Reproductive BioMedicine Online (2015) 30, 415–420
w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m
ARTICLE
Analysis of WNT4 polymorphism in Chinese Han women with endometriosis Zhangying Wu, Ming Yuan, Yan Li, Fangfang Fu, Wenqinq Ma, Haixia Li, Wenwen Wang *, Shixuan Wang * Cancer Biology Research Center, Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Street 1095#, Wuhan, Hubei 430030, PR China * Corresponding authors.
E-mail addresses: [email protected] (W Wang); [email protected] (S Wang). Shi-Xuan Wang, Professor of Gynaecology and Gynaecological Oncology, is Director of the Division of Gynecology and Vice Director of the Cancer Biology Research Centre in Tongji hospital, Huazhong University of Science and Technology. He received his PhD at Tongji Medical University, Wuhan, China in 1996. From 2002 to 2004, he devoted himself to a tumour immunology programme as a postdoctoral researcher at UT Southwestern Medical Centre, Dallas, Texas, USA. His research interests are gynaecology and gynaecological oncology.
Endometriosis is a complex disease that is influenced by genetic and environmental factors. In endometriosis, WNT4 plays a likely role owing to its biological functions. In this study, the TaqMan allelic discrimination technique was used to investigate the relationship between endometriosis and four single nucleotide polymorphisms in WNT4 (rs7521902 [A/C], rs16826658 [G/T], rs7515106 [C/T] and rs2235529 [A/G]) in Chinese Han women. A total of 646 patients with endometriosis and 766 normal controls were recurited. Regression analyses revealed that rs2235529 was a risk locus for endometriosis (P = 1.80E-03, OR, 95% CI = 1.311, 1.129 to 1.522), particularly in patients with stage III and IV disease. No significant association was found between endometriosis and rs7521902 (A/C), rs16826658 (G/T), or rs7515106 (C/T). For each of the four single nucleotide polymorphisms, no association was found between patients with endometriosis-related infertility or primary infertility and the controls. The results demonstrated that WNT4 rs2235529 is associated with endometriosis in Chinese Han women, which may result in aberrant expression of WNT4, leading to the pathogenesis of endometriosis.
Abstract
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: endometriosis, infertility, polymorphism, WNT4
http://dx.doi.org/10.1016/j.rbmo.2014.12.010 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
416
Introduction
Z Wu et al. endometriosis), genital tract anomalies and histories of malignant disease were excluded. The control patients underwent surgery for uterine leiomyomas (231/766 [30.2%]) or infertility (535/766 [69.8%]). The postoperative diagnosis of infertility was hydrosalpinx. According to the revised American Fertility Society classification (Anon., 1985), 102 (15.8%) stage I-II patients and 544 (84.2%) patients had moderate to severe endometriosis (stage III-IV). In our sample, 39.8% (257/646) women had endometriosis-associated infertility, and 22.5% (172/766) controls had primary infertility. Peripheral blood samples were preoperatively collected into tubes treated with ethylenediaminetetraacetic acid. All patients provided signed informed consent. The study was approved by Tongji Medical College, Huazhong University of Science and Technology on 26 July 2011 (reference number S462).
Endometriosis is defined by the presence of endometrial tissue outside the uterus, and it affects 5–10% of women of reproductive age and up to 50% of infertile women (Eskenazi and Warner, 1997; Giudice and Kao, 2004; Meuleman et al., 2009). It often results in dysmenorrhoea, chronic pelvic pain, dyspareunia and infertility (Giudice and Kao, 2004). Epidemiological studies have determined that the risk of endometriosis in women with a family history of the diseaseisincreased by approximately five-fold (Stefansson et al., 2002; Treloar et al., 1999). Although the pathogenesis of endometriosis is largely unclear, it is considered to be a multifactorial disease involving environmental and genetic factors and interactions between these factors (Montgomery et al., 2008). The wingless-type MMTV integration site family member 4 (WNT4) is a protein-coding gene that plays a crucial role in the development of the female reproductive tract, human endometrial stromal cell differentiation and embryonic implantation (Kobayashi and Behringer, 2003; Li et al., 2013; Tulac et al., 2003; Vainio et al., 1999). A previous study observed abnormal expression of the WNT4 gene in eutopic endometrium from women with endometriosis (Pabona et al., 2012). Recently, several genome-wide association studies (GWAS) of endometriosis have detected some endometriosisrelated genetic markers in the region close to or within an intron of WNT4 (Nyholt et al., 2012; Painter et al., 2011; Uno et al., 2010). A Japanese GWAS reported an association between endometriosis and rs7521902(A/C) and rs16826658(G/ T) (Nyholt et al., 2012; Uno et al., 2010). In addition, a significant association between rs7521902(A/C) and endometriosis was confirmed in the study by Painter et al. (2011), as well as an association between rs7515106(C/T) and endometriosis. Some studies have repeatedly investigated associations between these single nucleotide polymorphisms (SNPs) and endometriosis in women of different races (Albertsen et al., 2013; Rahmioglu et al., 2014; Sundqvist et al., 2013). These results, however, are inconsistent. In a recent study by Albertsen et al. (2013), an association between one novel SNP (rs2235529[A/G]) and endometriosis was observed in American women. In the present study, therefore, three controversial SNPs were studied (rs7521902[A/C], rs16826658[G/ T], and rs7515106[C/T]) and one novel SNP (rs2235529[A/ G]) in Chinese Han women with endometriosis.
Four SNPs were selected for genotyping: rs7521902(A/C), rs16826658(G/T), rs7515106(C/T) and rs2235529(A/G). Detailed information is presented in Table 1. TaqMan allelic discrimination analysis was carried out using the Applied Biosystems protocol. A 384-well plate was prepared with a mixture of 1 × TaqMan SNP Genotyping assay (Applied Biosystems, USA), 1 × TaqMan Universal Master Mix (Applied Biosystems, USA) and 20 ng DNA per well. Polymerase chain reaction (PCR) was carried out using a 7900 HT Fast-RealTime PCR system (Applied Biosystems, USA). The PCR conditions were denaturation at 95°C for 10 min, followed by 40 cycles of 95°C denaturation for 15 s and a 60°C anneal for 1 min. After PCR, the plates were read, and the data analysed using SDS 2.4 (Applied Biosystems, USA). For quality control, a random 10% of the samples were repeatedly genotyped; the reproducibility rate was 100%. It was determined that a call rate of at least 97% met the standard criteria.
Materials and methods
Statistical analyses
Patients
The Statistical Package for Social Sciences (SPSS) version 18.0 for Windows (SPSS, Chicago, IL, USA) was used to conduct statistical analyses. Certain differences (i.e. current age, age at menarche, menstrual cycle and menstrual period) between the patients and controls were evaluated using the nonparametric Kruskal–Wallis test. The chi-squared test was used to test the infertility rate, Hardy–Weinberg equilibrium, and allele and genotype frequencies. Statistically significant values were corrected for multiple tests using the Bonferroni correction, and P < 0.05 was considered to be statistically significant. The odds ratio (OR) and 95% confidence intervals (CI) were calculated by binary logistic regression. A post-hoc
In this study, 1412 women were enrolled (646 endometriosis patients and 766 controls). All women had undergone surgery between 2007 and 2013 at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The following inclusion criteria were applied: members of the Chinese Han population, aged 25–45 years, preoperative 6 months without hormonal therapy, regular menstruation, laparoscopy or laparotomy and with a histologically confirmed presence of endometriosis or absence of endometriosis. Patients with ovarian malignant or benign cysts (except ovarian
DNA extraction Genomic DNA was extracted from leukocytes with the Quickgene DNA whole blood kit (Fuji Film, Japan), according to the manufacturer’s protocol.
WNT4 genotyping
WNT4 polymorphism in Chinese Han women with endometriosis Table 1
417
WNT4 single nucleotide polymorphisms in previous genome-wide association studies of endometriosis. Location
Allele
Distance to WNT4
P-value
OR (95% CI)
Ethnicity
Reference
rs7521902
Chr1:22164231
A/C
Upstream 21 kb
rs16826658 rs2235529 rs7515106
Chr1:22159278 Chr1:22123994 Chr1:22146917
G/T A/G C/T
Upstream 16 kb Intron 1 Upstream 3.9 kb
4.23E-05 8.90E-05 3.46E-05 1.36E-07 3.60E-05
1.25 (1.12 to1.39) 1.16 (1.08 to 1.25) 1.25 (1.12 to 1.39) 1.28 (1.16 to 1.41) 1.13 (1.07 to 1.05)
Japanese White White White White
Uno et al. (2010) Painter et al. (2011) Painter et al. (2011) Albertsen et al. (2013) Painter et al. (2011)
SNP
CI = confidence interval; OR = odds ratio; SNP = single nucleotide polymorphism.
Table 2
Characteristics of the study population.
Characteristic Number of participants Age (years) Age at menarche Menstrual cycle length Menstrual period Infertility
Endometriosis
Control
P-value
646 33.25 ± 7.55 13.24 ± 1.42 29.47 ± 4.23 5.93 ± 1.32 257/646 (39.8)
766 34.06 ± 7.46 13.40 ± 1.50 29.42 ± 4.41 5.59 ± 1.68 172/766 (22.5)
NS NS NS 0.019
w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m
ARTICLE
Analysis of WNT4 polymorphism in Chinese Han women with endometriosis Zhangying Wu, Ming Yuan, Yan Li, Fangfang Fu, Wenqinq Ma, Haixia Li, Wenwen Wang *, Shixuan Wang * Cancer Biology Research Center, Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Street 1095#, Wuhan, Hubei 430030, PR China * Corresponding authors.
E-mail addresses: [email protected] (W Wang); [email protected] (S Wang). Shi-Xuan Wang, Professor of Gynaecology and Gynaecological Oncology, is Director of the Division of Gynecology and Vice Director of the Cancer Biology Research Centre in Tongji hospital, Huazhong University of Science and Technology. He received his PhD at Tongji Medical University, Wuhan, China in 1996. From 2002 to 2004, he devoted himself to a tumour immunology programme as a postdoctoral researcher at UT Southwestern Medical Centre, Dallas, Texas, USA. His research interests are gynaecology and gynaecological oncology.
Endometriosis is a complex disease that is influenced by genetic and environmental factors. In endometriosis, WNT4 plays a likely role owing to its biological functions. In this study, the TaqMan allelic discrimination technique was used to investigate the relationship between endometriosis and four single nucleotide polymorphisms in WNT4 (rs7521902 [A/C], rs16826658 [G/T], rs7515106 [C/T] and rs2235529 [A/G]) in Chinese Han women. A total of 646 patients with endometriosis and 766 normal controls were recurited. Regression analyses revealed that rs2235529 was a risk locus for endometriosis (P = 1.80E-03, OR, 95% CI = 1.311, 1.129 to 1.522), particularly in patients with stage III and IV disease. No significant association was found between endometriosis and rs7521902 (A/C), rs16826658 (G/T), or rs7515106 (C/T). For each of the four single nucleotide polymorphisms, no association was found between patients with endometriosis-related infertility or primary infertility and the controls. The results demonstrated that WNT4 rs2235529 is associated with endometriosis in Chinese Han women, which may result in aberrant expression of WNT4, leading to the pathogenesis of endometriosis.
Abstract
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: endometriosis, infertility, polymorphism, WNT4
http://dx.doi.org/10.1016/j.rbmo.2014.12.010 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
416
Introduction
Z Wu et al. endometriosis), genital tract anomalies and histories of malignant disease were excluded. The control patients underwent surgery for uterine leiomyomas (231/766 [30.2%]) or infertility (535/766 [69.8%]). The postoperative diagnosis of infertility was hydrosalpinx. According to the revised American Fertility Society classification (Anon., 1985), 102 (15.8%) stage I-II patients and 544 (84.2%) patients had moderate to severe endometriosis (stage III-IV). In our sample, 39.8% (257/646) women had endometriosis-associated infertility, and 22.5% (172/766) controls had primary infertility. Peripheral blood samples were preoperatively collected into tubes treated with ethylenediaminetetraacetic acid. All patients provided signed informed consent. The study was approved by Tongji Medical College, Huazhong University of Science and Technology on 26 July 2011 (reference number S462).
Endometriosis is defined by the presence of endometrial tissue outside the uterus, and it affects 5–10% of women of reproductive age and up to 50% of infertile women (Eskenazi and Warner, 1997; Giudice and Kao, 2004; Meuleman et al., 2009). It often results in dysmenorrhoea, chronic pelvic pain, dyspareunia and infertility (Giudice and Kao, 2004). Epidemiological studies have determined that the risk of endometriosis in women with a family history of the diseaseisincreased by approximately five-fold (Stefansson et al., 2002; Treloar et al., 1999). Although the pathogenesis of endometriosis is largely unclear, it is considered to be a multifactorial disease involving environmental and genetic factors and interactions between these factors (Montgomery et al., 2008). The wingless-type MMTV integration site family member 4 (WNT4) is a protein-coding gene that plays a crucial role in the development of the female reproductive tract, human endometrial stromal cell differentiation and embryonic implantation (Kobayashi and Behringer, 2003; Li et al., 2013; Tulac et al., 2003; Vainio et al., 1999). A previous study observed abnormal expression of the WNT4 gene in eutopic endometrium from women with endometriosis (Pabona et al., 2012). Recently, several genome-wide association studies (GWAS) of endometriosis have detected some endometriosisrelated genetic markers in the region close to or within an intron of WNT4 (Nyholt et al., 2012; Painter et al., 2011; Uno et al., 2010). A Japanese GWAS reported an association between endometriosis and rs7521902(A/C) and rs16826658(G/ T) (Nyholt et al., 2012; Uno et al., 2010). In addition, a significant association between rs7521902(A/C) and endometriosis was confirmed in the study by Painter et al. (2011), as well as an association between rs7515106(C/T) and endometriosis. Some studies have repeatedly investigated associations between these single nucleotide polymorphisms (SNPs) and endometriosis in women of different races (Albertsen et al., 2013; Rahmioglu et al., 2014; Sundqvist et al., 2013). These results, however, are inconsistent. In a recent study by Albertsen et al. (2013), an association between one novel SNP (rs2235529[A/G]) and endometriosis was observed in American women. In the present study, therefore, three controversial SNPs were studied (rs7521902[A/C], rs16826658[G/ T], and rs7515106[C/T]) and one novel SNP (rs2235529[A/ G]) in Chinese Han women with endometriosis.
Four SNPs were selected for genotyping: rs7521902(A/C), rs16826658(G/T), rs7515106(C/T) and rs2235529(A/G). Detailed information is presented in Table 1. TaqMan allelic discrimination analysis was carried out using the Applied Biosystems protocol. A 384-well plate was prepared with a mixture of 1 × TaqMan SNP Genotyping assay (Applied Biosystems, USA), 1 × TaqMan Universal Master Mix (Applied Biosystems, USA) and 20 ng DNA per well. Polymerase chain reaction (PCR) was carried out using a 7900 HT Fast-RealTime PCR system (Applied Biosystems, USA). The PCR conditions were denaturation at 95°C for 10 min, followed by 40 cycles of 95°C denaturation for 15 s and a 60°C anneal for 1 min. After PCR, the plates were read, and the data analysed using SDS 2.4 (Applied Biosystems, USA). For quality control, a random 10% of the samples were repeatedly genotyped; the reproducibility rate was 100%. It was determined that a call rate of at least 97% met the standard criteria.
Materials and methods
Statistical analyses
Patients
The Statistical Package for Social Sciences (SPSS) version 18.0 for Windows (SPSS, Chicago, IL, USA) was used to conduct statistical analyses. Certain differences (i.e. current age, age at menarche, menstrual cycle and menstrual period) between the patients and controls were evaluated using the nonparametric Kruskal–Wallis test. The chi-squared test was used to test the infertility rate, Hardy–Weinberg equilibrium, and allele and genotype frequencies. Statistically significant values were corrected for multiple tests using the Bonferroni correction, and P < 0.05 was considered to be statistically significant. The odds ratio (OR) and 95% confidence intervals (CI) were calculated by binary logistic regression. A post-hoc
In this study, 1412 women were enrolled (646 endometriosis patients and 766 controls). All women had undergone surgery between 2007 and 2013 at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The following inclusion criteria were applied: members of the Chinese Han population, aged 25–45 years, preoperative 6 months without hormonal therapy, regular menstruation, laparoscopy or laparotomy and with a histologically confirmed presence of endometriosis or absence of endometriosis. Patients with ovarian malignant or benign cysts (except ovarian
DNA extraction Genomic DNA was extracted from leukocytes with the Quickgene DNA whole blood kit (Fuji Film, Japan), according to the manufacturer’s protocol.
WNT4 genotyping
WNT4 polymorphism in Chinese Han women with endometriosis Table 1
417
WNT4 single nucleotide polymorphisms in previous genome-wide association studies of endometriosis. Location
Allele
Distance to WNT4
P-value
OR (95% CI)
Ethnicity
Reference
rs7521902
Chr1:22164231
A/C
Upstream 21 kb
rs16826658 rs2235529 rs7515106
Chr1:22159278 Chr1:22123994 Chr1:22146917
G/T A/G C/T
Upstream 16 kb Intron 1 Upstream 3.9 kb
4.23E-05 8.90E-05 3.46E-05 1.36E-07 3.60E-05
1.25 (1.12 to1.39) 1.16 (1.08 to 1.25) 1.25 (1.12 to 1.39) 1.28 (1.16 to 1.41) 1.13 (1.07 to 1.05)
Japanese White White White White
Uno et al. (2010) Painter et al. (2011) Painter et al. (2011) Albertsen et al. (2013) Painter et al. (2011)
SNP
CI = confidence interval; OR = odds ratio; SNP = single nucleotide polymorphism.
Table 2
Characteristics of the study population.
Characteristic Number of participants Age (years) Age at menarche Menstrual cycle length Menstrual period Infertility
Endometriosis
Control
P-value
646 33.25 ± 7.55 13.24 ± 1.42 29.47 ± 4.23 5.93 ± 1.32 257/646 (39.8)
766 34.06 ± 7.46 13.40 ± 1.50 29.42 ± 4.41 5.59 ± 1.68 172/766 (22.5)
NS NS NS 0.019
