesion molecule (EPCAM) gene, causing transcription read-through resulting in silencing of MSH2 through hypermethylation of its promoter. The tumor spectrum of such families appears to differ from classical LS.

METHODS

Our study of two large families (USA Family R and Dutch Family A) with an EPCAM deletion was carried out using each institution's standard family study protocol. DNA was extracted from peripheral blood and EPCAM deletion analysis was performed.

RESULTS

Both families were found to harbor the same deletion at the 3' end of EPCAM. Analysis showed that the deletion originated from a common ancestor. Family R and Family A members showed segregation of CRC with the presence of this EPCAM mutation. Compared with classic LS, there were almost no extracolonic cancers.

CONCLUSIONS

Members of Family R and Family A, all with the same EPCAM deletion, predominantly presented with CRC but no LS-associated endometrial cancer, confirming findings seen in other, smaller, LS families with EPCAM mutations. In these EPCAM mutation carriers, cancer surveillance should be focused on CRC.

Heterozygous germ-line mutations in DNA mismatch repair (MMR) genes predispose individuals to hereditary nonpolyposis colorectal cancer (HNPCC), whereas with homozygous MMR gene mutations children are diagnosed at an early age with de novo neurofibromatosis type 1 (NF1) and/or hematological malignancies. Here, we describe a mutation, MLH1 P648S, which was found in a typical HNPCC family, with one homozygous child displaying mild features of NF1 and no hematological cancers. To evaluate the pathogenicity of the mutation, we studied both the expression and the function of the mutated protein. It generally has been assumed that the predisposing mutations prevent the production of a functional protein. The mutated MLH1 P648S protein was found to be unstable but still functional in mismatch repair, suggesting that the cancer susceptibility in the family and possibly also the mild disease phenotype in the homozygous individual are linked to shortage of the functional protein.

OBJECTIVE

Genetic instability related to defective DNA mismatch repair genes may be involved in the pathogenesis of carcinoma in hereditary nonpolyposis colon cancer (HNPCC). However, nonneoplastic tissues from patients inheriting defects in human MSH2 or human MLH1 do not show significant genetic instability. The aim of this study was to determine whether acquisition of genetic instability at the adenoma stage promotes malignant transformation by studying adenoma-carcinoma progression in HNPCC.

METHODS

Dinucleotide repeat loci were analyzed by polymerase chain reaction from microdissected adenoma and/or carcinoma stages from formalin-fixed paraffin-embedded HNPCC tumors.

RESULTS

Although genetic instability was observed at some loci in almost all cases, the proportion of microsatellite loci altered was significantly less (P < 0.01) in completely benign adenomas (24%) than in benign areas of adenomas with malignancy (54%). Molecular fingerprints indicated intratumor heterogeneity, with evolution of related subclones of neoplastic cells. However, in all cases of tumor progression, at least one subclone from the adenoma stage was closely related to the carcinoma.

CONCLUSIONS

Some genetic instability develops at the benign adenoma stage in most HNPCC tumors. Adenomas with a greater rate of genetic instability are more likely to progress to carcinoma. Topographic genotyping data provides evidence supporting the hypothesis of adenoma-carcinoma progression in HNPCC.

The first-degree relatives of patients affected by colorectal cancer, who do not belong to familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer families, have a doubled risk of developing tumors of the large intestine. We have previously demonstrated that subjects with a single first-degree relative (SFDR) with colon cancer have a doubled risk for developing colorectal adenomas, and in these cases, polyps recur more frequently. The mechanism underlying this predisposition has not been clarified. In this study, we evaluated the frequency of microsatellite instability (MSI) using the five markers suggested by the National Cancer Institute workshop, target gene mutations, hMLH1 and hMSH2 expression, and hMLH1 promoter hypermethylation in the adenomas of patients with and without a SFDR affected by colon cancer. Seventy polyps were obtained from 70 patients: 27 with a single FDR with colon cancer and 43 without such a history. Of the 70 polyps, 12 were MSI-H (17.1%), 20 were MSI-L (28.6%), and 30 were microsatellite stable (42.9%). Of the 27 patients with positive family history, 8 polyps (29.6%) were MSI-H compared with those with negative history in which 4 polyps (9.3%) were MSI-H (P < 0.02). Of the 12 MSI-H polyps, all of the polyps obtained from patients with positive family history had loss of hMLH1 immunostaining versus one with negative family history (P < 0.02). Of the MSI-H polyps, 2 had a somatic frameshift mutation of the MBD4 gene, 1 of MSH6, 1 of BAX, and 2 of transforming growth factor betaRII. Furthermore, 6 of 8 polyps from patients with positive family history with MSI-H and loss of MLH1 had hypermethylation of the MLH1promoter versus none of the MSI-H with negative family history (P < 0.02). All 6 polyps of the 27 from SFDR positive subjects, with hMLH1 promoter hypermethylation loss of hMLH1 and MSI, were located in the right colon (P < 0.02). Hypermethylation of the promoter of hMLH1, consequent loss of hMLH1 expression, and MSI are at the basis of approximately 25% of adenomatous polyps developed in subjects with a SFDR affected by colorectal cancer.

Germline PTEN mutations cause Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRR), two hamartoma-tumor syndromes with an increased risk of breast, thyroid and endometrial cancers. Somatic genetic and epigenetic inactivation of PTEN is involved in as high as 93% of sporadic endometrial carcinomas (EC), irrespective of microsatellite status, and can occur in the earliest precancers. EC is the most frequent extra-colonic cancer in patients with hereditary non-polyposis colon cancer syndrome (HNPCC), characterized by germline mutations in the mismatch repair (MMR) genes and by microsatellite instability (MSI) in component tumors. To determine whether PTEN is involved in the pathogenesis of EC arising in HNPCC cases, and whether PTEN inactivation precedes MMR deficiency, we obtained 41 ECs from 29 MLH1 or MSH2 mutation positive HNPCC families and subjected them to PTEN expression and mutation analysis. Immunohistochemical analysis revealed 68% (28/41) of the HNPCC-related ECs with absent or weak PTEN expression. The remaining 27% (11/41) of tumors had normal expression and 5% (2/41) with mixed populations showing weak/absent as well as normal expression. Mutation analysis of 20 aberrant PTEN-expressing tumors revealed that 17 (85%) harbored 18 somatic PTEN mutations. All mutations were frameshift, 10 (56%) of which involved the 6(A) tracts in exon 7 or 8. These results suggest that PTEN plays a significant pathogenic role in both HNPCC and sporadic endometrial carcinogenesis, unlike the scenarios for colorectal cancer. Furthermore, we have shown that somatic PTEN mutation, especially frameshift, is a consequence of profound MMR deficiency in HNPCC-related ECs. In contrast, among 60 previously reported MSI+ sporadic ECs with 70 somatic mutations in PTEN, 39 (56%) were frameshift, of which only eight (21%) were affecting the 6(A) tracts in exon 7 or 8 (P = 0.01), suggesting that PTEN mutations may precede MMR deficiency.

Our understanding of somatic alterations in colon cancer has evolved from a concept of a series of events taking place in a single sequence to a recognition of multiple pathways. An oncogenetic tree is a model intended to describe the pathways and sequence of somatic alterations in carcinogenesis without assuming that tumors will fall in mutually exclusive categories. We applied this model to data on colon tumor somatic alterations. An oncogenetic tree model was built using data on mutations of TP53, KRAS2, APC, and BRAF genes, methylation at CpG sites of MLH1 and TP16 genes, methylation in tumor (MINT) markers, and microsatellite instability (MSI) for 971 colon tumors from a population-based series. Oncogenetic tree analysis resulted in a reproducible tree with three branches. The model represents methylation of MINT markers as initiating a branch and predisposing to MSI, methylation of MHL1 and TP16, and BRAF mutation. APC mutation is the first alteration in an independent branch and is followed by TP53 mutation. KRAS2 mutation was placed a third independent branch, implying that it neither depends on, nor predisposes to, the other alterations. Individual tumors were observed to have alteration patterns representing every combination of one, two, or all three branches. The oncogenetic tree model assumptions are appropriate for the observed heterogeneity of colon tumors, and the model produces a useful visual schematic of the sequence of events in pathways of colon carcinogenesis.

OBJECTIVE

Lynch syndrome is a genetic disease that predisposes to colorectal tumors, caused by mutation in mismatch repair genes. The use of genetic tests to identify mutation carriers does not always give perfectly clear results, as happens when an unclassified variant is found. This study aimed to define the pathogenic role of 35 variants present in MSH2, MLH1, MSH6, and PMS2 genes identified in our 15-year case study.

METHODS

We collected clinical and molecular data of all carriers, and then we analyzed the variants pathogenic role with web tools and molecular analyses. Using a Bayesian approach, we derived a posterior probability of pathogenicity and classified each variant according to a standardized five-class system.

RESULTS

The MSH2 p.Pro349Arg, p.Met688Arg, the MLH1 p.Gly67Arg, p.Thr82Ala, p.Lys618Ala, the MSH6 p.Ala1236Pro, and the PMS2 p.Arg20Gln were classified as pathogenic, and the MSH2 p.Cys697Arg and the PMS2 p.Ser46Ile were classified as likely pathogenic. Seven variants were likely nonpathogenic, 3 were nonpathogenic, and 16 remained uncertain.

CONCLUSIONS

Quantitative assessment of several parameters and their integration in a multifactorial likelihood model is the method of choice for classifying the variants. As such classifications can be associated with surveillance and testing recommendations, the results and the method developed in our study can be useful for helping laboratory geneticists in evaluation of genetic tests and clinicians in the management of carriers.

An accurate algorithm is essential for effective molecular diagnosis of hereditary colorectal cancer (CRC). Here, we have extended the analysis of 71 CRC cases suspected to be Lynch syndrome cases for MSH2, MLH1, MSH6, and PMS2 gene defects. All cases were screened for mutations in MSH2, MLH1, and MSH6, and all cases where tumors were available were screened for microsatellite instability (MSI) and expression of MSH2 and MLH1. Subsequently, mutation-negative cases were screened for MLH1 methylation and mutations in PMS2. Of the MSI-high (MSI-H) cases, 96% had a mismatch repair (MMR) gene defect, mostly involving MSH2 or MLH1; one PMS2 mutation, one MLH1 epimutation, and no MSH6 mutations were found. Four of the 28 MSI-H cases, including one Amsterdam criteria case, had biallelic tumor MLH1 methylation, indicating that sporadic cases can be admixed in with Lynch syndrome cases, even those meeting the strongest criteria for Lynch syndrome. MMR gene defects were found in similar frequency in cases where tumors were and were not available. One MLH1 and one MSH2 deletion mutation were found in MSI-stable/low cases, indicating that MSI testing can exclude cases with pathogenic mutations. Our analysis supports a diagnostic algorithm where cases are selected for analysis based on clinical criteria or prediction models; isolated sporadic young-onset cases can be prescreened by tumor testing, whereas familial cases may be directly subjected to molecular analysis for mutations in MMR genes followed by MSI, protein expression, and DNA methylation analysis to aid in the resolution of mutation-negative cases.

BACKGROUND

We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability.

RESULTS

We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohistochemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value = 0.72 vs. 2.3×10(-4) when the SNP was examined alone).

CONCLUSIONS

The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

Although early detection of Lynch syndrome (LS) is important, a considerable proportion of patients with LS remains unrecognized. We aimed to study the yield of LS detection by routine molecular analyses in colorectal cancer (CRC) patients until 70 years of age. We prospectively included consecutive CRC patients ≤70 years. Tumour specimens were analysed for microsatellite instability (MSI), immunohistochemical mismatch-repair protein expression and MLH1-promoter methylation. Tumours were classified as either: (a) likely caused by LS; (b) sporadic microsatellite-unstable (MSI-H); or (c) microsatellite-stable (MSS). Predictors of LS were determined by multivariable logistic regression. A total of 1117 CRC patients (57% males, median age 61 years) were included. Fifty patients (4.5%, 95% CI 3.4-5.9) were likely to have LS, and 71 had a sporadic MSI-H tumour (6.4%, 95% CI 5.1-8.0). Thirty-five patients likely to have LS (70%) were aged>> 50 years. A molecular profile compatible with LS was detected in 10% (15/144) of patients aged ≤50, in 4% (15/377) of those aged 51-60 and in 3% (20/596) of patients>> 61 years. Compared to MSS cases, patients likely to have LS were significantly younger (OR 3.9, 95% CI 1.7-8.7) and more often had right-sided CRCs (OR 14, 95% CI 6.0-34). In conclusion, molecular screening for LS in CRC patients ≤70 years leads to identification of a molecular profile compatible with LS in 4.5% of patients, with most of them not fulfilling the age criterion (≤50 years) routinely used for LS assessment. Routine use of MSI testing may be considered in CRC patients up to the age of 70 years, with a central role for the pathologist in the selection of patients.

Mutation analysis of large genes, such as MSH2 and MLH1, is time-consuming and expensive. We investigated the sensitivity and specificity of DHPLC analysis for the detection of mutations within both MSH2 and MLH1. Studies included a series of 46 patients affected by colorectal cancer from HNPCC families. We confirmed 19 changes previously identified by DNA sequencing and, in a blind study, an additional 16 rare alterations including four mutations not previously described. Generally, false negative results were not observed. Elution profiles were highly characteristic for a given change and in 98.5% cases allowed the distinction between novel alterations and previously identified mutations and polymorphisms. For the detection of changes in almost all amplicons, it was sufficient to use just one denaturing temperature. DHPLC was confirmed to be highly sensitive, specific and a cost-effective technique with particularly high potential for the detection of MSH2 and MLH1 gene mutations in the diagnostic setting.

BACKGROUND

Microsatellite instability (MSI) has been identified as a factor with good prognosis and chemosensitivity in stage III C colon cancer. The purpose of this study was to evaluate the routine use of immunohistochemical analysis (immunohistochemical staining of MSH2 and MLH1) to identify T3N0M0 (stage II) colon cancer with MSI and assess the prognostic value of this analysis. The study was conducted in a large cohort of patients in a single institution who had a curatively resected T3N0M0 colon cancer and were not receiving adjuvant therapy.

METHODS

Between June 1995 and December 2001, 142 patients (77 females) with a mean age of 68 years, suffering from T3N0M0 colon cancer curatively resected and not receiving adjuvant therapy, were checked in terms of their follow up status. The results of colonoscopy, hepatic ultrasonography, chest x ray, and blood carcinoembryological antigen were noted. All tumours were immunohistochemically stained for MSH2 and MLH1. Perineural invasion, lymphovascular invasion, and the presence of vascular neoplastic emboli were assessed.

RESULTS

Twenty four patients (17%) had MSI tumours. Patients with MSI and microsatellite stable (MSS) tumours did not differ in terms of age, perineural or lymphovascular invasion, or the presence of vascular neoplastic emboli. Patients with MSI tumours were more frequently female (18/24 v 60/118; p = 0.001) and more frequently suffered from right sided cancer (19/24 v 58/118; p<0.001). Patients with MSI tumours exhibited significantly better recurrence free survival than those with MSS tumours (p = 0.02). Cox analysis identified age and MSI determined by immunohistochemistry as independent predictive factors of good prognosis (p = 0.009, odds ratio 1.04 (1.01-1.08); p = 0.04, odds ratio 7.9 (1.05-59.6)).

CONCLUSIONS

MSI determined by immunohistochemistry is an independent predictive factor of good prognosis in T3N0M0 colon cancer. The prognosis for MSI T3N0M0 colon cancer is excellent and chemotherapy should not be proposed in these patients as immunohistochemical analysis produces rapid results.

Both genetic and epigenetic events have been implicated in the stepwise histological progression involving adenoma-carcinoma and hyperplastic polyp/serrated adenoma-carcinoma sequences in the development of colorectal cancer. Genetic changes have been observed at each step in the initiation and progression of polyps to adenocarcinomas. Epigenetic changes also occur at each step in the pathogenesis of colorectal cancers and include CpG island DNA hypermethylation in the promoter region of genes resulting in transcriptional silencing through associated changes in chromatin structure and effects on binding of transcription factors, and DNA global hypomethylation which leads to chromosomal instability. Recent studies on MLH1 and APC genes indicate that epigenetic and genetic changes cooperate to facilitate tumor initiation and progression. Since aberrant CGI DNA promoter hypermethylation can be detected not only in colorectal polyps and cancers, but also in sera and stool, hypermethylated genes may serve as molecular markers for early detection, risk assessment and diagnosis. In addition, silenced genes caused by CGI DNA promoter hypermethylation can be reactivated by demethylating agents and also by both the inhibitors of DNA methyltransferases and histone deacetylases. Therefore, these epigenetically acting drugs should be evaluated for their chemopreventive and therapeutic potential for colorectal cancers.

OBJECTIVE

Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer (CRC). We sought to determine the frequency of high-level microsatellite instability (MSI-H) and the mutational and methylation profile of MSI-H IBD-related neoplasms (IBDNs).

METHODS

A total of 124 IBDNs (81 cancers, 43 dysplasias) from 78 patients were studied for the frequency of MSI-H and hypermethylation of 3 target genes: MLH1 , HPP1 , and RAB-32 . Fifteen MSI-H IBDNs were characterized according to their profile of frameshift mutations in 28 mononucleotide repeats and compared with 46 sporadic MSI-H CRCs.

RESULTS

Nineteen of 124 IBDNs were MSI-H. The frequency of frameshift mutations in coding mononucleotide repeats was significantly lower in MSI-H IBDNs than in sporadic MSI-H CRCs for TGFBR2 (7 of 14 vs 34 of 43 samples; P = .047) and ACVR2 (3 of 14 vs 25 of 43 samples; P = .029). In contrast, ICA1 was mutated in 3 of 9 MSI-H IBDNs vs 2 of 54 sporadic MSI-H CRCs ( P = .028). HPP1 and RAB32 methylation was independent of MSI status and was observed in 4 of 59 and 0 of 64 nondysplastic mucosae, 20 of 38 and 1 of 25 dysplasias, and 28 of 61 and 20 of 60 carcinomas, respectively.

CONCLUSIONS

The profiles of coding microsatellite mutations (instabilotypes) differ significantly between MSI-H IBDNs and MSI-H sporadic CRCs. Specifically, TGFBR2 and ACVR2 mutations are significantly rarer in MSI-H IBDNs than in MSI-H sporadic CRCs. Furthermore, HPP1 methylation occurs early, in 7% of nondysplastic and approximately half of dysplastic mucosae, whereas RAB32 methylation occurs at the transition to invasive growth, being rarer in dysplasias.

A small set of gastric adenocarcinomas (9%) harbor Epstein-Barr virus (EBV) DNA within malignant cells, and the virus is not an innocent bystander but rather is intimately linked to pathogenesis and tumor maintenance. Evidence comes from unique genomic features of host DNA, mRNA, microRNA and CpG methylation profiles as revealed by recent comprehensive genomic analysis by The Cancer Genome Atlas Network. Their data show that gastric cancer is not one disease but rather comprises four major classes: EBV-positive, microsatellite instability (MSI), genomically stable and chromosome instability. The EBV-positive class has even more marked CpG methylation than does the MSI class, and viral cancers have a unique pattern of methylation linked to the downregulation of CDKN2A (p16) but not MLH1. EBV-positive cancers often have mutated PIK3CA and ARID1A and an amplified 9p24.1 locus linked to overexpression of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2). Multiple noncoding viral RNAs are highly expressed. Patients who fail standard therapy may qualify for enrollment in clinical trials targeting cancer-related human gene pathways or promoting destruction of infected cells through lytic induction of EBV genes. Genomic tests such as the GastroGenus Gastric Cancer Classifier are available to identify actionable variants in formalin-fixed cancer tissue of affected patients.

OBJECTIVE

High level of microsatellite instability (MSI-H) in colorectal cancer (CRC) is caused by the inactivation of mismatch repair (MMR) genes; however, it is unknown for tumors with low level MSI (MSI-L). The protein complex involving MSH3 preferentially recognizes insertion/deletion loops (IDLs) of two to eight bases and di- and tetranucleotide repeats are affected in the majority of MSI-L CRC.

METHODS

We selected 10 and eight MSI-L CRCs from 228 and 204 patients with sporadic and hereditary disease, respectively. The tumors were analyzed for protein expression of MSH3, MSH2, MSH6, MLH1, and PMS2, and for mutations and loss of heterozygosity (LOH) in MSH3.

RESULTS

Four tumors showed a markedly reduced MSH3 expression, whereas all 18 tumors had normal expression of the remaining MMR proteins. Twenty-five different sequence variants were identified. None of these results in a truncated protein, though L902W represents the first constitutional missense mutation in MSH3 predicted to be functional based on conservation among mutS homologues. All variants have also been found in normal DNA of the patients and in controls. LOH intragenic to MSH3 was evident for 12 of 16 (75%) informative tumors.

CONCLUSIONS

Occurrence of sequence variants in normal DNA of the patients and in controls excludes somatic mutations and mutations specific to the CRC patient population, respectively. In contrast, the high frequency of LOH as well as the aberrant protein expression in some tumors indicates an involvement of MSH3 impairment in MSI-L CRC.

To date, immunocytology has been used in humans to detect a limited number of meiotic proteins: components of the synaptonemal complex (SCP1 and SCP3) and some proteins known to participate in recombination events, such as MLH1 or RAD51. However, the colocalization or coexistence of proteins known to participate during the different stages of human meiosis remains largely unstudied, and these studies could provide important clues about the mechanics of recombination. This work reports the relative timing and localization of five different meiotic proteins that have previously been implicated in human homologous recombination [RAD51, replication protein A (RPA), MSH4, MLH1 and MLH3]. MSH4 foci appear concurrently with synapsis initiation at zygotene, shortly after the first RAD51 foci are detected. The presence of RPA in MSH4 foci was noted, suggesting that these two proteins may act co-operatively. Both RPA and MSH4 foci reach maximal numbers at the end of zygotene, when synapsis is concluding. From this point, RPA foci all but disappear by the end of pachytene, whereas MSH4 foci decline to a stable number at mid-pachytene, where they localize with MLH1/MLH3 recombination sites. We discuss a possible role for MSH4 in synapsis initiation and/or maintenance.

Inherited mutations in 1 of 4 known mismatch repair genes (MLH1, MSH2, MSH6, PMS2) are associated with various cancer risks collectively referred to as Lynch syndrome. Roughly 3 of every 100 new colorectal cancers (CRCs) have an underlying Lynch mutation. Tumor-based screening for Lynch among all patients with newly diagnosed CRC could theoretically improve the ability to identify Lynch and prevent cancer among at-risk family members, but the patient-level and social implications of this approach must be carefully considered before adopting this strategy. Poorly addressed issues include the role/timing of informed consent for testing, access and cost barriers associated with genetic counseling and DNA testing, psychosocial burdens to the thousands of middle-aged and elderly patients with CRC coping with surgical and chemotherapy treatments and poor prognosis, the need for providers to warn third-party relatives of risk for Lynch syndrome, limited effectiveness of screening, and the cost burden to society when poor DNA testing uptake, test limitations, and modest screening compliance are considered. Diverse barriers to the success of a population-based Lynch screening program in the United States remain (e.g., clinical resource needs, financial limitations, clinical expertise gaps, educational deficits). Data supporting clinical efficacy (feasibility) and effectiveness (real-life performance) are critical before important policy changes are adopted, especially where issues of hereditary cancer risk and genetic privacy are involved.

OBJECTIVE

There is a well known association between obesity and endometrial cancer. We sought to examine the relationships between body mass index (BMI), as a measure of obesity, and known demographic, clinical, and molecular characteristics of microsatellite instability and MLH1 promoter methylation in a cohort of patients with endometrial cancer.

METHODS

Corpus cancer specimens were prospectively obtained from 473 consecutively enrolled patients between 1992 and 2004. Clinical and pathologic data were extracted from review of the medical record. Microsatellite instability (MSI) was evaluated in all tumors, and methylation of the MLH1 promoter was determined for MSI positive tumors.

RESULTS

The median (SD) age and BMI were 64.8 years (11.9) and 33.5 (9.4), respectively. Histology included 376 endometrioid (79%), 69 serous/clear cell or mixed (15%), and 28 sarcomas (6%). Median BMI was 32.4 for endometrioid, 31.0 for serous/clear cell or mixed, and 27.8 for sarcomas (p=0.14). BMI was negatively associated with age at surgery (p<0.01). The remainder of analyses excluded sarcoma histology. BMI was associated with stage of disease; patients with stage I/II disease had significantly higher BMI than those with stage III/IV disease (32.6 vs. 30.6; p=0.02). In relation to molecular features of endometrial cancer, BMI was significantly different between MSI positive tumors compared to MSI negative tumors (30.3 vs. 32.7; p=0.02). MSI was also significantly different between tumor histology, occurring with a higher frequency in Type I than Type II tumors (p<0.01).

CONCLUSIONS

The majority of endometrial cancer patients are obese. Those with higher BMI are more likely to be younger, present with early stage disease, and have MSI negative tumors.

An increasing number of mismatch-repair (MMR) gene mutations have been identified in hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. This study presents the population-based Danish MMR gene mutation profile, which contains 138 different MMR gene alterations. Among these, 88 mutations in 164 families are considered pathogenic and an additional 50 variants from 76 families are considered to represent variants of unknown pathogenicity. The different MMR genes contribute to 40% (MSH2), 29% (MLH1), and 22% (MSH6) of the mutations and the Danish population thus shows a considerably higher frequency of MSH6 mutations than previously described. Although 69/88 (78%) pathogenic mutations were present in a single family, previously recognized recurrent/founder mutations were causative in 75/137 (55%) MLH1/MSH2 mutant families. In addition, the Danish MLH1 founder mutation c.1667+2_1667_+8TAAATCAdelinsATTT was identified in 14/58 (24%) MLH1 mutant families. The Danish Lynch syndrome population thus demonstrates that MSH6 mutations and recurrent/founder mutations have a larger contribution than previously recognized, which implies that the MSH6 gene should be included in routine diagnostics and suggests that directed analysis of recurrent/founder mutations may be feasible e.g. in families were diagnostic material is restricted to archival tissue.

Arsenic is carcinogenic, possibly partly through epigenetic mechanisms. We evaluated the effects of arsenic exposure and metabolism on DNA methylation. Arsenic exposure and methylation efficiency in 202 women in the Argentinean Andes were assessed from concentrations of arsenic metabolites in urine (inorganic arsenic, methylarsonic acid [MMA], and dimethylarsinic acid [DMA]), measured by HPLC-ICPMS. Methylation of CpGs of the tumor suppressor gene p16, the DNA repair gene MLH1, and the repetitive elements LINE1 was measured by PCR pyrosequencing of blood DNA. Genotyping (N = 172) for AS3MT was performed using Sequenom™, and gene expression (N = 90) using Illumina DirectHyb HumanHT-12 v3.0. Median arsenic concentration in urine was 230 μg L(-1) (range 10.1-1251). In linear regression analysis, log(2)-transformed urinary arsenic concentrations were positively associated with methylation of p16 (β = 0.14, P = 0.0028) and MLH1 (β = 0.28, P = 0.0011), but not with LINE1. Arsenic concentrations were of borderline significance negatively correlated with expression of p16 (r(s) = -0.20; P = 0.066)), but not with MLH1. The fraction of inorganic arsenic was positively (β = 0.026; P = 0.010) and DMA was negatively (β = -0.017, P = 0.043) associated with p16 methylation with no effect of MMA. Carriers of the slow-metabolizing AS3MT haplotype were associated with more p16 methylation (P = 0.022). Arsenic exposure was correlated with increased methylation, in blood, of genes encoding enzymes that suppress carcinogenesis, and the arsenic metabolism efficiency modified the degree of epigenetic alterations.

To prevent genome instability, recombination between sequences that contain mismatches (homeologous recombination) is suppressed by the mismatch repair (MMR) pathway. To understand the interactions necessary for this regulation, the genetic requirements for the inhibition of homeologous recombination were examined using mutants in the RAD52 epistasis group of Saccharomyces cerevisiae. The use of a chromosomal inverted-repeat recombination assay to measure spontaneous recombination between 91 and 100% identical sequences demonstrated differences in the fidelity of recombination in pathways defined by their dependence on RAD51 and RAD59. In addition, the regulation of homeologous recombination in rad51 and rad59 mutants displayed distinct patterns of inhibition by different members of the MMR pathway. Whereas the requirements for the MutS homolog, MSH2, and the MutL homolog, MLH1, in the suppression of homeologous recombination were similar in rad51 strains, the loss of MSH2 caused a greater loss in homeologous recombination suppression than did the loss of MLH1 in a rad59 strain. The nonequivalence of the regulatory patterns in the wild-type and mutant strains suggests an overlap between the roles of the RAD51 and RAD59 gene products in potential cooperative recombination mechanisms used in wild-type cells.