Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Defective mismatch repair (MMR) in humans causes hereditary nonpolyposis colorectal cancer. This genetic predisposition to colon cancer is linked to heterozygous familial mutations, and loss-of-heterozygosity is necessary for tumor development. In contrast, the rare cases with biallelic MMR mutations are juvenile patients with brain tumors, skin neurofibromas, and café-au-lait spots, resembling the neurofibromatosis syndrome. Many of them also display lymphomas and leukemias, which phenotypically resembles the frequent lymphoma development in mouse MMR knockouts. Here, we describe the identification and characterization of novel knockout mutants of the three major MMR genes, mlh1, msh2, and msh6, in zebrafish and show that they develop tumors at low frequencies. Predominantly, neurofibromas/malignant peripheral nerve sheath tumors were observed; however, a range of other tumor types was also observed. Our findings indicate that zebrafish mimic distinct features of the human disease and are complementary to mouse models.

We report on a nonconsanguineous family in which two siblings with cutaneous manifestations leading to a diagnosis of neurofibromatosis type 1 (NF1) developed CNS tumors at an early age. In addition, one of them developed a T-cell lymphoma. Neither parent had NF1. The mother was known to be heterozygous for a MSH6 mutation, and the father was found to be heterozygous for a different MSH6 mutation. Screening of MSH2, MLH1, MSH6, PMS1, PMS2, and MLH3 in the affected children disclosed that they both were compound heterozygote for the MSH6 mutations of their parents. Most recently, about a dozen other cases of inherited bi-allelic deficiency of mismatch repair (MMR) genes associated with early onset CNS tumors, hematologic malignancy, gastrointestinal neoplasia, café-au-lait spots, and other NF1 features have been reported. In the present study, we summarize the clinical findings of 27 individuals homozygous or compound heterozygous for an MMR gene mutation reported in the medical literature. We suggest that biparentally inherited mutations of one of the MMR genes should be considered in children with multiple café-au-lait spots who have early-onset CNS tumors, hematologic malignancies, or early onset gastrointestinal neoplasia.

BACKGROUND

After breast-conserving radiation therapy most patients experience acute skin toxicity to some degree. This may impair patients' quality of life, cause pain and discomfort. In this study, we investigated treatment and patient-related factors, including genetic polymorphisms, that can modify the risk for severe radiation-induced skin toxicity in breast cancer patients.

METHODS

We studied 377 patients treated at Ghent University Hospital and at ST.-Elisabeth Clinic and Maternity in Namur, with adjuvant intensity modulated radiotherapy (IMRT) after breast-conserving surgery for breast cancer. Women were treated in a prone or supine position with normofractionated (25 × 2 Gy) or hypofractionated (15 × 2.67 Gy) IMRT alone or in combination with other adjuvant therapies. Patient- and treatment-related factors and genetic markers in regulatory regions of radioresponsive genes and in LIG3, MLH1 and XRCC3 genes were considered as variables. Acute dermatitis was scored using the CTCAEv3.0 scoring system. Desquamation was scored separately on a 3-point scale (0-none, 1-dry, 2-moist).

RESULTS

Two-hundred and twenty patients (58%) developed G2+ dermatitis whereas moist desquamation occurred in 56 patients (15%). Normofractionation (both p < 0.001), high body mass index (BMI) (p = 0.003 and p < 0.001), bra cup size ≥ D (p = 0.001 and p = 0.043) and concurrent hormone therapy (p = 0.001 and p = 0.037) were significantly associated with occurrence of acute dermatitis and moist desquamation, respectively. Additional factors associated with an increased risk of acute dermatitis were the genetic variation in MLH1 rs1800734 (p=0.008), smoking during RT (p = 0.010) and supine IMRT (p = 0.004). Patients receiving trastuzumab showed decreased risk of acute dermatitis (p < 0.001).

CONCLUSIONS

The normofractionation schedule, supine IMRT, concomitant hormone treatment and patient related factors (high BMI, large breast, smoking during treatment and the genetic variation in MLH1 rs1800734) were associated with increased acute skin toxicity in patients receiving radiation therapy after breast-conserving surgery. Trastuzumab seemed to be protective.

OBJECTIVE

Lynch syndrome (hereditary nonpolyposis colorectal cancer; HNPCC) is an autosomal-dominant cancer predisposition syndrome that increases risk for multiple cancers, including colon, endometrial, and ovarian cancer. Revised Bethesda Criteria recommend that patients with two HNPCC-associated cancers undergo molecular evaluation to determine whether they have a mismatch repair (MMR) defect associated with HNPCC. The purpose of our study was to determine the likelihood of MMR defects (MSH2, MSH6, MLH1) in women with synchronous endometrial and ovarian cancer.

METHODS

Between 1989 and 2004, 102 women with synchronous endometrial and ovarian cancers were identified; 59 patients had tumor blocks available for analysis. Patients were divided into risk groups based on family history: high (met Amsterdam criteria), medium (personal history or first-degree relative with an HNPCC-associated cancer), and low (all others). Protein expression for MSH2, MSH6, and MLH1 was evaluated by immunohistochemistry. Microsatellite instability and MLH1 promoter methylation analyses were performed on a subset of cases.

RESULTS

Median age was 50 years. Two patients met Amsterdam criteria for HNPCC. Five additional patients, all medium-risk, had molecular findings consistent with a germline mutation of either MSH2 or MLH1. None of the low-risk patients had molecular results consistent with a germline mutation.

CONCLUSIONS

Overall, 7% of women in our cohort met either clinical or molecular criteria for Lynch syndrome. All of these women had a prior history or a first-degree relative with an HNPCC-associated cancer. Limiting genetic evaluation to women with synchronous endometrial and ovarian cancer who have a family history suggestive of HNPCC may appropriately identify women with Lynch syndrome.

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Lynch syndrome is one of the most common cancer susceptibility syndromes. Individuals with Lynch syndrome have a 50%-70% lifetime risk of colorectal cancer, 40%-60% risk of endometrial cancer, and increased risks of several other malignancies. It is caused by germline mutations in the DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2. In a subset of patients, Lynch syndrome is caused by 3' end deletions of the EPCAM gene, which can lead to epigenetic silencing of the closely linked MSH2. Relying solely on age and family history based criteria inaccurately identifies eligibility for Lynch syndrome screening or testing in 25%-70% of cases. There has been a steady increase in Lynch syndrome tumor screening programs since 2000 and institutions are rapidly adopting a universal screening approach to identify the patients that would benefit from genetic counseling and germline testing. These include microsatellite instability testing and/or immunohistochemical testing to identify tumor mismatch repair deficiencies. However, universal screening is not standard across institutions. Furthermore, variation exists regarding the optimum method for tracking and disclosing results. In this review, we summarize traditional screening criteria for Lynch syndrome, and discuss universal screening methods. International guidelines are necessary to standardize Lynch syndrome high-risk clinics.

Bisphenol A (BPA) is a 'weak' endocrine disruptor. The effect of BPA on human reproduction is controversial but has been related to meiotic anomalies, recurrent spontaneous abortion, abnormal karyotypes, the diminishing of oocyte survival, delay in meiotic progression and an elevated rate of MLH1 foci in vitro. The aim of this study is to characterize the gene expression of human fetal oocytes in culture as well as to evaluate the effect of BPA in cultured human oocytes. To accomplish our objective, 12 ovaries from 6 euploid fetuses were used. The ovarian fetal tissue was cultivated in two groups: control group and BPA group (BPA30 µM). The cultures were analyzed at T0 and after 7 (T7), 14 (T14) and 21 (T21) days of culture. Evaluation of gene expression was performed by real-time PCR (RT-PCR), with the evaluated genes being: Smc1β, Sycp1 (pairing-synapsis), Spo11, Rpa, H2ax, Mlh1 and Blm [double-strand break (DSBs) generation, signaling and repair], Erα, Erβ and Errγ (estrogen receptors), Stra8 and Nalp5 (markers of meiotic progression). Oocytes from ovaries cultured and treated with BPA show changes in the expression of Spo11, H2ax and Blm genes, with a significant increase from 3- to 5-fold (P≤ 0.05). Finally, Rpa, showed a 100-fold increment (P≤ 0.01). Erα, Erβ and Errγ genes showed a BPA up-regulation of 2-4-fold in all of the culture times (P≤ 0.05). Oocytes exposed to BPA showed an up-regulation of genes involved in DSB generation, signaling and repair except by Mlh1. Thus, BPA can modify the gene expression pattern, which may explain the effects of BPA on female germ cells.

The aim of this study was to determine the prognostic value of APAF-1 in colorectal cancer (CRC). Immunohistochemistry for APAF-1 was performed on a tissue microarray of 1015 mismatch-repair (MMR) proficient and 130 sporadic MLH1-negative CRCs. The association of APAF-1 with clinico-pathological features including 10-year survival time was analysed. Methylation specific PCR was performed on a subset of MMR-proficient and MLH1-negative CRC. Loss of APAF-1 was associated with advanced T stage (p-value=0.022), N stage (p-value=0.009), vascular invasion (p-value=0.001) and worse survival (p-value=0.017) in MMR-proficient CRC. In MLH1-negative CRC, loss of APAF-1 was associated with metastasis (p-value=0.041), worse prognosis (p-value<0.001) and independently predicted shorter survival time (p-value<0.001). No methylation was found in the selected region of APAF-1. APAF-1 is a marker of tumour progression in MMR-proficient CRC and an independent adverse prognostic factor in MLH1-negative CRC.

The pathogenic role for heritable mutations in the DNA sequence of tumor suppressor and DNA repair genes has been well established in familial cancer syndromes. These germ line mutations confer a high risk of developing particular types of cancer, according to the gene affected, at a young age of onset when compared to sporadically arising cancers of a similar type. The widespread role for epigenetic dysregulation in the development and progression of sporadic cancers is also well recognized. However, it has only become apparent in recent years that epigenetic aberrations can also occur constitutionally to confer a similar cancer phenotype as a genetic mutation within the same gene. These epigenetic errors are termed "constitutional epimutations" and are characterized by promoter methylation and transcriptional silencing of a single allele of the gene in normal somatic tissues in the absence of a sequence mutation within the affected locus. This is best exemplified in Lynch syndrome, which is an autosomal dominant cancer susceptibility syndrome characterized by the early development of colorectal, uterine, and additional cancers exhibiting microsatellite instability due to impaired mismatch repair. Lynch syndrome is usually caused by heterozygous loss-of-function germ line mutations of the mismatch repair genes, namely MLH1, MSH2, MSH6, and PMS2. Tumors develop following an acquired somatic loss of the remaining functional allele. However, a subset of Lynch syndrome cases without genetic mutations instead has a constitutional epimutation of MLH1 or MSH2. These epimutations are associated with distinct patterns of inheritance depending on the nature of the mechanisms underlying them.

Colorectal cancer is a heterogeneous disease at the histomorphological, clinical and molecular level. Approximately 20% of cases may progress through the "serrated" pathway characterized by BRAF mutation and high-level CpG Island Methylator Phenotype (CIMP). A large subgroup are additionally microsatellite instable (MSI) and demonstrate significant loss of tumor suppressor Cdx2. The aim of this study is to determine the specificity of Cdx2 protein expression and CpG promoter hypermethylation for BRAF(V600E) and high-level CIMP in colorectal cancer. Cdx2, Mlh1, Msh2, Msh6, and Pms2 were analyzed by immunohistochemistry using a multi-punch tissue microarray (TMA; n = 220 patients). KRAS and BRAF(V600E) mutation analysis, CDX2 methylation and CIMP were investigated. Loss of Cdx2 was correlated with larger tumor size (P = 0.0154), right-sided location (P = 0.0014), higher tumor grade (P < 0.0001), more advanced pT (P = 0.0234) and lymphatic invasion (P = 0.0351). Specificity was 100% for mismatch repair (MMR)-deficiency (P < 0.0001), 92.2% (P < 0.0001) for BRAF(V600E) and 91.8% for CIMP-high. Combined analysis of BRAF(V600E)/CIMP identified Cdx2 loss as sensitive (80%) and specific (91.5%) for mutation/high status. These results were validated on eight well-established colorectal cancer cell lines. CDX2 methylation correlated with BRAF(V600E) (P = 0.0184) and with Cdx2 protein loss (P = 0.0028). These results seem to indicate that Cdx2 may play a role in the serrated pathway to colorectal cancer as underlined by strong relationships with BRAF(V600E), CIMP-high and MMR-deficiency. Whether this protein can only be used as a "surrogate" marker, or is functionally involved in the progression of these tumors remains to be elucidated.

Lynch syndrome (clinically referred to as HNPCC - Hereditary Non-Polyposis Colorectal Cancer) is a frequent, autosomal, dominantly-inherited cancer predisposition syndrome caused by various germline alterations that affect DNA mismatch repair genes, mainly MLH1 and MSH2. Patients inheriting this predisposition are susceptible to colorectal, endometrial and other extracolonic tumors. It has recently been shown that germline deletions of the last few exons of the EPCAM gene are involved in the etiology of Lynch syndrome. Such constitutional mutations lead to subsequent epigenetic silencing of a neighbouring gene, here, MSH2, causing Lynch syndrome. Thus, deletions of the last few exons of EPCAM constitute a distinct class of mutations associated with HNPCC. Worldwide, several investigators have reported families with EPCAM 3'end deletions. The risk of colorectal cancer in carriers of EPCAM deletions is comparable to situations when patients are MSH2 mutation carriers, and is associated with high expression levels of EPCAM in colorectal cancer stem cells. A lower risk of endometrial cancer was also reported. Until now the standard diagnostic tests for Lynch syndrome have contained analyses such as immunohistochemistry and tests for microsatellite instability of mismatch repair genes. The identification of EPCAM deletions or larger EPCAM-MSH2 deletions should be included in routine mutation screening, as this has implications for cancer predisposition.

In most eukaryotes, recombination of homologous chromosomes during meiosis is necessary for proper chromosome pairing and subsequent segregation. The molecular mechanisms of meiosis are still relatively unknown, but numerous genes are known to be involved, among which are many mismatch repair genes. One of them, mlh1, colocalizes with presumptive sites of crossing over, but its exact action remains unclear. We studied meiotic processes in a knockout line for mlh1 in zebrafish. Male mlh1 mutants are sterile and display an arrest in spermatogenesis at metaphase I, resulting in increased testis weight due to accumulation of prophase I spermatocytes. In contrast, females are fully fertile, but their progeny shows high rates of dysmorphology and mortality within the first days of development. SNP-based chromosome analysis shows that this is caused by aneuploidy, resulting from meiosis I chromosomal missegregation. Surprisingly, the small percentage of progeny that develops normally has a complete triploid genome, consisting of both sets of maternal and one set of paternal chromosomes. As adults, these triploid fish are infertile males with wild-type appearance. The frequency of triploid progeny of mlh1 mutant females is much higher than could be expected for random chromosome segregation. Together, these results show that multiple solutions exist for meiotic crossover/segregation problems.

Hyperplastic polyposis syndrome (HPS) is characterized by the presence of multiple colorectal serrated polyps and is associated with an increased colorectal cancer (CRC) risk. The mixture of distinct precursor lesion types and malignancies in HPS provides a unique model to study the canonical pathway and a proposed serrated CRC pathway in humans. To establish which CRC pathways play a role in HPS and to obtain new support for the serrated CRC pathway, we assessed the molecular characteristics of polyps (n = 84) and CRCs (n = 19) in 17 patients with HPS versus control groups of various sporadic polyps (n = 59) and sporadic microsatellite-stable CRCs (n = 16). In HPS and sporadic polyps, APC mutations were exclusively identified in adenomas, whereas BRAF mutations were confined to serrated polyps. Six of 19 HPS CRCs (32%) were identified in a serrated polyp. Mutation analysis performed in the CRC and the serrated component of these lesions showed identical BRAF mutations. One HPS CRC was located in an adenoma, both components harboring an identical APC mutation. Overall, 10 of 19 HPS CRCs (53%) carried a BRAF mutation versus none in control group CRCs (P = 0.001). Six BRAF-mutated HPS CRCs (60%) were microsatellite unstable owing to MLH1 methylation. These findings provide novel supporting evidence for the existence of a predominant serrated CRC pathway in HPS, generating microsatellite-stable and microsatellite-instable CRCs.

OBJECTIVE

The mismatch repair (MMR) genes are in charge of maintaining genomic integrity. Mutations in the MMR genes are at the origin of a familial form of colorectal cancer (CRC). This syndrome accounts for only a small proportion of the excess familial risk of CRC. The characteristics of the alleles that account for the remainder of cases are unknown. To assess the putative associations between common variants in MMR genes and CRC, we performed a genetic case-control study using a single-nucleotide polymorphism (SNP) tagging approach.

METHODS

A total of 2299 cases and 2284 unrelated controls were genotyped for 68 tagging SNPs in seven MMR genes (MLH1, MLH3, MSH2, MSH3, MSH6, PMS1 and PMS2). Genotype frequencies were measured in cases and controls and analysed using univariate analysis. Haplotypes were constructed and analysed using logistic regression. We also carried out a two-locus interaction analysis and a global test analysis.

RESULTS

Genotype frequencies were found to be marginally different in cases and controls for MSH3 rs26279 with a rare homozygote OR = 1.31 [95% confidence interval (CI) 1.05 to 1.62], p(trend) = 0.04. We found a rare MLH1 (frequency <5%) haplotype, increasing the risk of colorectal cancer: (OR = 9.76; 95% CI, 1.25 to 76.29; p = 0.03). The two-locus interaction analysis has exhibited signs of interaction between SNPs located in genes MSH6 and MSH2. Global testing has showed no evidence of interaction.

CONCLUSIONS

It is unlikely that common variants in MMR genes contribute significantly to colorectal cancer.

The frequency and distribution of the mismatch repair protein MLH1 was analyzed on synaptonemal complex spreads of chicken oocytes using indirect immunofluorescence. MLH1 foci appeared in late zygotene and their number remains constant throughout pachytene. The average number of foci on autosomal synaptonemal complexes (65.02 +/- 4.02) is in agreement with the number of chiasmata estimated from lampbrush chromosomes. The distribution of foci along the synaptonemal complexes is shown to be nonrandom and nonuniform in terms of the distances between them. It is concluded that MLH1 foci are good markers of crossing over in bird (chicken) meiocytes.

In the yeast Saccharomyces cerevisiae, mismatch repair (MMR) is initiated by the binding of heterodimeric MutS homolog (MSH) complexes to mismatches that include single nucleotide and loop insertion/deletion mispairs. In in vitro experiments, the mismatch binding specificity of the MSH2-MSH6 heterodimer is eliminated if ATP is present. However, addition of the MutL homolog complex MLH1-PMS1 to binding reactions containing MSH2-MSH6, ATP, and mismatched substrate results in the formation of a stable ternary complex. The stability of this complex suggests that it represents an intermediate in MMR that is subsequently acted upon by other MMR factors. In support of this idea, we found that the replication processivity factor proliferating cell nuclear antigen (PCNA), which plays a critical role in MMR at step(s) prior to DNA resynthesis, disrupted preformed ternary complexes. These observations, in conjunction with experiments performed with streptavidin end-blocked mismatch substrates, suggested that PCNA interacts with an MSH-MLH complex formed on DNA mispairs.

Familial juvenile polyposis (FJP) is a hamartomatouspolyposis syndrome in which affected family members develop upper and lower gastrointestinal juvenile polyps and are at increased risk for gastrointestinal cancer. A genetic locus for FJP has not yet been identified by linkage; therefore, the objective of this study was to perform a focused genome screen in a large family segregating FJP. No evidence for linkage was found with markers near MSH2, MLH1, MCC, APC, HMPS, CDKN2A, JP1, PTEN, KRAS2, TP53, or LKB1. Linkage to FJP was established with several markers from chromosome 18q21.1. The maximum LOD score was 5.00, with marker D18S1099 (recombination fraction of .001). Analysis of critical recombinants places the FJP gene in an 11.9-cM interval bounded by D18S1118 and D18S487, a region that also contains the tumor-suppressor genes DCC and DPC4. These data demonstrate localization of a gene for FJP to chromosome 18q21.1 by linkage, and they raise the possibility that either DCC or DPC4 could be responsible for FJP.

BACKGROUND

In colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island methylator phenotype (CIMP). Explaining why studies on CIMP and survival yield conflicting results is essential. Most experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines into uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched cryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE) samples using pyrosequencing.

METHODS

Couples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed. Rates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured.

RESULTS

For the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD) was 1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and 57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On frozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA showing a total bisulfite conversion, 8 couples (27.6%) for LINE-1, 4 couples (15.4%) for MLH1 and 8 couples (25.8%) for MGMT displayed significant differences in methylation levels.

CONCLUSIONS

Frozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE samples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation levels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended. This can partly explain the conflicting results on the prognosis of CIMP colon cancers.

Missense alterations of the mismatch repair gene MLH1 have been identified in a significant proportion of individuals suspected of having Lynch syndrome, a hereditary syndrome that predisposes for cancer of colon and endometrium. The pathogenicity of many of these alterations, however, is unclear. A number of MLH1 alterations are located in the C-terminal domain (CTD) of MLH1, which is responsible for constitutive dimerization with PMS2. We analyzed which alterations may result in pathogenic effects due to interference with dimerization. We used a structural model of CTD of MLH1-PMS2 heterodimer to select 19 MLH1 alterations located inside and outside two candidate dimerization interfaces in the MLH1-CTD. Three alterations (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) caused decreased coexpression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that these alterations interfere with dimerization. All three alterations are located within the dimerization interface suggested by our model. They also compromised mismatch repair, suggesting that defects in dimerization abrogate repair and confirming that all three alterations are pathogenic. Additionally, we provided biochemical evidence that four alterations with uncertain pathogenicity (p.Ala586Pro, p.Leu636Pro, p.Thr662Pro, and p.Arg755Trp) are deleterious because of poor expression or poor repair efficiency, and confirm the deleterious effect of eight further alterations.

Hexavalent chromium [Cr(VI)] is a well-recognized human lung carcinogen. In order to gain further insight into Cr(VI)-induced carcinogenesis, we have established an adequate in vitro cellular model for the study of this process. To this end, BEAS-2B cells were used. Chronic exposure of cells to 1 microM Cr(VI) induced changes in the cells' ploidy and a decrease in cloning efficiency, although cultures continued to progress to confluence. After prolonged exposure (12 passages), the culture became heterogeneous, exhibiting areas where apparently normal epithelial cells and morphologically altered cells coexisted. Subsequent culture at a very low density strongly accentuated the Cr(VI)-induced changes in morphology and pattern of growth. Three individual colonies were then ring-cloned and expanded into three subclonal aneuploid cell lines. These subclonal cell lines showed changes in growth pattern and morphology, as well as a karyotype drift concomitant with the overexpression of genes commonly involved in malignant transformation (c-MYC, EGFR, HIF-1alpha and LDH-A). Moreover, when one of these cell lines (RenG2) was injected into nude mice, it showed the ability to induce tumors. This cell line revealed no microsatellite instability (MSI), which points to the expression of a functional MLH1 protein and an active mismatch repair (MMR) system. Therefore, the progression to malignancy of the BEAS-2B cells involved Cr(VI)-induced transformants that retained the ability to repair DNA damage, suggesting that genotoxicity underlies the ongoing carcinogenic process.

To better understand the forces affecting individual alleles, we introduce a method for finding the joint distribution of the frequency of a neutral allele and the extent of variability at closely linked marker loci (the intraallelic variability). We model three types of intraallelic variability: (a) the number of nonrecombinants at a linked biallelic marker locus, (b) the length of a conserved haplotype, and (c) the number of mutations at a linked marker locus. If the population growth rate is known, the joint distribution provides the basis for a test of neutrality by testing whether the observed level of intraallelic variability is consistent with the observed allele frequency. If the population growth rate is unknown but neutrality can be assumed, the joint distribution provides the likelihood of the growth rate and leads to a maximum-likelihood estimate. We apply the method to data from published data sets for four loci in humans. We conclude that the Delta32 allele at CCR5 and a disease-associated allele at MLH1 arose recently and have been subject to strong selection. Alleles at PAH appear to be neutral and we estimate the recent growth rate of the European population to be approximately 0.027 per generation with a support interval of (0.017-0.037). Four of the relatively common alleles at CFTR also appear to be neutral but DeltaF508 appears to be significantly advantageous to heterozygous carriers.

Constitutional epimutations of DNA mismatch repair (MMR) genes have been recently reported as a possible cause of Lynch syndrome. However, little is known about their prevalence, the risk of transmission through the germline and the risk for carriers to develop cancers. In this study, we evaluated the contribution of constitutional epimutations of MMR genes in Lynch syndrome. A cohort of 134 unrelated Lynch syndrome-suspected patients without MMR germline mutation was screened for constitutional epimutations of MLH1 and MSH2 by quantitative bisulfite pyrosequencing. Patients were also screened for the presence of EPCAM deletions, a possible cause of MSH2 methylation. Tumors from patients with constitutional epimutations were extensively analyzed. We identified a constitutional MLH1 epimutation in two proband patients. For one of them, we report for the first time evidence of transmission to two children who also developed early colonic tumors, indicating that constitutional MLH1 epimutations are associated to a real risk of transgenerational inheritance of cancer susceptibility. Moreover, a somatic BRAF mutation was detected in one affected child, indicating that tumors from patients carrying constitutional MLH1 epimutation can mimic MSI-high sporadic tumors. These findings may have important implications for future diagnostic strategies and genetic counseling.

Considerable evidence demonstrates that genetic factors are important in the development and aggressiveness of prostate cancer. To identify genetic variants that predispose to prostate cancer we tested candidate SNPs from genomic regions that show linkage to prostate cancer susceptibility and/or aggressiveness, as well as genes that show a significant difference in mRNA expression level between tumor and normal tissue. Cases had histologically verified prostate cancer. Controls were at least 65 years old, never registered a PSA above 2.5 ng/ml, always had digital rectal examinations that were not suspicious for cancer, and have no known family history of prostate cancer. Thirty-nine coding SNPs and nine non-coding SNPs were tested in up to 590 cases and 556 controls resulting in over 40,000 SNP genotypes. Significant differences in allele frequencies between cases and controls were observed for ID3 (inhibitor of DNA binding), p = 0.05, HPN (hepsin), p = 0.009, BCAS1 (breast carcinoma amplified sequence 1), p = 0.007, CAV2 (caveolin 2), p = 0.007, EMP3 (epithelial membrane protein 3), p < 0.0001, and MLH1 (mutL homolog 1), p < 0.0001. SNPs in three of these genes (BCAS1, EMP3 and MLH1) remained significant in an age-matched subsample.