Somatic mutations in MLH1 gene and MSI status as molecular characteristics of sporadic colorectal cancer


Fabián Čaja (1, 2, 3), Lucie Schwarzová (1), Michaela Schneiderová (1, 4), Petr Lněnička (1), Jitka Štekrová (1), Pavel Vodička (1, 2), Milada Kohoutová (1)


  1. Institute of Biology and Medical Genetics, First Faculty of Medicine and General Teaching Hospital, Charles University in Prague, Czech Republic
  2. Institute of Experimental Medicine, Academy of Sciences, Czech Republic
  3. Department of Cell Biology, Faculty of Science, Charles University in Prague, Prague, Czech Republic
  4. 1st Surgical Department, First Faculty of Medicine, Charles University in Prague and General Teaching Hospital in Prague, Czech Republic

Colorectal carcinoma (CRC) is one of the most prevalent malignancies in the Czech Republic. In general there are two molecular pathways leading to CRC. One pathway is characterized by chromosomal instability, the other by the deficiency in DNA mismatch repair (MMR) genes. MLH1 gene is one of the MMR gene-family members and represents a key component of the MMR system, responsible for recognition of nucleotide mismatches occurring during DNA replication, and for the recruitment of repair proteins to correct the replication errors. Germline mutations in MLH1 are responsible for Lynch syndrome or hereditary non-polyposis colorectal cancer (HNPCC) that accounts for 2-3 % of all CRC. Somatic mutations in MLH1 gene hallmark sporadic, MMR deficient, CRC cases.
Our aim was to detect somatic mutations in MLH1 and therefore we have analysed MSI status and MLH1 gene mutations in DNA samples from 100 CRC patients from General teaching hospital in Prague. The mutations have been screened by high resolution melting (HRM) curve analysis. HRM involves precise monitoring of the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it denatures at high temperatures. Positive cases in each run were subsequently controlled by automatic sequencing.
In summary, we have found only few mutations in MLH1 gene. The prevailing one represents polymorphism (c. 655 A>G, p. Ile219Val), located in exon 8 and detected in 50 patients. Its role in CRC risk has been recently discussed in literature. We have found three novel mutations, in exon 2 (c. 204 C>G, p. Ile68Met) and in exon 11 (c. 973 C>T, p. Arg325Trp). The third one is located in intronic region at position c. 1731+19 T>C. These findings require verification on a larger scale of CRC patients. Four previously described mutations in exon 16 (c. 1733 A>G, p. Glu578Gly), in exon 19 (c. 2146 G>A, p. Val716Met), in exon 17 (c. 1959 G>T, p. Leu653Leu) and c. 1558+14 G>A have been detected . All detected mutations are germ-line, except of that in exon 11 (c. 973 C>T, p. Arg325Trp) which seems to be sporadic. MSI-H status was determined in 9 cases, the other tumours (91 cases) were considered as MSI-stable. Further task will be the investigation of the role of epigenetic mechanisms, mainly methylation in promoter region of MMR genes, in relation to the development of CRC.
In conclusion, we have found three new mutations in MLH1 gene (c. 204C>G, p. Ile68Met in exon 2; c. 973C>T, p. Arg325Trp in exon 11 and intronic one c. 1731+19 T>C) on the cohort of 100 patients so far. It seems that somatic mutations play minor role in the development of sporadic form of CRC and different mechanisms play the main role, probably epigenetic changes or promoter polymorphisms.

Supported by the VZ MSM0021620808 and by GACR 310/07/1430 of the Czech Republic.

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