Analyzing the correlation of TLR-4 Asp299Gly andThr399Ile polymorphism in women with Bacterial Vaginosis in Hillah city.

 

Ilham A. Bunyan¹, Bushra J. Umran², Zahraa Kais Salman^3*

¹Department of Microbiology, College of Medicine, University of Babylon, Hillah, Iraq.

²Department of Gynecology and Obstetrics, College of  Medicine, University of Babylon, Hillah, Iraq.

³Department of Microbiology, College of medicine, University of Babylon, Hillah, Iraq.

 

ABSTRACT:

Women in fecundity age have different vaginal infections , the commonly is bacterial vaginosis. Different microorganisms causes bacterial vaginosis, One of them were anaerobes that considered as vaginal flora and there presents in large number due to decrease in Lactobacilli growth lead to causing vaginal infections, where toll-like receptor-4 (TLR-4) can be stimulating by induces of immune response with these microbes.

The association of TLR4polymorphism with the incidence of bacterial vaginosis among women was investigated in this study.

High vaginal swabs and blood samples were obtained from 150 married women plagued from bacterial vaginosis and 150 unailing ladies as control. Human DNA was extracted, and TLR4 gene was amplified using specific primers. Restriction fragment length polymorphism technique was used for genotyping. Two single nucleotide polymorphisms (SNPs) (Asp299Gly and Thr399Ile) were investigated their relation with bacterial vaginosis.

There is insignificant association between Thr399Ile  and bacterial vaginosis  (OR=0.765, 95%CI= 0.319-1.834 , p= 0.001).

The SNPs of Thr399Ile canbe indicated to the susceptibility to get bacterial vaginosis.

 

 

 

INTRODUCTION:

One of the dominant gynecological infections is vaginal infection, which represents a significant public health problem because of the majority of cases around the world causing different consequences and most prevalence of these infections affecting women in the generative age[1,2]. The massive growth of normal anaerobes present in the vaginal inviron due to loss of lactobacilli, lead to increase the pH after elevation of proteolytic carboxylase amounts, which disintegrate vaginal peptides into different amines that are volatile, malodorous, and associated with increased vaginal diverting and squamous epithelial cell exfoliation, giving the typical clinical features observed in patients with BV. Toll-like receptor 4 is activated by, LPS, lipoglycans and/or other microbial that subsequently induce cytokine production, inflammation, phagocytosis and innate immune defense responses. Single nucleotide polymorphism (SNP) in the coding and promoter regions of human TLR4 have been linked to an increased incidence of certain infectious and inflammatory diseases, such as Gram-negative infections[3]. Different functional effective polymorphisms for Asp299Gly and Thr399Ile genes have been described that are associated with impaired lipopolysaccharide signal transduction [4]. Two single nucleotide polymorphisms in TLR4 gene; Asp299Gly and Thr399Ile have been shown to cause hyporesponsiveness to LPS, reduced epithelial TLR4 density, and reduced response of inflammatory cytokines [5].

 

This study aimed to investigate the association of these two SNPs with the incidence of bacterial vaginosis in women.

 

MATERIAL AND METHODS:

Specimens collection:

High vaginal swabs were taken from 150 married women who attended to the out-patient clinics of Gynecology and Obstetrics, in two hospitals in Babylon Province: Babylon Maternity and Pediatrics Teaching Hospital, and Al-Hillah General Teaching Hospital during the period from February to October 2016.These women were diagnosed suffering from bacterial vaginosis depending on [6], their age range from 15 to 45years, they represent patients group. Blood samples collected by taken five milliliters of venous blood from patients and other 150 women as control then kept in EDTA tubes until be used for DNA extraction.

 

DNA extraction and PCR Analysis:

DNA was extracted from blood samples using Blood/Cell DNA Mini Kit (GB100, GB300) (Geneaid/UK) according to the manufacturer's instructions. The primers used for amplification ofPCR was used as a diagnostic technique. Primers used in this study illustrated in Table (1).

 

Table (1) Sequence of primers, amplicon size and their references

 

The reaction mixture was 12.5µlMaster Mix, 2.5µl Forward Primer, 2.5µlReverse Primer, 5µlTemplate DNA and 2.5µlNuclease -Free Water.

 

The programs used for PCR analysis and there references mentioned in Table (2) and Restriction Enzymes and there cutting sites used illustrates in Table (3).

Table (2) The programs used in PCR analysis and there references

 

Table (3) Restriction Enzymes and there cutting sites.

 

Polymerase Chain Reaction (PCR) - Restriction Fragment Length Polymorphism (RFLP) Analysis of Single Nucleotide Polymorphisms (SNPs):

Genetic susceptibility for bacterial vaginosis were studied by PCR-RFLP analysis for SNPs genes listed in Table (1), coding for TLR-4 position, Asp299Gly G/A SNP and Thr399Ile T/C. After amplification of these primers according to the PCR conditions listed at Table (2), and in which extracted DNA from 150 patients and 150 control fresh blood samples were used for each primer set. For Asp299Gly G/A, and Thr399Ile T/C, NcoI and HinfI restriction endonuclease enzyme were utilized respectively for PCR products digestion according to company protocol .Detection of amplified products by agarose gel electrophoresis and genotyping examined according to [7].

 

Statistical analysis:

The Statistical Package for the Social sciences (SPSS, version 18.0) was used for statistical analysis. Risk association between the genotype and Bacterial Vaginosis susceptibility was estimated by the calculation of odds ratio and 95% confidence intervals. Genotype and allele frequency differences between the groups were tested using Chi-square test with one degree of freedom. A p-value 0.05was considered statistically significant.

 

RESULTS AND DISCUSSION:                                                                      

Amsel's clinical criteria was considered as one of the most standard method for Bacterial Vaginosis diagnosis [6]. Amsel's Criteria revealed that all 150 of women patients gave positive results.

Analysis of the Toll-like receptor -4 genes by PCR-RFLPs Technology:

Products of PCR were subjected to RFLP technique, as the digestion was done by the use of NcoI and Hinf I restriction enzymes respectively. Once more, digestion products were separated on agarose gel against UV light.

 

As NcoI, a type I restriction endonuclease gathered from Nocardia corallina; digestion with this enzyme was resulting in three genotypes related to this SNP, appeared as three bands with size 188, 168, 20 bp respectively representing the heterozygous genotype (AG). While the mutant homozygous genotype (GG) was cut to produce two bands with size 168, 20 bp and the smallest band 20 bp was not visible in both cases due to its small size .On the other hand, the wild homozygous genotype (AA) appeared as one band with size of 188 bp, as shown in Figure (1),(2).

 

Figure (1): 3% Gel electrophoresis for TLR-4 Asp299Gly PCR products visualized under UV light after staining with ethidiume bromide. M: marker (2000-100bp), lane (1-10) PCR product without restriction for the Asp299Gly gene.

 

Figure (2) : 3% Gel electrophoresis for TLR-4 Asp299Gly PCR products visualized under UV light after staining with ethidiume bromide. M: marker (2000-100bp), lane (1-10) restricted fragments with NcoI restriction enzyme.

 

As Hin.fI a type I restriction endonuclease gathered from Haemophilus influenzae localizes its recognition site for cleavage of double stranded DNA containing a copy of this site, digestion with this enzyme was resulting in three genotypes related to this SNP, appeared as three bands with size 124, 98, 26 bp representing the heterozygous genotype (CT), While the mutant homozygous genotype (TT) was cut to produce two bands with size 98, 26 bp, also the smallest band 26 bp was not visible due its small size .On the other hand, the wild homozygous genotype (CC) appeared as one band with size of 124 bp respectively, as shown in Figure (3), (4).

 

Figure (3) : 3% Gel electrophoresis for TLR-4 Thr399Ile PCR products visualized under UV light after staining with ethidiume bromide. M: marker (2000-100bp), lane (1-10) PCR product without restriction for the Thr 399Ile gene.

 

Figure (4) : 3% Gel electrophoresis for TLR-4 Thr399Ile PCR products visualized under UV light after staining with ethidiume bromide. M: marker (2000-100bp), lane (1-10) restricted fragments with HinfI restriction enzyme

 

 

Table (4) showed that the genotype frequency revealing that the mutant homozygous GG genotype frequency was found more occurrence whereby gave (50.0%) in BV group and (59.3%) in the healthy group, with Odds Ratio 1.575, 95%CI (0.981 – 2.527),whereas wild homozygous AA genotype frequency was found (46.0%) in BV group and (34.7%) in the healthy group and heterozygous GA genotype frequency was found to be (4.0%) in BV group and (6.0%) in the healthy group with Odds Ratio 1.99, 95%CI (0.667– 5.943) and there is no significant relationship between the Asp299Thr polymorphism and bacterial vaginosis .

 

Table (4) Prevalence of Toll-like receptor 4 allele (Asp299Gly) in individuals with bacterial vaginosis and control group and their statistical analysis

*P-value≤0.05 is significant.

 

The A allele frequency was 96 (48%) in patient group and 75 (38%) in control group whereas G allele was more frequency than A allele where was 104 (52%) in patient group and 125 (62%) in control group, with significant P-value (0.034) among them as shown in Table (5).

 

Table (5): Allele frequencies of A and G alleles in patients and control groups

Allele	Allele frequency of patients %	Allele frequency of control %
A(wilde)	96 (48%)	75 (38%)
G(mutant)	104 (52%)	125 (62%)
P -value	0.034*

*P-value≤0.05 is significant.

The results in Table (6) showed the genotype frequency revealing that the heterozygous TC genotype frequency was found (12.0%) in BV group and (6.0%) in the healthy group, whereas wild homozygous CC genotype frequency was found(36.0%) in BV group and (60.0%) in the healthy group with Odds Ratio 2.549, 95%CI (1.564 – 4.154) and mutant homozygous TT genotype frequency was found to be (52.0%) in BV group and (34.0%) in the healthy group, Odds Ratio 0.765, 95% CI (0.319 -1.834) and there is significant relationship between Thr399Ile polymorphism and bacterial vaginosis where the P-value was 0.001*.

 

 

Table (6) Prevalence of Toll-like receptor 4 allele (Thr399Ile) in individuals with bacterial vaginosis and control group and their statistical analysis .

*P-value≤0.05 is significant.

 

Table (7): Allele frequencies of T and C alleles in patients and control groups

Allele	Allele frequency of patients %	Allele frequency of control %
T(mutant)	116 (58%)	74 (37%)
C(wilde)	84 (42%)	126 (63%)
P -value	<0.001*

*P-value≤0.05 is significant.

 

The T allele frequency was 116 (58%) in patient group and 74(37%) in control group whereas C allele was less frequency where was 84 (42%) in patient group and 126 (63%) in control group, with significant P-value (0.001) among them as shown in Table (7).

 

The values in the Table (5) and (7) revealed the mutant alleles T and G are the highest frequencies among patient and control groups additively that these genes are mutant ones, so we suggesting that TLR-4 polymorphisms are related mostly with these alleles.

 

Our results explained that the TLR-4 Thr399Ile polymorphism linked with susceptibility to bacterial vaginosis BV, the TLR-4 Thr399Ile polymorphism showed a significant correlation with the incidence of BV (P = 0·001 by Fisher's) while the TLR-4 Asp299Gly polymorphism did not (P = 0·123 by Fisher's).

 

These SNPs are located in the coding sequence, affect TLR-4 extracellular domain and result in amino acid exchanges, an Aspartic acid for a Glycine at position 299 (Asp299Gly) and a Threonine for an Isoleucine at position 399 (Thr399Ile). Individuals possessing a co-segregating polymorphism in TLR-4 (Asp299Gly and Thr399Ile) are hyporesponsive to LPS and are more susceptible to Gram-negative bacterial infections [8].

 

These SNPs have been reported to be associated with a wide infectious and non-infectious diseases where the ability of individuals to respond properly to TLR ligands may be impaired by single nucleotide polymorphisms (SNPs) within TLR genes due to two cosegregating SNPs-Asp299gly in the risk allele is (G) and Thr399Ile in the risk allele is (T) -within the gene encoding TLR4 that is the receptor for bacterial lipopolysaccharide [9].

 

The more prevalent nonsynonymous SNPs are an A/G transition that causes the substitution of aspartic acid by glycine at amino acid position 299 (Asp299Gly) and a C/T transition that causes the substitution of threonine by isoleucine at amino acid position 399 (Thr399Ile) were an association with susceptibility to various conditions and diseases, including infections with gram-negative bacteria and bacterial vaginosis as mentioned by [10].

 

While [11] reported that two single nucleotide polymorphisms in TLR4 gene; Asp299Gly and Thr399Ilehave been shown to cause hyporesponsiveness to LPS, reduced epithelial TLR4 density, reduced inflammatory cytokines response that increased susceptibility to infection.

 

CONCLUSIONS:

There was significant association between TLR-4 Thr399Ile polymorphism and the susceptibility to infection with bacterial vaginosis and other Gram negative bacterial infections.

 

REFERENCES:

1.       Mascarenhas, R., E., M., Machado, M., S., C., Silva, B., F., B., C, Pimentel, R., F., W., Ferreira, T., T., FernandaMaria Silva Leoni, F., M., S. and Grassi, M., F., R. Prevalence and Risk Factors for Bacterial Vaginosis and Other Vulvovaginitis in a Population of Sexually Active Adolescents from Salvador, Bahia, Brazil. Infectious Diseases in Obstetrics and Gynecolog. 2012, 5(2), pp: 101-109.

2.       Mastromarino, P. Evaluation of vaginal pH for detection of bacterial vaginosis. Indian J Med Res.2013, (138), pp: 354-359.

3.       Nishimori J. H.; Newman T. N.; Oppong G. O.; Rapsinski G. J.; Yen J. H.; Biesecker S. G. Microbial amyloids induce interleukin 17A and IL-22 via TLR2 activation. Infect. Immun.80; 2012: 4398–4408.

4.       Agnese DM.; Calvano JE.; Hahm SJ.; Coyle SM.; Corbett SA.; Calvano SE. and Lowry SF. Human toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of gram-negative infections. J Infect Dis.186; 2002:1522–1525.

5.       M. Folwaczny; J Glas; H-P Torok; O Limbersky and C Folwaczny. Toll-like receptor (TLR) 2 and 4 mutations in periodontal disease.Clin Exp Immunol.135 (2); 2004: 330–335.

6.       Spiegel, C, Amsel, R and Holmes, A. Diagnosis of bacterial vaginosis by direct Gram stain of vaginal fluid. Journal of Clinical Microbiology.18; 1983:170-177.

7.       Schroder, N. W. and Schumann, R. R. Single nucleotide polymorphism of toll like receptors and susceptibility to infectious disease. Lancet Infec. Dis. 5; 2005:156-164.

8.       Jing Chen and Jianping Xie. Role and regulation of bacterial LuxR-like regulators. Journal of Cellular Biochemistry.112 (10); 2011: 2694-2702.

9.       Carvalho A.; Pasqualotto AC.; Pitzurra L.; Romani L. Denning DW and Rodrigues F. Polymorphisms in toll-like receptor genes and susceptibility to pulmonary aspergillosis. J Infect Dis.197 (4); 2008:618-21.

10.     D Santini; S Angeletti; A Ruzzo; G Dicuonzo; S Galluzzo; B Vincenzi; A Calvieri; F Pizzagalli; N Graziano; E Ferraro; G Lorino; A Altomare; M Magnani; F Graziano and G Tonini. Toll-like receptor- 4 Asp299Gly and Thr399Ile polymorphisms in gastric cancer of intestinal and diffuse histotypes. Clin Exp Immunol.154 (3); 2008:360–364.

11.     Schroder, N. W. and Schumann, R. R. Single nucleotide polymorphism of toll like receptors and susceptibility to infectious disease. Lancet Infec. Dis.5; 2005:156-164.

 

 

 

Accepted on 09.10.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(12): 4178-4182.