A rare variant in theMARVELD2 gene is associated with Chinese samples with ovarian endometriosis

Objectives: Endometriosis is a common gynecological disease affecting up to ~10% of women at reproductive age. Prior combined studies implied that MARVELD2 might be involved in the pathogenesis of certain malignancies. Here, 211 Han Chinese samples with ovarian endometriosis were analyzed for the presence ofMARVELD2 mutations. Methods: We analyze the potential presence ofMARVELD2 mutations by direct DNA sequencing. Results: A total of 7 variants, 5 missense and 2 synonymous variants, were identified in our 211 ovarian endometriosis samples with different frequencies. Among the 5 missense variant, a missense rare variant p.V198M (c.592G>A), was identified in 10 out of our 211 samples (4.74%). This rare variant was identified with extremely low frequency in 766 control samples from 766 Chinese women without endometriosis (0.13%, 1/766) and control samples in the public databases. The evolutionary conservation analysis results suggested that theMARVELD2 rare variant lead to highly conserved amino acid substitutions among 14 vertebrate species from Human to Snake. Furthermore, both the SIFT and Polyphen-2 programs predicted this rare variant to be ‘disease causing’. However, we failed to observe any statistical significance between the MARVELD2 rare variant and the available clinical data. Conclusions: We identified a potential pathogenic rare variant in the MARVELD2 gene in Chinese samples with ovarian endometriosis, indicating theMARVELD2 rare variant might play an active role in the pathogenesis of endometriosis.


Introduction
Endometriosis is a common gynecological disease, in spite of the subject of many scientific researches, the detailed molecular etiology remains still unclear [1]. Among which, the 'Sampsons hypothesis' is the most extensively admissive interpretation, which implies that endometriosis takes place owing to retrograde menstruation, where endometrial tissue passes through the fallopian tube into the peritoneal and pelvic cavity where it implants [2]. The implantation theory indicates the formation of endometriosis in the peritoneal cavity and ovary needs endometrial tissue or cells fulfilling a process of adhesion, invasion and proliferation [3].
Tricellulin, a major component of tight junctions, encoded by the MARVELD2 gene [4], was the first identified protein to be uniquely localized at the tri-epithelial junctions [5]. Prior studies have revealed that MARVELD2 mutations could cause nonsyndromic deafness, and these MARVELD2 mutations were shown to cause tricellulin defects and deletion, thereby affecting the adhesion and the tight junction function between epithelial cells [6][7][8]. Fur-thermore, dysregulated MARVELD2 expression was associated with patients' prognosis in certain cancer types [9][10][11] and could promote cell migration [12]. Endometriosis is similar to cancer and exhibits enhanced invasion and migration [13][14][15]. This disorder is considered as a precancerous lesion and harbored a variety of mutations in certain oncogene and tumor suppressor genes [16][17][18][19]. A large-scale sequencing effort has revealed that epithelial cells harbored cancer-associated mutations promoting the pathogenesis of endometriosis [20]. In addition, increased expression of tricellulin could promote the invasions and metastasis of certain human tumor [21].
Prior studies have revealed that certain germline mutations, including single nucleotide polymorphism (SNP), played important roles in the pathogenesis of endometriosis via candidate gene or large-scale sequencing strategies [22,23]. In addition, somatic mutations in certain genes, were also shown to confer the risk of endometriosis [16][17][18]24]. Prior studies have found that dysregulated expression of MARVELD2 was associated with patients' prognosis in certain cancer types [9][10][11] and could promote cell migra-  tion [12]. Considering the important role of tight junction played in cell invasion and migration [24], and endometriosis was a premalignant disorder and harbored a number of mutations [25,26], we thus hypothesized that MARVELD2 mutations might also existed in endometriosis samples.
Here, we recruited and analyzed a total of 211 Chinese patients with ovarian endometriosis for the presence of MARVELD2 mutations. A rare variant in the MARVELD2 gene was identified in 10 out of 211 samples (4.74%).

Samples
A total of 211 Chinese patients with ovarian endometriosis, as well as control samples from 766 Chinese women without endometriosis were also collected from Jiangxi Provincial Maternal and Child Health Hospital (Nanchang, China). Written informed consent was obtained from each sample prior to this study, and the present study was performed according to the tenets of the Helsinki Declaration and was approved by the Institutional Review Board of Jiangxi Provincial Maternal and Child Health Hospital.

Clinical data
The clinical data for the participating women with ovarian endometriosis was collected at the time of sampling, including age, age at menarche, the laboratorial data included serum hemoglobin, free triiodothyronine (FT3), free thyroxine (FT4), thyroid stimulating hormone (TSH), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), squamous cell carcinoma antigen (SCCA) and α-fetoprotein (AFP) were determined on day 3 of the menstrual cycle by radioimmunoassay, as described previously [17,18]. In addition, the number of white blood cells, lymphocytes, eosinophil granulocytes, neutrophil granulocytes, mononuclear cells, platelet, and neutrophil granulocyte proportion was analyzed by an automated hematology analyzer XN-3000 (Sysmex Corporation, Kobe, Japan) (Table 1), as described previously [17,18].

DNA extraction and mutation analysis
The genomic DNA was extracted from the peripheral blood of our samples. Omega Blood DNA kit (OMEGA Bio-tek Inc., Doraville, GA) was used to isolate the genomic DNA for our samples, as described previously [17]. The entire coding regions of the MARVELD2 gene

In silico analysis of the MARVELD2 rare variant
We used SIFT [28] and Polyphen-2 [29] online programs to predict the potential pathogenicity of these missense variants of MARVELD2. These programs could automatically assess this rare variant to be damaging or benign.

Statistical analysis
We used two-sided Student's t-test and Mann-Whitney's method to analyze the potential association of numerical and continuous variables between ovarian endometriosis samples with and without MARVELD2 mutations, respectively; Fisher's exact test was used to analyze the allele frequency of MARVELD2 rare variant between the cases and controls; p value less than 0.05 was considered statistically significant. All statistical analyses were performed by the software SPSS 19.0 (SPSS, Inc., Chicago, IL, USA).

In silico and Evolutionary conservation analyses of the MARVELD2 missensevariants
Both the SIFT and Polyphen-2 programs were used to predict the potential pathogenicities of these variants, the predicted results showed that the p.V198M variant was 'disease causing'; while other four variants were benign. The evolutionary conservation analysis result suggested that the MARVELD2 rare variant lead to highly conserved amino acid substitution from valine to methionine at the 198th codon (V198M), among the 14 vertebrate species from Human to Snake (Fig. 2).

Association between MARVELD2 rare variant and clinical data
The clinical data between the ovarian endometriosis samples with and without MARVELD2 rare variant (p.V198M) was analyzed, including patients' age, age of menarche, FT3, FT4, TSH, hemoglobin, CA125, SCCA, AFP, CEA, white blood cell count, eosinophil granulocyte, lymphocyte cell count, neutrophil cell count, mononuclear cell count, platelet and neutrophil cell proportion. However, we failed to get any significant association between MARVELD2 rare variant and these clinical parameters (Table 1).

Discussion
MARVELD2, formerly referred to as TRIC-a, encodes tricellulin, a tricellular tight junction protein [4]. Tricellulin is mainly localized in tricellular cell contacts, while in bicellular tight junctions to a lesser extent [30]. To date, it's deemed that tricellulin expression ubiquitously exists in epithelial junctions of tissues and organs throughout the body [6], as well as in ovarian epithelia [31].
Prior large-scale sequencing studies have revealed that common variants in certain genes played important role in the pathogenesis of endometriosis, including vascular endothelial growth factor receptor 2 (VEGFR2), mitogenactivated protein kinase kinase kinase 4 (MAP3K4), and Wnt family member 4 (WNT4) [32][33][34]. Recently, increasing evidences have suggested that rare variants also facilitate the initiation and development of endometriosis [35,36]. In the present study, we have screened a total of 211 Han Chinese samples with ovarian endometriosis for the presence of MARVELD2 mutations, via sequencing the whole coding region and the exon-intron boundaries of the MARVELD2 gene. Here, we identified a MARVELD2 missense rare variant, p.V198M (c.592G>A), in 10 out of 211 Han Chinese samples with ovarian endometriosis (4.74%), the frequency of this rare variant is significant higher than that either in the local control women without endometriosis or in the control samples in the 1000 genome and EXAC databases (p < 0.01). The evolutionary conservation anal-ysis result showed that the MARVELD2 rare variant caused highly conserved amino acid substitution among 14 vertebrate species. Furthermore, the bioinformatic programs prediction result showed this rare variant might be damaging.
MARVELD2 mutations could cause nonsyndromic deafness [6][7][8] and the molecular mechanism involved in affecting the paracellular permeability, leading to a toxicity for cochlear hair cells [37]. Subsequent studies found that dysregulated expression of MARVELD2 could change the capacities of cell invasion and migration in diverse cancer types, including colorectal and gastric cancers, and the potential underlying molecular mechanism involved in actin and cytoskeletal reorganization, as well as epithelialmesenchymal transitions (EMT) process [21,38]. As a premalignant condition, endometriosis usually exhibited with dysregulation of cell invasion and migration, we thus speculated that the MARVELD2 rare variant identified in the present study might play active role in the pathogenesis of endometriosis.
On the other hand, we failed to observe any positive association between the MARVELD2 rare variant and the available clinical data. Of note, our sample size of ovarian endometriosis was relatively small, we will continue to obtain additional samples in order to re-analyze the potential association.

Conclusions
We identified a relatively high frequency of MAR-VELD2 rare variant in our samples with ovarian endometriosis, indicating this rare variant might play positive role in the pathogenesis of this disease.

Ethics approval and consent to participate
All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Jiangxi Provincial Maternal and Child Health Hospital (approval number: JXSFYBJYY2020KJK015).