Introduction:
Etiological factors of early pregnancy loss are numerous and diverse. Fetal karyotype abnormalities can be registered in case of development termination of normal pregnancy and also in pregnancies after assisted reproductive technologies.(1-3) However the causes for occurrence of mutations still yet to find out. Some data shows that polymorphic variants of Mad1, Mad2, Bub1, Bub3 genes which are responsible for meiotic chromosome segregation can be one of the causes.(4) Abnormal chromosome structure may originate from defects in the checkpoint mechanisms. Early stages of individual development are defined by active cell division and differentiation processes coordinated in time and collocation. Intercellular signaling molecules as well as proliferation control and DNA stabilizing factors act as basic functional groups. Changes in genome stability maintaining genes functioning can cause the mutation number increase during blastomeric cleavage and differentiation.
Nucleotide changes in DNA sequence should be detected by cell cycle control proteins which act as a DNA damage sensors (for example specific protein kinases ATM and ATR) passing a signal to checkpoint kinases (CHEK 1, 2). Checkpoint kinases suppress cyclin-dependent kinases (CDK) activity. This results in cell cycle termination and increases time needed for DNA damage reparation previous to mitosis.(5,6) Lack of damage reparation launches apoptosis program.(7,8)
Polymorphic site Pro72Arg of TP53 gene lays within proline-rich domain which is involved in apoptosis induction. It is shown that Arg72 variant induce apoptosis more effectively than Pro72 allele.(9,10) Orsted D. and colleagues (11) confirmed the data showed that Arg72 allele enhances the ability to induce cell death while 72Pro demonstrates low apoptosis activation capacity and increase of cell cycle arrest frequency in G1 phase.
Apurinic apyrimidinic endonuclease repairs apurinic sites by cleaving the phosphodiester bond near apurinic or apyrimidinic site. This endonuclease also regulates gene transcription. It is shown that APEX1 influences the activity of transcriptional factors including p53 and hypoxia-inducible factor 1, which is functionally active on early stages of blastocyst development and implantation. (12) APEX1 deficit induces mice embryonic lethality.(13)
Systems of excision reparation having universal functions take a central position among all DNA reparation systems. Excision DNA-repair protein ERCC2 encoded by XPD gene is involved in excision repair while despiralizing DNA near damaged region and also is involved RNA transcription while attaching CDK-activating kinase complex to transcriptional factor core complex. The protein regulates vitamin D receptor activity and is involved in chromosome segregation process being a part of the mitotic spindle complex. Also ERCC2 protein could play a role in aging and skin cancer development.(14–16)
Allele polymorphism of reparation system and cell cycle control genes has been studied extensively in connection with etiology and pathogenesis of different cancer diseases; however implication of these genes to pregnancy pathology development is poorly understood.(17–19)
Functional efficiency of cell cycle and DNA reparation control proteins can affect on successful proceeding of early stages of embryogenesis and individual development. Thereby the goal of current work was to investigate the frequencies of cell cycle and DNA reparation control genes polymorphic variants: Pro72Arg in TP53, Asp148Glu in APEX1 and Lys751Gln in ERCC2 (XPD) in women with normally progressing pregnancies and in women with first trimester pregnancy loss.
Materials and Methods
Prior to inclusion in the study, all subjects underwent a standard diagnostic work-up. The women were examined using transvaginal ultrasonography for the absence of uterine abnormalities and polycystic ovary syndrome. Women with previously diagnosed arterial hypertension, diabetes, thyroid diseases, autoimmune pathology and infections during pregnancy were excluded from studied population. Women contacting with exogenous risk factors, such as alcohol, electromagnetic radiation, industrial noise, vibration, chemical pollutants were also excluded.
The study was approved by the Southern Federal University Bioethics Committee. The participants willingly signed the informed consent. After approval by institutional review board, 151 women (mean age 29) with spontaneous abortion in 5-11 week of gestation and 134 women (mean age 30) with normally progressing pregnancies and without any history of spontaneous or missed abortion were studied.
Genomic DNA was isolated from the EDTA-anticoagulated peripheral blood using the commercial kit “DNA Express” (thermo-coagulation method, Lytech, Russia).
Polymorphisms Pro72Arg in TP53 (MIM*191170, rs1042522), Asp148Glu in APEX1 (MIM*107748, rs1130409) and Lys751Gln in ERCC2 (XPD) (MIM*126340, rs1052559) was detected by allele-specific polymerase chain reaction method using SNP-express reaction kits (Lytech, Russia). The PCR products were analyzed by horizontal 3% agarose gel electrophoresis. Gel images were captured using GelDoc XR system (Bio-Rad, USA).
Hardy-Weinberg equilibrium analysis was performed using Hardy-Weinberg equilibrium calculator in www.oege.org/software/Hardy-Weinberg.(20) Differences in distribution of allele variants between studied groups were assessed by χ2-analysis. Individuals that carried more than one risk allele or genotype may have a higher risk of pregnancy loss. Therefore gene-gene interactions were explored. We analyzed gene-gene interactions among 3 polymorphisms using the multifactor dimensionality reduction (MDR) method (MDR software, version 2.0) and a P value of less than 0.05 was considered statistically significant.
Results
The distributions of all genotypes and alleles of reparation and cell cycle control system genes polymorphic variants in each group of pregnant women were in Hardy-Weinberg equilibrium. The data on frequencies of polymorphic variants (Asp148Gl in ?PEX1, Lys751Gln in ERCC2 (XPD) and Pro72Arg in TP53) are shown in Table 1. The distributions of genotype and allele frequency between women with early pregnancy loss and the controls (normally progressing pregnancies) were equal for APEX1, ERCC2 (XPD) and TP53 gene polymorphism.
Table 1: Distribution of TP53, APEX1, ERCC2 genotypes among 151 cases with early pregnancy loss (EPL) and 134 controls |
Genotype, allele |
Control (n = 134) |
EPL (n = 151) |
p |
TP53 Pro72Arg rs1042522 |
ProPro |
11 (8.2%) |
10 (6.6%) |
0.8 |
ProArg |
43 (32.1%) |
53 (35.1%) |
ArgArg |
80 (59.7%) |
88 (58.3%) |
72Arg allele |
0.757 |
0.758 |
0.98 |
APEX1 Asp148Glu rs1130409 |
AspAsp |
28 (20.9%) |
35 (23.2%) |
0.85 |
AspGlu |
71 (53.0%) |
80 (53.0%) |
Glu/Glu |
35 (26.1%) |
36 (23.8%) |
148Glu allele |
0.526 |
0.503 |
0.59 |
ERCC2 (XPD) Lys751Gln rs1052559 |
LysLys |
42 (31.3%) |
36 (23.8%) |
0.18 |
LysGln |
73 (54.5%) |
83 (55.0%) |
GlnGln |
19 (14.2%) |
32 (21.2%) |
781Gln allele |
0.414 |
0.487 |
0.08 |
Gene-gene interactions were analyzed among 3 polymorphisms of these 3 genes using the MDR method. After cross-validation and permutation tests of the gene-gene interactions in relation to the EPL group, the best model included a three-marker model (rs1042522, rs1130409, rs1052559) with accuracy of 51% and a maximum cross-validation (CV) consistency of 10 out of 10. (Table 2).
| Table 2: MDR analysis of gene-gene interaction in relation to EPL |
Best model |
Testing accuracy |
Cross-validation consistency |
p |
Testing χ2 |
Testing OR |
rs1042522, rs1130409, rs1052559 |
0.51 |
10/10 |
0.0004 |
12.5 |
6.75
(2.1 – 21.3) |
Thus we found combination of SNPs APEX1 rs1130409, ERCC2 rs1052559, and TP53 rs1042522 to be the best interaction model for predicting early pregnancy loss.
Discussion:
Reparation processes depend on intracellular signaling cascades which are activated by DNA damage. Signal molecules activate reparation system genes as well as cell cycle arrest and cell cycle stage changes genes. At the same time reparation genes transcription is activated while cyclin genes and replication fork stabilization proteins expression is inhibited. Dysregulation in reactions mentioned above results in fixation of DNA damage and genome changes stabilization in cell populations.
The first trimester of pregnancy is characterized by active fetal and maternal cells division that inevitably leads to mistakes which normally are repaired by DNA damage control system. It's obvious that the presence of polymorphic gene variants in reparation system and cell cycle control genes changes the functional activity of BER (base excision repair)/NER (nucleotide excision repair) - proteins and specific protein kinases. This can effect on pregnancy pathology development because of lack in DNA damage correction mechanism, occurrence of genome instability and induction of apoptosis in maternal and fetal cells.
P53 is involved in apoptosis induction. Furthermore p53 protein plays an important role in expression regulation of leukemia inhibitory factor (LIF) which is involved in blastocyst implantation processes.(21) P53 protein polymorphic variant isn’t capable to activate effectively the cytokines of interleukin 6 family, in particular LIF, which is essential for implantation. It is shown that p53 deficient mice have decreased LIF expression levels.(22) There is evidence that Arg72 TP53 has higher transcriptional activity toward a particular subset of p53 target genes, including LIF, than Pro72.(23) P53 protein functional activity change can result in inadequate endometrial decidualization that leads to incomplete or poor cytotrophoblast invasion and suppresses physiological gestation changes in uterine and placental arteries with blood flow decrease.
Several studies have shown the association between Pro72Arg polymorphism in TP53 gene with in vitro fertilization failure, infertility and endometriosis.(24–28) Literature data on the association of TP53 gene polymorphism with pregnancy loss are poor and incomplete.(29–31) Fraga and colleagues (32) stated that TP53 Arg/Arg (rs1042522) genotype frequency in group of women with recurrent pregnancy loss is equal to control group. Therefore the combination of current genotype with MDM TT (rs2279744) genotype results in pregnancy loss risk increase (OR was 2.58).(32)
Polymorphic site in APEX1 gene that causes amino acid substitution in 148 position of protein molecule is located in fifth exon. It encodes C-terminal domain with ß-structure. Such amino acid substitution doesn’t affect enzyme endonuclease activity. APEX1 is included into big group of proteins capable for regulation of DNA repair processes (33), DNA recombination, DNA demethylation (34), cellular redox homeostasis, DNA-dependent transcription regulation, mRNA stability regulation (35). It is shown that 148Glu allele is associated with mitotic block after ionizing radiation. (36) Aminoacid substitution in APEX1 molecule caused by allele gene variant doesn’t affect on protein’s enzyme activity but it can cause different effects on the cooperation between other components of gene regulatory network. This can result in common changes in reparation regulation efficiency, transcriptional factors activity and mRNA stability.(37)
Polymorphic site of ERCC2 (XPD) geneis located in 23th exon and not in the helicase/ATPase domain. But current amino acid residue (Lys751Gln) is located in highly evolutionary conserved sequence.(38) There is the association between the presence of XPD gene polymorphic variant and decrease of effectiveness and activity of DNA repair among group of older persons.(39,40)
There is the data that mutations in C-terminal domain and in region responsible for XPD helicase activity can be associated with placental insufficiency and with higher risk of preeclampsia development. The reason may be found in changes of binding efficiency between XPD, CDK-activating kinase and p44 subunit of IIH transcriptional factor. Mutations in XPD gene cause the disruption in protein-protein interactions of transcription factor with its targets.(18,37)
Down regulation of the ability to interact with other molecular components of the system can lead to decrease of repair processes efficiency. Moslehi R. with colleagues (17) showed the association between repair system genes (XRCC1, XPD6, XPD23) allele variants and high DNA fragmentation index.
The MDR method was introduced to identify gene-gene interactions that are associated with early pregnancy loss. The MDR method reduces high-dimensional genetic data into a single dimension. This permits gene-gene interactions to be detected in relatively small sample sizes.
Several loci usually contribute to the phenotypes expressed in complex diseases, including pregnancy loss. Therefore, it is important to identify gene–gene interactions, because they may more accurately predict the risk of pathology than single genes. In the present study, an MDR analysis was used to predict potential gene–gene interactions that may partly determine the early pregnancy loss. We found that rs1042522, rs1130409, and rs1052559 were associated with EPL.
The presence of more than one polymorphism in DNA damage repair genes can cause the remarkable decrease in repair system efficiency. Thus it may influence on pregnancy pathology development by disrupting the processes of cell cycle control and apoptosis, inducing genome instability in maternal and fetal tissues. All changes mentioned above may result in implantation defects and shallow trophoblast invasion of the decidua leading to pregnancy loss.
Conclusion
The combination of SNPs APEX1 rs1130409, ERCC2 rs1052559, and TP53 rs1042522 is the best interaction model for predicting early pregnancy loss. According to model of gene-gene interaction the genotype combination of polymorphic variants of repair system and cell cycle control genes increases the relative risk of pregnancy loss.
Acknowledgement
This study was supported by the federal assignment No. 6.98.2014/K from Russian Ministry of Science and Education.
Conflict of Interest: None.
References
- Simon C, Rubio C, Vidal F, et al. Increased chromosome abnormalities in human preimplantation embryos after in-vitro fertilization in patients with recurrent miscarriage. Reprod Fert Development. 1998;10:87-92.
- Ljunger E, Cnattingius S, Lundin C, et al. Chromosomal anomalies in first-trimester miscarriages. Acta Obst Gyn Scand. 2005;84:1103-7.
- Morales C, Sanchez A, Bruguera J, et al. Cytogenetic study of spontaneous abortions using semi-direct analysis of chorionic villi samples detects the broadest spectrum of chromosome abnormalities. Am J Med Genet. 2008;146A:66–70.
- Musacchio A, Salmon E. The spindle-assembly checkpoint in space and time. Nature Reviews. Mol Cell Biol. 2007;8:379-93.
- Poehlmann A, Roessner A. Importance of DNA damage checkpoints in the pathogenesis of human cancers. Path Res Practice. 2010;206:591–601.
- Richardson E, Stockwell S, Aherne W, et al. Mechanism-based screen establishes signalling framework for DNA damage-associated G1 checkpoint response. Publ Library Sci. 2012;7(2):e31627.
- Zhou B, Elledge S. The DNA damage response: putting checkpoints in perspective. Nature. 2000;408:433–9.
- Jackson S, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009; 461:1071–8.
- Dumont P, Leu J, Pietra A, et al. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nature Genetics. 2003;33:357-65.
- Vousden K, Prives C. Blinded by the light: the growing complexity of p53. Cell. 2009;137:413-31.
- Orsted D, Bojesen S, Tybjaerg-Hansen A. et al. Tumor suppressor p53 Arg72Pro polymorphism and longevity, cancer survival, and risk of cancer in the general population. J Exp Med. 2007;204:1295–301.
- Bhakat K, Mantha A, Mitra S. Transcriptional regulatory functions of mammalian AP-endonuclease (APE1/Ref-1), an essential multifunctional protein. Antiox Redox Signaling. 2009;11:621-38.
- Guikema J, Linehan E, Tsuchimoto D, et al. APE1- and APE2-dependent DNA breaks in immunoglobulin class switch recombination. J Exp Med. 2007;204:3017-26.
- Tirode F, Busso D, Coin F, et al. Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7. Mol Cell. 1999;3:87–95.
- Drane P, Compe E, Catez P, et al. Selective regulation of vitamin D receptor-responsive genes by TFIIH. Molecular Cell. 2004;16:187–97.
- Ito S, Tan L, Andoh D, et al. MMXD, a TFIIH-independent XPD-MMS19 protein complex involved in chromosome segregation. Molecular Cell. 2010;39:632-40.
- Moslehi R, Kumar A, Mills J, et al. Phenotype-Specific adverse effects of XPD mutations on human prenatal development implicate impairment of TFIIH-mediated functions in placenta. Eur J Hum Genet. 2012;20:626–31.
- Rull K, Nagirnaja L, Laan M. Genetics of recurrent miscarriage: challenges, current knowledge, future directions. Frontiers in Genetics. 2012;3:34. DOI: 10.3389/fgene.2012.00034
- Tamura D, Khan S, Merideth M, et al. Effects of mutations in XPD (ERCC2) on pregnancy and prenatal development in mothers of patients with trichothiodystrophy or xerodermapigmentosum. Eur J Hum Genet. 2012;20:1308–10.
- Rodriguez S, Gaunt T, Day I. Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies. Am J Epid Adv Acc. 2009; DOI: 10.1093/aje/kwn359
- Hu W, Feng Z, Teresky A, et al. p53 regulates maternal reproduction through LIF. Nature. 2007;450:721–4.
- Hu W, Feng Z, Atwal G, et al. p53: a new player in reproduction. Cell Cycle. 2008;7:848–52.
- Feng Z, Zhang C, Kang H, et al. Regulation of female reproduction by p53 and its family members. J Fed Am Soc Exp Biol. 2011;25:2245–55.
- Chang C, Hsieh Y, Tsai F, et al. The proline form of p53 codon 72 polymorphism is associated with endometriosis. Fertility and Sterility. 2002;77:43–5.
- Ammendola M, Gloria-Bottini F, Sesti F, et al. Association of p53 codon 72 polymorphism with endometriosis. Fertility and Sterility. 2008;90:406–8.
- Kang H, Feng Z, Sun Y, et al. Single-nucleotide polymorphisms in the p53 pathway regulate fertility in humans. Proc Nat Acad Sci USA. 2009;106:9761–6.
- Ribeiro C, Arruda J, Silva C, et al. Analysis of p53 codon 72 gene polymorphism in Brazilian patients with endometriosis. Genet Mol Res. 2009;8:494–9.
- Paskulin D, Cunha-Filho J, Souza C, et al. TP53 PIN3 and PEX4 polymorphisms and infertility associated with endometriosis or with post-in vitro fertilization implantation failure. Cell Death and Disease. 2012;3:e392. DOI: 10.1038/cddis.2012.116
- Pietrowski D, Bettendorf H, Riener E, et al. Recurrent pregnancy failure is associated with a polymorphism in the p53 tumor suppressor gene. Hum Reprod. 2005;20:848–51.
- Kay C, Jeyendran R, Coulam C. p53 tumor suppressor gene polymorphism is associated with recurrent implantation failure. Reprod Biomed Online. 2006;13:492–6.
- Goodman C, Jeyendran R, Coulam C. P53 tumor suppressor factor, plasminogen activator inhibitor, and vascular endothelial growth factor gene polymorphisms and recurrent implantation failure. Fertility and Sterility. 2009;92:494–8.
- Fraga L, Dutra C, Boquett J, et al. p53 signaling pathway polymorphisms associated to recurrent pregnancy loss. Mol Biol Rep. 2014;41:1871-7.
- Bennett R, Wilson D, Wong D, Demple B. Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway. Proc Nat Acad Sci USA. 1997;94:7166-9.
- Guo J, Su Y, Zhong C, et al. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell. 2011;145:423-34.
- Barnes T, Kim W, Mantha A, et al. Identification of apurinic/apyrimidinic endonuclease 1 (APE1) as the endoribonuclease that cleaves c-myc mRNA. Nucleic Acid Research. 2009;37:3946-58.
- Hu J, Smith T, Miller M, et al. Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity. Carcinogenesis. 2001;22:917-22.
- Kasahara M, Osawa K, Yoshida K, et al. Association of MUTYH Gln324His and APEX1 Asp148Glu with colorectal cancer and smoking in a Japanese population. J Exp Clin Cancer Res. 2008;27:1–49.
- Spitz M, Wu X, Wang Y, et al. Modulation of nucleotide excision repair capacity by XPD polymorphisms in lung cancer patients. Cancer Res. 2001;61:1354-7.
- Hemminki K, Xu G, Angelini S, et al. XPD exon 10 and 23 polymorphisms and DNA repair in human skin in situ. Carcinogenesis. 2001;22:1185-8.
- Chi X, Liu Y, Shi S, et al. XRCC1 and XPD genetic polymorphisms and susceptibility to age-related cataract: a meta-analysis. Molecular Vision. 2015;21:335-46.
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