Vol. 60, No 2/2013

Detection of mutations in families with a hereditary predisposition to colon cancer gives an opportunity to precisely define the high-risk group. 36 patients operated on for colon cancer, with familiar prevalence of this malignancy, were investigated using the DNA microarrays method with the potential detection of 170 mutations in MLH1, MSH2, MSH6, CHEK2, and NOD2 genes. In microarrays analysis of DNA in 9 patients (25% of the investigated group), 6 different mutations were found. The effectiveness of genetic screening using the microarray method is comparable to the effectiveness of other, much more expensive and time-consuming methods.


INTRoDucTIoN
In25%of cases of patients with colorectal cancer positive familial history is recognized.The most commonly inherited colon cancer predisposition syndrome is hereditary non-polyposis colorectal cancer (HNPCC), also called the Lynch syndrome (LS).This disorder with autosomal dominant inheritance pattern and high penetrance accounts for 2-3% of all CRC diagnoses and is caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 (Wijnen et al., 1993;Hampel et al., 2005;Kauff et al., 2007;Lu et al., 2007).
Detection of mutations in families with a hereditary predisposition to colon cancer gives an opportunity to precisely define the high-risk group by cost-effective carrier screening.The mutation carriers should be subject to regular control examinations, whereas the non-carriers bear only the population risk of the colon cancer, therefore can be considered as a general, risk-free population.Due to several genes and large number of mutations involved (http://www.insight-group.org),genetic testing of HNPCC is challenging, and in practice preceded by pedigree analysis, microsatellite instability assay, and/or immunohistochemistry for MMR proteins, despite the fact that detection of the MMR gene mutation alone is enough to confirm a LS diagnosis.Thus, simple and effective methods for genetic screening are still investigated.

MATeRIAl AND MeThoDs:
Patients.In our pilot study the group of 36 adults after the surgery for colon cancer, with familiar prevalence of this malignancy, were investigated (minimum 2 family members with colorectal/endometrial cancer in 2 generations).In this group, in 6 patients, hereditary non-polyposis colorectal cancer (HNPCC) was recognized based on the Amsterdam criteria (Vasen et al., 1991).All patients received a collection kit for samples and a questionnaire.Genomic DNA was extracted from buccal mucosa samples collected by each patient him/ herself and sent back to the genetic laboratory.
Genetic examination.Mutations were detected using the DNA microarrays SNP method in APEX technology (INNO GENE S.A., Poland), with the potential detection of 169 unique mutations in MLH1, MSH2, MSH6, CHEK2, and NOD2 genes (see Table 1 for details).
In the group of patients with recognized HNPCC the following mutations: 83C>T (MLH1) and 1077-10T>C (MSH2) were found in 2 cases (33%).The cost of a single microarray assay was about 367 EUR/477 USD; whereas the cost of detection of one mutation in the examined group was 1468 EUR/1908 USD.

DIscussIoN
Genetic diagnostics may provide efficient and costeffective tools for testing patients with genetically related colorectal cancer, if all costs are considered (Ladabaum et al., 2011;Wang et al., 2012).In the group of patients with hereditary colon cancer, or suspicion of hereditary symptoms, screening and pre-symptomatic clinical examination of all family members is recommended.It is a very effective method in the secondary prophylaxis of the malignant transformation.In the analysis of mutations, the DNA sequencing of the following 4 MMR genes: MLH1, MSH2, MSH6, and PMS2, may be considered as a 'gold standard'.Performance of this strategy is difficult to estimate, and it is not known if laboratory proficiency testing will be an adequate validity measure (Bonis et al., 2007; Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group 2009; Palomaki et al., 2009).Because of the high lifetime, colorectal cancer risk for the Lynch syndrome patients (reaching 80%) (Chung et al., 2003;Brodersen et al., 2004), the effectiveness of screening in this group of patients is well supported (Järvinen et al., 2000;Dove-Edvin et al., 2005).Despite this, there is suboptimal uptake of screening by high-risk individuals (Bleiker et al., 2005;Geary et al., 2007;Rees et al., 2008).The discovery of cancer-causing germline mutations has proved to be highly advantageous in determining patients' lifetime risk status (Lynch et al., 2009).The knowledge about the colorectal cancer risk can determine the patients' and their physicians' decision-making regarding surveillance and management (Watson et al., 2003).The localization of the mutation also gives an opportunity to predict the clinical follow-up of the disease, as for example the lower risk of extra colonic cancer (such as endometrial cancer) in the type 2 Lynch syndrome (MLH1-related) or later onset CRCs and a greater number of endometrial carcinomas in Lynch syndrome-MSH6 type (Lynch et al., 2010).The DNA microarray method, based on the DNA hybridization seems to be a simple and effective method for genetic testing.The application of DNA microarrays for fundamental biomedical research has recently been reviewed elsewhere.(Schulze et al., 2001;Smyth et al., 2003;Egeland et al., 2005;Chagovetz et al., 2009).There are very promising indications for using this method in cancer research (Wadlow et al., 2005;Perez-Cabornero et al., 2009;van Roon et al., 2011).The main benefits of the microarray method are: large scale screening (>100 mutations); short turn around time processing (days), low cost (< 500 EUR/test), ease of upgrading the open platform (new mutations).The frequency of the mutation's detection using DNA microarrays seems to be similar when compared to other studies.
Differences in the frequency of the mutation detection rate are observed between HNPCC cohorts, depending on the inclusion criteria and the investigated population.In Spaepen et al. (Spaepen et al., 2006), study of patients with HNPCC, pathogenic mutations were found in 11% -25 out of 225 investigated patients.In colorectal cancer patients without preselection and regardless of family history, 38 pathogenic mutations among 870 participants (4%) were found (Barnetson et al., 2006).In 281 patients diagnosed with CRC before the age of 50 years or with CRC and at least one additional HNPCC-associated cancer, 25 pathogenic mutations (8.9%) were detected (Niessen et al., 2006).In 93 unrelated Taiwanese families that fulfilled the Amsterdam criteria II 38 pathogenic mutations in the MSH2 or MLH1 genes were identified in 61 families (Tang et al., 2009).In another study of 76 Chinese probands from HNPCC families the overall mutation rate was 33%, and 22 different mutations were found in the MLH1 and MSH2 genes (Fu et al., 2008).The mutation detection rate with our DNA microarray assay was 25% in patients with familial history (14% if only MMR mutations were considered), and 33% in HNPCC patients, which is similar to those presented in other studies.
In all HNPCC families with mutations detected, the recognition of high-risk carriers is easy, and can decrease the number of investigated persons by about 50% and reduce the cost, as well as psychological stress of unaffected family members.
The effectiveness of genetic screening using the microarray method is similar to the effectiveness of other approaches, that are much more expensive and timeconsuming.The method needs to be validated in further studies among larger group of patients; however, we believe that the assay can be widely used as a simple, accepted, and cost-effective method in colorectal cancer screening programs.
conflicts of Interest statementKaszuba M, Sikorski A, Wojciechowicz J -collaboration with INNO GENE SA Poland.Banasiewicz Thonoraria for the lecture about DNA microarray analysis (paid by INNO GENE SA Poland).There are no more conflicts of interest of the other authors.