ARTICLE IN PRESS Reproductive BioMedicine Online (2014) ■■, ■■–■■
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
Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome Yuqian Cui a,b,c,d,e,f, Shigang Zhao a,b,c,d,e, Han Zhao b,c,d,e, Yue Lv b,c,d,e, Mengru Yu b,c,d,e, Yu Wang g, Zi-Jiang Chen a,b,c,d,e,* a
Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, 200127, China; b Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, 324 Jingwu Road, Jinan 250021, China; c National Research Center for Assisted Reproductive Technology and Reproductive Genetics, 324 Jingwu Road, Jinan 250021, China; d The Key Laboratory for Reproductive Endocrinology of Ministry of Education, 324 Jingwu Road, Jinan 250021, China; e Shandong Provincial Key Laboratory of Reproductive Medicine, 324 Jingwu Road, Jinan 250021, China; f Center for Reproductive Medicine, Qilu Hospital of Shandong University, 107 Wenhuaxi Road, Jinan 250012, China; g Institute of Obstetrics and Gynecologic Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China * Corresponding author. E-mail address: [email protected] (Z-J Chen). Dr Yuqian Cui received her MD and PhD from the School of Medicine, Shandong University in 2012. She is now pursuing her postdoctoral training at the center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. Yuqian Cui has received both clinical and laboratory training. Her current academic focus is on the pathogenesis and mechanism of polycystic ovary syndrome.
A previous genome-wide association study of polycystic ovary syndrome (PCOS) identified several susceptibility loci. TOX3 is the nearest gene to signal rs4784165. In the present study, all exons and exon–intron boundaries of TOX3 were amplified and sequenced in 200 Chinese women with PCOS. A 3-bp nucleotide deletion of CAG repeat and two known single nucleotide polymorphisms were identified. No plausible pathogenic mutations were detected. The results suggest that mutations in TOX3 are not common in Chinese Han women with PCOS. Abstract
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Keywords:, mutation, TOX3, polycystic ovary syndrome, single-nucleotide polymorphism
http://dx.doi.org/10.1016/j.rbmo.2014.08.004 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yuqian Cui, et al., Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome, Reproductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.08.004
ARTICLE IN PRESS 2
Y Cui et al.
Introduction Polycystic ovary syndrome (PCOS) is a heterogeneous disease, and the clinical presentation may vary from patient to patient (Alexander et al., 2009). Familiar aggregation and heritable tendencies of PCOS suggest that genetic factors might participate in the development of the disease (Urbanek, 2007; Vink et al., 2006). To unravel underlying common genetic risk factors for PCOS, this study group conducted a large genome-wide association study (Shi et al., 2012), and found a suggestive significant association between PCOS and a novel genetic locus on 16q12.1 (rs4784165, OR = 1.15, P = 3.64 × 10−11). TOX3 is the nearest gene to this signal. The following genotype-phenotype correlations of rs4784165 in a large cohort of Han Chinese women showed that it was associated with hyperandrogenaemia (unpublished data), and that GT + TT genotypes had significantly higher values for modified Ferriman–Gallwey score than did the GG genotype (P < 0.01). The underlying cause, however, remains unexplained in the pathogenesis of PCOS. The TOX3 gene, also known as TNRC9 or CAGF9, was first identified in a screen for transcripts containing trinucleotide (CAG) repeat expansions (Margolis et al., 1997). It contains a nuclear localization signal and a high-mobility group box domain followed by a C-terminal polyglutamine stretch. The high-mobility group box proteins can modify chromatin structure by bending and unwinding DNA (O’Flaherty and Kaye, 2003). It is involved in the regulation of calcium-dependent transcription and interacts with cAMP-response-elementbinding protein CREB and CBP (CREB-binding protein) (Yuan et al., 2009). Additionally, TOX3 can interact with CITED1 and increase transcription (Dittmer et al., 2010). CITED is a transcription co-regulator that enhances the activity of transcription factor such as oestrogen receptor (Yahata et al., 2001) and SMAD4 (Shioda et al., 1998). In a study that screened TOX3 for mutations in 133 breast tumours, four mutations were identified (three missense, one in-frame deletion of 30 base pairs) (Jones et al., 2013). TOX3 coding region was screened in 200 Chinese women with PCOS to identify the presence of a mutation that alters the amino acid sequence.
Materials and methods A total of 200 Chinese women with PCOS were recruited from the center for Reproductive Medicine, Provincial Hospital affiliated to Shandong University. The 2003 Rotterdam criteria were used for diagnosing PCOS (Rotterdam ESHRE/ASRM- Sponsored PCOS Consensus Workshop Group, 2004), with at least two of the following three features required for diagnosis: oligomenorrhoea or amenorrhoea; clinical or biochemical hyperandrogenism; and polycystic ovaries on ultrasound. Other related diseases, such as adrenal congenital hyperplasia, Cushing syndrome and androgensecreting tumours were excluded. The study was approved by the Institutional Review Board of Reproductive Medicine of Shandong University on 20 January 2014 (IRB reference number 12). Each patient signed a written informed consent. Genomic DNA was obtained from peripheral blood samples using a DNA blood kit (Qiagen, USA) according to the
manufacturer’s protocol. The coding regions of TOX3 were amplified using polymerase chain reaction (PCR) with seven pairs of TOX3 specific primers. Direct sequencing was carried out using an automated sequencer (ABI PRISM 310; Applied Biosystems). Primer sequences and PCR conditions are presented in Supplementary Tables S1 and 2. Chi-squared test was used for statistical analysis, using the Statistical Package for Social Sciences version 13.0 (SPSS Inc., USA). Statistical differences were considered significant when P < 0.05. The HapMap project (http:// www.asia.ensembl.org/Homo_sapiens/Info/Index) was used to compare the PCOS population with the general population of Chinese women.
Results All seven exons and exon–intron boundaries of the TOX3 gene were screened for variations. No significant mutations were found in exons 1 to 5 compared with the normal sequence available in the National Center for Biotechnology database. Two known single-nucleotide polymorphisms (SNP), rs3743796 and rs3743797 in exon 6, and a 3-bp nucleotide deletion of CAG repeat in exon 7, were identified. Both of the two SNP are known in the National Center for Biotechnology of Information SNP database (www.ncbi.nlm.nih.gov/snp). They do not alter the amino acid sequence. Comparisons of genotype and allelic frequencies between PCOS cases and the general population of Chinese women showed no significant differences for either SNP (Table 1). The 3-bp nucleotide deletion of CAG repeat in exon 7 did not cause a typical frame-shift mutation but resulted in the deletion of one amino acid (Gln) after translation (Figure 1). This is not predicted to change the folding of TOX3 according to PSIPRED (Protein Structure Prediction Server; http:// bioinf.cs.ucl.ac.uk/psipred/) and SIFT (http://sift.jcvi.org/ www/SIFT_chr_coords_submit.html).
Discussion As far as is known, the present study is the first to investigate mutations of TOX3 in women with PCOS. The results identified two known SNP, and 3-bp nucleotide deletion of CAG repeat. None of these, however, offers an explanation of the disease. The allele and genotype frequencies of the two SNP (rs3743796 and rs3743797) in TOX3 were not significantly different in this study. A 3-bp nucleotide deletion of CAG repeat in exon 7 was identified in one out of 200 PCOS patients. The carrier was a 35-year-old woman referred to our hospital with primary infertility. According to the Rotterdam criteria, she was diagnosed with PCOS. After two cycles of standard long protocol ovarian stimulation, she conceived. Since the early 1990s, a new class of molecular disease has been characterized based upon the presence of unstable and abnormal expansions of DNA-triplets (trinucleotides). The first triplet disease to be identified was fragile X syndrome. Expansion of polyglutamine-encoding CAG trinucleotide repeats has been identified as the pathogenic mutation in genes
Please cite this article in press as: Yuqian Cui, et al., Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome, Reproductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.08.004
ARTICLE IN PRESS Mutational analysis of TOX3
3
Table 1 Genotype frequencies of single nucleotide polymorphisms rs3743796 and rs3743797 in Chinese women with polycystic ovary syndrome (n = 200). Genotype frequency
Allele frequency
dbSNP ID
PCOS (n, %)a
CHW (%)b
PCOS (%)a
Chinese women (%)b
Exon 6
rs3743796
G(84.47) A(15.53)
rs3743797
72.82 23.31 3.88 72.82 23.31 3.88
G(78.00) A(22.00)
Exon 6
GG(120, 60.00) GA(72, 36.00) AA(8, 4.00) GG(120, 60.00) GA(72, 36.00) AA(8, 4.00)
G(78.00) A(22.00)
G(84.47) A(15.53)
a
Present study. International HapMap project database (CSHL-HAPMAP). PCOS = polycystic ovary syndrome.
b
associated with neurodegenerative disorders (Butland et al., 2007). Shorter androgen receptor CAG number is indicative of increased androgen sensitivity and ovarian hyperandrogenism (Ibáñez et al., 2003; Vottero et al., 2006). Although the deletion of CAG repeat in this study did not cause a typical frame-shift mutation, it does result in the deletion of one amino acid (Gln) after translation. The deletion could prove significant once future samples in other ethnic groups are studied. Mutations in the coding region of TOX3 are not common in Chinese Han women with PCOS. Future studies in larger cohorts from different ethnic populations are necessary to determine the role played by TOX3 in PCOS.
Acknowledgements This study was supported by a grant from the National Basic Research Program of China (973 program: 2012CB944700); the National Natural Science Foundation of China (81000236, 81300459); China Postdoctoral Science Foundation (2012M520909). The authors thank Guangyu Li, Shizhen Su, Zhao Wang and Changming Zhang from Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University for their technical guidance. They also appreciate the patients’ participation and their colleagues for patient examination and sample collection.
Figure 1 (A) Deletion and single nucleotide polymorphisms in TOX3 exons. (A) The 3-bp deletion in TOX3 sequencing test result. The deleted base sequence is shown above the chromatogram; the normal sequence (wild sequence) is shown beneath the chromatogram; (B) Localization of the single nucleotide polymorphisms and deletion in the TOX3 gene coding sequence. The two SNPs were located on exon 6 and 3-bp deletion was located on exon 7 of TOX3 gene.
Please cite this article in press as: Yuqian Cui, et al., Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome, Reproductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.08.004
ARTICLE IN PRESS 4
Y Cui et al.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.rbmo.2014.08.004.
References Alexander, C.J., Tangchitnob, E.P., Lepro, N.E., 2009. Polycystic ovary syndrome: a major unrecognized cardiovascular risk factor in women. Rev. Obstet Gynecol. 2, 232–239. Butland, S.L., Devon, R.S., Huang, Y., Mead, C.L., Meynert, A.M., Neal, S.J., Lee, S.S., Wikinson, A., Yang, G.S., Yuen, M.M., Hayden, M.R., Holt, R.A., Leavitt, B.P., Ouellette, B.F., 2007. CAG-encoded polyglutamine length polymorphism in the human genome. BMC Genomics 8, 126. Dittmer, S., Kovacs, Z., Yuan, S.H., Siszler, G., Kögl, M., Summer, H., Geerts, A., Golz, S., Shioda, T., Methner, A., 2010. TOX3 is a neuronal survival factor that induces transcription depending on the presence of CITED1 or phosphorylated CREB in the transcriptionally active complex. J. Cell Sci. 124, 252–260. Ibáñez, L., Ong, K.K., Mongan, N., Jääskeläinen, J., Marcos, M.V., Hughes, I.A., De Zegher, F., Dunger, D.B., 2003. Androgen receptor gene CAG repeat polymorphism in the development of ovarian hyperandrogenism. J. Clin. Endocrinol. Metab. 88, 3333– 3338. Jones, J.O., Chin, S.F., Wong-Taylor, L.A., Leaford, D., Ponder, B.A., Caldas, C., Maia, A.T., 2013. TOX3 mutations in breast cancer. PLoS ONE 8, e74102. Margolis, R.L., Abraham, M.R., Gatchell, S.B., Li, S.H., Kidwai, A.S., Breschel, T.S., Stine, O.C., Callahan, C., McLnnis, M.G., Ross, C.A., 1997. cDNAs with long CAG trinucleotide repeats from human brain. Hum. Genet. 100, 114–122. O’Flaherty, E., Kaye, J., 2003. TOX defines a conserved subfamily of HMG-box proteins. BMC Genomics 4, 13. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004. Revised 2003 consensus on diagnostic criteria and longterm health risks related to polycystic ovary syndrome. Fertil. Steril. 81, 19–25.
Shi, Y., Zhao, H., Shi, Y., Cao, Y., Yang, D., Li, Z., Zhang B., Liang X., Li T., Chen J., Shen J., Zhao J., You L., Gao X., Zhu D., Zhao X., Yan Y., Qin Y., Li W., Yan J., Wang Q., Zhao J., Geng L., Ma J., Zhao Y., He G., Zhang A., Zou S., Yang A., Liu J., Li W., Li B., Wan C., Qin Y., Shi J., Yang J., Jiang H., Xu J.E., Qi X., Sun Y., Zhang Y., Hao C., Ju X., Zhao D., Ren C.E., Li X., Zhang W., Zhang Y., Zhang J., Wu D., Zhang C., He L., Chen Z.J., 2012. Genome-wide association study identifies eight new risk loci for polycystic ovary syndrome. Nat. Genet. 44, 1020– 1025. Shioda, T., Lechleider, R.J., Dunwoodie, S.L., Li, H., Yahata, T., de Caestecker, M.P., Fenner, M.H., Roberts, A.B. 1998. Transcriptional activating activity of Smad4: roles of SMAD heterooligomerization and enhancement by an associating transactivator. Proc Natl Acad Sci USA. 95, 9785–9790. Urbanek, M., 2007. The genetics of the polycystic ovary syndrome. Nat. Clin. Pract. Endocrinol. Metab. 3, 3–111. Vink, J., Sadrzadeh, S.M., Lambalk, C.B., Boomsma, D.I., 2006. Heritability of polycystic ovary syndrome in a Dutch twin-family study. J. Clin. Endocrinol. Metab. 91, 2100–2104. Vottero, A., Capelletti, M., Giuliodori, S., Viani, I., Ziveri, M., Neri, T.M., Bernasconi, S., Ghizzoni, L., 2006. Decreased androgen receptor gene methylation in premature pubarche: a novel pathogenetic mechanism? J. Clin. Endocrinol. Metab. 91, 968– 972. Yahata, T., Shao, W., Endoh, H., Hur, J., Coser, K.R., Sun, H., Ueda Y., Kato S., Isselbacher K.J., Brown M., Shioda T., 2001. Selective coactivation of estrogen-dependent transcription by CITED CBP/p300-binding protein. GenesDev. 15, 2598–2612. Yuan, S.H., Qiu, Z., Ghosh, A., 2009. TOX3 regulates calciumdependent transcription in neurons. Proc Natl Acad Sci USA. 106, 2909–2914.
Declaration: The authors report no financial or commercial conflicts of interest.
Received 30 March 2014; refereed 25 July 2014; accepted 12 August 2014.
Please cite this article in press as: Yuqian Cui, et al., Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome, Reproductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.08.004
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
Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome Yuqian Cui a,b,c,d,e,f, Shigang Zhao a,b,c,d,e, Han Zhao b,c,d,e, Yue Lv b,c,d,e, Mengru Yu b,c,d,e, Yu Wang g, Zi-Jiang Chen a,b,c,d,e,* a
Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, 200127, China; b Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, 324 Jingwu Road, Jinan 250021, China; c National Research Center for Assisted Reproductive Technology and Reproductive Genetics, 324 Jingwu Road, Jinan 250021, China; d The Key Laboratory for Reproductive Endocrinology of Ministry of Education, 324 Jingwu Road, Jinan 250021, China; e Shandong Provincial Key Laboratory of Reproductive Medicine, 324 Jingwu Road, Jinan 250021, China; f Center for Reproductive Medicine, Qilu Hospital of Shandong University, 107 Wenhuaxi Road, Jinan 250012, China; g Institute of Obstetrics and Gynecologic Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China * Corresponding author. E-mail address: [email protected] (Z-J Chen). Dr Yuqian Cui received her MD and PhD from the School of Medicine, Shandong University in 2012. She is now pursuing her postdoctoral training at the center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. Yuqian Cui has received both clinical and laboratory training. Her current academic focus is on the pathogenesis and mechanism of polycystic ovary syndrome.
A previous genome-wide association study of polycystic ovary syndrome (PCOS) identified several susceptibility loci. TOX3 is the nearest gene to signal rs4784165. In the present study, all exons and exon–intron boundaries of TOX3 were amplified and sequenced in 200 Chinese women with PCOS. A 3-bp nucleotide deletion of CAG repeat and two known single nucleotide polymorphisms were identified. No plausible pathogenic mutations were detected. The results suggest that mutations in TOX3 are not common in Chinese Han women with PCOS. Abstract
© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Keywords:, mutation, TOX3, polycystic ovary syndrome, single-nucleotide polymorphism
http://dx.doi.org/10.1016/j.rbmo.2014.08.004 1472-6483/© 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yuqian Cui, et al., Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome, Reproductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.08.004
ARTICLE IN PRESS 2
Y Cui et al.
Introduction Polycystic ovary syndrome (PCOS) is a heterogeneous disease, and the clinical presentation may vary from patient to patient (Alexander et al., 2009). Familiar aggregation and heritable tendencies of PCOS suggest that genetic factors might participate in the development of the disease (Urbanek, 2007; Vink et al., 2006). To unravel underlying common genetic risk factors for PCOS, this study group conducted a large genome-wide association study (Shi et al., 2012), and found a suggestive significant association between PCOS and a novel genetic locus on 16q12.1 (rs4784165, OR = 1.15, P = 3.64 × 10−11). TOX3 is the nearest gene to this signal. The following genotype-phenotype correlations of rs4784165 in a large cohort of Han Chinese women showed that it was associated with hyperandrogenaemia (unpublished data), and that GT + TT genotypes had significantly higher values for modified Ferriman–Gallwey score than did the GG genotype (P < 0.01). The underlying cause, however, remains unexplained in the pathogenesis of PCOS. The TOX3 gene, also known as TNRC9 or CAGF9, was first identified in a screen for transcripts containing trinucleotide (CAG) repeat expansions (Margolis et al., 1997). It contains a nuclear localization signal and a high-mobility group box domain followed by a C-terminal polyglutamine stretch. The high-mobility group box proteins can modify chromatin structure by bending and unwinding DNA (O’Flaherty and Kaye, 2003). It is involved in the regulation of calcium-dependent transcription and interacts with cAMP-response-elementbinding protein CREB and CBP (CREB-binding protein) (Yuan et al., 2009). Additionally, TOX3 can interact with CITED1 and increase transcription (Dittmer et al., 2010). CITED is a transcription co-regulator that enhances the activity of transcription factor such as oestrogen receptor (Yahata et al., 2001) and SMAD4 (Shioda et al., 1998). In a study that screened TOX3 for mutations in 133 breast tumours, four mutations were identified (three missense, one in-frame deletion of 30 base pairs) (Jones et al., 2013). TOX3 coding region was screened in 200 Chinese women with PCOS to identify the presence of a mutation that alters the amino acid sequence.
Materials and methods A total of 200 Chinese women with PCOS were recruited from the center for Reproductive Medicine, Provincial Hospital affiliated to Shandong University. The 2003 Rotterdam criteria were used for diagnosing PCOS (Rotterdam ESHRE/ASRM- Sponsored PCOS Consensus Workshop Group, 2004), with at least two of the following three features required for diagnosis: oligomenorrhoea or amenorrhoea; clinical or biochemical hyperandrogenism; and polycystic ovaries on ultrasound. Other related diseases, such as adrenal congenital hyperplasia, Cushing syndrome and androgensecreting tumours were excluded. The study was approved by the Institutional Review Board of Reproductive Medicine of Shandong University on 20 January 2014 (IRB reference number 12). Each patient signed a written informed consent. Genomic DNA was obtained from peripheral blood samples using a DNA blood kit (Qiagen, USA) according to the
manufacturer’s protocol. The coding regions of TOX3 were amplified using polymerase chain reaction (PCR) with seven pairs of TOX3 specific primers. Direct sequencing was carried out using an automated sequencer (ABI PRISM 310; Applied Biosystems). Primer sequences and PCR conditions are presented in Supplementary Tables S1 and 2. Chi-squared test was used for statistical analysis, using the Statistical Package for Social Sciences version 13.0 (SPSS Inc., USA). Statistical differences were considered significant when P < 0.05. The HapMap project (http:// www.asia.ensembl.org/Homo_sapiens/Info/Index) was used to compare the PCOS population with the general population of Chinese women.
Results All seven exons and exon–intron boundaries of the TOX3 gene were screened for variations. No significant mutations were found in exons 1 to 5 compared with the normal sequence available in the National Center for Biotechnology database. Two known single-nucleotide polymorphisms (SNP), rs3743796 and rs3743797 in exon 6, and a 3-bp nucleotide deletion of CAG repeat in exon 7, were identified. Both of the two SNP are known in the National Center for Biotechnology of Information SNP database (www.ncbi.nlm.nih.gov/snp). They do not alter the amino acid sequence. Comparisons of genotype and allelic frequencies between PCOS cases and the general population of Chinese women showed no significant differences for either SNP (Table 1). The 3-bp nucleotide deletion of CAG repeat in exon 7 did not cause a typical frame-shift mutation but resulted in the deletion of one amino acid (Gln) after translation (Figure 1). This is not predicted to change the folding of TOX3 according to PSIPRED (Protein Structure Prediction Server; http:// bioinf.cs.ucl.ac.uk/psipred/) and SIFT (http://sift.jcvi.org/ www/SIFT_chr_coords_submit.html).
Discussion As far as is known, the present study is the first to investigate mutations of TOX3 in women with PCOS. The results identified two known SNP, and 3-bp nucleotide deletion of CAG repeat. None of these, however, offers an explanation of the disease. The allele and genotype frequencies of the two SNP (rs3743796 and rs3743797) in TOX3 were not significantly different in this study. A 3-bp nucleotide deletion of CAG repeat in exon 7 was identified in one out of 200 PCOS patients. The carrier was a 35-year-old woman referred to our hospital with primary infertility. According to the Rotterdam criteria, she was diagnosed with PCOS. After two cycles of standard long protocol ovarian stimulation, she conceived. Since the early 1990s, a new class of molecular disease has been characterized based upon the presence of unstable and abnormal expansions of DNA-triplets (trinucleotides). The first triplet disease to be identified was fragile X syndrome. Expansion of polyglutamine-encoding CAG trinucleotide repeats has been identified as the pathogenic mutation in genes
Please cite this article in press as: Yuqian Cui, et al., Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome, Reproductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.08.004
ARTICLE IN PRESS Mutational analysis of TOX3
3
Table 1 Genotype frequencies of single nucleotide polymorphisms rs3743796 and rs3743797 in Chinese women with polycystic ovary syndrome (n = 200). Genotype frequency
Allele frequency
dbSNP ID
PCOS (n, %)a
CHW (%)b
PCOS (%)a
Chinese women (%)b
Exon 6
rs3743796
G(84.47) A(15.53)
rs3743797
72.82 23.31 3.88 72.82 23.31 3.88
G(78.00) A(22.00)
Exon 6
GG(120, 60.00) GA(72, 36.00) AA(8, 4.00) GG(120, 60.00) GA(72, 36.00) AA(8, 4.00)
G(78.00) A(22.00)
G(84.47) A(15.53)
a
Present study. International HapMap project database (CSHL-HAPMAP). PCOS = polycystic ovary syndrome.
b
associated with neurodegenerative disorders (Butland et al., 2007). Shorter androgen receptor CAG number is indicative of increased androgen sensitivity and ovarian hyperandrogenism (Ibáñez et al., 2003; Vottero et al., 2006). Although the deletion of CAG repeat in this study did not cause a typical frame-shift mutation, it does result in the deletion of one amino acid (Gln) after translation. The deletion could prove significant once future samples in other ethnic groups are studied. Mutations in the coding region of TOX3 are not common in Chinese Han women with PCOS. Future studies in larger cohorts from different ethnic populations are necessary to determine the role played by TOX3 in PCOS.
Acknowledgements This study was supported by a grant from the National Basic Research Program of China (973 program: 2012CB944700); the National Natural Science Foundation of China (81000236, 81300459); China Postdoctoral Science Foundation (2012M520909). The authors thank Guangyu Li, Shizhen Su, Zhao Wang and Changming Zhang from Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University for their technical guidance. They also appreciate the patients’ participation and their colleagues for patient examination and sample collection.
Figure 1 (A) Deletion and single nucleotide polymorphisms in TOX3 exons. (A) The 3-bp deletion in TOX3 sequencing test result. The deleted base sequence is shown above the chromatogram; the normal sequence (wild sequence) is shown beneath the chromatogram; (B) Localization of the single nucleotide polymorphisms and deletion in the TOX3 gene coding sequence. The two SNPs were located on exon 6 and 3-bp deletion was located on exon 7 of TOX3 gene.
Please cite this article in press as: Yuqian Cui, et al., Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome, Reproductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.08.004
ARTICLE IN PRESS 4
Y Cui et al.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.rbmo.2014.08.004.
References Alexander, C.J., Tangchitnob, E.P., Lepro, N.E., 2009. Polycystic ovary syndrome: a major unrecognized cardiovascular risk factor in women. Rev. Obstet Gynecol. 2, 232–239. Butland, S.L., Devon, R.S., Huang, Y., Mead, C.L., Meynert, A.M., Neal, S.J., Lee, S.S., Wikinson, A., Yang, G.S., Yuen, M.M., Hayden, M.R., Holt, R.A., Leavitt, B.P., Ouellette, B.F., 2007. CAG-encoded polyglutamine length polymorphism in the human genome. BMC Genomics 8, 126. Dittmer, S., Kovacs, Z., Yuan, S.H., Siszler, G., Kögl, M., Summer, H., Geerts, A., Golz, S., Shioda, T., Methner, A., 2010. TOX3 is a neuronal survival factor that induces transcription depending on the presence of CITED1 or phosphorylated CREB in the transcriptionally active complex. J. Cell Sci. 124, 252–260. Ibáñez, L., Ong, K.K., Mongan, N., Jääskeläinen, J., Marcos, M.V., Hughes, I.A., De Zegher, F., Dunger, D.B., 2003. Androgen receptor gene CAG repeat polymorphism in the development of ovarian hyperandrogenism. J. Clin. Endocrinol. Metab. 88, 3333– 3338. Jones, J.O., Chin, S.F., Wong-Taylor, L.A., Leaford, D., Ponder, B.A., Caldas, C., Maia, A.T., 2013. TOX3 mutations in breast cancer. PLoS ONE 8, e74102. Margolis, R.L., Abraham, M.R., Gatchell, S.B., Li, S.H., Kidwai, A.S., Breschel, T.S., Stine, O.C., Callahan, C., McLnnis, M.G., Ross, C.A., 1997. cDNAs with long CAG trinucleotide repeats from human brain. Hum. Genet. 100, 114–122. O’Flaherty, E., Kaye, J., 2003. TOX defines a conserved subfamily of HMG-box proteins. BMC Genomics 4, 13. Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004. Revised 2003 consensus on diagnostic criteria and longterm health risks related to polycystic ovary syndrome. Fertil. Steril. 81, 19–25.
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Declaration: The authors report no financial or commercial conflicts of interest.
Received 30 March 2014; refereed 25 July 2014; accepted 12 August 2014.
Please cite this article in press as: Yuqian Cui, et al., Mutational analysis of TOX3 in Chinese Han women with polycystic ovary syndrome, Reproductive BioMedicine Online (2014), doi: 10.1016/j.rbmo.2014.08.004
