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Background Imprinting Control Regions (ICRs) are CpG-rich sequences acquiring differential methylation in the female and male germline and maintaining it in a parental origin-specific manner in somatic cells. Despite their expected high mutation rate due to spontaneous deamination of methylated cytosines, ICRs show conservation of CpG-richness and CpG-containing transcription factor binding sites in mammalian species. However, little is known about the mechanisms contributing to the maintenance of a high density of methyl CpGs at these loci. Results To gain functional insights into the mechanisms for maintaining CpG methylation, we sought to identify the proteins binding the methylated allele of the ICRs by determining the interactors of ZFP57 that recognizes a methylated hexanucleotide motif of these DNA regions in mouse ESCs. By using a tagged approach coupled to LC–MS/MS analysis, we identified several proteins, including factors involved in mRNA processing/splicing, chromosome organization, transcription and DNA repair processes. The presence of the post-replicative mismatch-repair (MMR) complex components MSH2 and MSH6 among the identified ZFP57 interactors prompted us to investigate their DNA binding profile by chromatin immunoprecipitation and sequencing. We demonstrated that MSH2 was enriched at gene promoters overlapping unmethylated CpG islands and at repeats. We also found that both MSH2 and MSH6 interacted with the methylated allele of the ICRs, where their binding to DNA was mediated by the ZFP57/KAP1 complex. Conclusions Our findings show that the MMR complex is concentrated on gene promoters and repeats in mouse ESCs, suggesting that maintaining the integrity of these regions is a primary function of highly proliferating cells. Furthermore, the demonstration that MSH2/MSH6 are recruited to the methylated allele of the ICRs through interaction with ZFP57/KAP1 suggests a role of the MMR complex in the maintenance of the integrity of these regulatory regions and evolution of genomic imprinting in mammalian species.

Lynch syndrome (LS) is a dominantly inherited cancer susceptibility syndrome defined by presence of pathogenic variants in DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2, or in deletions of the EPCAM gene. Although LS is present in about 1 in 400 people in the UK, it estimated that only 5% of people with this condition are aware of the diagnosis. Therefore, testing for LS in all new diagnoses of colorectal or endometrial cancers is now recommended in the UK, and gastroenterologists can offer ‘mainstreamed’ genetic testing for LS to patients with cancer. Because LS results in a high lifetime risk of colorectal, endometrial, gastric, ovarian, hepatobiliary, brain and other cancers, the lifelong care of affected individuals and their families requires a coordinated multidisciplinary approach. Interventions such as high-quality 2-yearly colonoscopy, prophylactic gynaecological surgery, and aspirin are proven to prevent and facilitate early diagnosis and prevention of cancers in this population, and improve patient outcomes. Recently, an appreciation of the mechanism of carcinogenesis in LS-associated cancers has contributed to the development of novel therapeutic and diagnostic approaches, with a gene-specific approach to disease management, with potential cancer-preventing vaccines in development. An adaptive approach to surgical or oncological management of LS-related cancers may be considered, including an important role for novel checkpoint inhibitor immunotherapy in locally advanced or metastatic disease. Therefore, a personalised approach to lifelong gene-specific management for people with LS provides many opportunities for cancer prevention and treatment which we outline in this review.

Genomic and chromosomal instability are hallmarks of cancer and shape the genomic composition of cancer cells, thereby determining their behavior and response to treatment. Various genetic and epigenetic alterations in cancer have been linked to genomic instability, including DNA repair defects, oncogene-induced replication stress, and spindle assembly checkpoint malfunction. A consequence of genomic and chromosomal instability is the leakage of DNA from the nucleus into the cytoplasm, either directly or through the formation and subsequent rupture of micronuclei. Cytoplasmic DNA subsequently activates cytoplasmic DNA sensors, triggering downstream pathways, including a type I interferon response. This inflammatory signaling has pleiotropic effects, including enhanced anti-tumor immunity and potentially results in sensitization of cancer cells to immune checkpoint inhibitors. However, cancers frequently evolve mechanisms to avoid immune clearance, including suppression of inflammatory signaling. In this review, we summarize inflammatory signaling pathways induced by various sources of genomic instability, adaptation mechanisms that suppress inflammatory signaling, and implications for cancer immunotherapy.

Context Adrenocortical carcinoma (ACC) has a heterogeneous prognosis, and current medical therapies have limited efficacy in its advanced stages. Genome-wide multiomics studies identified molecular patterns associated with clinical outcome. Objective Here, we aimed at identifying a molecular signature useful for both personalized prognostic stratification and druggable targets, using methods applicable in clinical routine. Design In total, 117 tumor samples from 107 patients with ACC were analyzed. Targeted next-generation sequencing of 160 genes and pyrosequencing of 4 genes were applied to formalin-fixed, paraffin-embedded (FFPE) specimens to detect point mutations, copy number alterations, and promoter region methylation. Molecular results were combined with clinical/histopathological parameters (tumor stage, age, symptoms, resection status, and Ki-67) to predict progression-free survival (PFS). Results In addition to known driver mutations, we detected recurrent alterations in genes not previously associated with ACC (e.g., NOTCH1, CIC, KDM6A, BRCA1, BRCA2). Best prediction of PFS was obtained integrating molecular results (more than one somatic mutation, alterations in Wnt/β-catenin and p53 pathways, high methylation pattern) and clinical/histopathological parameters into a combined score (P < 0.0001, χ2 = 68.6). Accuracy of prediction for early disease progress was 83.3% (area under the receiver operating characteristic curve: 0.872, 95% confidence interval 0.80 to 0.94). Furthermore, 17 potentially targetable alterations were found in 64 patients (e.g., in CDK4, NOTCH1, NF1, MDM2, and EGFR and in DNA repair system). Conclusions This study demonstrates that molecular profiling of FFPE tumor samples improves prognostication of ACC beyond clinical/histopathological parameters and identifies new potential drug targets. These findings pave the way to precision medicine in this rare disease.

Genomic and chromosomal instability are hallmarks of cancer and shape the genomic composition of cancer cells, thereby determining their behavior and response to treatment. Various genetic and epigenetic alterations in cancer have been linked to genomic instability, including DNA repair defects, oncogene-induced replication stress, and spindle assembly checkpoint malfunction. A consequence of genomic and chromosomal instability is the leakage of DNA from the nucleus into the cytoplasm, either directly or through the formation and subsequent rupture of micronuclei. Cytoplasmic DNA subsequently activates cytoplasmic DNA sensors, triggering downstream pathways, including a type I interferon response. This inflammatory signaling has pleiotropic effects, including enhanced anti-tumor immunity and potentially results in sensitization of cancer cells to immune checkpoint inhibitors. However, cancers frequently evolve mechanisms to avoid immune clearance, including suppression of inflammatory signaling. In this review, we summarize inflammatory signaling pathways induced by various sources of genomic instability, adaptation mechanisms that suppress inflammatory signaling, and implications for cancer immunotherapy.

Aims:Beta2-Microglobulin (B2M) forms part of the HLA class I complex and plays a role in metastatic biology. B2M mutations occur frequently in mismatch repair-deficient colorectal cancer (dMMR CRC) with limited data suggesting they may protect against recurrence. Our experimental study tested this hypothesis by investigating B2M mutation status and B2M protein expression and recurrence in patients in the stage II QUASAR clinical trial. Methods:Sanger sequencing was performed for the three coding exons of B2M on 121 dMMR and a subsample of 108 pMMR tumours; 52 with recurrence and 56 without. B2M protein expression was assessed by immunohistochemistry. Mutation status and protein expression were correlated with recurrence and compared to proficient mismatch repair (pMMR) CRCs. Results:Of 121 dMMR CRCs, deleterious B2M mutations were detected in 39 (32%). Five tumours contained missense B2M-variants of unknown significance, and were thus excluded from further analyses, leaving 116 dMMR tumours. With median follow-up 7.4 years, none of the 39 B2M-mutant tumours recurred, compared with 14/77 (18%) B2M-wildtype tumours (p=0.005); six at local and eight at distant sites. Sensitivity and specificity of IHC in detecting B2M mutations was 87% and 71% respectively. Significantly (p

Objective The aim of this study was to validate a molecular classification of colorectal cancer (CRC) based on microsatellite instability (MSI), CpG island methylator phenotype (CIMP) status, BRAF, and KRAS and investigate each subtype's response to chemotherapy. Design This retrospective observational study included a population-based cohort of 878 CRC patients. We classified tumours into five different subtypes based on BRAF and KRAS mutation, CIMP status, and MSI. Patients with advanced stage II (T4N0M0) and stage III tumours received 5-fluoruracil (5-FU)-based chemotherapy or no adjuvant treatment based on clinical criteria. The main outcome was disease-free survival (DFS). Results Patients with the combination of microsatellite stable (MSS) tumours, BRAF mutation and CIMP positive exhibited the worst prognosis in univariate (log rank P < 0.0001) and multivariate analyses (hazard ratio 1.75, 95% CI 1.05-2.93, P = 0.03) after adjusting for age, sex, chemotherapy, and TNM stage. Treatment with 5-FU-based regimens improved prognosis in patients with the combination of MSS tumours, KRAS mutation and CIMP negative (log rank P = 0.003) as well as in patients with MSS tumours plus BRAF and KRAS wild-type and CIMP negative (log-rank P < 0.001). After adjusting for age, sex, and TNM stage in the multivariate analysis, only patients with the latter molecular combination had independently improved prognosis after adjuvant chemotherapy (hazard ratio 2.06, 95% CI 1.24-3.44, P = 0.005). Conclusion We confirmed the prognostic value of stratifying CRC according to molecular subtypes using MSI, CIMP status, and somatic KRAS and BRAF mutation. Patients with traditional chromosomally unstable tumours obtained the best benefit from adjuvant 5-FU-based chemotherapy. This work was supported by the Instituto de Salud Carlos III (PI08/0726, INT-09/208, PI11/2630, INT-12-078, INT13-196, PI14/01386), FISABIO-ISABIAL foundation (UGP-13-221, UGP-14-265), and the Asociación Española contra el Cáncer (Fundación Científica GCB13131592CAST). Eva Hernández-Illán received a grant from Instituto de Salud Carlos III (FI12/00233). Mar Giner-Calabuig received a grant from VALi+d. EXP ACIF/2016/002. Miren Alustiza received a predoctoral grant from ISABIAL (UGP-16-138). Other financial support was obtained from Asociación Española de Gastroenterología grants (Gonzalo Miño 2014, Tamarite 2015, Grupo de endoscopia 2016).

Microsatellite instability (MSI) is a molecular hallmark for some colorectal cancers (CRCs) in which short tandem repeats are prone to mutations along with DNA sequences. It is due to DNA-mismatch-repair system deficiency because of a germline/somatic mutation in mismatch-repair (MMR) genes. The germline mutations lead to Lynch syndrome (LS) while epigenetic gene silencing results in sporadic CRC tumors. We discuss in our paper the most important clinical aspects of MSI testing in CRCs. We reviewed the most reliable relevant studies and clinical trials according to their high-quality methods, particularly within two recent decades. MSI testing is used to classify CRC tumors as MSI-high (MSI-H), MSI-low, and microsatellite stable tumors. MSI-H or MMR deficient tumors have shown the best prognosis among all CRCs, so MSI testing is considered as a good prognostic marker. Moreover, it is used to identify LS among familial CRC patients. There is a diagnostic mutation in BRAF gene (V600E) by which sporadic CRCs could be distinguished from LS associated CRCs, due to its concordance with sporadic CRCs not LS. Although, some previous studies had demonstrated a predictive role for MSI testing in chemotherapy process, emerging some controversial findings in recent studies has not convinced many authors to recommend it as a routine examination to evaluate therapeutic response. Though emerging new molecular findings have opened novel windows to develop clinical management of CRC, MSI testing has remained as an excellent prognostic and diagnostic tool for CRC tumors.

The reliability of microbial (starter) strains in terms of quality, functional properties, growth performance and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing such as acid, osmotic, temperature, and oxidative stress. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution towards survival under these stress conditions. However, this may lead to the accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effect of specific fermentation or processing conditions on the mutation frequencies is an important step towards preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (NTG) and by controlled expression of endogenous candidate mutator genes (e.g. truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, in low pH conditions, at high salt concentrations or in starvation conditions did not result in a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide showed a 100-fold increase of the mutation frequency when compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42 degrees C, prior to the treatment with sublethal levels of hydrogen peroxide, this induced a 10-fold increase in peroxide treatment survival, with a concomitant 50-fold decrease of the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be achieved by pre-treatment of cultures with other, non-mutagenic stress conditions

Purpose: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease characterized by the clustering of colorectal cancer, endometrial cancer, and various other cancers. The disease is caused by mutations in DNA-mismatch-repair (MMR) genes, most frequently in MLH1, MSH2, and MSH6. The aims of the present study were to compare the risk of developing colorectal, endometrial, and other cancers between families with the various MMR-gene mutations.Patients and Methods: Clinical and pathologic data were collected from 138 families with HNPCC. Mutation analyses were performed for all families. Survival analysis was used to calculate the cumulative risk of developing cancer in the various subsets of relatives.Results: Mutations were identified in 79 families: 34 in MLH1, 40 in MSH2 and five in MSH6. The lifetime risk of developing cancer at any site was significantly higher for MSH2 mutation carriers than, for MLH1 mutation carriers (P Conclusion: Pending large prospective studies, the extension of the current surveillance program in MSH2 mutation carriers with the inclusion of the urinary tract should be considered.

Contains fulltext : 185698.pdf (Publisher’s version ) (Open Access) PURPOSE: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease characterized by the clustering of colorectal cancer, endometrial cancer, and various other cancers. The disease is caused by mutations in DNA-mismatch-repair (MMR) genes, most frequently in MLH1, MSH2, and MSH6. The aims of the present study were to compare the risk of developing colorectal, endometrial, and other cancers between families with the various MMR-gene mutations. PATIENTS AND METHODS: Clinical and pathologic data were collected from 138 families with HNPCC. Mutation analyses were performed for all families. Survival analysis was used to calculate the cumulative risk of developing cancer in the various subsets of relatives. RESULTS: Mutations were identified in 79 families: 34 in MLH1, 40 in MSH2, and five in MSH6. The lifetime risk of developing cancer at any site was significantly higher for MSH2 mutation carriers than for MLH1 mutation carriers (P < .01). The risk of developing colorectal or endometrial cancer was higher in MSH2 mutation carriers than in MLH1 mutation carriers, but the difference was not significant (P = .13 and P = .057, respectively). MSH2 mutation carriers were found to have a significantly higher risk of developing cancer of the urinary tract (P < .05). The risk of developing cancer of the ovaries, stomach, and brain was also higher in the MSH2 mutation carriers than in the MLH1 mutation carriers, but the difference was not statistically significant. CONCLUSION: Pending large prospective studies, the extension of the current surveillance program in MSH2 mutation carriers with the inclusion of the urinary tract should be considered.

To study the involvement of DNA mismatch-repair genes in sporadic breast cancer, matched normal and tumoral DNA samples of 22 patients were analysed for genetic instability and loss of heterozygosity (LOH) with 42 microsatellites at or linked to hMLH1 (3p21), hMSH2 (2p16), hMSH3 (5q11–q13), hMSH6 (2p16), hPMS1 (2q32) and hPMS2 (7p22) loci. Chromosomal regions 3p21 and 5q11–q13 were found hemizygously deleted in 46% and 23% of patients respectively. Half of the patients deleted at hMLH1 were also deleted at hMSH3. The shortest regions of overlapping (SRO) deletions were delimited by markers D3S1298 and D3S1266 at 3p21 and by D5S647 and D5S418 at 5q11–q13. Currently, the genes hMLH1 (3p21) and hMSH3 (5q11–q13) are the only known candidates located within these regions. The consequence of these allelic losses is still unclear because none of the breast cancers examined displayed microsatellite instability, a hallmark of mismatch-repair defect during replication error correction. We suggest that hMLH1 and hMSH3 could be involved in breast tumorigenesis through cellular functions other than replication error correction. © 1999 Cancer Research Campaign

Various mutations were introduced in a conserved helicase domain (motif VI) of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) by site-directed mutagenesis. These mutations affected the helicase activity and the ATP-dependent exonuclease activity on double-stranded DNA (dsDNA) as the substrate to various degrees, but had hardly any effect on the exonuclease activity on single-stranded DNA (ssDNA), suggesting that exonuclease activity on dsDNA of the enzyme requires unwinding of the DNA. This idea was supported by the finding that, initially, the rate and extent of unwinding of the DNA were higher than those of its degradation to acid-soluble products by the exonucleolytic activity. The effects of the mutations on DNA repair and recombination correlated strongly with their effects on helicase activity. Taken together, these results suggest that motif VI is essential for the helicase activity, and that this activity is required for DNA repair and recombination.

Trinucleotide repeat expansions cause 17 heritable human neurological disorders. In some diseases, somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited. This finding stimulated significant interest in replication-independent expansion mechanisms. Aberrant DNA repair is a likely source, based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutS beta (Msh2-Msh3 complex). Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats. The findings included expansions on one strand but not the other, or processing of DNA hairpin structures thought to be important intermediates in the expansion process. However, it has been difficult to recapitulate complete expansions in vitro, and the biochemical role of MutS beta remains controversial. Here, we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication. The extract promotes a size range of expansions that is similar to certain diseases, and triplet repeat length and sequence govern expansions in vitro as in vivo. MutS beta stimulates expansions in the extract, consistent with aberrant repair of endogenous DNA damage as a source of expansions. Overall, this biochemical system retains the key characteristics of somatic expansions in humans and mice, suggesting that this important mutagenic process can be restored in the test tube.

Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington's disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutS beta (MSH2-MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (similar to 30-40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG center dot CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutS beta subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutS alpha subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutS beta, but not MutS alpha, was enriched at the TNR. These findings imply a direct role for MutS beta in promoting expansion of threshold-length CTG center dot CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutS beta, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs.

The reliability of microbial (starter) strains in terms of quality, functional properties, growth performance and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing such as acid, osmotic, temperature, and oxidative stress. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution towards survival under these stress conditions. However, this may lead to the accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effect of specific fermentation or processing conditions on the mutation frequencies is an important step towards preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (NTG) and by controlled expression of endogenous candidate mutator genes (e.g. truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, in low pH conditions, at high salt concentrations or in starvation conditions did not result in a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide showed a 100-fold increase of the mutation frequency when compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42 degrees C, prior to the treatment with sublethal levels of hydrogen peroxide, this induced a 10-fold increase in peroxide treatment survival, with a concomitant 50-fold decrease of the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be achieved by pre-treatment of cultures with other, non-mutagenic stress conditions

Hereditary non-polyposis colorectal cancer (HNPCC) is a common autosomal dominant condition characterized by early onset colorectal cancer as well as other tumour types at different anatomical sites1. HNPCC tumours often display a high level of genomic instability, characterized by changes in repeat numbers of simple repetitive sequences (microsatellite instability, MSI), which reflects the malfunction of the DNA mismatch repair machinery2, 3. Accordingly, HNPCC was shown to be caused by germline mutations in the DNA mismatch repair genes (MMR) MSH2, MLH1, PMS1, PMS2 and MSH6 (refs 3, 4, 5, 6). So far, more than 220 predisposing mutations have been identified, most in MSH2 and MLH1 and in families complying with the clinical Amsterdam criteria3, 7, 8 (AMS+). Many HNPCC families, however, do not fully comply with these criteria, and in most cases the causative mutations are unknown.

Trinucleotide repeats frequently expand and contract in humans and model organisms. Protein factors that modulate this process have been found by candidate gene approaches or mutant screens for increased expansion rates. To extend this effort, Saccharomyces cerevisiae mutants with higher CAG.CTG repeat contraction rates were sought using a disruption library. This screen identified Mrc1, the homolog of human Claspin, which mediates the replication and DNA damage checkpoints, and also couples the replicative helicase and polymerase. Genetic analysis showed that Mrc1, along with Tof1 and Csm3, inhibits instability in two distinct ways. Contraction rates Of (CAG)(20) tracts are elevated by loss of Mrc1, Tof1 or Csm3, but not by defects in most replication checkpoint or DNA damage checkpoint proteins. The three proteins likely inhibit contractions primarily through their coupling activity, which would prevent accumulation of single-strand template DNA prior to the formation of aberrant secondary structure. In contrast, expansion rates of (CTG)(13) are elevated in strains defective for Mrc1, Tof1, Csm3, Mec1, Ddc2, Rad24, Ddc1, Mec3, Rad17, Rad9, Rac53 or Chk1, suggesting that the DNA damage checkpoint inhibits expansions after formation of repeat-dependent structures. Together, these results indicate that at least two Mrc1-dependent mechanisms function to reduce CAG.CTG repeat instability. Published by Elsevier B.V.

Hereditary non-polyposis colorectal cancer is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Here we describe the application of a novel technique for the detection of genomic deletions in MLH1 and MSH2. This method, called multiplex ligation-dependent probe amplification, is a quantitative multiplex PCR approach to determine the relative copy number of each MLH1 and MSH2 exon. Mutation screening of genes was performed in 126 colorectal cancer families selected on the basis of clinical criteria and in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumours. Thirty-eight germline mutations were detected in 37 (29.4%) of these kindreds, 31 of which have a predicted pathogenic effect. Among families with MSI-high tumours 65.7% harboured germline gene defects. Genomic deletions accounted for 54.8% of the pathogenic mutations. A complete deletion of the MLH1 gene was detected in two families. The multiplex ligation-dependent probe amplification approach is a rapid method for the detection of genomic deletions in MLH1 and MSH2. In addition, it reveals alterations that might escape detection using conventional diagnostic techniques. Multiplex ligation-dependent probe amplification might be considered as an early step in the molecular diagnosis of hereditary non-polyposis colorectal cancer. British Journal of Cancer (2002) 87, 892–897. doi:10.1038/sj.bjc.6600565 www.bjcancer.com © 2002 Cancer Research UK

The presence and functionality of DNA repair mechanisms in Campylobacter jejuni are largely unknown. In silico analysis of the complete translated genome of C. jejuni NCTC 11168 suggests the presence of genes involved in methyl-directed mismatch repair (MMR), nucleotide excision repair, base excision repair (BER), and recombinational repair. To assess the functionality of these putative repair mechanisms in C. jejuni , mutS , uvrB , ung , and recA knockout mutants were constructed and analyzed for their ability to repair spontaneous point mutations, UV irradiation-induced DNA damage, and nicked DNA. Inactivation of the different putative DNA repair genes did not alter the spontaneous mutation frequency. Disruption of the UvrB and RecA orthologues, but not the putative MutS or Ung proteins, resulted in a significant reduction in viability after exposure to UV irradiation. Assays performed with uracil-containing plasmid DNA showed that the putative uracil-DNA glycosylase (Ung) protein, important for initiation of the BER pathway, is also functional in C. jejuni . Inactivation of recA also resulted in a loss of natural transformation. Overall, the data indicate that C. jejuni has multiple functional DNA repair systems that may protect against DNA damage and limit the generation of genetic diversity. On the other hand, the apparent absence of a functional MMR pathway may enhance the frequency of on-and-off switching of phase variable genes typical for C. jejuni and may contribute to the genetic heterogeneity of the C. jejuni population.