Endometriosis is a heritable, benign, chronic, estrogen-dependent, inflammatory, common gynecological disorder, affecting up to 10% of the female population during their reproductive years, representing a considerable cause of pain and infertility (1). Endometriosis is associated with chronic pelvic pain, dyspareunia, dysmenorrhea and infertility, and in women with this condition, the growth of endometrial tissue occurs in sites other than the uterine cavity, most commonly in the pelvic cavity, including the ovaries, the uterosacral ligaments, and the pouch of Douglas (2).

Endometriosis is characterized by a multifactorial pattern of inheritance, influenced by multiple genetic and environmental factors (3–5); however, the exact genomic basis of endometriosis remains unclear. Monozygotic twin-based and family studies have demonstrated an approximately 50% heritability for endometriosis (6,7), while a strong familial aggregation has also been reported in a large pedigree of non-human primates (8). Previous linkage and candidate genetic studies on endometriosis did not succeed in identifying genetic variants strongly associated with risk of the disease (9), probably due to the small effect size for individual risk factors of the disease, which required studies based on large cohorts. Contrarily, genome-wide association studies (GWAS) and meta-analyses conducted in several series of samples have led to the identification of disease-susceptibility loci implicated in matrix remodeling, cell cycle regulation and signaling, cell adhesion, transcription regulation and inflammation, as well as immune, oxidative stress processes, as well as hormone receptors and metabolism (10–12). The list of novel endometriosis-associated loci is still being enriched due to novel GWAS and meta-analyses that are currently in progress. Importantly, these data only account for approximately 5% of the disease variance (11). Of note, the role of epigenetics in the development of endometriosis, through interactions with environmental changes, has been investigated and the DNA methylation and histone post-translational modifications have been thoroughly investigated (13,14).

Meta-analysis of European ancestry GWAS have identified 27 genome-wide significant loci associated with endometriosis (15). Several studies have genotyped key single nucleotide polymorphisms (SNPs) from GWAS to replicate the association in different samples and ethnic groups. Thus, the association of rs10859871 SNP, close to the Vezatin gene (VEZT) (16), CDKN2BAS rs1333049, rs7521902 close to Wnt family member 4 (WNT4), rs12700667 in an inter-genic region on 7p15.2 and fibronectin 1 (FN1) rs1250248 with endometriosis has been successfully replicated in an Italian ancestry population (16). Similarly, eight interleukin (IL)1A SNPs were successfully replicated in different ethnic populations (17). However, the chances of significant replication depend on the relative sample size in the replication and discovery samples.

To gain a better understanding of the genetic architecture of endometriosis in the Greek population, and encouraged by the successful replication of Wnt family member 4 (WNT4), VEZT and follicle stimulating hormone subunit beta (FSHB) gene association with endometriosis in this population (18), we sought to substantially replicate more data from recently conducted GWAS for endometriosis (11,19). In particular, in the present study, we aimed to investigate whether rs1250248 and rs11674184 SNPs, mapping to fibronectin (FN1) and growth regulation by estrogen in breast cancer 1 (GREB1) gene loci, respectively, previously reported to be associated with endometriosis (11,20), were associated with an increased risk of developing endometriosis and/or with progression to the minimal/mild (I/II) or moderate/severe stages (III–IV) of this condition in the Greek population. Furthermore, we attempted to detect any ethnic specific differences regarding the genetic association of these SNPs with endometriosis, considering that population differences for endometriosis have been reported previously in terms of genetic susceptibility and disease manifestations in Greek women with endometriosis (18,21).

Nowadays, a better understanding of the genetic risk factors associated with endometriosis has been achieved by using advanced technological applications, including GWAS and next generation sequencing (NGS). However, accumulated data point out the existence of a number of differences in genetic associations with the disease across different world populations (24) and, therefore, it is evident that replication studies in multiple populations have to be conducted in order for the genetic basis of endometriosis to be clarified.

In the present study, we identified that the rs1250248 SNP was significantly associated with endometriosis in the Greek population in ‘all endometriosis’ cases, as well as in the subgroup of stages I and II. Notably, previous studies have demonstrated a genome-wide significant association with the rs1250248 SNP of FN1 locus, particularly with moderate-to-severe endometriosis (20,25). A meta-analysis based on four independent GWAS and four replication datasets, confirmed consistency of effects across datasets and populations for rs1250248 in FN1, providing evidence close to the genome-wide significance threshold of P<5×10⁻⁸ (26). This SNP is located in an intronic region between exon 10 and exon 11 in FN1 and, in particular, on a transcription factor-binding site according to ENCODE (27). FN1 is a glycoprotein of the extracellular matrix, which is also present in plasma, and at the cell surface. It mediates various cellular interactions with the extracellular matrix and has important roles in cell adhesion and migration processes, host defense, would healing, growth, differentiation and metastasis (28,29). Furthermore, it has been shown that SOX2, encoding a transcription factor that targets FN1, is a key gene regulating cell migration in ovarian cancer (30). FN1 is involved in the sex steroid hormone pathway and in a recent study, we demonstrated the replication of rs11031006, associated with endometriosis in the FSHB gene, which is also involved in the regulation of the sex steroid hormone pathway in the Greek population (18). However, the effects of the variants in underlying biological pathways need to be determined (25). Taking into consideration that rs1250248 SNP is located within the FN1 gene, it can be assumed that its biological consequences may act through this gene. However, functional studies are required to better understand its role in endometriosis disease etiology. These studies will aid towards the understanding of how the confirmed gene variant is involved in the disease phenotype, including gene expression and protein levels in different tissues, relevant to endometriosis, such as the eutopic and ectopic endometrium.

It is worth noting that the number of the loci identified by GWAS is increasing as the proportion of cases analyzed are limited to more severe disease stages (stage III/IV endometriosis) rather than stage I/II, thus indicating that moderate to severe endometriosis cases have greater genetic burden relative to minimal or mild disease (25). However, as regards the rs1250248 FN1 SNP, in the present study, the milder disease stages (stage I/II endometriosis) were found to be associated with endometriosis rather than the more severe stages (III/IV). Evidently, detailed surgical data from the cases will confer to a better assessment of the genetic variants associated with different stages of the disease. In an attempt to develop new options dealing with the better treatment and the therapy of women with endometriosis, focusing on subtypes of the diseases, it is necessary to understand the fundamental pathogenic mechanisms underlying the disease and gain novel insights from the risk gene loci detected through genetic studies. To this direction, the small effects of various identified SNPs should not to be underestimated, given that their targets may have a remarkable therapeutic value (31).

Previous research has identified genome-wide, endometriosis-associated significant variants, namely rs11674184 and rs13394619 in GREB1 on 2p25.1 (24). However, the present study did not reveal any association of the rs11674184 SNP investigated in the Greek population. The analyzed rs11674184 SNP has a minor allele frequency (MAF) of 0.47 in the Greek population; however, this SNP has a MAF of 0.61 in Australians, as reported by Fung et al (32). Notably, the same SNP was found to have a MAF of 0.61 in a previous meta-analysis (11). GREB1 is an estrogen-regulated gene that was first identified in breast cancer cell lines and tumors (33). Its increased expression in peritoneal eutopic endom triotic lesions probably reflects a potential role in estrogen-dependent growth of endometriosis (34). Nevertheless, the underlying biological mechanism through which GREB1 plays a role in estrogen-stimulated cell proliferation in endometriosis remains still elusive.

The rs1250248 of FN1 has a MAF of 0.24 in the Greek population, as observed in the present study (Table II). Notably, this SNP had a MAF of 0.29 in a meta-analysis conducted by Sapkota et al (11), 0.33 in an Icelandic population (11) and 0.21 in a meta-analysis performed by Rahmioglu et al (25). The current study has some limitations, considering that only the single lead SNPs from the FN1 and GREB1 genes were genotyped. However, these lead SNPs are not necessarily the causal variants, but they ‘tag’ the causal SNPs. Fine-mapping studies are required in order to identify the disease-causing SNPs. Apparently, the failure to replicate previous results regarding the rs11674184 SNP of GREB1 does not eliminate the fact that there could be other genetic variants in/around the GREB1 locus that are associated with endometriosis in the Greek population. Hence, better coverage of the common variation in/around the reported by GWAS endometriosis-predisposing SNPs is required in the Greek population. Notably, given that the replication Greek sample was smaller than the (original) discovery sample, the lack of significant replication does not constitute a real failure to replicate the original finding. Furthermore, genotyping in large case-control groups is necessary for the identification of more robust findings.

In conclusion, this study demonstrated that the FN1 but not the GREB1 polymorphisms analyzed may be involved in the development of endometriosis in Greek women and emphasize the importance of assessing genetic variants in different populations, even within Europe, aiming to define the genetic architecture of endometriosis. Moreover, further investigations are required to clarify the possible role of the FN1 SNP in the clinical course of endometriosis and elucidate the mechanism through which rs1250248 increases the risk of developing this disease.