NAT2 gene polymorphisms and endometriosis risk: A PRISMA-compliant meta-analysis

Objective Endometriosis is a common chronic, gynecological disease. Despite many studies on the role of N-acetyltransferase 2 (NAT2) in endometriosis, its clinical significance is unclear. In this study, associations between NAT2 phenotypes as well as single nucleotide polymorphisms (SNPs) within NAT2 (i.e. rs1799929, rs1799930, rs1208, and rs1799931) and endometriosis risk were evaluated using a meta-analysis approach. Methods Embase, PubMed, ClinicalTrials.gov, CNKI (China National Knowledge Infrastructure), Wanfang databases, Cochrane Library for clinical trials, and Web of Science were searched to identify relevant articles. ORs (odds ratios) and 95% CIs (95% confidence intervals) were used to estimate the associations between NAT2 polymorphisms and endometriosis risk. Heterogeneity among included studies was also assessed. In addition, a subgroup analysis of NAT2 phenotypes and endometriosis risk based on ethnicity was performed. Results Nine case-control studies met the inclusion criteria. The odds ratio was 2.30 (95% CI: 1.61–3.28) for the NAT2 slow acetylation phenotype versus the intermediate + fast acetylation phenotype in the Asian population. These results suggest that Asian individuals with the NAT2 slow acetylation phenotype have a 130% increased risk of endometriosis. A significant association was also found for rs1799930 (OR = 0.74; 95% CI, 0.59–0.92), suggesting that individuals with this mutant genotype have a 26% decreased risk of endometriosis. Conclusions The rs1799930 mutant genotypes are associated with a decreased risk of endometriosis. No statistically significant associations were found between rs1799931, rs1208, or rs1799929 and endometriosis. Based on a subgroup analysis based on ethnicity, the NAT2 slow acetylation phenotype was found to increase the risk of endometriosis in Asians. No statistically significant associations were found between the NAT2 slow acetylation phenotype and endometriosis risk in Caucasians. Accordingly, NAT2 phenotypes and SNPs are potential biomarkers for the diagnosis and treatment of endometriosis.


Introduction
Endometriosis is a common estrogen-dependent chronic gynecological disease affecting 5-10% of women of reproductive age [1]. It occurs when the endometrium grows outside of the uterine corpus, which causes inflammation, pelvic pain, dysmenorrhea, menstrual disorders, ectopic bleeding, bladder symptoms, infertility, and further malignant transformation [2]. It is strongly associated with significant social and physical debility. The etiology of this disease is unclear. However, the main hypotheses include classical theories of blood reflux, blood and lymphatic dissemination, coelomic metaplasia, immunology, endocrinology, and genetics, but none of these can satisfactorily explain the occurrence of endometriosis [3].
With increasing evidence for a role of genetic factors, many scholars are trying to find genes related to the pathogenesis of endometriosis [4]. Recently, toxic metabolic enzyme genes have been a focus of research. Genes encoding many metabolic enzymes such as cytochrome P4501A1 (CYP1A1), catechol-O-methyltransferase (COMT), N-acetyltransferase 2 (NAT2), and glutathione S-transferase P1 (GSTP1) are associated with polymorphisms that can distinguish between populations. Mutations might be related to the decreased function or changes in the function of these enzymes including enzymes involved in detoxification, resulting in differences in the risk of endometriosis and other diseases [5]. Recent meta-analysis-based studies have investigated the relationships between polymorphisms in GSTP1 [6], COMT [7], and CYP1A1 [8] and the risk of endometriosis. Here, we further analyzed the relationship among NAT2 phenotypes, genotypes, and endometriosis.
NAT2 is the product of a single, intron-less gene comprising an 870-bp open reading frame that encodes 290 amino acids. The gene is located on chromosome 8p21.323.1 [9]. NAT2 nucleotide substitutions can change the protein structure and cause reductions in substrate affinity, protein stability, and/or catalytic activity for the recombinant N-acetyltransferase allozyme. Recombinant human NAT2 clusters catalyze N-, O-, and N, O-acetyltransferase activities at slow speeds compare to that with wild-type phenotype [10,11].
Endometriosis is associated with a range of environmental factors (hormonal/reproductive, lifestyle) and genetic factors. It is well established that high levels of natural and man-made chemicals are present in the environment and play a role in the pathogenesis of endometriosis. For example, dioxin-like PCBs (polychlorinated biphenyls) promote the development of endometriosis through the stimulation of endocrine-inflammation interactions. Toxicant dioxins (dioxin and dioxin-like chemicals) have an adverse effect on growth factors, cytokines, hormones, and the immune system. Exposure to dioxins is a potential factor for the development of endometriosis [18,19]. Because endometriosis is a hormone-dependent disease, polymorphisms in genes encoding detoxification enzymes might contribute to its development [20][21][22][23]. NAT2 plays a key role in xenobiotic metabolism. Other xenobiotic metabolic enzymes like aromatase P450 family members CYP1A1 and CYP19A1 [24] also have a fundamental role in the pathogenesis of endometriosis [23,25], and have been widely studied and applied to endometriosis treatment. However, the role of NAT2 polymorphisms in endometriosis remains to be discovered. Specifically, previous studies have evaluated the relationship between NAT2 polymorphisms and the risk of endometriosis; however, contradictory results have been obtained [14,26,27]. Accordingly, in this study, a meta-analysis was performed to clarify whether different NAT2 phenotypes or the SNPs rs1799929, rs1799930, rs1208, and rs1799931 are associated with susceptibility to endometriosis worldwide.

Materials and methods
The detailed protocol, which followed the template of the Cochrane review is available in the PROSPERO registry (No. CRD42018111924). This meta-analysis was prepared according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines.
Declaration of human rights: no formal consent was required for this type of study.

Search strategy
PubMed, Embase, Web of science, Cochrane Library for clinical trials, ClinicalTrials.gov, CNKI (China National Knowledge Infrastructure), and Wanfang databases were searched extensively (the last search was updated on June 20, 2019). The search words and strategy included the following: (NAT2 OR "N-acetyltransferase 2" OR "Arylamine-N-acetyltransferase" OR "NAT" OR "dioxin detoxification enzymes") AND (mutation OR variant OR polymorphism) AND (endometriosis OR endometriosis OR Mulleriosis OR Mullerianosis OR EM OR EMT OR EMS OR Mullerianosis).

Inclusion and exclusion criteria
Inclusion criteria were as follows. (1) The relationship between NAT2 polymorphisms and endometriosis risk was evaluated.
(2) Only case-control studies that included both endometriosis cases and endometriosis-free controls were included. (3) Sufficient and procurable data for both cases and controls were required to estimate the odds ratio (OR) and 95% confidence interval (95% CI). (4) Endometriosis diagnosis in accordance with the Revised American Society for Reproductive Medicine classification was required. The exclusion criteria were as follows: (1) abstracts, case reports, letters, reviews, or single-arm studies; (2) phenotype/genotype frequency and endometriosis risk were not reported; (3) incomplete data for the calculation of ORs and 95% CIs.

Data extraction
All potentially relevant studies were checked by two investigators (M-M.Z. and Z-M.W.) independently, and a third investigator (X-L.F.) resolved discrepancies. The following items were extracted: year of publication, first author, diagnostic standard, NAT2 phenotypes, target genotypes, diagnostic methods, genotyping methods, case age, ethnicity, features of the controls, endometriosis stage, genotype distributions in cases and controls, and the total number of cases and controls. The corresponding authors of the original studies were contacted when further data were needed.

Statistical analysis
Subgroup analysis by ethnicity, χ 2 tests, Begg's funnel plots, and Egger's tests were performed. In addition, the Newcastle-Ottawa scale (NOS), OR, 95% CI, and I 2 statistic were estimated. The NOS criteria were used to assess the methodological quality of all studies. Studies with NOS scores � 7 were regarded as good quality (range, 0-8) [28]. To determine whether genotypes were in accordance with the Hardy-Weinberg equilibrium, an internet-based program was used (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) [29].

Summary of study characteristics
A total of 617 articles were retrieved from the electronic search. Among these, 604 were excluded based on titles and abstracts. The full texts of the remaining 13 articles were screened. One review article was excluded [25]. Two articles were excluded for not reporting exact genotypes [20,31]. One article was excluded for being a single-arm study [32]. Finally, nine casecontrol studies [14,17,26,27,[33][34][35][36][37] met the inclusion criteria. The details of the study selection process are presented in Fig 1. All article titles excluded-with reason-are shown in S1 File. The characteristics and quality of the included studies are summarized in Tables 1 and 2.
Quantitative data analysis NAT2 phenotype and endometriosis risk. In the primary model (slow vs. intermediate + fast), heterogeneity was high (χ 2 = 44.09, I 2 = 82%; Fig 2). Accordingly, we performed a subgroup analysis focusing on different ethnicities, mainly a Caucasian group and an Asian group. In the Asian group, the heterogeneity was low (χ 2 = 3.18, I 2 = 6%) and the OR was 2.30 (95% CI, 1.59-3.32; P < 0.001). This result suggests that individuals with the NAT2 slow acetylation phenotype have a 130% increased risk of endometriosis in the Asian population. Results for other ethnicity groups are summarized in Table 3.
Publication bias. Begg's funnel plot and Egger's test were used to evaluate publication bias. The symmetry detected in Begg's funnel plot indicated low publication bias in these statistically significant models (Fig 5).
Sensitivity analysis. By sequentially excluding individual studies, the outcomes were consistent with the overall study results, indicating that our results showed good stability and reliability. After excluding each study, the changes in OR with 95% CIs are presented in Table 5.

Discussion
Endometriosis is one of the most common diseases in women [38]. However, its etiology and pathogenesis have not been fully elucidated [39]. It has been proven that endometriosis exhibits polygenic inheritance. Evidence suggests that its occurrence and development involve genetic variation, abnormal regulation, or mutation accumulation, but the exact genetic basis remains to be discovered. Accordingly, studies of genetic susceptibility to endometriosis and screening for susceptibility genes are increasing [40].
Recently, some scholars have proposed a theory for the inheritance and induction of endometriosis, suggesting that it is a multi-factorial genetic disease caused by the cumulative effects of mutations at multiple loci and environmental factors [41]. In 1993, Rier et al. first reported that environmental toxins such as dioxins are potential risk factors for endometriosis [22];  [19,21]. As one of the links between environmental factors and genetic factors, toxin-metabolizing enzymes have crucial roles in the pathogenesis of endometriosis. With increasing research focused on polymorphisms in genes encoding toxin-metabolizing enzymes, more and more attention has been paid to the relationships between these enzymes and genetic susceptibility to endometriosis.
NAT2 is an important two-phase detoxifying enzyme for the in vivo transformation of exogenous chemicals and is involved in the metabolism of various environmental toxicants [21]. Twenty-seven alleles have been reported, and the C481T, G590A, A803G, and G857A SNPs cause decreased enzyme activity and impaired acetylation activity, resulting in differences in susceptibility to cancer, diabetes, and allergic diseases [12,42]. Some researchers have speculated that changes in NAT2 activity increase the risk of endometriosis or that there is a link between an imbalance in NAT2 gene polymorphisms and susceptibility to endometriosis. In 1999, Baranvo et al. reported that stage I-II endometriosis in the French population is associated with slow acetylation [27]. However, in 2001, Nakago et al. reported that NAT2 polymorphisms have no relationship with the risk of endometriosis in the UK population [34]. In  [14]. Thus, there is no consensus regarding whether the NAT2 gene is associated with endometriosis. Previously, Guo et al. reviewed the literature on this topic. This study comprised a metaanalysis of the NAT2 slow acetylation phenotype and endometriosis. No significant associations were found at that time [25]. Here, we included four additional new studies on this topic, including one study of Caucasians and three studies of Asians. We also performed subgroup analysis and added a meta-analysis between different SNPs and endometriosis risk. To our knowledge, this meta-analysis includes the largest sample size of studies on this topic and provides new insights on the clinical significance of NAT2.
We comprehensively evaluated NAT2 phenotypes with respect to the risk of endometriosis, including a subgroup analysis according to ethnic groups. We concluded that the NAT2 slow acetylation phenotype is a risk factor for endometriosis in the Asian population. We also found that there is no association between the slow, intermediate, or fast acetylation NAT2 phenotypes and endometriosis in Caucasian individuals. Furthermore, we provide additional evidence that the G590A SNP might act as a protective factor. Studies on this SNP exhibit low heterogeneity. No statistically significant results were found between rs1799931, rs1208, or rs1799929 and endometriosis. Sensitivity analyses were also consistent with the overall study results, establishing the stability and reliability of our results. From this analysis, we found that  It is worth mentioning that an increasing number of genome-wide association studies have identified polymorphisms associated with endometriosis including rs10859871, rs10965235, rs1270667, rs13394619, rs1537377, rs758316, rs7739264, rs7521902, and rs16826658. However, these studies have not conclusively supported an association between rs1799930 (G590A) or rs1799931 (G857A) and endometriosis [43][44][45][46]. Therefore, more elaborate hierarchical genome-wide association studies are needed to examine the relationships between these SNPs and endometriosis with respect to a variety of ethnicities and stages and to eliminate confounding factors like dioxin exposure, pollution exposure, lifestyle, diabetes, smoking, coffee intake, breast feeding time, or other genetic factors [47].  There was substantial heterogeneity among results with respect to the association between the NAT2 phenotype and endometriosis risk, and these differences might be related to race and geographical differences. NAT2 genotype frequencies were significantly different among races and regions. More than 53% of Western Caucasians harbored the slow acetylation type, whereas the Eastern Asians predominantly had the fast acetylation type [36]. Because the pathogenesis of endometriosis is highly complex, the underlying genetic mechanism might differ among populations. These differences among studies suggest differences in the underlying genetic factors, further validating the genetic diversity of endometriosis. Based on a subgroup analysis, heterogeneity was effectively eliminated. Furthermore, endometriosis is a polygenic disease, involving many pathogenic genes and environmental factors. Various environmental conditions like smoking, drinking, age, economic status, medical history, dioxin exposure, pollution exposure, lifestyle, diabetes, coffee intake, breast feeding time, or other genetic factors should be considered and controlled for in both case and control groups. As one of the studies included in this meta-analysis, Matsuzaka et al. reported the smoking history and BMI distribution and Baranova et al. reported the distribution of clinical symptoms relative to NAT2 genotypes. Further, a recent study revealed that breast feeding time could significantly account for endometriosis risk. However, no studies have been performed adjusting for these factors, and thus, more elaborate studies are needed on this topic.
Some limitations of this study should be mentioned. First, it only considered certain databases and unpublished negative results or ongoing research might be missing, thereby affecting our results. Second, only data for Asian and Caucasian populations are reported in the databases, and gene frequency distributions for other ethnicities like African or Latin American populations are unavailable. Third, heterogeneity in the NAT2 phenotype between Asian and Caucasian individuals should not be ignored. When we performed subgroup analysis of different ethnicities, heterogeneity was effectively eliminated, but more precise studies of different regions or ethnicities should also be performed. Fourth, the heterogeneity among existing studies might be explained by sampling errors and the small number of samples in some studies. More samples and data are urgently needed, and analyses should be subdivided according to the stages of endometriosis. However, despite these limitations, based on an elaborately designed protocol and using comprehensive methods to evaluate previous studies, we conclude that there is an association between NAT2 polymorphisms and endometriosis. Our research suggests that there is still room for improvement with respect to studies on the genetic basis of endometriosis.

Conclusion
In summary, the rs1799930 mutant genotypes are associated with a decreased risk of endometriosis. No statistically significant results were found between rs1799931, rs1208, or rs1799929 and endometriosis. In a subgroup analysis based on ethnicity, the NAT2 slow acetylation phenotype was found to increase the risk of endometriosis in Asians. However, no statistically significant results were found between the NAT2 slow acetylation phenotype and endometriosis risk in Caucasians. These SNPs and NAT2 phenotype are potential biomarkers for the diagnosis and treatment of endometriosis. Further large-scale case-control studies are needed, with specific designs to account for the disease stage, for a more in-depth and thorough exploration of the relationship between NAT2 polymorphisms and endometriosis.