Balkan Journal of Medical Genetics

CLINICAL RELEVANCE OF CHEK2 AND NBN MUTATIONS IN THE MACEDONIAN POPULATION
Maleva Kostovska I, Jakimovska M, Kubelka-Sabit K, Karadjozov M, Arsovski A, Stojanovska L, Plaseska-Karanfilska D1,
*Corresponding Author: Dijana Plaseska-Karanfilska, M.D., Ph.D., Research Centre for Genetic Engineering and Biotechnology “Geogi D. Efremov,” Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia. Tel: +389-2-3235-410. Fax: +389-2-3155-434. E-mail: dijana@manu.edu.mk
page: 47

MATERIALS AND METHODS

Study Participants. The study population consisted of 300 unselected breast cancer patients. Median age at diagnosis was 50 years, and 30.0% of patients reported a first degree family history of breast cancer. The patient series had been used previously to determine the frequency of 11 selected mutations in BRCA1and BRCA2 genes [15]. The general population controls included 283 female volunteers. Informed written consent was obtained from each participant. The study was approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia. Genomic DNA was isolated from peripheral EDTA blood samples following standard phenol-chloroform extraction procedures. Genotyping. Polymerase chain reaction (PCR) primers were designed to give a variety of PCR fragment sizes for multiplex PCR (Table 1). The PCR multiplexes were performed in a final volume of 25 μL, in 1 × B2 reaction buffer, 200 mM dinucleotide triphosphate, 2.5 mM MgCl2, 10 pmol of each primer, 1 unit of HOT FIRE Pol DNÀ polymerase (Solis Bio- Dyne, Tartu, Estonia) and 100 ng of genomic DNA. Thirty-three cycles of PCR amplification were performed with denaturation at 95 °C, annealing at 58 °C and extension at 72 °C. An aliquot of the completed reaction was treated with Exonuclease I and Shrimp Alkaline Phosphatase (ExoSAP-IT) (Affymetrix, Santa Clara, CA, USA) to eliminate unincorporated nucleotide triphosphates and excess PCR primers overnight at 37 °C, followed by 15 min. at 85 °C to inactivate the enzyme. The purified PCR products were directly used as templates in a primer extension reaction containing the mutation specific primer mixture (Table 1). The mini sequencing primers were 5’-tailed with poly-C sequences of various sizes to produce extension products of 27-54 nucleotides to allow for separation by capillary electrophoresis. For the extension reaction, we used the ABI PRISM™ SNaPshot Multiplex Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. The extension products were treated with Shrimp Alkaline Phosphatase (Affymetrix). An aliquot of the SAP-inactivated single-nucleotide extension reaction was diluted in 12 μl HiDi Formamide (Life Technologies), supplied with GeneScan 120 LIZ Size Standard (Life Technologies), heat-denatured, snap-cooled on ice and loaded onto an ABI PRISM™ 3010 Genetic Analyzer (Life Technologies). Extension products were visualized and called automatically using GeneScan 4.0 (Life Technologies). Detection of the 5395 bp deletion spanning exons 9 and 10 of CHEK2 was performed using an allelespecific duplex PCR assay [10]. Briefly, two primer pairs were used specifically for detection of the large deletion in a single PCR. The first pair flanked the breakpoint site in intron 8, while the second flanked the breakpoint site in intron 10. The PCR products were analyzed by agarose gel electrophoresis. Mutation- negative cases produced two PCR fragments of 379 and 522 bp from the wild-type allele, while in the presence of the mutation, the forward primer of the first pair and the reverse primer of the second pair amplified a 450 bp long PCR product. Statistical Analyses. Fisher’s exact test (http:// vassar stats.net/) was used to determine the significant difference in the mutation frequencies between the two groups. Associations with a p value lower than 0.05 were considered to be significant.

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