A rare variant in the IL22RA2 gene is associated with unexplained recurrent pregnancy loss in Han Chinese women

Introduction: Recurrent pregnancy loss (RPL) occurred in ~1-2% of reproductive women. Previous studies have implicated that altered expression and polymorphisms of interleukin-related genes might be involved in RPL. The aim of this study was to explore the potential presence of interleukin-22 receptor subunit alpha-2 (IL22RA2) mutations in Han Chinese women with unexplained recurrent pregnancy loss (URPL). Material and methods: A total of 328 Han Chinese women with URPL were analyzed for the potential mutations in the IL22RA2 gene by sequencing all of the exons. Furthermore, 615 Han Chinese control women without miscarriage history were also analyzed. Evolutionary conservation and in silico analyses were performed to predict the potential pathogenicity of IL22RA2 mutations. Results: A rare variant, p.Arg204* (c.610C>T), was identified in 4 out of 328 URPL samples. In contrast, this rare variant was absent in 615 Han Chinese control women without miscarriage history and had a significantly higher mutation frequency when compared with 10588 Chinese control samples from The China Metabolic Analytics Project (ChinaMAP), 4245 East Asians, or 60051 individuals across the world from the Exome Aggregation Consortium (ExAC) database. Evolutionary conservation analysis indicated this rare variant was highly conserved from Human to Zebrafish, in silico analysis implicated this rare variant might be pathogenic. Conclusions: An enrichment of a potentially pathogenic rare variant in the IL22RA2 gene was observed in URPL patients, for the first time, implicating this rare variant might be associated with the development of URPL. However, further functional Pr ep rin t assays would be performed to confirm the potential role of this rare variant in URPL.


Introduction
Recurrent pregnancy loss (RPL) is a highly heterogeneous condition and defined as two or more sequential abortions before the 20th week of gestation [1,2]. This condition affects approximately 1-2% of fertile couples, and becomes a common public reproductive concern causing both physical and emotional burdens [1,3]. A number of risk factors have been proposed to be associated with RPL, including anatomic abnormalities of the female reproductive tract, immunological and endocrine aberrations, genetic factors, coagulation protein defects and environmental factors [4][5][6][7]. However, ~50% RPL cases are not attributable to these known risk factors and these cases are classified as unexplained recurrent pregnancy loss (URPL) [8][9][10].
Successful pregnancies required the induction of maternal tolerance to fetal tissues, where natural killer (NK) cells migrated and adjusted the secretion of cytokines, to adjust the invasion of trophoblast cells into the uterus [11][12][13]. Among the complex URPL-related factors, those involved in abnormal activation of inflammatory processes in immune rejection and suppression of immune regulators attracted much attention [14][15][16]. Multiple studies have suggested that genetic variants in immune-associated genes conferred increased risk for URPL. A genetic variant in the interleukin 1 beta (IL1B) gene (-511T>C) was shown to be associated with RPL women in South Korea, and the -511C allele cases possessed higher proportion of NK cells than -511T carriers [17]. Another study found that tumor necrosis factor-alpha (TNF-α) rs1800630 (-863C>A) variant could confer an increased risk of RPL women P r e p r i n t in South Korea [18]. Likewise, certain genetic variations in pro-inflammatory or anti-inflammatory genes were also shown to affect the risk for RPL in different ethnic populations of women, including the rs2275913 polymorphism in interleukin 17 (IL-17) [19], rs187238 polymorphism in interleukin 18 (IL-18) [20], rs1518111 and rs1800871 polymorphisms in interleukin 10 (IL-10) [21] and rs1800796 polymorphism in interleukin 6 (IL-6) [22].
Prior study has found that IL-22 level was increased after inflammatory stimulus at the maternal-fetal interface in pregnant mice, indicating a potential involvement of IL-22 in protecting from inflammation-induced preterm birth [23]. Furthermore, a recent study showed that the expression of IL-22 and its soluble IL-22 receptor, IL22RA2, was downregulated in human villi tissues and trophoblasts in RPL women, and the decreased IL-22/IL22RA2 expression could suppress the growth and proliferation of trophoblast [24], implicating that IL-22/IL22RA2 signaling pathway might play crucial roles in the development of RPL.
Due to the potential role of IL-22 signaling in URPL [25][26][27], we thus hypothesized that mutations or aberrant expression of IL22RA2, the inhibitor of IL22, might existed in URPL women. Here, we analyzed the potential mutations in the IL22RA2 gene in a cohort of 328 Han Chinese women with URPL, and a potential pathogenic mutation was identified in 4 out of these samples.

DNA extraction and mutation analysis
Blood samples from both control and URPL subjects were collected and stored at

Statistical analysis
The statistical analysis was performed with the statistical software package SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA). Fisher's exact test was used to compare the age and number of pregnancy loss between URPL samples with and without IL22RA2 mutation, and the frequency difference of IL22RA2 rare variant between our URPL samples and local control women, and control samples from the ChinaMAP and ExAC databases. All tests were two-sided and a P-value <0.05 was considered statistically significant.

Sample characteristics
The age of these samples was 30.6 ± 5.6 years (range, 18-45), and the average pregnancy loss was 2.4 (range, 2-7). All of these samples had no childbearing history.
In addition, the age of the 615 control women was 29.2 ± 6.3 years (range, 22-39), all of these controls had childbirth history while no history of pregnancy loss.

P r e p r i n t
The age (P=0.56) and number of pregnancy loss (P=0.87) in IL22RA2-mutated URPL samples exhibit no significant differences with that of URPL samples without IL22RA2 mutation.

In silico functional annotation of the IL22RA2 mutation
Two different software programs were used to predict the potential pathogenicity of IL22RA2 p.Arg204* mutation. This mutation was predicted to be "Disease causing" and "Deleterious" by MutationTaster and SIFT, respectively. Evolutionary conservation analysis indicated that the "Arg" residue at 204 in the IL22RA2 protein was highly conserved among the 17 vertebrate species from Human to Zebrafish (Fig.   2).

Discussion
Prior studies have found that IL-22 level was increased after inflammatory stimulus at the maternal-fetal interface in pregnant mice, indicating a potential involvement of IL-22 in protecting from inflammation-induced preterm birth [23].
Furthermore, decreased expression of IL-22/IL22RA2 in trophoblasts in RPL patients could suppress the growth and proliferation of trophoblast, implicating that IL-22/IL22RA2 signaling pathway might play important roles in URPL [24].
In the present study, we analyzed all exons of the IL22RA2 gene in 328 Han Chinese URPL women and 615 Han Chinese control women without miscarriage history. A rare variant in IL22RA2, p.Arg204* (c.610C>T), predicted to be pathogenic with in silico programs, was found to be associated with Han Chinese URPL women in our sample cohort. Also, the frequency of p.Arg204* (c.610C>T) in URPL women P r e p r i n t