DNA repair genes are involved in the maintenance of genomes integration. Defects in DNA repair genes may contribute to the development of various types of cancers (1-4). X-ray repair cross-complementing group 2 (XRCC2), is one of the DNA repair genes which works in the homologous repair pathway (5). There are plenty of reported SNPs in this gene (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=7516) where R188H (rs3218536) is the most studied (6). This non-synonymous polymorphism (G to A) causes a non-conservative change in the level of protein expression which may alter its DNA repair functional activity (7). Many studies have been conducted to show possible associations of this polymorphism with various cancers (8-10). Various techniques have been used for R188H genotyping but most of them include mutation detection screening methods, such as PCR-RFLP and TaqMan allele discrimination techniques. However, these techniques have some potential disadvantages: PCR-RFLP technique is time consuming and in this case uses a relatively expensive restriction enzyme. Taqman probes are expensive and setting up the technique may require considerable effort. As a large number of samples should be tested in molecular-epidemiological studies, high throughput techniques are needed. To address this demand, we used a high resolution melting (HRM) method to discriminate R188H genotypes. This rapid and relatively new mutation scanning method has some advantages over similar gel based scanning methods (i.e. DGGE, SSCP and CSGE). For example, compared to a modified and rapid CSGE technique (11, 12), HRM method is much faster. Also, HRM has been successfully used as a mutation detection screening technique in other genes (13).
Assessment of HRM method to discriminate R188H genotypes as a fast and high throughput screening method and compare it to PCR-RFLP method.
3. Materials and Methods
In this study 350 human genomic DNA samples were genotyped for R188H polymorphism in XRCC2 gene using PCR-HRM method and the results were compared with PCR-RFLP results obtained from the same samples. Samples were collected from healthy volunteers from two academic centers. After signing the genetic test’s consent form by all candidates, peripheral blood samples were collected in EDTA tubes and genomic DNA of white blood cells were extracted using a previously described salting out method (14). R188H Genotyping: R188H alleles of XRCC2 gene from each sample were screened using polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis using Rotor- Gene™ 6000 real time rotary analyzer (Corbett Research, Qiagen). Four previously known genotype samples were included in each run (two for each 188RR and 188RH genotypes). These control samples were used as unique analysts in every run to test reproducibility. Forward and reverse primers were designed. A 104 bp fragment of exon 3 from XRCC2 gene containing codon 188 was amplified using GGA AAT GTT CTC AGT GCT TAG AG and TTC TTC TGA TGA GCT CGA GG primers. The PCR-HRM reactions were carried out in a 10 μl volume using Type-it HRM PCR Kit (Qiagen) containing 10 pmol of each primer and 20-50 ng genomic DNA. The PCR cycling temperature was: 95°C for 5 min; 45X (95°C 10 s, 59°C 30 s, 72°C 10 s); 72°C for 4 min; followed by Pre-HRM heteroduplex enrichment: 95°C for 10 s; 65°C for 5 min and HRM protocol was: Ramp, from 65°C to 95°C; increasing by 0.05°C at each step and waiting for 10 s in each round. After PCR-HRM, the same samples were treated with SexA1 restriction enzyme (RE) (Fermentas) using the manufacturer’s protocol and were subjected to 3.5% agarose gel electrophoresis and ethidium bromide visualization.
After RE treatment, PCR products carrying the wild type allele (188R) remained intact, while presence of the 188H allele produced two fragments of 61 bp and 53 bp. Simultaneously, based on melting profiles, three main groups were retrieved (see below) and four samples from each group were randomly selected and subjected to DNA sequencing. The third group consisted of two samples and both samples were sequenced. HRM runs for the 65 samples are shown in Figure 1A, B. Three different melting profiles could be recognized (Figure 1B). The profile of each control sample fell in a separate group. DNA sequencing of a few samples, each from a control-containing group confirmed R188H heterozyosity and 188R homozygosity in the same relevant group (Figure 2A). Also, DNA sequencing of the samples belonging to the third group showed 188H homozygosity. PCR-RFLP results are shown in Figure 2B. No discrepancy was seen between the resultant alleles from PCR-HRM and PCR-RFLP techniques.
The importance of DNA repair genes in modifying the risk of developing cancer is not a subject that could be neglected. There are more than 20 DNA repair genes in different DNA repair pathways (15) in which plenty of single nucleotide polymorphisms are recognized. Therefore, using rapid SNP detection methods is crucial in genotyping studies. R188H polymorphism in XRCC2 gene is one of the changes and its association with different cancers has been shown to lead to various outcomes. For example presence of the 188RH allele has been associated with decreased risk of breast cancer (16), but an increased risk of pancreatic cancer (9). In spite of the general view that HRM should be used as a scanning method for the detection of an unknown DNA polymorphism, we suggest that it can also be used as a screening method for the detection of specific polymorphisms, where no frequent SNPs have been reported in their proximity. Accordingly, we found that R188H polymorphism in XRCC2 gene was a good candidate. Indeed, we designed primers to amplify a small PCR fragment to minimize the chance for the presence of other polymorphisms around R188H. In the present study we analyzed 350 genomic DNA for R188H polymorphisms by PCR-HRM technique. To our knowledge, this was the first time that the HRM technique was implicated for genotyping of R188H changes in XRCC2 gene. No discrepancy was found between these results and those obtained from the PCR-RFLP method. The simplicity, speed and accuracy of the PCR-HRM method convinced us that it could be used for screening R188H polymorphism in XRCC2 gene. Moreover, the probable power of the PCR-HRM method for screening of other single nucleotide polymorphisms in other sites of the genome is a subject not far from imagination.
This project was supported by the Pasteur Institute of Iran and Tehran University of Medical Sciences. The authors do not have any conflicts of interest to report for this manuscript.
Implication for health policy/practice/research/medical education Early detection of cancer and cancer researchers.
Please cite this paper as Development of a High-Resolution Melting Method for Screening R188H Polymorphism in XRCC2 Gene. Iran J Biotech. 2013:11(2): in press. DOI: 10.5812/ijb.11450
Authors’ Contribution Pezhman Fard-Esfahani designed and directed this study, Shima Fayaz did HRM and RFLP analysis and wrote the manuscript, Shahnaz Khaghani contributed to data analysis.
Financial Disclosure The authors stated that they had no interests which might be posing a conflict.
Funding/ Support This study was partly supported by Pasteur Institute of Iran and partly by Tehran University of Medical Sciences.
16. Pooley KA, Baynes C, Driver KE, Tyrer J, Azzato EM, Pharoah PD, et al. Common single-nucleotide polymorphisms in DNA double-strand break repair genes and breast cancer risk. Cancer Epidemiol Biomarkers Prev. 2008;17(12):3482-9. [DOI] [PubMed]