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Lack of association of WNT5A mutations with Müllerian duct abnormalities
Reproductive BioMedicine Online (2013) 26, 164– 167
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ARTICLE
Lack of association of WNT5A mutations with ¨llerian duct abnormalities Mu Keliang Wu a,b,1, Xinyue Chang a,b,1, Deying Wei c, Chengyan Xu Yingying Qin a,b,*, Zi-Jiang Chen a,b,d
a,b
,
a Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, China; b The Key laboratory for Reproductive Endocrinology of Ministry of Education, Shandong Provincial Key Laboratory of Reproductive Medicine China, Jinan, China; c Department of Obstetrics and Gynecology, Provincial Hospital Affiliated to Shandong University, Jinan, China; d Center for Reproductive Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
* Corresponding author. E-mail address: [email protected] (Y Qin). 1 These authors contribute equally to this work.
Keliang Wu was born on 24 November 1981 in Yancheng, Jiangsu, PR China. In 2007, he graduated from Shandong University with a Masters in science majored in developmental biology. After graduation, he worked as an embryologist in Center for Reproductive Medicine, Provincial Hospital affiliated to Shandong University. From 2007 to 2010, he applied five national patents from China referring to assisted reproduction technology.
Abstract The aim of this study was to determine if mutations in WNT5A contribute to the aetiology of Mu ¨llerian duct abnormalities
(MDA) in 189 Chinese women. Three novel single-nucleotide polymorphisms (SNP; IVS2 115G > A, IVS4 + 66T > A, IVS4 + 102G > T) in introns 2 and 4 as well as one known SNP (rs62620048) in exon 6 were detected, but no causal mutations were observed. The results suggested that mutations in WNT5A may be not responsible for MDA in Chinese women. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: Mu ¨llerian duct abnormalities, mutation, single-nucleotide polymorphisms, WNT5A
Introduction The WNT family (wingless-type MMTV integration site family) is the major genetic focus for finding genes whose perturbations cause uterus anomalies. Nineteen major WNT genes have been identified and it has been reported that mutations in WNT4, WNT5A, WNT7A and WNT9B may lead to Mu ¨llerian duct abnormalities (MDA) (Timmreck et al., 2003).
As one member of WNT family, WNT5A is crucial for the patterning and cytodifferentiation of the female reproductive tract as well as the limbs and craniofacial and skeletal anatomy collectively and development of the anterior– posterior axis. It signals through the noncanonical WNT5A/ ROR2 transduction pathway (Schambony and Wedlich, 2007). In mice, Wnt5a is expressed in the genital primordia at 10.5 days post coitum at a high level and is restricted to
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.10.015
Mutations in WNT5A may be not responsible for MDA in Chinese women the mesenchyme of the genital tract later (17 days post coitum) until birth (Pavlova et al., 1994). Wnt5a null mice exhibit phenotypes grossly similar to those found in humans (Yamaguchi et al., 1999). One missense mutation in exon 4 of WNT5A was detected in five females with genital hypoplasia affected with autosomal dominant Robinow syndrome (Person et al., 2010). In humans, MDA present in women as congenital absence or severe hyperplasia of the genital tract, due to embryonic Mu ¨llerian dysplasias or improper Mu ¨llerian fusion, including congenital vagina and uterus defect (MRKH syndrome), unicornuate uterus, bicornuate uterus, didelphic uterus, septate uterus and vaginal septum (Creatsas et al., 2010; Parelkar et al., 2009; Tiker et al., 2000). Previous genetic studies have shown that mutations in candidate genes, including PAX2 (Fryns et al., 1998) and LXH1 (Bernardini et al., 2009; Cheroki et al., 2006), would result in MDA, whereas mutations in WNT4 may be related to atypical MRKH syndrome (Biason et al., 2004, 2007; Philibert et al., 2008, 2011). Although extensive efforts have been channelled into elucidating the mechanisms of MDA, the majority of cases are still unexplained at the molecular level. It is presumed that other genes might contribute to the aetiology of MDA. The aim of the present study was to identify whether perturbation in WNT5A was causative for patients with MDA in Chinese women.
Materials and methods During 2005–2011, 189 Chinese women with MDA were recruited, including those with MRKH syndrome with hyperadrogenism (n = 1), uterine agenesis (n = 14), uterus didelphys (n = 37), septate uterus (n = 40), bicornuate uterus (n = 43) and unicornuate uterus (n = 54). The diagnosis was confirmed in all patients by pelvic/abdominal ultrasonography, hysteroscopy and hysterosalpingogram. Renal malformation was detected in four patients: ectopic kidney (n = 1) and unilateral renal agenesis (n = 3). As controls, 198 women without MDA confirmed through ultrasonography and hysterosalpingTable 1
165
ogram, who asked for infertility treatment because of tubal factor or male factor, were recruited. All participants had normal female secondary sexual characteristics and 46, XX karyotype. The study was approved by the Institutional Review Board of Reproductive Medicine of Shandong University (IRB reference No. 10, approval granted 3 January 2012). Informed consent was obtained from all subjects. After extracting genomic DNA from peripheral blood samples, the six exons of WNT5A were amplified by PCR and then sequenced directly by an automated sequencer (PRISM 310; Applied Biosystems). Details of PCR conditions and primers are available in Supplementary Table 1 (available online only). Statistical analyses were performed using the chi-squared test and Fisher‘s Exact test with the Statistical Package for Social Sciences version 16.0 (SPSS, USA). Statistical differences were considered statistically significant when P < 0.05.
Results Throughout the six exons and exon–intron boundaries of WNT5A, four variants were detected: three novel variants (IVS2 115G > A, IVS4 + 66T > A, IVS4 + 102G > T) and one known SNP (rs62620048) in exon 6. Comparison of genotype and allelic frequencies of IVS2 115G > A, which was found in a patient who presented with unicornuate uterus, showed significant differences (P < 0.01) compared with controls; however, the clinical meaning of this difference is not clear. The other two intronic variants IVS4 + 66T > A and IVS4 + 102G > T were found in controls at frequencies that were not significantly different from the patients (Table 1).
Discussion The present study is the first to screen variants of WNT5A in a larger cohort of women with MDA. No causative perturbations were found. Three novel variants in introns and one known SNP were identified but unlikely to be causative.
Variants of WNT5A found in Chinese women with MDA and controls.
Location and variant
Intron 2: IVS2 (novel)
115G > A
Intron 4: IVS4 + 66T > A (novel) Intron 4: IVS4 + 102G>T (novel) Exon 6: c.1181G > A (rs62620048)
Genotype frequency
Allele frequency
Genotype
MDA (n = 189)
Control (n = 198)
P
Allele
MDA (n = 189)
Control (n = 198)
P
GG GA AA TT AT AA GG GT TT GG GA AA
188 (99.5) 1 (0.5) 0 (0) 186 (98.4) 3 (1.6) 0 (0) 181 (95.8) 8 (4.2) 0 (0) 187 (98.9) 2 (1.1) 0 (0)
198 (100) 0 (0) 0 (0) 189 (95.5) 8 (4.0) 1 (0.5) 191 (96.5) 7 (3.5) 0 (0) – – –
<0.01
G A
377 (99.7) 1 (0.3)
396 (100) 0 (0)
<0.01
NS
T A
375 (99.2) 3 (0.8)
386 (97.5) 10 (2.5)
NS
NS
C A
370 (97.9) 8 (2.1)
389 (98.2) 7 (1.8)
NS
NS
G A
376 (99.5) 2 (0.5)
– –
NS
Values are n (%). MDA = Mu ¨llerian duct abnormalities; NS = not statistically significant; novel = not reported before.
166 Despite sequencing all exons, negative searches were found in WNT4 (Chang et al., 2012), as well as LHX1 (Xia et al., 2012), WNT7A (Dang et al., 2012) and PBX1 (Ma et al., 2011) in Chinese women with MDA. Given this failure to find association with any of the candidate genes potentially involved in reproductive system development, these findings also underscore the likelihood of considerable genetic and aetiological heterogeneity in MDA. The clinical significance of being heterozygous for IVS2 115G > A in MDA is unclear, and further functional studies are needed to determine its effect, if any, on Mu ¨llerian duct development.
Authors’ contributions Keliang Wu and Xinyue Chang drafted and critically revised the manuscript, performed the statistical analyses, contributed to the interpretation of the data. Deying Wei and Chengyan Xu coordinated the blood collection and storage, performed the sequencing, performed the statistical analyses. Yingying Qing contributed to the study design, interpretation of the data, drafted and critically revised the manuscript. Zi-Jiang Chen collected the clinical data, critically revised the manuscript, contributed to the study design, analysis, and supervised the research.
Conflict of interest K.W. has nothing to disclose. X.C. has nothing to disclose. D.Y. has nothing to disclose. C.X. has nothing to disclose. Y.Q. has nothing to disclose. Z.-J.C. has nothing to disclose. Financial support for this project was the National Basic Research Program of China (973 program-2011CB944502, 2012CB944700, 2010CB945002), the National Natural Science Foundation of China (81000236, 30973170), Foundation for the Author of National Excellent Doctoral Dissertation of PR China (201078), and Independent Innovation Foundation of Shandong University, IIFSDU (2012TS130). The authors report no financial or commercial conflicts of interest.
Acknowledgements This study was supported by the National Basic Research Program of China (973 program-2011CB944502, 2012CB944700, 2010CB945002), the National Natural Science Foundation of China (81000236, 30973170), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (201078) and Independent Innovation Foundation of Shandong University, IIFSDU (2012TS130).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.rbmo.2012.10.015.
References Bernardini, L., Gimelli, S., Gervasini, C., Carella, M., Baban, A., Frontino, G., Barbano, G., Divizia, M.T., Fedele, L., Novelli, A.,
K Wu et al. Be ´na, Lalatta, F., Miozzo, M., Dallapiccola, B., 2009. Recurrent microdeletion at 17q12 as a cause of Mayer– Rokitansky–Kuster–Hauser (MRKH) syndrome: two case reports. Orphanet J. Rare Dis. 4, 25. Biason, L.A., Konrad, D., Navratil, F., Schoenle, E.J., 2004. WNT4 mutation associated with Mu ¨llerian-duct regression and virilization in a 46, XX woman. N. Engl. J. Med. 351, 792–798. Biason, L.A., De Filippo, G., Konrad, D., Scarano, G., Nazzaro, A., Schoenle, E.J., 2007. WNT4 deficiency-a clinical phenotype distinct from the classic Mayer–Rokitansky–Kuster–Hauser syndrome: a case report. Hum. Reprod. 22, 224–229. Chang, X., Qin, Y., Xu, C., Li, G., Zhao, X., Chen, Z.J., 2012. Mutations in WNT4 are not responsible for Mu ¨llerian duct abnormalities in Chinese women. Reprod. Biomed. Online 24, 630–633. Cheroki, C., Krepischi-Santos, A.C., Rosenberg, C., Jehee, F.S., Mingroni-Netto, R.C., Pavanello Filho, I., Zanforlin Filho, S., Kim, C.A., Bagnoli, V.R., Mendonc ¸a, B.B., Szuhai, K., Otto, P.A., 2006. Report of a del22q11 in a patient with Mayer– Rokitansky–Ku ¨ster–Hauser (MRKH) anomaly and exclusion of WNT-4, RAR-gamma, and RXR-alpha as major genes determining MRKH anomaly in a study of 25 affected women. Am. J. Med. Genet. A. 140, 1339–1342. Creatsas, G., Deligeoroglou, E., Christopoulos, P., 2010. Creation of a neovagina after creatsas modification of Williams vaginoplasty for the treatment of 200 patients with Mayer–Rokitansky– Kuster–Hauser syndrome. Fertil. Steril. 94, 1848–1852. Dang, Y., Qin, Y., Tang, R., Mu, Y., Li, G., Xia, M., Chen, Z.J., 2012. Variants of the WNT7A gene in Chinese patients with Mu ¨llerian duct abnormalities. Fertil. Steril. 97, 391–394. Fryns, J.P., Lingen, C.V., Devriendt, K., Legius, E., Raus, P., 1998. Two adult females with a distinct familial mental retardation syndrome: non-progressive neurological symptoms with ataxia and hypotonia, similar facial appearance, hypergonadotrophic hypogonadism, and retinal dystrophy. J. Med. Genet. 35, 333–335. Ma, J., Qin, Y., Liu, W., Duan, H., Xia, M., Chen, Z.J., 2011. Analysis of PBX1 mutations in 192 Chinese women with Mu ¨llerian duct abnormalities. Fertil. Steril. 95, 2615–2617. Parelkar, S.V., Gupta, R.K., Oak, S., Sanghvi, B., Kaltari, D., Patil, R.S., Prakash, A., Shimoga, P., 2009. Laparoscopic management of persistent mu ¨llerian duct syndrome. J. Pediatr. Surg. 44, 1–3. Pavlova, A., Boutin, E., Cunha, G., Sassoon, D., 1994. Msx1 (Hox-7, 1) in the adult mouse uterus:cellular interactions underlying regulation of expression. Development 120, 335–345. Person, A.D., Beiraghi, S., Sieben, C.M., Hermanson, S., Neumann, A.N., Robu, M.E., Schleiffarth, J.R., Billington Jr, C.J., van Bokhoven, H., Hoogeboom, J.M., Mazzeu, J.F., Petryk, A., Schimmenti, L.A., Brunner, H.G., Ekker, S.C., Lohr, J.L., 2010. WNT5A mutations in patients with autosomal dominant Robinow syndrome. Dev. Dyn. 239, 327–337. Philibert, P., Biason, L.A., Rouzier, R., Pienkowski, C., Paris, F., Konrad, D., Schoenle, E., Sultan, C., 2008. Identification and functional analysis of a new WNT4 gene mutation among 28 adolescent girls with primary amenorrhea and Mu ¨llerian duct abnormalities: a French collaborative study. J. Clin. Endocrinol. Metab. 93, 895–900. Philibert, P., Biason, L.A., Gueorguieva, I., Stuckens, C., Pienkowski, C., Lebon-Labich, B., Paris, F., Sultan, C., 2011. Molecular analysis of WNT4 gene in four adolescent girls with mu ¨llerian duct abnormality and hyperandrogenism (atypical Mayer–Rokitansky–Kuster–Hausersyndrome). Fertil. Steril. 95, 2683– 2686. Schambony, A., Wedlich, D., 2007. Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway. Dev. Cell 12, 779–792.
Mutations in WNT5A may be not responsible for MDA in Chinese women ˘isß, T., 2000. Familial Tiker, F., Yildirim, S.V., Barutc ¸u, O., Bag mu ¨llerian agenesis. Turk. J. Pediatr. 42, 322–324. Timmreck, L.S., Pan, H.A., Reindollar, R.H., Gray, M.R., 2003. WNT7A mutations in patients with Mullerian duct abnormalities. J. Pediatr. Adolesc. Gynecol. 16, 217–221. Xia, M., Zhao, H., Qin, Y., Mu, Y., Wang, J., Bian, Y., Ma, J., Chen, Z.J., 2012. LHX1 mutation screening in 96 patients with mu ¨llerian duct abnormalities. Fertil. Steril. 97, 682–685. Yamaguchi, T.P., Bradley, A., McMahon, A.P., Jones, S., 1999. A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development 126, 1211–1223.
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Declaration: We declare that this paper is our original unpublished work and it has not been submitted to any other journal for reviews. Its publication is approved by all authors and explicitly by the responsible authorities where the work was carried out and will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyright-holder. Received 21 July 2012; refereed 15 October 2012; accepted 16 October 2012.
www.sciencedirect.com www.rbmonline.com
ARTICLE
Lack of association of WNT5A mutations with ¨llerian duct abnormalities Mu Keliang Wu a,b,1, Xinyue Chang a,b,1, Deying Wei c, Chengyan Xu Yingying Qin a,b,*, Zi-Jiang Chen a,b,d
a,b
,
a Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, China; b The Key laboratory for Reproductive Endocrinology of Ministry of Education, Shandong Provincial Key Laboratory of Reproductive Medicine China, Jinan, China; c Department of Obstetrics and Gynecology, Provincial Hospital Affiliated to Shandong University, Jinan, China; d Center for Reproductive Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
* Corresponding author. E-mail address: [email protected] (Y Qin). 1 These authors contribute equally to this work.
Keliang Wu was born on 24 November 1981 in Yancheng, Jiangsu, PR China. In 2007, he graduated from Shandong University with a Masters in science majored in developmental biology. After graduation, he worked as an embryologist in Center for Reproductive Medicine, Provincial Hospital affiliated to Shandong University. From 2007 to 2010, he applied five national patents from China referring to assisted reproduction technology.
Abstract The aim of this study was to determine if mutations in WNT5A contribute to the aetiology of Mu ¨llerian duct abnormalities
(MDA) in 189 Chinese women. Three novel single-nucleotide polymorphisms (SNP; IVS2 115G > A, IVS4 + 66T > A, IVS4 + 102G > T) in introns 2 and 4 as well as one known SNP (rs62620048) in exon 6 were detected, but no causal mutations were observed. The results suggested that mutations in WNT5A may be not responsible for MDA in Chinese women. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: Mu ¨llerian duct abnormalities, mutation, single-nucleotide polymorphisms, WNT5A
Introduction The WNT family (wingless-type MMTV integration site family) is the major genetic focus for finding genes whose perturbations cause uterus anomalies. Nineteen major WNT genes have been identified and it has been reported that mutations in WNT4, WNT5A, WNT7A and WNT9B may lead to Mu ¨llerian duct abnormalities (MDA) (Timmreck et al., 2003).
As one member of WNT family, WNT5A is crucial for the patterning and cytodifferentiation of the female reproductive tract as well as the limbs and craniofacial and skeletal anatomy collectively and development of the anterior– posterior axis. It signals through the noncanonical WNT5A/ ROR2 transduction pathway (Schambony and Wedlich, 2007). In mice, Wnt5a is expressed in the genital primordia at 10.5 days post coitum at a high level and is restricted to
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.10.015
Mutations in WNT5A may be not responsible for MDA in Chinese women the mesenchyme of the genital tract later (17 days post coitum) until birth (Pavlova et al., 1994). Wnt5a null mice exhibit phenotypes grossly similar to those found in humans (Yamaguchi et al., 1999). One missense mutation in exon 4 of WNT5A was detected in five females with genital hypoplasia affected with autosomal dominant Robinow syndrome (Person et al., 2010). In humans, MDA present in women as congenital absence or severe hyperplasia of the genital tract, due to embryonic Mu ¨llerian dysplasias or improper Mu ¨llerian fusion, including congenital vagina and uterus defect (MRKH syndrome), unicornuate uterus, bicornuate uterus, didelphic uterus, septate uterus and vaginal septum (Creatsas et al., 2010; Parelkar et al., 2009; Tiker et al., 2000). Previous genetic studies have shown that mutations in candidate genes, including PAX2 (Fryns et al., 1998) and LXH1 (Bernardini et al., 2009; Cheroki et al., 2006), would result in MDA, whereas mutations in WNT4 may be related to atypical MRKH syndrome (Biason et al., 2004, 2007; Philibert et al., 2008, 2011). Although extensive efforts have been channelled into elucidating the mechanisms of MDA, the majority of cases are still unexplained at the molecular level. It is presumed that other genes might contribute to the aetiology of MDA. The aim of the present study was to identify whether perturbation in WNT5A was causative for patients with MDA in Chinese women.
Materials and methods During 2005–2011, 189 Chinese women with MDA were recruited, including those with MRKH syndrome with hyperadrogenism (n = 1), uterine agenesis (n = 14), uterus didelphys (n = 37), septate uterus (n = 40), bicornuate uterus (n = 43) and unicornuate uterus (n = 54). The diagnosis was confirmed in all patients by pelvic/abdominal ultrasonography, hysteroscopy and hysterosalpingogram. Renal malformation was detected in four patients: ectopic kidney (n = 1) and unilateral renal agenesis (n = 3). As controls, 198 women without MDA confirmed through ultrasonography and hysterosalpingTable 1
165
ogram, who asked for infertility treatment because of tubal factor or male factor, were recruited. All participants had normal female secondary sexual characteristics and 46, XX karyotype. The study was approved by the Institutional Review Board of Reproductive Medicine of Shandong University (IRB reference No. 10, approval granted 3 January 2012). Informed consent was obtained from all subjects. After extracting genomic DNA from peripheral blood samples, the six exons of WNT5A were amplified by PCR and then sequenced directly by an automated sequencer (PRISM 310; Applied Biosystems). Details of PCR conditions and primers are available in Supplementary Table 1 (available online only). Statistical analyses were performed using the chi-squared test and Fisher‘s Exact test with the Statistical Package for Social Sciences version 16.0 (SPSS, USA). Statistical differences were considered statistically significant when P < 0.05.
Results Throughout the six exons and exon–intron boundaries of WNT5A, four variants were detected: three novel variants (IVS2 115G > A, IVS4 + 66T > A, IVS4 + 102G > T) and one known SNP (rs62620048) in exon 6. Comparison of genotype and allelic frequencies of IVS2 115G > A, which was found in a patient who presented with unicornuate uterus, showed significant differences (P < 0.01) compared with controls; however, the clinical meaning of this difference is not clear. The other two intronic variants IVS4 + 66T > A and IVS4 + 102G > T were found in controls at frequencies that were not significantly different from the patients (Table 1).
Discussion The present study is the first to screen variants of WNT5A in a larger cohort of women with MDA. No causative perturbations were found. Three novel variants in introns and one known SNP were identified but unlikely to be causative.
Variants of WNT5A found in Chinese women with MDA and controls.
Location and variant
Intron 2: IVS2 (novel)
115G > A
Intron 4: IVS4 + 66T > A (novel) Intron 4: IVS4 + 102G>T (novel) Exon 6: c.1181G > A (rs62620048)
Genotype frequency
Allele frequency
Genotype
MDA (n = 189)
Control (n = 198)
P
Allele
MDA (n = 189)
Control (n = 198)
P
GG GA AA TT AT AA GG GT TT GG GA AA
188 (99.5) 1 (0.5) 0 (0) 186 (98.4) 3 (1.6) 0 (0) 181 (95.8) 8 (4.2) 0 (0) 187 (98.9) 2 (1.1) 0 (0)
198 (100) 0 (0) 0 (0) 189 (95.5) 8 (4.0) 1 (0.5) 191 (96.5) 7 (3.5) 0 (0) – – –
<0.01
G A
377 (99.7) 1 (0.3)
396 (100) 0 (0)
<0.01
NS
T A
375 (99.2) 3 (0.8)
386 (97.5) 10 (2.5)
NS
NS
C A
370 (97.9) 8 (2.1)
389 (98.2) 7 (1.8)
NS
NS
G A
376 (99.5) 2 (0.5)
– –
NS
Values are n (%). MDA = Mu ¨llerian duct abnormalities; NS = not statistically significant; novel = not reported before.
166 Despite sequencing all exons, negative searches were found in WNT4 (Chang et al., 2012), as well as LHX1 (Xia et al., 2012), WNT7A (Dang et al., 2012) and PBX1 (Ma et al., 2011) in Chinese women with MDA. Given this failure to find association with any of the candidate genes potentially involved in reproductive system development, these findings also underscore the likelihood of considerable genetic and aetiological heterogeneity in MDA. The clinical significance of being heterozygous for IVS2 115G > A in MDA is unclear, and further functional studies are needed to determine its effect, if any, on Mu ¨llerian duct development.
Authors’ contributions Keliang Wu and Xinyue Chang drafted and critically revised the manuscript, performed the statistical analyses, contributed to the interpretation of the data. Deying Wei and Chengyan Xu coordinated the blood collection and storage, performed the sequencing, performed the statistical analyses. Yingying Qing contributed to the study design, interpretation of the data, drafted and critically revised the manuscript. Zi-Jiang Chen collected the clinical data, critically revised the manuscript, contributed to the study design, analysis, and supervised the research.
Conflict of interest K.W. has nothing to disclose. X.C. has nothing to disclose. D.Y. has nothing to disclose. C.X. has nothing to disclose. Y.Q. has nothing to disclose. Z.-J.C. has nothing to disclose. Financial support for this project was the National Basic Research Program of China (973 program-2011CB944502, 2012CB944700, 2010CB945002), the National Natural Science Foundation of China (81000236, 30973170), Foundation for the Author of National Excellent Doctoral Dissertation of PR China (201078), and Independent Innovation Foundation of Shandong University, IIFSDU (2012TS130). The authors report no financial or commercial conflicts of interest.
Acknowledgements This study was supported by the National Basic Research Program of China (973 program-2011CB944502, 2012CB944700, 2010CB945002), the National Natural Science Foundation of China (81000236, 30973170), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (201078) and Independent Innovation Foundation of Shandong University, IIFSDU (2012TS130).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.rbmo.2012.10.015.
References Bernardini, L., Gimelli, S., Gervasini, C., Carella, M., Baban, A., Frontino, G., Barbano, G., Divizia, M.T., Fedele, L., Novelli, A.,
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Declaration: We declare that this paper is our original unpublished work and it has not been submitted to any other journal for reviews. Its publication is approved by all authors and explicitly by the responsible authorities where the work was carried out and will not be published elsewhere including electronically in the same form, in English or in any other language, without the written consent of the copyright-holder. Received 21 July 2012; refereed 15 October 2012; accepted 16 October 2012.