Dagmara Dymerska

DNA and RNA analyses in detection of genetic predisposition to cancer

During the past decade many new molecular methods for DNA and RNA analysis have emerged. The most popular thus far have been SSCP, HET, CMC, DGGE, RFLP or ASA, which have now been replaced by methods that are more cost effective and less time consuming. Real-time amplification techniques and particularly those with the capacity of multiplexing have become commonly used in laboratory practice. Novel screening methods enable the very rapid examination of large patients series. Use of liquid handling robotics applied to the isolation of DNA or RNA, the normalisation of sample concentration, and standardization of target amplification by PCR have also contributed to a reduced risk of sample contamination and have resulted in laboratory analysis being easier and faster.The aim of this study is the introduction of a few modern techniques, most commonly used in detection of genetic predisposition to cancer.

Cumulative Small Effect Genetic Markers and the Risk of Colorectal Cancer in Poland, Estonia, Lithuania, and Latvia.

The continued identification of new low-penetrance genetic variants for colorectal cancer (CRC) raises the question of their potential cumulative effect among compound carriers. We focused on 6 SNPs (rs380284, rs4464148, rs4779584, rs4939827, rs6983267, and rs10795668), already described as risk markers, and tested their possible independent and combined contribution to CRC predisposition. Material and Methods. DNA was collected and genotyped from 2330 unselected consecutive CRC cases and controls from Estonia (166 cases and controls), Latvia (81 cases and controls), Lithuania (123 cases and controls), and Poland (795 cases and controls). Results. Beyond individual effects, the analysis revealed statistically significant linear cumulative effects for these 6 markers for all samples except of the Latvian one (corrected P value = 0.018 for the Estonian, corrected P value = 0.0034 for the Lithuanian, and corrected P value = 0.0076 for the Polish sample). Conclusions. The significant linear cumulative effects demonstrated here support the idea of using sets of low-risk markers for delimiting new groups with high-risk of CRC in clinical practice that are not carriers of the usual CRC high-risk markers.

Lynch syndrome mutations shared by the Baltic States and Poland.

To the Editor : The definite diagnosis of Lynch syndrome (LS) is based on the identification of a mutation within one of the DNA mismatch repair (MMR) genes: MLH1 , MSH2 , MSH6 and PMS2 . To date, hundreds of pathogenic and suspected-pathogenic DNA variants in the MMR genes have been described worldwide (1). Large rearrangements account for about 10% of all MMR changes and can be easily detected by multiplex ligation-dependent probe amplification (MLPA), whereas substitutions, small deletions and insertions account for about 90% (2) and only full screening using either denaturing high-performance liquid chromatography (DHPLC), high resolution melting (HRM) or direct DNA sequencing seems to be the option for genetic analysis. Despite significant progress in the detection of gene mutations full screening remains expensive and time-consuming. According to the previous studies, majority of Polish families are affected by recurrent mutations found at least twice in our own series (2). That tendency provided an opportunity to design a common inexpensive assay for recurrent mutations, an approach that has been successfully applied as prescreening method (3). Founder mutations have been reported from a number of countries and geographical regions and are thought to be a result of population bottlenecks occurring throughout history. It is well recognized that populations from neighboring countries can share genetic changes and as such the probability of finding common mutation is greater. With respect to the regions bounded by Estonia, Latvia, Lithuania and Poland there are reports of common mutations occurring in these populations (4, 5).

New EPCAM founder deletion in Polish population

It is well known that founder mutations associated with cancer risk have useful implications for molecular diagnostics. We report the presence of a founder mutation in EPCAM involved in the etiology of Lynch syndrome (LS). The mutation extends nearly 8.7 kb (c.858 + 2478_*4507del) and is shared by 8 Polish families. Family members suffered almost exclusively from colorectal cancer; however, pancreatic and gastric cancers were also apparent. Next to mutations c. 2041G>A in MLH1 gene and c.942+3A>T in MSH2, the deletion mutation encompassing EPCAM is one of the most common causative changes responsible for LS in Poland.

Lynch syndrome mutations in Poland and the Baltic States

Lynch syndrome is a hereditary cancer syndrome that accounts for up to 15% of all colorectal cancers diagnosed at a young age. The disease is caused by germinal mutations in mismatch repair genes. To date, hundreds of small pathogenic and possibly pathogenic DNA variants (small insertions, deletions and substitutions) have been described for Lynch syndrome worldwide. Because of the variety of small changes in mismatch repair genes only full screening (exon by exon) by DHPLC/sequencing or HRM/sequencing seems to be the appropriate screening option. However, full screening is still too expensive and time consuming. A solution that uses the opportunity to reduce costs and improve diagnosis is the detection of recurrent mutations. This approach has been successfully applied as a pre-screening method. We have compared all identified changes in MMR genes detected to date from populations emanating from Estonia, Latvia, Lithuania and Poland to ascertain if there are any similarities in the mutation spectra that can be used in designing a rapid diagnostic test for recurrent mutations characteristic for the Eastern Baltic Sea region.

Fast diagnostic test for the identification of an increased genetic predisposition to colon cancer (exemplified on a DNA test for recurrent mutations of the gene MMR)

Hereditary nonpolyposis colorectal cancer (Lynch Syndrome, LS) is a genetic disorder, where family members are at high risk of developing cancer of the colon, endometrium, small intestine and urinary tract. The cause for LS is due to constitutional mutations in several mismatch repair genes (MMR) mainly in MLH1, MSH2 and MSH6. The Polish population has been well characterized by a large study comprising ~1000 LS-suspected families. Most nucleotide substitutions (about 90%) were discovered mainly by DHPLC (denaturing high-performance liquid chromatography) or sequencing, whereas deletion of one or more exons (about 10% of all mutations) were detected mainly by MLPA (multiplex ligation-dependent probe amplification) [1,2]. Over 60% families of the former study, which were affected by recurrent MMR mutations, were taken as a basis to design the following iPLEX/TaqMan test, that allows simultaneous testing of almost all recurrent mutations in only analysis [3] . The drawback of this kind of analysis are the high costs of the machines (Sequenom), for which a reasonable cost-effectiveness is achieved only for large series of probes. Thus the approach is convenient only for high-throughput laboratories. However the present approach is cost-effective even for testing individual patients. The present test, based on a Taqman PCR analysis, allows a fast identification of the 20 most frequent mutations of the genes MLH1 and MSH2. An analogous test for recurrent mutations of the gene APC could, in a similar way, accelerate the molecular diagnostic of predisposition to FAP.

Combined iPLEX and TaqMan assays to screen for 45 common mutations in Lynch syndrome and FAP patients

Mutations of genes associated with the mismatch repair mechanism and mutations of the APC gene are the most frequent causes of hereditary colorectal cancer. An iPLEX test combined with TaqMan genotyping assays was therefore developed to identify common recurrent mutations of those genes in the Polish population. We analyzed 349 DNA samples from 95 positive controls previously identified by sequencing and 254 unexamined individuals. The iPLEX test included two plexes, which comprised seven mutations of the APC gene and 29 mutations of three of the mismatch repair genes. TaqMan assays were designed for nine mutations not covered by the iPLEX assays: one mutation in the APC gene and eight mutations in the mismatch repair genes. Results were then verified independently by sequencing. Our combination method allowed detection of all recurrent mutations occurring in group of patients, followed by full analysis by DNA sequencing. With the exception of one false positive in the iPLEX test in the positive control group that could be assigned to contamination from neighboring wells rather than a detection error, given sufficient DNA concentration and quality, the designed iPLEX/TaqMan test had an accuracy of 100% for the designed assays. These results suggest that the combined iPLEX/TaqMan test is an outstanding tool for identification of recurrent mutations among hereditary colorectal cancer patients.