{"paper_id":"c7a545b6-f794-49b5-8bb6-d95448e42568","body_text":"Endometriosis, a benign gynecological and estrogen dependent disease, is the growth of endometrium outside the uterine cavity or myometrium, usually in the peritoneal cavity  1 – 3 . The most important symptoms of endometriosis are back pain, infertility, dyspareunia, dysmenorrheal, dyschezia and pelvic pain  4 . Several factors such as genetic, immune, endocrine and environmental factors have been associated with the pathogenesis of endometriosis  1 , 5 – 10 .\nThe human arylamine N-acetyltransferase 2  (NAT2)  gene, on chromosomal region 8p22  11 , has a key role in conjugation of xenobiotic substances.  NAT2  is involved in the initial biotransformation metabolism of aromatic amines and hydrazines, and catalyzes the transfer of an acetyl group from acetyl CoA to the nitrogen of the substrate  12 . Some polymorphisms in  NAT2  lead to amino acid substitution, which may result in impairment of the enzyme activity. Regarding  NAT2 , individuals are classified as rapid metabolizers if they have one or two wild-type alleles such as  NAT2  *4 which is the most common allele. On the other hand, individuals are classified as slow metabolizers only if they carry two slow metabolizer variants such as  NAT2  *5,*6 or *7 alleles. In a paper by Hein  et al , they determined the NAT gene nomenclature  13 . The alleles themselves are effectively haplotypes composed of several  NAT2  polymorphisms, most typically assigned according to the status of the seven polymorphisms.  NAT2  slow acetylation has been reported as a risk factor for bladder cancer  14 , 15 , while fast acetylation has been implicated as a risk factor for colon cancer  16 . Smelt and Mardon showed that there was a linkage disequilibrium between NAT1 and  NAT2  genes  17 .\nThe results of previous studies that evaluated the  NAT2  polymorphisms with the risk of endometriosis are controversial. Therefore, in the current study, it was decided to investigate the association of  NAT2  C481T (rs1799929), G590A (rs1799930), A803G (rs1208) and G857A (rs1799931) polymorphisms with endometriosis in an Iranian population.\n\nIn this case-control study, 141 Iranian patients with diagnosis of endometriosis were included. The diagnosis of endometriosis was accurately predicted preoperatively by clinicians. The severity of the disease (Stage I–IV) was scored according to the revised American Society for Reproductive Medicine (ASRM) classification. As the control group, 158 age-matched healthy women without endometriosis were selected. Absence of the endometriosis in the control group was confirmed by laparoscopic abdominal examination to inspect conditions other than endometriosis. Cases without confirmation of the disease by laparoscopy were excluded. Approval from Avicenna Research Institute’s Ethics Committee was obtained. Written informed consent was received from the patients and controls. The study was done at Avicenna Research Institute, Tehran, Iran.\nDNA was extracted from peripheral blood samples according to the salting out procedure. Genotyping of the four polymorphisms were performed using PCR-RFLP methods according to the previous study  16 . Briefly, 546  bp  fragment from exon 2 of  NAT2  was amplified using the forward primer 5′-GCTGGTCTGG AAGCTCCTC-3′ and reverse primer 5′-TTGGGTGA TACATACACAAGGG-3′. Following PCR amplification, digestion with the restriction enzymes KpnI, Dd-eI, BamHI and TaqI were performed to detect genotypes of the 481C/T, 803A/G, 857G/A and 590G/A polymorphisms, respectively. All enzymes were obtained from New England BioLabs and were used according to the manufacturer’s instructions.\nNAT2  acetylator phenotypes (slow, intermediate and fast) were predicted using the NAT2PRED Web server available at  http://nat2pred.rit.albany.edu \n 18 . NAT2P-RED is a web-server that predicts  NAT2  acetylator phenotype from six polymorphisms of  NAT2  C282T, T341C, C481T, A803G, G857A and G590A, without taking the extra step of reconstructing haplotypes for each individual  19 . Because the genotypes for the  NAT2  C282T and T341C were not determined, the wild type genotype for these polymorphisms was selected in the NAT2PRED web server.\nStatistical analysis was performed with SPSS for Windows, version 13 (Chicago, IL, USA). Analyses of the allele frequencies and genotype distributions for the  NAT2  polymorphisms in case and control groups were performed by  χ 2  test and logistic regression analysis. All p-values were reported following two tailed statistical tests and values less than 0.05 were considered statistically significant. Each polymorphism was tested for Hardy-Weinberg equilibrium in the case and control population using  χ 2  test. The SNPStat online software was used to estimate pairwise linkage disequilibrium (LD) and haplotype frequency ( https://www.snp-stats.net/snpstats ).\n\nOur results showed that of 141 endometriosis patients, 100 (71.0%) and 25 (17.7%) had stage I–II and III–IV of the disease, respectively; on the other hand, in 16 (11.3%) patients, the severity of the disease was not determined. The mean age of healthy and endometriosis group was 29±5 (range 19–44) and 31±6 (19–50) years, respectively that showed significant differences (p=0.03). The Body Mass Index (BMI) comparison between two groups shows significant differences (p= 0.01). The differences of age and BMI between groups could affect the other results and therefore the genotype analysis was modified with regression logistic analysis based on age and BMI.\nThe distributions of genotype using  χ 2  shown in both case and control groups for the  NAT2  C481T, G590A, A803G and G857A polymorphisms were in Hardy-Weinberg equilibrium (p>0.05).\nThe genotype distributions and allele frequencies of the  NAT2  C481T, G590A, A803G and G857A polymorphisms have been shown in  table 1 . Our results revealed that there was a significant difference in the genotype distributions and allele frequencies of  NAT2  G590A polymorphism between the case and control groups. The  NAT2  590 GA genotype was significantly lower (p=0.001; OR=0.42, 95% CI: 0.25–0.71) in the patients (38.3%) than the control group (55.1%). The  NAT2  590A allele was significantly lower (p=0.033; OR=0.69, 95% CI: 0.49–0.79) in the patients (31.2%) compared with the controls (39.6%). This finding suggested that the  NAT2  G590A polymorphism may be associated with susceptibility to endometriosis and the 590A allele may have a protective role in the development of endometriosis in Iranian women.\nAllele and genotype frequencies of  NAT2  C481T, A803G, G857A and G590A polymorphisms in endometriosis patients and controls\nAdjusted by age and BMI\nIn contrast, no significant difference in the genotype and allele frequencies of the  NAT2  C481T, A803G and G857A polymorphisms was found between the case and control groups.\nThe frequency of  NAT2  acetylator phenotypes are shown in  table 2 . Slow phenotype was prevalent in the control group. Statistical analysis showed that the frequency of rapid acetylator phenotype was significantly higher in the endometriosis (34.8%) than the control (27.8%) group (p=0.025; OR=2.19, 95% CI: 1.10–4.36). Analysis of haplotypes showed that among different combinations of the four  NAT2  SNPs ( Table 3 ), only haplotype (481C, 803A, 590A, 587A) was significantly different between the case and control women (p=0.029; OR=3.11, 95% CI: 1.13–8.52).\nThe  NAT2  acetylator phenotypes in cases and controls\nHaplotypes frequency and association with endometriosis (n=299)\n\nC481T, G590A, A803G and G857A polymorphisms in exon 2 of  NAT2  with the risk of endometriosis were investigated in an Iranian population. Our results revealed that there was significant difference in the genotype distributions and allele frequencies of  NAT2  G590A polymorphism between the women with endometriosis and control groups. The  NAT2  590 GA genotype and  NAT2  590A allele were significantly lower in the patients than the controls (p=0.001 and p=0.033, respectively). This finding suggested that the polymorphism may be associated with susceptibility to endometriosis and  NAT2  590A allele may have a protective role in the progression of endometriosis in Iranian women. On the other hand, the frequency of rapid acetylator phenotype was two-fold higher in the endometriosis than the normal controls.\nThe global Minor Allele Frequency (MAF) in the NCBI dbSNP database of the  NAT2  C481T, G590A, A803G and G857A polymorphisms were T=0.27, A= 0.27, G=0.32 and A=0.08, respectively. According to these frequencies, G857A is a relatively rare polymorphism.\nThe results of a few studies that evaluated the association of  NAT2  polymorphisms and acetylator phenotypes with the endometriosis are controversial  19 – 23 . There may be several explanations for the observed discrepancies. For example, in some studies only endometriosis patients with stage I–II or III–IV have been included. On the other hand, in some studies the acetylator phenotype for  NAT2  enzyme was classified as slow and fast, while in the others, a three-category classification as slow, intermediate and fast has been used.\nBaranova  et al  have reported that a higher proportion of women with stage I–II endometriosis were slow acetylators compared with controls, who were women undergoing termination of pregnancy with no evidence of endometriosis on physical or ultrasound examination (69  vs . 39%)  21 . There was no statistically significant difference in acetylator status between women with stage III–IV endometriosis and the controls  20 . Bischoff  et al  only investigated women with stage III–IV endometriosis and reported that 16/29 (55%) had the slow-acetylator phenotype  20 .\nNakago  et al  investigated the relationship between endometriosis and  NAT2  polymorphisms in a UK population  18 . Homozygotes for the  NAT2  *4 wild type allele are fast  NAT2  acetylators, while heterozygotes with one wild-type allele and a variant  NAT2  *5, *6 or *7 allele have reduced enzyme activity, and individuals with two variant alleles are slow acetylators. The  NAT2  *4/*6 genotype was significantly more common among endometriosis patients (35.2%) than the controls (8.1%) or unaffected women (4.2%). Fast acetylators phenotype was significantly prevalent in endometriosis group (57.4%) than the controls (32.3%) or unaffected women (33.3%). Their results suggest that altered  NAT2  enzyme activity may be a predisposition factor in endometriosis  18 .\nBabu  et al  investigated the  NAT2  polymorphisms in 252 unrelated women with endometriosis and 264 controls of South Indian women  22 . They found no differences between the frequencies of fast and slow acetylators in cases (34.9 and 65.1%) and controls (33.3 and 66.7%). Deguchi  et al  investigated the association between endometriosis and polymorphisms in the  NAT1  and  NAT2  in a Japanese population  23 . Their results showed that the distribution of  NAT1  and  NAT2  allele and genotype frequencies were not significantly different between Japanese cases and controls.\n\nInvestigation of  NAT2  polymorphisms (C481T, A803-G, G857A and G590A) showed that there may be an association between G590A polymorphism and risk for development of endometriosis in Iranian population, and the 590A allele may have a protective role in development of endometriosis. The  NAT2  481C, 803A, 590A, 587A haplotype was associated with a higher risk of endometriosis.","source_license":"CC0","license_restricted":false}