Abstract 4569: A simple method to screen patients for SNPs in NAT1 gene for prostate cancer risk
Authors: Gomes James , Emaimem Melody , Zuric Maja , Long Maitland 
Published: August 2015

Single nucleotide polymorphisms (SNPs) are single base differences in DNA and are suitable for genotyping markers of risk in human disease. With emerging bio-technology and a cross-platform application of techniques improvements can be made in methodology. Although a number of high-throughput SNP genotyping systems are available the technology is expensive for it requires both trained personnel and dedicated platforms. The method reported here is a simple qPCR based High Resolution Method (HRM) based method and can be cost effectively used in screening patients in a general molecular biology and clinical laboratories. The NAT1 gene directs N-acetylation and O-acetylation of toxins including heterocyclic amines through the phase II xenobiotic metabolizing enzymes toxicity pathway. The purpose was to develop a screening tool to screen individuals with SNPs at locations 190, 445, 560 and 640.

A modified qPCR approach was used with HRM analyses. Primers were designed using an in-house protocol; the rs sequence for the site-specific mutation was obtained from SNP database and fed to Primer Quest and having diligently identified the location of the mutation primers were designed and assessed using OligoAnalyzer Tool. The Tm for the forward and reverse were kept as close as possible and G was kept between -1 and -9 and the GC content greater than 50%. The following sequences were used rs58379106 (190C&gt;T); rs4987076 (445G&gt;A); rs4986782 (560G&gt;A) and rs4986783 (640T&gt;G). A RT qPCR 10μl reaction was designed using iTAQ Supermix (BioRad, CA), forward and reverse primers and genomic DNA obtained from prostate cancer cases and controls. Standard cycling conditions described for iTAQ were observed for 44 cycles. The data collected was analysed using HRM software (BioRad, CA) as per the guidelines described by the software.

The data shows that when all the conditions are normalized and benchmarked the shape of the HRM curves are typical and characteristic of the SNP. The figures show the symmetry and similarity in the HRM. The similarity from the standard curve (Fig.1) and standard curve with unknown SNP (Fig. 2) and curves with a complementary SNP at 445 along with SNP at 640 (Fig. 3).

Fig. 1 Std. curve with NAT1640 Fig. 2 Std. curve with unknown samples Fig. 3 Unknown samples with 640 and 445 This data shows that with this approach it is possible to identify site specific mutation and the presence of other mutation in the vicinity of the SNP of interest. The presence or absence of mutations among cases and controls can be assessed using this approach.

Note: This abstract was not presented at the meeting.

Citation Format: James Gomes, Melody Emaimem, Maja Zuric, Maitland Long. A simple method to screen patients for SNPs in NAT1 gene for prostate cancer risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4569. doi:10.1158/1538-7445.AM2015-4569