Rapid multiplex high resolution melting method to analyze inflammatory related SNPs in preterm birth

Abstract

BackgroundComplex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs) from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM) analysis is a new method in which polymerase chain reaction (PCR) and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB) is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB.MethodsHere, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism.ResultsWe were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB.ConclusionsSeveral gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing their association to PTB. It can easily be used for simultaneously analyze any set of SNPs, either as the first choice for new association studies or as a complement to large-scale genotyping analysis. Given that inflammatory pathway is in the base of several diseases, it is potentially useful to analyze a broad range of disorders.

QMC-PCRx: a novel method for rapid mutation detection

“…While Seipp et al 17 used GC-rich and AT-rich tailed primers as well as primers incorporating locked nucleic acids to achieve ample gaps between the melt curves of the target amplicons, Pereyra et al 18 designed conventional, non-modified primer pairs to amplify targets of 50–200 bp long, simply using product sizes to achieve adequate separation of melt curve Tms. These have been reasonably successful but they are single-stage reactions which increases the risk of inappropriate primer interaction.…”

Polymorphisms in GJA1 and their association with growth traits in chicken

“…Previous studies have shown that transcription factors can bind a few specific sites located in the promoter and intron regions of genes to influence gene expression (Myers et al, 2007;Pereyra et al, 2012) and protein translation (Down et al, 2012). However, the exact functions of SNPs in non-coding regulatory regions are not clear, but are predicted to influence the binding affinity of transcription factors (Kim et al, 2008).…”

The SEPS1 G-105A Polymorphism Is Associated with Risk of Spontaneous Preterm Birth in a Chinese Population

“…It is well established that inflammation is associated with SPTB [10], [11], [12]. During recent years, the link between polymorphisms in genes encoding cytokines involved in inflammatory mechanisms has received increased attention from researchers [4], [5], [6], [7]. However, no study has yet explored association between SPTB and SEPS1, which is an upstream regulatory factor of inflammatory cytokine [14].…”

“…Moreover, there are significant racial/ethnic disparities in the incidence of SPTB, with African Americans running more than twice as great a risk of SPTB as European-American women. In addition, association studies have identified a number of genetic polymorphisms related to infection, inflammation and innate immune systems that are risk factors for SPTB [4], [5], [6], [7], [8], [9]. Despite these advances, the alleles accounting for the bulk of genetic susceptibility to SPTB remain undiscovered, particularly in Chinese populations.…”