DETECTION OF ALLELIC VARIANTS OF THE POLE AND POLD1 GENES IN COLORECTAL CANCER PATIENTS
Pätzold LA, Bērziņa D, Daneberga Z, Gardovskis J, Miklaševičs E*
*Corresponding Author: Professor Dr. Edvīns Miklaševičs, Institute of Oncology, Riga Stradiņš University, Dzirciema iela 16, Riga LV1007, Latvia. Tel: +371-6770-4028. Fax: +371-6706-9545. E-mail: edvins. miklasevics@rsu.lv
page: 83

MATERIALS AND METHODS

Study Group. One thousand, seven hundred and forty-nine DNA samples from colorectal cancer patients were collected from 2002 to 2013. The patients’ age at diagnosis ranges from 20 to 93 with a mean age of 67.09 ± 10.96. The hereditary colorectal cancer group consisted of 230 patients corresponding to Amsterdam I and II criteria and the Bethesda criteria. The age at diagnosis in this group ranged from 24 to 88 with a mean age of 67.5. The second group of hereditary cancer syndromes including hereditary breast/ovarian cancer, hereditary endometrial cancer, hereditary stomach cancer and cancer aggregation in the family consisted of 163 patients. The age at diagnosis ranged from 35 to 82 years with a mean age of 67. The rest of the samples consisted of 1356 patients with no history or unknown history of cancer in the family. The age of diagnosis ranged from 20 to 93 with a mean age of 70. All samples belonging to the HNPCC group were tested and found negative for pathogenic allelic variants of the MLH1, MSH2 and MSH6 genes. None of those samples had been screened for genetic variants of the POLE or POLD1 genes before. Informed consent was obtained from all individual participants included in the study. Methods. DNA was extracted from whole blood using Flexi gene DNA kit (Qiagen GmbH, Hilden, Germany) and polymerase chain reaction (PCR) was done using a novel proofreading polymerase Optimase, from thermophilic Archaeal bacterium manufactured by Transgenomic Inc. (Omaha, NE, USA). The DNA samples were screened for allelic variants of POLE rs483352909 and POLD1 rs397514632. The primers were self-designed and in the case for POLD1, they were designed to exclude a common heterozygous variant in preferred PCR fragment. Primers for POLE rs483352909 were forward 5’-ATG TCC TCC GGG TCT AGC TC-3’ and reverse 5’-GGT GCC TGT TAG GAA CTT GC-3’; for POLD1 rs397514632, forward 5’-ACT CTT CAT TCC AGT CCA AG-3’, and reverse 5’-TGA TGA TGC TGT GCT GC- 3’. The annealing temperature was 58 °C for POLE and 59.5 °C for POLD1 primers. The elongation time was set to 1 min. as the proofreading polymerase needs approximately twice as long for elongation. After PCR, the samples were denaturated from 95 °C to 25 °C ramping 1.5 °C per min. The PCR products were then analyzed by denaturing high performance liquid chromatography (DHPLC). The DHPLC analysis was performed using the WAVE® system (model 4500; Transgenomic Inc.), which is equipped with DNASep®HT Cartridge, UV Detector (model L-2400), Oven (model L-2310T), autosampler (model L-2200) and pump (model L-2310) (all from Transgenomic Inc.). The DNA was detected using the cartridge in combination with the buffers A (0.1M TEAA aqueous solution) and B (0.1M TEAA aqueous solution with 25.0% acetonitrile). The conditions for DHPLC were established using Transgenomic Navigator™ software and adjusted by adding positive and negative controls in each sample set (Figure 1). The DHPLC analysis of one sample with the rapid DNA method takes 3.5 min. The optimal temperature was found by titrating it at ±1.5 °C and the necessary TEAA and acetonitrile concentrations were calculated according to the constitution of the fragment by the Navigator™ software. The partial denaturation temperature for POLE was 62 °C and for POLD1 63 °C. This method was validated and deviations from wildtype curves seen in DHPLC were then analyzed by Sanger sequencing using capillary electrophoresis with ABI PRISM™ 3130 (Applied Biosystems, Foster City, CA, USA) (Figure 2).



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