Polymorphic Variants of FOXP3 Gene (rs 3761548) and (rs 3761549) are Significantly Associated with the Risk for Recurrent Pregnancy Losses. A Study in High Incidence Zone (Kashmir, North India)

Recurrent Pregnancy Loss (RPL), presently defined as the failure of two or more clinically recognised pregnancies before the 20^th week of gestation [1]. It is a significant pregnancy complication affecting 1–3% of couples attempting to conceive [2]. RPL is a multifactorial, although many known causes were shown to contribute to its pathogenesis, including anatomic, infectious, hormonal, immunological and genetic predisposing aspects, while around 40–50 % of the causes remain idiopathic [3]. It is worth mentioning that present studies have indicated the role of miRNAs in the regulation of genes involved in the pivotal processes for the maintenance of a successful pregnancy outcome [4,5]. In addition, previous reports have suggested that RPL is associated with the failure of foetal-maternal immunologic tolerance, and disturbed immune response has been suggested as a pivotal mechanism to understand the underlying aetiology [6].

Regulatory T cells (Tregs) play a crucial role in the induction and development of an immune-tolerant environment at the foetal-maternal interface, thus supporting the maintenance of pregnancy [7]. It has been found that RPL patients have reduced rates of CD4+CD25+Treg cells in peripheral blood as well as in decidua [8], which is strongly associated with reduced expression of the FOXP3 gene [8]. FOXP3 plays a key role in the development and functioning of Treg cells, while decreased expression of the FOXP3 gene impairs the suppressive function of Treg cells by affecting the development of CD4+CD25+ Tregs [9]. This reduction in Tregs (CD4+CD25+) in RPL patients is closely linked to the immunological barrier disruption at the foetal-maternal interface [8]. The human FOXP3 gene is positioned on chromosome X(Xp11.23)-a member of the Forkhead/winged-helix family, and is also known as “Scurfin”, which encodes a transcription factor required for the formation and activity of CD4+CD25+Tregs [10]. Previous reports have found polymorphisms in the promoter region of FOXP3 gene affect transcription initiation and thus, gene expression. Furthermore, autoimmune diseases like systemic lupus erythematosus (SLE), autoimmune thyroid diseases (AITDs) [11], and type I diabetes (TID) [12] have been linked to polymorphisms in the FOXP3 gene. Studies in different populations have also observed the association between RPL and FOXP3 gene polymorphisms, including the Intron 1 rs2232365 (− 924 G > A), rs3761548 (− 3279C > A), and rs5902434 (− 6054) and rs2294021 [14–13]. In view of the plausible role between FOXP3 gene and RPL, this study was carried out for the first time in the Kashmiri population to assess the association of the FOXP3 genetic variants and RPL to analyse the difference in genotype and allele frequencies among cases and controls to rule out the any possible association and risk thereof.

The cases and controls were frequency matched in terms of their age and dwelling. The mean age of RPL cases and controls were 29.83 ± 4.47 and 29.58 ± 3.98 years respectively. The clinico-pathological characteristics of study subjects are shown in Table 1. In addition to that, the number of live births was found to be significantly lower in women with RPL as compared to the control population (p < 0.0001). Family history (p = 0.03) and consanguinity (p = 0.01) were also significantly related between cases and the control group. Age, body mass, smoking and irregularity in menstrual functions were comparable between RPL cases and control women.

For the successful implantation and maintenance of the fetus, Treg cells play a vital role in foetal-maternal immunologic tolerance. It is evident that the FOXP3 controls the successful development of Treg cells, which are believed to be one of the most prominent immunological factors investigated in RPL [15]. Dysregulated immune response was considered as a potential underlying cause for RPL [16,17] that substantiates its crucial role to regulate immune response to support a favourable pregnancy outcome [18]. There are evidences which show defective number of Tregs (CD4+,CD 25+) in peripheral blood as well as the decidua of RPL patients and its association with decreased expression of FOXP3 in the women with poor pregnancy outcome [8]. Polymorphisms in the promotor region of the FOXP3 gene could result in altered gene expression by affecting the initiation of transcription caused by inefficient binding of transcription factor and thus leads to immune system imbalance and mediate clinically serious human disease development like recurrent abortions.

In this study, we observe that among Kashmiri women population, the frequency of variant genotype AA in FOXP3- 3279 C/A (rs 3761548) was significantly higher in cases than control population (0.11% vs. 0.8%: p < 0.05) and clearly demonstrates a significant association of FOXP3 −3279 C/A genotype with the susceptibility of subjects to develop the risk for RPL in our population. Our findings agree with the study by Jaber et al. [19], where AA genotype and the A allele frequency were significantly higher in RPL women (OR = 2.86 and OR = 1.75) respectively. The results of our study were also in accordance to the findings of Wu et al. [20] and Sharif et al. [21] that reported a substantial contribution of FOXP3 −3279 C/A gene polymorphism with recurrent pregnancy losses in different countries of Asian ethnicity. Our findings were further corroborated by the observations of Saxena et al. [14] and Dirsipam et al. [22] in the Indian population, wherein the hazard of RPL was indicated as 2- 3 fold in patients with polymorphic variants of SNP rs3761548 C/A in the FOXP3 gene. A meta-analysis done by Bamba et al. [23] observed SNP rs3761548 C/A as the most common variant among various Asian countries. Thus, our data provided more evidence in supporting the predisposing role of FOXP3 polymorphism in RPL. On the contrary, a previous study by Hadinedoshan et al. [24] on the Iranian population concluded unavailing results for FOXP3 –3267 C/A (rs3761548) polymorphisms in women with RPL. In addition, the investigation done by Mishra et al. [25] for rs3761548 C/A was also in disagreement with the earlier studies. In this study for rs3761548, the observed frequency of the C allele was 56.6% and 43.3% for A allele in cases vs. 69% and 31% in controls respectively. In line with our study, Zaigui et al. [20] found the frequency of C and A alleles as 29.45% vs. 42.40% and 70.55% vs. 57.60% in cases and controls, respectively (p = 0.003). Thus, polymorphic variations in FOXP3 gene may result in the dysregulated expression of FOXP3 and significantly impact the modulation of suppressive capabilities of Tregs [26]. The abated function of regulatory T cells, in turn, plays a key role in the abnormal expression of various immune cells, causing an unfavourable pregnancy outcome. Our study revealed a greater proportion of variant alleles in the age group ≤ 30 years in cases than control group (45% vs 22%: p = 0.001). Furthermore, we observed significant association of minor allele A in patients without any family history and non-consanguinity as compared to control population (p < 0.05). As there is a limited number of investigations regarding this subject, the discussed parameters need to be studied further in detail to come up with more authentic results.

For FOXP3 gene, rs3761549 C/T, genotypic and allelic distribution portrayed more than 3-fold elevated risk of RPL in women carrying TT genotype than CC genotype (p = 0.04) but showing borderline significance for T allele between the two groups (cases vs. controls: Table 2). However, it is worth mentioning that the presence of T allele has been observed to be significantly higher in patients with lung carcinoma, thus suggesting its role in susceptibility to the disease [27]. There is ample evidence for suggesting the association between FOXP3-2383 C/T (rs3761549) polymorphism and some autoimmune diseases. One of the earlier studies has elucidated a genetic association between the FOXP3 gene and Psoriasis in the Chinese majority population, suggesting the T allele as a contributing factor to the disease [28]. Furthermore, Lan et al. [29] attributed the association between FOXP3-2383 SNP and genetic susceptibility to systematic lupus erythematosus in Guangxi Zhuang population signifying relative risk of suffering as 1.71 times in T allele carriers (p <0.05). Interestingly, these results are also consistent with the other studies done by Andre et al. [30] for single marker analysis of FOXP3 polymorphisms (rs3761549) revealing a possible association with endometriosis in Brazilian population (P= 0.03). Nevertheless, stratification of genotypic and allelic analysis of this SNP have failed to detect any implication in hepatocellular carcinoma and breast carcinoma [31] in Asian ethnicity. FOXP3 rs3761549 polymorphisms have not previously been investigated in women with RPL. This is the first prospective study in this region to actively follow women for recurrent abortions. Our study has demonstrated the possible role of TT genotype in FOXP3-2383 C/T SNP indicating immunopathogenesis in patients with RPL. Overall keeping in view the importance of this gene, many studies across the globe that explored the association between FOXP3 variants and RPL came upon inconclusive and inconsistent but interesting results and our study has further strengthened its importance (Table 4). Thus, further study of FOXP3 polymorphic variations may contribute to a better understanding of their implications in conferring risk to RPL.

Limitations of the study include a slightly smaller sample size, keeping in view the high incidence of RPL in our region. Besides, study augments for the expression analysis of FOXP3 gene and its association with genotypes. Future studies that incorporate longitudinal FOXP3 protein levels, functional genomics, and epigenetic modifications would be pivotal in demonstrating a more reliable knowledge of FOXP3-mediated role in etiopathogenesis of RPL. In addition, further studies are required with the inclusion of more FOXP3 polymorphic variations to analyse the linkage disequilibrium. Furthermore, the data of the study underscores the need for different ethnic based studies to confirm significant association of the genetic variants with respect to the RPL.

The study concludes that FOXP3 gene, C/A (−3279) and C/T (−2383) are significantly associated with an increased risk for RPL. Also, the exhibition of FOXP3 variants infers a more pronounced risk in the lower age group. Our data suggests a need to authenticate the results through expressional epigenetic regulation and functional validation studies in a series of cohort studies to confirm its possible implications for RPL.