Preterm births contribute significantly to neonatal mortality and childhood morbidity in developing countries [1]. Globally, the incidence of preterm birth is highest in India at 23.6% [2]. There is a need to find etiological causes of preterm birth, including its genetic associates and biomarkers of inflammation. TLR-4 and TNF-a are the strong candidate genes of inflammatory pathway. TLR-4 has leucine rich repeats or pattern recognition receptors that help to identify the molecular pattern of several pathogens which initiates the activation of cascade of inflammatory pathway [2]. The objective of this study was to assess the association of the SNP at the rs4986790 and rs4986791 of the TLR4 gene and rs361525 of TNF-a gene and the expression analysis of TLR-4 gene with preterm birth.

Both the polymorphisms of TLR4, SNP rs4986790 (A+896 G) and rs4986791 (C+1196T) are located in the coding region which further regulate the transcription of the gene. These polymorphic sites cause the exchange of an amino acid on alteration with the rare allele: an aspartic acid in exchange for glycine at position 299 i.e. Asp299Gly and threonine for an isoleucine at position 399: Thr399IIl [2]. Alternatively, TNF-a (rs361525) SNP is located on the promoter region of the gene and is reported to be associated with elevated expression of the gene [2]. The details of primers, PCR condition, restriction enzymes, and their products are given in Web Table I. Total RNA was extracted the peripheral blood with the help of QIAGEN kit. All the reverse transcription was carried out by Fermentas reverse transcriptase kit using oligo dT priming.

Real-time PCR was performed with 1 µL cDNA, Syber green universal PCR mix, and 20X primer (Applied Biosystems, Foster City, CA), in a 7000 Sequence Detection System (ABI Prism, Applied Biosystems).The 18S rRNA gene was used as an endogenous control. Results were evaluated using pfaffa method: the delta-delta Ct method, where delta Ct was calculated as (TLR4Ct - 18sRNA Ct), and the relative quantity of TLR4 mRNA expression was calculated by the delta–delta Ct as 2" [(case delta Ct) " (control sample delta Ct)] [3].

Statistical analysis: All the analyses was carried out by SPSS (version 22.0). The genotypic and expression analysis was done blinded toward the patient‘s status. For categorical variables chi-square test was used. Student‘s t-test was used to determine the association of preterm birth with other factors. We calculated genotypic distribution by Pearson‘s c2 test taking 95% of confidence interval (C.I.) in consideration. P £0.05 was considered significant.

A total of 559 cases and 559 controls were recruited. Table I contains the demographic and anthropometric details of neonates and mothers. Table II depicts genotypic and allelic frequency of TLR4 Asp 299 Gly, Thr 399IIl and TNF-a (-308G/A) in association with preterm birth. The prevalence of genotype TLR4 Asp 299 Gly, Thr 399IIl did not differ significantly in cases and controls. Increased risk of preterm birth was found with AA genotype of TNF-a (OR 1.5; CI 1.02, 2.20; P=0.03; i.e. AA Vs GA+GG). TLR4 mRNA expression (Fig 1) was found to be higher (P<0.001) in cases [0.7 (0.04)] than in controls [0.5 (0.04)].

TABLE I	Details of the Neonates and Mothers Enrolled 

TABLE II	Genotypic Frequency of TLR4 Asp 299Gly, Thr 399 Ile and TNF-a Polymorphisms

Fig.1 Quantitative Real time-PCR analysis of TLR-4 gene expression which showed 2.5 fold increase in cases as compared to controls; the y axis of 2(–DCt) represents the relative gene expression of TLR-4.

Discussion

In this study, increased expression of TLR4 mRNA levels was reported in cases as compared with controls. However, no association was found between TLR4 SNP rs4986790 (A+896 G) and rs4986791 (C+1196T) and TNF-a SNP rs361525 with preterm birth.

A study conducted in Netherland by Krediet, et al. [10] found no association of TLR4 (Thr 399 IIl) polymorphism with the gestational age. While Lorenz, et al. [11] reported that TLR4 T allele was higher in singleton preterm neonates as compared to multiple preterm neonates and term neonates. Elovitz, et al. [12] reported that TLR4 G allele (Asp 299 Gly) populates are more at risk of premature rupture of membrane before or at 33 weeks. In case of TNF-a (-308G/A), no association was found in our study; similar results are seen in few earlier studies [13-15]. A study conducted on maternal-fetal genotype interface showed that mother carrying TNF-a-308GA genotype and fetus with the carrier of TNF-a-308GG genotype are at risk of preterm birth [16]. Speer, et al. [17] also supported the higher incidence of inflammation associated preterm birth in fetus with TNF-a-308GG genotype.