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Background: The hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) is the most frequent hereditary colorectal cancer syndrome. It has been associated with different types of cancers besides the early onset of the disease. To define HNPCC the Amsterdam or Bethesda criteria have to be met. Causative for the Lynch syndrome is a germline mutation of one of the four mismatch repair genes MLH1, MSH2, MSH6 and PMS2. The detection of pathogenic alterations is of vital importance for the patient as well as for their relatives. The analyses for the PMS2 gene have been severely complicated by the presence of multiple pseudogenes with high homologies to the gene especially in its 5 ́-region. The PMS2CL pseudogene has a high homology to the 3 ́-region of PMS2, in particular the exons 9 and 11- 15, which result in recombination events between the paralogues. Objectives: In this study, we established the newly developed technique of long-range-PCR for mutation detection within the PMS2 gene. Using this method we could clearly discriminate gene from pseudogene which makes it a significant advancement in the detection of pathogenic mutations in PMS2 compared to previously used methods. Until recently mutation detections had been performed with a method, which is now obsolete. It has been the aim of this study to implement the new method of long-range-PCR in the daily diagnostic routine. However, within the study we examined only exons 11-15 of PMS2 equal to the long-range-PCR product 3, because this region contains the highly homologous region to the PMS2CL pseudogene and is known to undergo recombination. Furthermore, it was the aim of this study to make a statement about occuring recombination events and their nature, frequency and consequences. The sequence of the pseudogene has been amplified as well. Methods: Patients were selected based on an isolated loss for PMS2 in the immunohisto- chemistry of their tumor. The long-range-PCR was performed on DNA from blood leukocytes. The amplicons of both the gene and pseudogene were confirmed on gel electrophoresis and used as template for ensuing exon-specific nested-PCR prior to sequencing. Multiplex ligation-depend amplification (MLPA) was performed to screen for deleterious alterations. Results: The study comprised 23 patients. We identified pathogenic mutations in 14 patients (61 %). Four of these mutations are located in the 5 ́-region (upstream exon 8), ten mutations were found in the 3 ́-region (downstream exon 9). Those mutations were nearly evenly distrib- uted over the exons 11-14, whereas two mutations in exon 14 were large deletions. There was no mutation found in exon 15. Moreover we identified one putative pathogenic mutation in exon 12. The second part of this study aimed at the analysis of recombination events between PMS2 and PMS2CL. We identified 27 paralogous sequence variants, which were classified by un- derlying recombination patterns. We found patients without any recombination as well as re- combination events encompassing the exons 13-15. In 14 of 23 patients (61 %) a recombination event could be confirmed occurring primarily in the exons 13-15. The overall number of recombination events are benign and consequences for cancer development are not clear, except one patient who presented with a malignant tumor and carried a sequence recombina- tion in exon 11, which had not been found in former studies. Conclusion: From now on the long-range-PCR should be gold standard for detection of path- ogene alterations in the PMS2 gene. The mechanism and the verification of recombination events encompassing the whole gene PMS2 should be point of interest in further studies.

BACKGROUND:Cancers often arise within an area of cells (e.g. an epithelial patch) that is predisposed to the development of cancer, i.e. a "field of cancerization" or "field defect." Sporadic colon cancer is characterized by an elevated mutation rate and genomic instability. If a field defect were deficient in DNA repair, DNA damages would tend to escape repair and give rise to carcinogenic mutations.PURPOSE:To determine whether reduced expression of DNA repair proteins Pms2, Ercc1 and Xpf (pairing partner of Ercc1) are early steps in progression to colon cancer.RESULTS:Tissue biopsies were taken during colonoscopies of 77 patients at 4 different risk levels for colon cancer, including 19 patients who had never had colonic neoplasia (who served as controls). In addition, 158 tissue samples were taken from tissues near or within colon cancers removed by resection and 16 tissue samples were taken near tubulovillous adenomas (TVAs) removed by resection. 568 triplicate tissue sections (a total of 1,704 tissue sections) from these tissue samples were evaluated by immunohistochemistry for 4 DNA repair proteins. Substantially reduced protein expression of Pms2, Ercc1 and Xpf occurred in field defects of up to 10 cm longitudinally distant from colon cancers or TVAs and within colon cancers. Expression of another DNA repair protein, Ku86, was infrequently reduced in these areas. When Pms2, Ercc1 or Xpf were reduced in protein expression, then either one or both of the other two proteins most often had reduced protein expression as well. The mean inner colon circumferences, from 32 resections, of the ascending, transverse and descending/sigmoid areas were measured as 6.6 cm, 5.8 cm and 6.3 cm, respectively. When combined with other measurements in the literature, this indicates the approximate mean number of colonic crypts in humans is 10 million.CONCLUSIONS:The substantial deficiencies in protein expression of DNA repair proteins Pms2, Ercc1 and Xpf in about 1 million crypts near cancers and TVAs suggests that the tumors arose in field defects that were deficient in DNA repair and that deficiencies in Pms2, Ercc1 and Xpf are early steps, often occurring together, in progression to colon cancer.