Rapid detection of functional gene polymorphisms of TLRs and IL-17 using high resolution melting analysis

Johanna Teräsjärvi,Jussi Mertsola,Matti Korppi,Qiushui He,Antti Hakanen,Kirsi Gröndahl-Yli-Hannuksela,Ville Peltola,Kirsi Nuolivirta

doi:10.1038/srep41522

Abstract

Genetic variations in toll-like receptors (TLRs) and IL-17A have been widely connected to different diseases. Associations between susceptibility and resistance to different infections and single nucleotide polymorphisms (SNPs) in TLR1 to TLR4 and IL17A have been found. In this study, we aimed to develop a rapid and high throughput method to detect functional SNPs of above mentioned proteins. The following most studied and clinically important SNPs: TLR1 (rs5743618), TLR2 (rs5743708), TLR3 (rs3775291), TLR4 (rs4986790) and IL17 (rs2275913) were tested. High resolution melting analysis (HRMA) based on real-time PCR combined with melting analysis of a saturating double stranded-DNA binding dye was developed and used. The obtained results were compared to the “standard” sequencing method. A total of 113 DNA samples with known genotypes were included. The HRMA method correctly identified all genotypes of these five SNPs. Co-efficient values of variation of intra- and inter-run precision repeatability ranged from 0.04 to 0.23%. The determined limit of qualification for testing samples was from 0.5 to 8.0 ng/μl. The identical genotyping result was obtained from the same sample with these concentrations. Compared to “standard” sequencing methods HRMA is cost-effective, rapid and simple. All the five SNPs can be analyzed separately or in combination.

Highlights

Nucleotide Amino acid Allele Frequency Allele Frequency Potential effect and reported change change (1000 Genome project)* of this study associations
Allele G, which is a minor allele in all other populations, except Europeans, is associated with increased susceptibility to asthma and allergic rhinitis after hospitalization due to bronchiolitis in infancy[13] and is a risk factor of tuberculosis[14,15,16]
high resolution melting analysis (HRMA) method has been applied for detection of TLR1, TLR2 and TLR4 SNPs35–37

Summary

Introduction

Nucleotide Amino acid Allele Frequency Allele Frequency Potential effect and reported change change (1000 Genome project)* of this study associations. The SNP 1805G >T (rs5743618) in TLR1 gene leads to substitution of amino acid at the position 602 from serine (Ser) to isoleucine (Ile) on the cytoplasmic side of the transmembrane domain of the receptor. It affects the cell surface trafficking in several cell types[4]. HRMA is ideal for clinical use because PCR amplification and melting curve analysis can be performed on the same plate and without any post-PCR processing This saves time and decreases the risk of contamination. HRMA method has been applied for detection of TLR1 (rs5743618), TLR2 (rs5743708) and TLR4 (rs4986790) SNPs35–37

Objectives

Methods

Results

Conclusion