Abstract
Cancers are the most deadly diseases in the world and their incidences continue to increase over time. Particularly, breast cancer in females places 1st rank among other types of cancers in term of cancer cases (23%) and death incidence (14%). Recent findings support the correlation between Ile655Val SNP in the HER2 gene with breast cancer risk. Moreover, the Ile655Val HER2 gene polymorphism could be a predictive factor in a neoadjuvant therapy setting. Precise detection of the Ile655Val HER2 gene SNP in early breast cancer patients will be beneficial in designing the most suitable treatment and in increasing the efficacy of anticancer drugs. Here we develop a rapid and inexpensive method for Ile655Val SNP detection in the HER2 gene based on allele-specific PCR technology. Two forward primers and one common reverse primer were designed to anneal specifically either on the HER2 gene fragment containing the GG genotype or to the HER2 gene fragment containing the AA genotype where one of these primers had been added with poly-GC at 5’ upstream. Moreover, to increase discrimination level, mismatch bases at the SNP site and the 3rd base of each forward primers from 3’end were added. To test the performance of the designed primers in discriminating a polymorphism and its annealing temperature, breast cancer specimen-derived genomic DNA (with GG genotype) and pGEM_HER2/AA (with AA genotype) were used as templates in the PCR reaction. The optimal annealing temperature for SNP detection was at 51.5°C as showed by the appearance of a 150 base pair (bp) band as AA genotype (pGEM_HER2/AA template), 116bp band as GG genotype (genomic DNA template), and both types of bands as AG genotype (mix of pGEM_HER2/AA and genomic DNA template). Allelic types of breast cancer patients were also determined using this optimized method compared to sanger sequencing. The 100% accordance was shown for all types of genotypes in both methods. The allele-specific PCR in this study may have application in determining polymorphisms of the breast cancers-originated Ile655Val HER2 gene.
Highlights
Breast cancer is one of common malignancy among women after cervical and ovary cancer and its incidence rate was 1.676.727 worldwide
Plenty of evidence have linked breast cancer risk with HER2 gene polymorphisms at codon 655, which is the conversion of amino acid isoleucine (ATC) to valine (GTC) [5,6,7,8,9,10,11,12]
Interesting finding published by Lu et al [18] who conducted comprehensive meta-analysis through SNP codon Ile655Val HER2 gene-related 27 published articles highlighted the dependency of Ile655Val HER2 polymorphisms as breast cancer risk factor to ethnicity
Summary
Breast cancer is one of common malignancy among women after cervical and ovary cancer and its incidence rate was 1.676.727 worldwide (http://www.cancerresearchuk.org). Available methods for allelic types discrimination of HER2 gene polymorphisms of breast cancer patients are (1) PCR-RLFP and (2) Real-Time PCR using Taq Man-based or dual colour-based [17]. Detection of HER2 Gene Polymorphism in Breast Cancer: PCR Optimization Study by looking at their size visually [5, 14].
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