Association between the rs2288947 polymorphism of the lncRNA TINCR gene and the risk of recurrent miscarriage in a Southern Chinese population

Abstract Studies have shown that many genes that regulate cell migration are associated with susceptibility to recurrent miscarriage. Terminal differentiation‐induced non‐coding RNA (TINCR) regulates the migration and invasion of a variety of tumor cells and is associated with susceptibility to various diseases. However, whether the lncRNA TINCR polymorphism is associated with susceptibility to recurrent miscarriage is unclear. Therefore, we investigated the relationship between the rs2288947 A > G polymorphism of the lncRNA TINCR and susceptibility to recurrent abortion. We recruited 248 recurrent spontaneous abortion patients and 392 healthy control subjects from the Southern Chinese population and used the TaqMan method for genotyping. There was no evidence that this polymorphism is associated with recurrent miscarriage (AG vs AA: adjusted OR = 0.904, 95% CI = 0.647‐1.264, P = 0.5552; GG and AA: adjusted OR = 0.871, 95% CI = 0.475‐1.597, P = 0.6542; dominant model: AG/GG vs AA: adjusted OR = 0.898, 95% CI = 0.653‐1.236, P = 0.5101; and recessive model: GG vs AA/AG: adjusted OR = 0.910, 95% CI = 0.505‐1.639, P = 0.7527). The stratified analysis also showed no significant associations. This study suggests that the rs2288947 A > G polymorphism of the lncRNA TINCR may not be associated with recurrent miscarriage in a Southern Chinese population. A larger multicenter study is needed to confirm our conclusions.


| INTRODUC TI ON
Recurrent miscarriage is a multifactorial disease, and the cause is unknown in approximately half of the patients with recurrent spontaneous abortion. 1 The occurrence of recurrent miscarriage is associated with genetics, uterine abnormalities, thrombotic tendency, hormone or metabolic disorders, infection, autoimmunity, age, and lifestyle problems. 1 Studies have found that susceptibility genes have an important relationship to the pathogenesis of recurrent spontaneous abortion. 2 In recent years, with the increase in research on recurrent abortion, an increasing number of susceptibility genes, such as MMP2, MMP9, CTLA4, FOXP3, and THBD, have been found to be associated with susceptibility to recurrent miscarriage. [3][4][5][6] In addition, studies have confirmed that some genes that regulate cell migration are associated with susceptibility to recurrent miscarriage 7,8 Studies have also found that long non-coding RNA (lncRNA) polymorphisms are also associated with recurrent miscarriage.
For example, in the early stage, we studied MALAT1, which regulates cell migration and is associated with susceptibility to recurrent miscarriage.
Therefore, exploring the relationship between lncRNA and the function of regulating cell migration and recurrent abortion provides an important reference for understanding the causes of recurrent miscarriage.
Terminal differentiation-induced non-coding RNA (TINCR) is a ln-cRNA that is located on human chromosome 19 and produces a 3.7-kb transcript. 9,10 Studies have shown that TINCR is involved in epidermal differentiation and somatic tissue differentiation. 10,11 Multiple studies have revealed that TINCR is involved in tumorigenesis and aberrant expression in multiple human cancers. [12][13][14] Moreover, TINCR has been shown to regulate proliferation, migration, and invasion of multiple tumor cells. [15][16][17][18] Recently, studies have found that genetic variation in TINCR was associated with a variety of diseases, such as the SNP rs2288947, which contributed to the susceptibility and progression of colorectal cancer and susceptibility to gastric cancer. 19,20 Furthermore, studies have found that some genes that regulate cell proliferation, invasion, and migration are associated with susceptibility to miscarriage; for example, H19 and p53 gene polymorphisms. 8,21 These studies suggest that TINCR gene polymorphisms may be associated with recurrent miscarriage. However, there have been no previous studies on the association between TINCR polymorphism rs2288947 A > G and recurrent miscarriage; therefore, we conducted this study.

| Ethics statement
The study was approved by the Ethics Review Committee of the Guangzhou Women and Children's Medical Center. Written informed consent was obtained from each participant.

| Subjects
From June 2017 to July 2018, 248 women diagnosed with recurrent miscarriage and 392 healthy controls were collected from the Department of Gynecology, Guangzhou Women and Children's Medical Center. Recurrent miscarriage was defined as two or more spontaneous abortions with unknown etiology. The control women had at least two normal pregnancies and no history of miscarriage.
Recurrent miscarriage patients and control women had no autoimmune, liver or kidney dysfunction; history of endocrine or metabolic disorders; arterial or venous thrombosis; abnormal uterine anomalies; hypertension; or embryo chromosomal abnormalities.

| Genotyping and DNA extraction
We isolated total genomic DNA from peripheral blood leukocytes using the TIANamp Blood DNA Kit (TianGen Biotech Co., Ltd.). Genotyping of SNPs rs2288947 was performed in 384-well plates by the TaqMan real-time polymerase chain reaction protocol on an ABI Q6 (Thermo Fisher Scientific). In addition, approximately 10% of samples were randomly selected for sequencing for quality control purposes and validation of genotyping results. The results were 100% concordant.

| Statistical analysis
Hardy-Weinberg equilibrium (HWE) of the control subjects was calculated by the goodness-of-fit chi-square test. Demographic and genotypic differences between cases and controls of recurrent spontaneous abortion were compared by the chi-square test. Unconditional univariate and multivariate logistic regression analyses were performed. Age-adjusted odds ratios (OR) and 95% confidence intervals (CI) were used to assess the strength of the association between this genetic polymorphism and the susceptibility to recurrent miscarriage. Stratified analysis of age and number of abortions was performed. A P-value of less than 0.05 was considered statistically significant, and all statistical tests were bilateral and calculated using SAS software (version 9.1; SAS Institute).

| Population characteristics
The demographic characteristics of recurrent spontaneous abortion and control groups are shown in Table 1. There was no statistically significant difference in age (31.00 ± 4.83 vs 31.44 ± 4.39 years, P = 0.7225) between the case and the control group. In recurrent miscarriage patients, approximately 68.15% of recurrent miscarriage patients experienced two or three spontaneous abortions, and more than 31.85% experienced four or more spontaneous abortions.

| Association between selected polymorphism and recurrent miscarriage susceptibility
The genotype frequencies of the TINCR gene rs2288947 polymorphism in patients with recurrent miscarriage and the control group are listed in Table 2. Genotype analysis of the TINCR gene rs2288947 showed no significant difference in HWE in the control group (P = 9731). We did not observe any significant association between the TINCR gene rs2288947 polymorphism and susceptibility to recurrent

| Stratified analysis of selected polymorphisms and recurrent miscarriage susceptibility
As shown in Table 3, we further evaluated the role of the TINCR gene rs2288947 polymorphism in cases and controls by strati-

| D ISCUSS I ON
In the current case-control study with 248 patients (with recurrent miscarriage) and 396 healthy controls from a Southern Chinese population, we did not observe a significant relationship between the TINCR gene rs2288947 A > G polymorphism and the recurrent spontaneous abortion susceptibility of women in Southern China. In addition, in the stratified analysis, we did not observe any significant associations in different age groups with recurrent spontaneous abortion or in the number of spontaneous abortions. To the best of our knowledge, this is the first study to investigate the relationship between the TINCR gene rs2288947 A > G polymorphism and the susceptibility to recurrent miscarriage.
Although the specific functions of most lncRNAs are still unknown, recent studies have shown that lncRNAs play an important role in the development and progression of abortion. 22

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
All authors contributed significantly to this work. DC and WD H devised the research plan. The data were analyzed by YF Y and QHL.
DC wrote the manuscript, and YQT, ZLL, and HZZ were responsible for performing the experiments. LP and LYF designed the experimental methods, and QFL modified and polished the manuscript. All authors support the publication of the manuscript.

DATA ACCE SS I B I LIT Y
Data sharing is not applicable to this article because no datasets were generated or analyzed during the current study. Please contact the author with any data requests.
TA B L E 3 Stratification analysis for associations between TINCR polymorphism and recurrent miscarriage risk in a south Chinese population