Association between single nucleotide polymorphism of immunoregulatory genes and preterm premature rupture of membranes in preterm labour

Aim. Investigation of the association between SNP genes of IL4 (rs2243250), IL10 (rs1800896 and rs1800872) and RLN2 (rs4742076 and rs3758239) and preterm premature rupture of membranes in 26–34 weeks of gestation in Zaporizhzhia population. Materials and methods. We have investigated markers of cytokines genes in 50 women with PPROM in 26–34 weeks of gestation period and 50 pregnant women with physiological pregnancy and term labour without complications. The genotyping using TaqMan tests was done on amplifier CFX96™ Real-Time PCR Detection Systems (Bio-Rad Laboratories, Inc., USA. The combined effect of the studied locus of the analyzed genes on the appearance of PPROM, PTL in the population was initiated using the Multifactory Dimensionality Reduction (MDR) method (software MDR ver. 3.0.2). Results. The distribution of the rs2243250 gene polymorphism alleles of the IL4 gene of the main study group – TT homozygotes were determined in 4 % cases, CT heterozygotes were found in 22 %, CC homozygotes – in 74 % cases. We have got statistically significant differences of rs4742076 polymorphisms (TT, CT, and CC) of the RLN2 gene in the study groups. We also detected statistically significant differences in all the alleles of rs3758239 polymorphism (AA, GG and AG) of the gene RLN2 – χ 2 = 23.86, OR( AA ) = 12.57; 95 % CI: 3.68–42.98; OR( GG )= 0.08; 95 % CI: 0.02–0.27; and OR( AG )= 0.11; 95 % CI: 0.03–0.42, respectively, P < 0.05, indicating the reliability of the received prognostic markers. The nature of interlocal interaction between the genes is at the level of “independent effects” of influence (IL4-IL10 rs1800896 = -23.08 %, IL10 rs1800896 – IL10 rs1800872 = -19.94 %, IL10 rs1800872-IL-4 = -19.34 %). The percentage of entropy of the investigated polymorphism of each gene for the case-control status was 14.15 % for the IL4 gene, 23.08 % for the IL10 rs1800896 gene and 9.34 % for the IL10 rs1800872 gene.


Background
Today in Ukraine the number of normal births is an average of 32.6 % of their total. The frequency of registration of the prematurity in different regions of the country ranges from 3 % to 12 %. Spontaneous preterm labor (PTL) and preterm premature rupture of membranes (PPROM) are major contributors to neonatal mortality and serious neonatal morbidity worldwide [3]. Despite all efforts to identify preventive measures and causative mechanisms, prematurity remains an unresolved issue worldwide [5].
In recent years a great attention has been paid to associative search of polymorphic markers with different diseases and as a result creation the genetic platform for personalized medicine. However, the genetic predisposition can contribute to PTL and PPROM [11]. The sequencing of the human genome and the discovery of the phenomenon of single nucleotide polymorphism (SNP) genes became a dawn of studying the effect of the genetic code on quantitative expression changes and further biological function of proteins [8,9]. Most of the SNP genes of the cytokines are found in the regulatory regions of the gene and directly affect their transcriptional activity and the concentration of the cytokine in the blood [4]. These genetic variations have an effect on the individual characteristics involved the immune response to infection and triggering PTL and PPROM [6,7]. Most of findings, however, are controversial, which demonstrates the importance of standardized methods and reproductive techniques as well as strictly performed evaluation adjusted for potential confounding factors [3]. Additionally, great part of inconsistencies found in the literature can be due to differences in genetic background and environmental exposures, parameters that vary greatly among distinct populations [3].
But information about the polymorphism of signaling molecules during PPROM in preterm pregnancy is small, scientific work in this direction can provide resources for analysis of polymorphic markers in associative studies, deepen the concept of the immune link of PPROM and PTL pathways, develop new effective methods of prediction these complications of pregnancy [10,12].

Aim
To investigate the association between SNP genes of IL4 (rs2243250), IL10 (rs1800896 and rs1800872) and RLN2 (rs4742076 and rs3758239) and preterm premature rupture of membranes in 26-34 weeks of gestation in Zaporizhzhia population.
These SNPs genes were selected based on existing evidence in the literature for a role in the pathogenesis of the studied conditions.

Materials and methods
We had done open cohort randomized research based on Regional Perinatal Center Zaporizhzhia city during period of 2015-2017 years. The case group consisted of 50 women with PPROM in preterm pregnancy, 26-34 weeks of gestation. For the control group we collected samples from 50 women with healthy term deliveries without previous history of PTL.
The genotyping using TaqMan tests was done on amplifier CFX96™ Real-Time PCR Detection Systems (Bio-Rad Laboratories, Inc., USA). Polymerase chain reaction (PCR) for TaqMan genotyping was performed according to the instructions of Applied Biosystems, USA. Statistical data processing was carried out using the software package Statistica 6.0 (StatSoft Inc, No. AXXR712D833214FAN5). The comparison of qualitative indices was carried out using the χ 2 criterion with Yates's correction and Fischer's exact test (F). In order to evaluate the contribution of gene polymorphism to the probability of development of PPROM in preterm pregnancy, odds ratios (OR) were calculated with 95 % confidence interval (CI). The differences were considered to be significant at P < 0.05.
The combined effect of the studied locus of the analyzed genes on the appearance of PPROM, PTL in the population was initiated using the Multifactory Dimensionality Reduction (MDR) method, a nonparametric statistical method that evaluates the main, independent and common effects of gene polymorphism, from deducting potential of the predicate (software MDR ver. 3.0.2). Also using the MDR method predicted the risks of PPROM, PTL with a graphic model of gene-gene interaction. The best gene-gene interaction patterns were characterized by the highest cross-validation consistency.

Results
Based on the genotyping results of rs1800896 gene polymorphism IL-10 gene TT homozygotes were detected in 37 (74 %) cases of the main study group, CT heterozygotes -in 2 (4 %) and CC homozygotes -in 11 (22 %) cases, consequently. In the study of polymorphism rs1800872 of the IL10 gene, in the main group of study homozygous TT were identified in 7 (14 %) cases, TG heterozygotes were found in 18 (36 %), GG homozygotes -in 25 (50 %) cases.
The distribution of the rs2243250 gene polymorphism alleles of the IL4 gene of the main study group -TT homozygotes were determined in 2 (4 %) cases, CT heterozygotes were found in 11 (22 %), CC homozygotes in 37 (74 %) cases. The prevalence of genotypes depending on term of PPROM is demonstrated in Fig. 1.
The analysis of the multiplicative model of inheritance has shown that the relative frequency of the investigated polymorphic variants of genes IL4 (rs2243250), IL10 (rs1800896 and rs1800872) and RLN2 (rs4742076 and rs3758239) was significantly different between the study groups ( Table 1). In general, the favorable СС polymorphic variant of the IL4 (rs2243250) gene over TT was dominant in the surveyed population (18.5 times more among patients (P < 0.05), 9.33 times -among healthy term deliveries (P < 0.05), T-alleles of the IL10 (rs2243250) gene over TC polymorphic variant (18.5 times among patients (P < 0.05), 5.5 times among healthy (P < 0.05), GG genotype of IL10 (rs1800872) gene over TT polymorphism (3.57 times among patients (P < 0.05), 18. 5  The best gene-gene interaction models including taking into account the balanced testing and training accuracy, the highest cross-validation consistency, high sensitivity and specifity are given in Table 2. Combinations of best MDR models associated with high and low risk of PPROM are graphically presented in Fig. 4 and in Fig. 6 -polymorphic variants of best MDR models in term deliveries.
The circular graph of the cluster analysis of the results of gene-gene interaction simulation using the MDR-method is shown in Figure 5. The nature of interlocal interaction between the genes is at the level of «independent effects» of influence (IL4-IL10 rs1800896 = -23.08 %, IL10 rs1800896 -IL10 rs1800872 = -19.94 %, IL10 rs1800872-IL-4 = -19.34 %). The percentage of entropy of the investigated polymorphism of each gene for the case-control status was 14.15 % for the IL4 gene, 23.08 % for the IL10 rs1800896 gene and 9.34 % for the IL10 rs1800872 gene, indicating the high significance of the all present genes and their significant effect on the appearance of PPROM in preterm pregnancies.
In control group with term healthy pregnancy and labour the percentage of entropy of the investigated polymorphism of each gene, based on the results of the cluster analysis, simulates gene-gene interaction, is high and makes for the gene IL4 -29.55 %, for IL10 rs1800896 -14.74 %, for IL10 rs1800872 -7.89 % (Figure 7), in the presence of an independent but sta-

Discussion
The data that we received in our work were compared with the results of other scientists. It should be note at once that the number of studies on genetic polymorphism in relation to PPROM and PTL has dramatically multiplied. In addition, the results of studies of the same gene polymorphisms are determined by the design of the study, the choice of patient groups, are significantly differentiated in different populations and ethnic groups, and the expression of these mutations depends on a combination of culturological, socioeconomic and semantic factors, which are determined by the style of life in general and the style of habitation, in particular. However, many of polymorphisms exhibit inconsistency and remain inconclusive.
Being a global problem preterm birth warrants global solutions. Recent genomic approaches are beginning to reveal the information for understanding the causes of PTL. To fully appreciate and understand the complexity of PPROM and PTL, further approaches using high-throughput genome sequencing methods such as whole-genome sequencing studies are essential. Therefore, the challenge is to identify high risk women and provide them a personalized medical care for reducing the burden of PTL.
Common biological pathways that play important role in the pathogenesis of PTL are infections and inflammation, maternal-fetal hypothalamopituitary adrenal activation, decidual defects and pathological uterine overdistension. PTL has multifactorial origins and often several systems in the body and many different factor classifications interact in increasing the risk of PTL. In the contrast, it is estimated that only 50% of PTLs can be assigned to a known risk or causative factor, while the rest remain idiopathic.
In connection with the above, there was a need for the study of frequent mutations of genes IL4 (rs2243250), IL10 (rs1800896 and rs1800872) and RLN2 (rs4742076 and rs3758239) in Zaporizhzhia population to determine their role in the pathogenesis of PPROM and PTL in order to identify patterns and mechanisms for the formation of PPROM, the division of high-risk groups, early diagnosis, prediction and prophylactics. In Ukraine, the study of such a combination of genes in the pathology of software at the start of this study was not conducted.
In the study performed by A. Heinzmann haplotypes IL13/IL4 in a German population were presented as associated with PTL [2]. We proved association of IL4 rs2243250 with high risk or PPROM in Zaporizhzhia region. IL10 is an anti-inflammatory cytokine and at- : an intensely pronounced antagonistic interaction; : moderately expressive antagonistic interaction.
tenuates the inflammatory response through effects on pro-inflammatory cytokines and reduces the function of host immune cells, such as neutrophils and macrophages. Stonek F., Metzenbauer M. in their studies have found no association of IL10 with PTL [1]. Results of our study are controversial to researches of F. Stonek. We evaluated the contribution of maternal IL10 rs1800896 and rs1800872 in the susceptibility to PPROM and PTL. Based on received reliable statistical data presented above this marker can be used for prognosis PPROM in PTL, using also for example method of Multifactory Dimensionality Reduction.
Multifactory Dimensionality Reduction program helped us to create graph gene-gene interaction models with the highest cross-validation consistency and established a complex «independent» effect of polymorphic locus of genes IL4 rs2243250, IL10 rs1800896 and rs1800872 for the appearance of PPROM and PTL in Ukrainian population. The classification ability of the cre-ated models, considering the high consistency (90 % or 100 %), confirmed the probability of a three-component model involving three abovementioned genes, which increase the risk of PPROM in PTL with an accuracy of 91.49 % and specificity of 82.98 % respectively.
RLN2 is produced by the corpus luteum and enters the systemic circulation in pregnancy. The action of RLN2 from the maternal decidua and fetal trophoblast is purely autocrine, it does not enter the systemic circulation. Women with spontaneous PTL have lower RLN levels in the early pregnancy but higher levels in later gestation, compared with women who deliver term. On the other hand, G. D. Bryant-Greenwood et al. analyzed that increased expression of intrauterine RLN is present in patients with PPROM without infection [9]. A recent study by I. Vogel with homogeneous Danish population improved that women who are homozygous for specific SNP in the promoter region of RLN2 have a genetic susceptibility for PTL [13].
In a study performed by Frederico G. Rocha et al. the contribution of SNP rs4742076 and rs 3758239 in the RLN2 promoter in Filipino population was shown [9]. We obtained high reliable data on SNP genes RLN2 rs4742076 and rs3758239 in Zaporizhzhia women. Therefore, multiplicative inheritance model on this sign could be used for pregnant women our Zaporizhzhia region.
Summarizing the data of repertoire of SNP genes involved in our study, we have got significant difference in all the markers, as evidenced by MDR-data, х2 and odds ratio. The presence of these alleles may disrupt the balance between pro-and anti-inflammatory cytokines, modify the inflammatory response, increasing the risk for PPROM and PTL.  : an intensely pronounced antagonistic interaction; : moderately expressive antagonistic interaction.