ORIGINAL ARTICLE
Polymorphisms in Genes Coding for the NK-Cell Receptor NKG2D and its Ligand MICA in Recurrent Miscarriage Sondes Hizem1*, Nabil Mtiraoui1,2*, Safia Massaoudi1, Catherine Fortier3, Wahid Boukouaci4, Amokrane Kahina3,4, Dominique Charron3,4, Touhami Mahjoub1, Ryad Tamouza3,4 1
Research Unit of Hematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia; Prince Fahd Bin Sultan Research Chair, Using Advance Technology for Diseases Detection and Treatment, Faculty of Applied Medical Sciences, University of Tabuk, Tabuk, Saudi Arabia; 3 Jean Dausset Laboratory, Saint Louis Hospital, Paris, France; 4 INSERM, U940, Saint Louis Hospital, Paris, France 2
Keywords Haplotypes, MICA gene, NKG gene, polymorphisms, recurrent miscarriage Correspondence Sondes Hizem, Research Unit of Hematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia. E-mail: [email protected] *These authors contributed equally to this work. Submission May 23, 2014; accepted August 6, 2014. Citation Hizem S, Mtiraoui N, Massaoudi S, Fortier C, Boukouaci W, Kahina A, Charron D, Mahjoub T, Tamouza R. Polymorphisms in genes coding for the NK-cell receptor NKG2D and its ligand MICA in recurrent miscarriage. Am J Reprod Immunol 2014 doi:10.1111/aji.12314
Problem To investigate the possible association of Natural Killer Group (NKG) receptors gene polymorphisms and MHC class I chain-related protein A (MICA) gene polymorphism with recurrent miscarriage (RM). Methods Seven SNPs in NKG2D gene (rs1049174, rs2255336, rs2617160, rs2617161, rs2246809, rs2617169, and rs2617170), one SNP in NKG2A gene (rs1983526), and one SNP in MICA gene (MICA129) were assessed by allelic discrimination (real-time PCR) in both patients and control women. Results The rs2617170 T/T genotype significantly protected against RM [OR (95%) = 0.63 (0.40–0.98)]. The NKG2D haplotypes analysis on the basis of pairwise LD revealed two haplotype blocks. In block1, we found an increased frequency of CAT (Pc = 0.007; OR = 2.13; 95% CI = 1.24– 3.68) and GGA haplotypes (Pc = 0.041; OR = 2.02; 95%CI = 1.03–3.96) and reduced frequency of CAA haplotype (Pc = 0.027; OR = 0.72; 95% CI = 0.54–0.96) in patients. In block2, increased frequency of GATG haplotype (Pc = 104; OR = 9.25; 95% CI = 3.04–28.12) and reduced frequency of ATTC haplotype (Pc = 0.035; OR = 0.69; 95%CI = 0.50– 0.97) were seen in patients. Conclusion The NKG2D gene polymorphisms may influence the success of pregnancy in Tunisian women.
Introduction Pregnancy is commonly admitted to be a semiallogenic graft under the control of immune-related establishment of tolerance allowing successful fetal implantation state. Dysregulation of such processes may induce recurrent miscarriage.1 American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Recurrent miscarriage (RM) is usually defined as the loss of three or more consecutive pregnancies until the 20th gestational week. It is a pregnancy complication affecting 1–2% of reproductive couples. Many etiologies have been proposed to contribute to pregnancy loss: chromosomal abnormalities, uterine malformations, endocrine defects, thrombophilias, 1
HIZEM ET AL.
autoimmune diseases, and infections. However, half of them are currently still undocumented.2,3 Interestingly, the number NK cells increases at the implantation site suggesting their important role in the maintenance of pregnancy. It was demonstrated that decidual NK cells produce a variety of cytokines that induce angiogenesis such as vascular endothelial growth factor (VEGF), placental growth factor (PLGF), TGF-b, angiopoietin (Ang) 1, and Ang 2, thus contributing to the uterine vascular remodeling.4 On the other hand, the maternal NK cells-mediated cytotoxicity has a positive role in placental formation by limiting excessive trophoblast invasion. The dysregulation of this cytotoxicity activity can induce recurrent miscarriage in pregnant women. Therefore, the balance between the activation and the regulation system in NK cells is crucial for the maintenance of the appropriate homeostasis at the normal fetomaternal interface and the fetal allograft during pregnancy.5,6 The mechanism, which strongly suggested for fetal escape from a potential cytotoxic attack by maternal NK was the downregulation of the activating NK-cell receptor NKG2D.7 Among 20 SNPs in NKG genes, eight SNPs were closely associated with natural cytotoxic activity. They are mostly located in the NKG2D gene region (NKC-3, NKC-4, NKC-7, NKC-9, NKC-10, NKC-11, and NKC-12) except for NKC-17 that is located in the promoter region of the NKG2A gene.8 The NKG2D receptor recognizes a variety of ligands such as the non-conventional MHC-I chain-related molecules (MICA and MICB). Some polymorphisms (SNPs) in NKG and MICA genes have been reported in cancer, viral infections, and autoimmune diseases but not in recurrent miscarriage. To support the hypothesis of an alloimmune etiology for miscarriage, we focused on the NKG2D receptor and on its ligand MICA, replicating the analysis of their gene variations already studied in other pathologies. We thus evaluated seven SNPs in NKG2D gene (rs1049174: NKC-3, rs2255336: NKC4, rs2617160: NKC-7, rs2246809: NKC-9, rs2617169: NKC-10, and rs2617170: NKC-11, rs2617171: NKC12), one SNP in NKG2A gene (rs1983526: NKC-17) and one SNP in MICA gene: MICA129 (rs1051792) by allelic discrimination (real-time PCR) in both patients and control women. To the best of our knowledge, this report is the first to investigate the association of these polymorphisms in a north African population. In addition to 2
identifying specific NKG2D alleles and haplotypes linked with RM, we also determine the overall RM risk associated with these variants, assuming additive, dominant, and recessive models of transmission. Materials and methods Patients and Controls This was a case–control retrospective study, performed at the Obstetrics and Gynecology Department of Hospital Farhat Hached in Sousse, Tunisia. Data collection procedures were the same for patients and control subjects. Patients were 312 fertile women (mean age: 32.44 5.7) with three or more unexplained pregnancy losses with the same partner. Exclusion criteria included first degree consanguinity, parental karyotype aberrations, Rh incompatibility, anatomic abnormalities, systemic autoimmune disease, arterial hypertension, endocrine diseases (diabetes mellitus, thyroid dysfunction), infections (toxoplasmosis, HCMV, rubella, HIV, Group B streptococci, Chlamydia trachomatis, hepatitis B and C, and bacterial vaginosis), previous venous or arterial thrombosis, or a family history of thromboembolism. All subjects were negative for coagulation defects, antiphospholipid syndrome and fetomaternal alloimmune thrombocytopenia. The control group consisted of 334 age matched (P = 0.433), multiparous and healthy women (mean age: 32.06 5.71). Who had at least two children, and no known personal or family history of RM or other pregnancy complications. In addition, controls were matched to patients according to body mass index (BMI) values and recruited from the same geographical area (The center of Tunisia) (Table I). Similar data collection procedures were used for patients and control subjects. Genetic study was performed on all patients and control subjects. Venous blood samples were collected in EDTA-containing tubes for genomic DNA extraction using salting out method. NKG2D and NKG2A Genotyping Genotyping was performed by the allelic discrimination method using VIC- and FAM-labeled primers. The reaction was performed in 20 lL as recommended by the manufacturer (Applied Biosystems, American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NK CELLS IN RECURRENT MISCARRIAGE
Table I Demographics of Recurrent Miscarriage Patients and Controls
c
Age at inclusion in study Body mass index (kg/m2)c Obesity (>30 kg/m2) Previous contraceptive users Number of live birthsc Number of abortionsc
Controlsa
Patients
Pb
OR (95% CI)
32.44 5.71 24.91 4.20 20 (11.4) 170 (51.0) 2.07 1.10 0.00 0.00
32.06 5.71 25.17 3.66 17 (10.1) 148 (47.4) 0.87 1.00 3.06 1.34
0.433 0.539 0.300 0.880 2.0; pink blocks, D0 < 1.0 with LOD < 2.0; numbers in blocks denoting D0 value. The genomic organization is depicted above the LD plot. D0 is calculated as per: D0 = (normalized LD measure; D) divided by the theoretical maximum for the observed allele frequencies.
frequent, and ATTC haplotype (Pc = 0.035; OR = 0.69; 95%CI = 0.50–0.97) which was less frequent among patients, thus conferring disease susceptibility and protective nature to these haplotypes, respectively.
6
The natural cytotoxicity receptors (NCRs) are unique markers that regulate NK-cell cytotoxicity and cytokine production. During pregnancy, the inhibitory signal dominates the activating signal to avoid killing fetal trophoblast cells.9 Dysregulation between the activating and inhibitoring NK cells receptors seems to be important for healthy pregnancy.6,10 In this case–control study, we investigated the possible association between the MICA, NKG2D, and NKG2A variants and RM risk among Tunisian women. The NKG2D is an activating and co-stimulatory receptor expressed on natural killer cells and T cells. The interaction between the activating NK-cell receptor NKG2D and their ligands MIC (MICA, MICB) and ULBP (ULBP1to ULBP6) mediates killing through perforin cytotoxic pathway.11,12 The recently described MICA amino acid substitution methionine (met) to valine (val) at position 129 of the a2-heavy chain domain (MICA-129) seems to affect its binding to NKG2D and would influence the effector cell function. This MICA-129 polymorphism (rs1051792) has been associated with a number of diseases such as ankylosing spondylitis, cancer, and chronic graft-versus-host disease (cGVHD).13–15 In this cohort, MICA129 (rs1051792) did not associate significantly with RM under any of the genetic models tested (additive, dominant, or recessive). This association has not been reported in the literature. However, it was suggested that the mechanism for fetal escape from
American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NK CELLS IN RECURRENT MISCARRIAGE
Table V Haplotype Frequencies Across NKG2D SNPs Analyzed Blocka
Haplotype
Frequency
Control:Patient frequencies
Pc valueb
aORc (95% CI)
Block 1
G–G-T C- A- A C– G -A C- A- T G–G-A C– G - T G–A-A G–A–C-G G–A–T–C A–T–T-C G–A–C-C G–A–T-G G–T–T-C A– A – T - C A–A–C-C
0.501 0.223 0.142 0.057 0.035 0.029 0.010 0.431 0.177 0.173 0.067 0.044 0.020 0.019 0.011
0.493:0.507 0.255:0.184 0.146:0.139 0.036:0.084 0.022:0.052 0.034:0.023 0.014:0.007 0.433:0.423 0.198:0.151 0.205:0.136 0.061:0.079 0.007:0.089 0.020:0.018 0.021:0.017 0.010:0.013
– 0.027 0.630 0.007 0.041 0.270 0.170 – 0.150 0.035 0.520 104 0.940 0.520 0.074
1.00 0.72 0.92 2.13 2.02 0.67 0.39 1.00 0.77 0.69 1.17 9.25 1.03 0.72 1.22
Block 2
(Reference) [0.54–0.96] [0.65–1.30] [1.24–3.68] [1.03–3.96] [0.33–1.36] [0.10–1.49] (Reference) [0.54–1.10] [0.50–0.97] [0.72–1.91] [3.04–28.12] [0.49–2.18] [0.27–1.95] [0.37–4.08]
a
block 1: rs1049174/rs2255336/rs2617160, block 2: rs2246809/rs2617169/rs2617170/rs2617171 haplotypes. aOR = adjusted odds ratio, adjusted for age and BMI. c Pc = corrected P according to Bonferroni correction. Underlined characters indicate minor allele. Bold values indicate significant association. b
a potential cytotoxic attack by maternal NK was the down-regulation of the activating NK-cell receptor NKG2D by the soluble MIC molecules. The soluble isoform of MICA (sMICA) results from the proteolytic shedding of the membrane-bound molecules by placental exosomes.16–18 The MICA-129 genotype did not influence sMICA levels.15 Several studies reported that NKG2D plays an important role through cytotoxicity and cytokine release in the pathogenesis of infections, autoimmune diseases, and GVHD.19–21 In our study, only NKG2D 11 (rs2617170) showed a significant association with RM (P = 0.043) under the additive but not dominant and recessive models, which remained significant after adjustment for the age and BMI. This association translate a low protective effect against RM [OR (95%) = 0.63 (0.40–0.98)] for the T/T genotype. The latter genotype may contribute to successful implantation in the Tunisian population. Hayashi et al.8 succeeded in identifying a haplotype constructed from five SNPs located in the NKG2D gene (rs1049174, rs2617160, rs2617170, rs2617171, and rs1983526) and which was associated with the natural cytotoxic activity. The T/T genotype in SNP rs2617170 was associated with a higher cytotoxic activity and a decreased risk of cancer development, while it is associated with successful pregnancy in this cohort. American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
The role of NK cells in allograft tolerance has been established suggesting their inability to kill the semiallogenic fetal cells.22,23 Our findings suggest that the variant NKG2D 11 (rs2617170) may influence the successful pregnancy outcome. The NK-cell cytotoxicity against trophoblast cells is tightly regulated by specific inhibitory and activating receptors contributing to immunomodulation at the maternal–fetal interface.24,25 Many studies reported associations between genetic polymorphisms in NKG2D gene and viral defense and autoimmune diseases. Both GG genotype in rs2255336 and TT genotype in rs2617160 conferred a high cytotoxic activity. They were associated, respectively, with an increased risk of SLE and chronic hepatitis B.20,26 In this current study, we identified two haplotype blocks from seven SNPs located in the NKG2D gene on the prevalence of individual SNPs and LD between them (Table IV). After adjustment for age and BMI, differences between patients and controls remain significant for CAT and GGA haplotypes suggesting their susceptibility contribution on the RM risk (aOR = 2.13 [1.24–3.68], aOR = 2.02 [1.03– 3.96], respectively. While, CAA haplotype seems to be protective against RM (aOR = 0.72 [0.54–0.92]). In block2, significant difference between GATG and ATTC haplotypes was seen, thus conferring disease 7
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susceptibility and protective nature to these haplotypes, respectively (Table V). Our results are different from those of Tomonori Hayashi et al. and J. Luis Espinoza et al., who found a correlation between NKG2D haplotype and phenotype (high or low natural cytotoxic activity) in the occurrence of cancer and the transplants outcomes. The HNK1 haplotype constructed from five SNPs (rs1049174, rs2617160, rs2617170, rs2617171, rs1983526) was significantly associated with reduced risk of cancer and better clinical outcome after transplantation.8,27 Discordance in results may be due to differences in NK cells functions in these different diseases. In pregnancy, other combined alleles and haplotypes seem to affect the cytotoxic activity of NK cells which contributes to fetal reject. On other hand, cytotoxicity of the maternal NK cells has also a positive role in placental formation by limiting excessive trophoblast invasion thus contributing in successful pregnancy.5,6 Kusakabe et al.28 reported that in the process of spontaneous abortion, complement C3 appears to be influential while the cytotoxicity of uterine NK cells is inhibited suggesting the benefic role of NK cells cytotoxicity in successful pregnancy. Conclusion In conclusion, our data support an effect of the NKG2D 11(rs2617170) variant on pregnancy loss risk in the Tunisian women, whereas the other variants tested were not found to play a major role in RM. The strength of our study lies in the large sample population (312 patients and 334 controls), the homogeneity of the ethnic backup (only Tunisian Arab women were included) and in the careful selection of controls, so as to avoid the contribution of confounding factors. It is noteworthy that the association of NKG2D genetic variants to RM risk should be regarded in the context of the cytokine environment in pregnancy, as well as likely participation of other polymorphisms in NKG2D and other modifying genes. Further studies, on other ethnic groups are needed to confirm the current findings and to reveal the mechanism by which NKG2D polymorphisms influence overall RM risk. Acknowledgments We especially thank all of the participants of this project who donated blood for this study. We also 8
especially thank the medical and non-medical staff from the Obstetrics and Gynecology Department of Hospital Farhat Hached in Sousse for collecting the samples involved in this work. Funding This study was supported by the Ministry of Education and Scientific Research in Tunisia and by INSERM, U940, Saint Louis Hospital, Paris, F75010, France. Conflict of interest None declared. References 1 Aruna M, Sudheer PS, Andal S, Tarakeswari S, Reddy AG, Thangaraj K, Singh L, Reddy BM: HLA-G polymorphism patterns show lack of detectable association with recurrent spontaneous abortion. Tissue Antigens 2010; 76:216–222. 2 Kwak-Kim J, Yang KM, Gilman-Sachs A: Recurrent pregnancy loss: a disease of inflammation and coagulation. J Obstet Gynaecol Res 2009; 35:609–622. 3 Branch DW, Gibson M, Silver RM: Clinical practice. Recurrent miscarriage. N Engl J Med 2010; 363:1740–1747. 4 Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V, Benharroch D, Porgador A, Keshet E, Yagel S, Mandelboim O: Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med 2006; 12:1065–1074. 5 Dosiou C, Giudice LC: Natural killer cells in pregnancy and recurrent pregnancy loss: endocrine and immunologic perspectives. Endocr Rev 2005; 26:44–62. 6 Saito S, Nakashima A, Myojo-Higuma S, Shiozaki A: The balance between cytotoxic NK cells and regulatory NK cells in human pregnancy. J Reprod Immunol 2008; 77:14–22. 7 Mincheva-Nilsson L, Nagaeva O, Chen T, Stendahl U, Antsiferova J, Mogren I, Hernestal J, Baranov V: Placenta-derived soluble MHC class I chain-related molecules down-regulate NKG2D receptor on peripheral blood mononuclear cells during human pregnancy: a possible novel immune escape mechanism for fetal survival. J Immunol 2006; 176:3585–3592. 8 Hayashi T, Kazue Imai K, Yukari Morishita Y, Kusunoki Y, Nakachi K: Identification of the NKG2D haplotypes associated with natural cytotoxic activity of peripheral blood lymphocytes and cancer immunosurveillance. Cancer Res 2006; 66:563–570. 9 Beaman KD, Ntrivalas E, Mallers TM, Jaiswal MK, Kwak-Kim J, Gilman-Sachs A: Immune etiology of recurrent pregnancy loss and its diagnosis. Kenneth D. Beaman, Evangelos Ntrivalas. Am J Reprod Immunol 2012; 67:319–325. 10 Dimasi N, Moretta L, Biassoni R: Structure of the Ly49 family of natural killer (NK) cell receptors and their interaction with MHC class I molecules. Immunol Res 2004; 30:95–104. 11 Eagle RA, Trowsdale J: Promiscuity and the single receptor: NKG2D. Nat Rev Immunol 2007; 7:737–744.
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12 Hedlund M, Stenqvist AC, Nagaeva O, Kjellberg L, Wulff M, Baranov V, Mincheva-Nilsson L: Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function. J Immunol 2009; 183:340–351. 13 Amroun H, Djoudi H, Busson M, Allat R, El Sherbini S, Sloma I, Ramasawmy R, Brun M, Dulphy N, Krishnamoorthy R, Toubert A, Charron D, Abbadi MC, Tamouza R: Early-onset ankylosing spondylitis is associated with a functional MICA polymorphism. Hum Immunol 2005; 66:1057–1061. 14 Douik H, Ben Chaaben A, Attia Romdhane N, Romdhane HB, Mamoghli T, Fortier C, Boukouaci W, Harzallah L, Ghanem A, Gritli S, Makni M, Charron D, Krishnamoorthy R, Guemira F, Tamouza R: Association of MICA-129 polymorphism with nasopharyngeal cancer risk in a Tunisian population. Hum Immunol 2009; 70:45–48. 15 Boukouaci W, Marc Busson M, Regis Peffault de Latour P, Rocha V, Suberbielle C, Bengoufa D, Dulphy N, Haas P, Scieux C, Amroun H, Gluckman E, Krishnamoorthy R, Toubert A, Charron D, Socie G, Tamouza R: MICA-129 genotype, soluble MICA, and anti-MICA antibodies as biomarkers of chronic graft-versus-host disease. Blood 2009; 114:5216–5224. 16 Porcu-Buisson G, Lambert M, Lyonnet L, Loundou A, Gamerre M, Camoin-Jau L, Dignat-George F, Caillat-Zucman S, Pascale Paul P: Soluble MHC Class I chain-related molecule serum levels are predictive markers of implantation failure and successful term pregnancies following IVF. Hum Reprod 2007; 22:2261–2266. 17 Choy MK, Phipps ME: MICA polymorphism: biology and importance in immunity and disease. Trends Mol Med 2010; 16:97–106. 18 Hang SY, Chang CH, Chen FP, Yu CL: The alteration of placentalderived soluble MHC class I chain-related protein A and B during pregnancy. Acta Obstet Gynecol Scand 2011; 90:802–807. 19 Park KS, Park JH, Song YW: Inhibitory NKG2A and activating NKG2D and NKG2C natural killer cell receptor genes: susceptibility for rheumatoid arthritis. Tissue Antigens 2008; 72:342–346.
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20 Kabalak G, Thomas RM, Martin J, Ortego-Centeno N, JimenezAlonso J, de Ram on E, Buyny S, Hamsen S, Gross WL, Schnarr S, Zeidler H, Gromnica-Ihle E, Schmidt RE, Witte T: Association of an NKG2D gene variant with systemic lupus erythematosus in two populations. Hum Immunol 2010; 71:74–78. 21 Zingoni A, Ardolino M, Santoni A, Cerboni C: NKG2D and DNAM1activating receptors and their ligands in NK–Tcell interactions: role in the NK cell mediated negative regulation of Tcell responses. Front Immunol 2013; 3:408. 22 Beilke JN, Kuhl NR, Van Kaer L, Gill RG: NK cells promote is let allograft tolerance via a perforin-dependent mechanism. Nat Med 2005; 11:1059–1065. 23 Yu G, Xu X, Vu MD, Kilpatrick ED, Li XC: NK cells promote transplant tolerance by killing donor antigen-presenting cells. J Exp Med 2006; 203:1851–1858. 24 Moffett-King A: Natural killer cells and pregnancy. Nat Rev Immunol 2002; 2:656–663. 25 Lash GE, Bulmer JN: Do uterine natural killer (uNK) cells contribute to female reproductive disorders? J Reprod Immunol 2011; 88:156–164. 26 Ma J, Guo X, Wu X, Li J, Zhu X, Li Z, Li J, Pan L, Li T, Li H, Liu Y: Association of NKG2D genetic polymorphism with susceptibility to chronic hepatitis B in a Han Chinese population. Med Virol 2010; 82:1501–1507. 27 Espinoza JL, Takami A, Onizuka A, Sao H, Akiyama H, Miyamura K, Okamoto S, Inoue M, Kanda Y, Ohtake S, Fukuda T, Morishima Y, Kodera Y, Shinji Nakao S: NKG2D gene polymorphism has a significant impact on transplant outcomes after HLA-fully-matched unrelated bone marrow transplantation for standard risk hematologic malignancies. Haematol 2009; 94:1427–1434. 28 Kusakabe K, Naka M, Yuko Ito Y, Eid N, Otsuki Y: Regulation of natural-killer cell cytotoxicity and enhancement of complement factors in the spontaneously aborted mouse placenta. Fertil Steril 2008; 90:1451–1459.
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Polymorphisms in Genes Coding for the NK-Cell Receptor NKG2D and its Ligand MICA in Recurrent Miscarriage Sondes Hizem1*, Nabil Mtiraoui1,2*, Safia Massaoudi1, Catherine Fortier3, Wahid Boukouaci4, Amokrane Kahina3,4, Dominique Charron3,4, Touhami Mahjoub1, Ryad Tamouza3,4 1
Research Unit of Hematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia; Prince Fahd Bin Sultan Research Chair, Using Advance Technology for Diseases Detection and Treatment, Faculty of Applied Medical Sciences, University of Tabuk, Tabuk, Saudi Arabia; 3 Jean Dausset Laboratory, Saint Louis Hospital, Paris, France; 4 INSERM, U940, Saint Louis Hospital, Paris, France 2
Keywords Haplotypes, MICA gene, NKG gene, polymorphisms, recurrent miscarriage Correspondence Sondes Hizem, Research Unit of Hematological and Autoimmune Diseases, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia. E-mail: [email protected] *These authors contributed equally to this work. Submission May 23, 2014; accepted August 6, 2014. Citation Hizem S, Mtiraoui N, Massaoudi S, Fortier C, Boukouaci W, Kahina A, Charron D, Mahjoub T, Tamouza R. Polymorphisms in genes coding for the NK-cell receptor NKG2D and its ligand MICA in recurrent miscarriage. Am J Reprod Immunol 2014 doi:10.1111/aji.12314
Problem To investigate the possible association of Natural Killer Group (NKG) receptors gene polymorphisms and MHC class I chain-related protein A (MICA) gene polymorphism with recurrent miscarriage (RM). Methods Seven SNPs in NKG2D gene (rs1049174, rs2255336, rs2617160, rs2617161, rs2246809, rs2617169, and rs2617170), one SNP in NKG2A gene (rs1983526), and one SNP in MICA gene (MICA129) were assessed by allelic discrimination (real-time PCR) in both patients and control women. Results The rs2617170 T/T genotype significantly protected against RM [OR (95%) = 0.63 (0.40–0.98)]. The NKG2D haplotypes analysis on the basis of pairwise LD revealed two haplotype blocks. In block1, we found an increased frequency of CAT (Pc = 0.007; OR = 2.13; 95% CI = 1.24– 3.68) and GGA haplotypes (Pc = 0.041; OR = 2.02; 95%CI = 1.03–3.96) and reduced frequency of CAA haplotype (Pc = 0.027; OR = 0.72; 95% CI = 0.54–0.96) in patients. In block2, increased frequency of GATG haplotype (Pc = 104; OR = 9.25; 95% CI = 3.04–28.12) and reduced frequency of ATTC haplotype (Pc = 0.035; OR = 0.69; 95%CI = 0.50– 0.97) were seen in patients. Conclusion The NKG2D gene polymorphisms may influence the success of pregnancy in Tunisian women.
Introduction Pregnancy is commonly admitted to be a semiallogenic graft under the control of immune-related establishment of tolerance allowing successful fetal implantation state. Dysregulation of such processes may induce recurrent miscarriage.1 American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Recurrent miscarriage (RM) is usually defined as the loss of three or more consecutive pregnancies until the 20th gestational week. It is a pregnancy complication affecting 1–2% of reproductive couples. Many etiologies have been proposed to contribute to pregnancy loss: chromosomal abnormalities, uterine malformations, endocrine defects, thrombophilias, 1
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autoimmune diseases, and infections. However, half of them are currently still undocumented.2,3 Interestingly, the number NK cells increases at the implantation site suggesting their important role in the maintenance of pregnancy. It was demonstrated that decidual NK cells produce a variety of cytokines that induce angiogenesis such as vascular endothelial growth factor (VEGF), placental growth factor (PLGF), TGF-b, angiopoietin (Ang) 1, and Ang 2, thus contributing to the uterine vascular remodeling.4 On the other hand, the maternal NK cells-mediated cytotoxicity has a positive role in placental formation by limiting excessive trophoblast invasion. The dysregulation of this cytotoxicity activity can induce recurrent miscarriage in pregnant women. Therefore, the balance between the activation and the regulation system in NK cells is crucial for the maintenance of the appropriate homeostasis at the normal fetomaternal interface and the fetal allograft during pregnancy.5,6 The mechanism, which strongly suggested for fetal escape from a potential cytotoxic attack by maternal NK was the downregulation of the activating NK-cell receptor NKG2D.7 Among 20 SNPs in NKG genes, eight SNPs were closely associated with natural cytotoxic activity. They are mostly located in the NKG2D gene region (NKC-3, NKC-4, NKC-7, NKC-9, NKC-10, NKC-11, and NKC-12) except for NKC-17 that is located in the promoter region of the NKG2A gene.8 The NKG2D receptor recognizes a variety of ligands such as the non-conventional MHC-I chain-related molecules (MICA and MICB). Some polymorphisms (SNPs) in NKG and MICA genes have been reported in cancer, viral infections, and autoimmune diseases but not in recurrent miscarriage. To support the hypothesis of an alloimmune etiology for miscarriage, we focused on the NKG2D receptor and on its ligand MICA, replicating the analysis of their gene variations already studied in other pathologies. We thus evaluated seven SNPs in NKG2D gene (rs1049174: NKC-3, rs2255336: NKC4, rs2617160: NKC-7, rs2246809: NKC-9, rs2617169: NKC-10, and rs2617170: NKC-11, rs2617171: NKC12), one SNP in NKG2A gene (rs1983526: NKC-17) and one SNP in MICA gene: MICA129 (rs1051792) by allelic discrimination (real-time PCR) in both patients and control women. To the best of our knowledge, this report is the first to investigate the association of these polymorphisms in a north African population. In addition to 2
identifying specific NKG2D alleles and haplotypes linked with RM, we also determine the overall RM risk associated with these variants, assuming additive, dominant, and recessive models of transmission. Materials and methods Patients and Controls This was a case–control retrospective study, performed at the Obstetrics and Gynecology Department of Hospital Farhat Hached in Sousse, Tunisia. Data collection procedures were the same for patients and control subjects. Patients were 312 fertile women (mean age: 32.44 5.7) with three or more unexplained pregnancy losses with the same partner. Exclusion criteria included first degree consanguinity, parental karyotype aberrations, Rh incompatibility, anatomic abnormalities, systemic autoimmune disease, arterial hypertension, endocrine diseases (diabetes mellitus, thyroid dysfunction), infections (toxoplasmosis, HCMV, rubella, HIV, Group B streptococci, Chlamydia trachomatis, hepatitis B and C, and bacterial vaginosis), previous venous or arterial thrombosis, or a family history of thromboembolism. All subjects were negative for coagulation defects, antiphospholipid syndrome and fetomaternal alloimmune thrombocytopenia. The control group consisted of 334 age matched (P = 0.433), multiparous and healthy women (mean age: 32.06 5.71). Who had at least two children, and no known personal or family history of RM or other pregnancy complications. In addition, controls were matched to patients according to body mass index (BMI) values and recruited from the same geographical area (The center of Tunisia) (Table I). Similar data collection procedures were used for patients and control subjects. Genetic study was performed on all patients and control subjects. Venous blood samples were collected in EDTA-containing tubes for genomic DNA extraction using salting out method. NKG2D and NKG2A Genotyping Genotyping was performed by the allelic discrimination method using VIC- and FAM-labeled primers. The reaction was performed in 20 lL as recommended by the manufacturer (Applied Biosystems, American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NK CELLS IN RECURRENT MISCARRIAGE
Table I Demographics of Recurrent Miscarriage Patients and Controls
c
Age at inclusion in study Body mass index (kg/m2)c Obesity (>30 kg/m2) Previous contraceptive users Number of live birthsc Number of abortionsc
Controlsa
Patients
Pb
OR (95% CI)
32.44 5.71 24.91 4.20 20 (11.4) 170 (51.0) 2.07 1.10 0.00 0.00
32.06 5.71 25.17 3.66 17 (10.1) 148 (47.4) 0.87 1.00 3.06 1.34
0.433 0.539 0.300 0.880 2.0; pink blocks, D0 < 1.0 with LOD < 2.0; numbers in blocks denoting D0 value. The genomic organization is depicted above the LD plot. D0 is calculated as per: D0 = (normalized LD measure; D) divided by the theoretical maximum for the observed allele frequencies.
frequent, and ATTC haplotype (Pc = 0.035; OR = 0.69; 95%CI = 0.50–0.97) which was less frequent among patients, thus conferring disease susceptibility and protective nature to these haplotypes, respectively.
6
The natural cytotoxicity receptors (NCRs) are unique markers that regulate NK-cell cytotoxicity and cytokine production. During pregnancy, the inhibitory signal dominates the activating signal to avoid killing fetal trophoblast cells.9 Dysregulation between the activating and inhibitoring NK cells receptors seems to be important for healthy pregnancy.6,10 In this case–control study, we investigated the possible association between the MICA, NKG2D, and NKG2A variants and RM risk among Tunisian women. The NKG2D is an activating and co-stimulatory receptor expressed on natural killer cells and T cells. The interaction between the activating NK-cell receptor NKG2D and their ligands MIC (MICA, MICB) and ULBP (ULBP1to ULBP6) mediates killing through perforin cytotoxic pathway.11,12 The recently described MICA amino acid substitution methionine (met) to valine (val) at position 129 of the a2-heavy chain domain (MICA-129) seems to affect its binding to NKG2D and would influence the effector cell function. This MICA-129 polymorphism (rs1051792) has been associated with a number of diseases such as ankylosing spondylitis, cancer, and chronic graft-versus-host disease (cGVHD).13–15 In this cohort, MICA129 (rs1051792) did not associate significantly with RM under any of the genetic models tested (additive, dominant, or recessive). This association has not been reported in the literature. However, it was suggested that the mechanism for fetal escape from
American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
NK CELLS IN RECURRENT MISCARRIAGE
Table V Haplotype Frequencies Across NKG2D SNPs Analyzed Blocka
Haplotype
Frequency
Control:Patient frequencies
Pc valueb
aORc (95% CI)
Block 1
G–G-T C- A- A C– G -A C- A- T G–G-A C– G - T G–A-A G–A–C-G G–A–T–C A–T–T-C G–A–C-C G–A–T-G G–T–T-C A– A – T - C A–A–C-C
0.501 0.223 0.142 0.057 0.035 0.029 0.010 0.431 0.177 0.173 0.067 0.044 0.020 0.019 0.011
0.493:0.507 0.255:0.184 0.146:0.139 0.036:0.084 0.022:0.052 0.034:0.023 0.014:0.007 0.433:0.423 0.198:0.151 0.205:0.136 0.061:0.079 0.007:0.089 0.020:0.018 0.021:0.017 0.010:0.013
– 0.027 0.630 0.007 0.041 0.270 0.170 – 0.150 0.035 0.520 104 0.940 0.520 0.074
1.00 0.72 0.92 2.13 2.02 0.67 0.39 1.00 0.77 0.69 1.17 9.25 1.03 0.72 1.22
Block 2
(Reference) [0.54–0.96] [0.65–1.30] [1.24–3.68] [1.03–3.96] [0.33–1.36] [0.10–1.49] (Reference) [0.54–1.10] [0.50–0.97] [0.72–1.91] [3.04–28.12] [0.49–2.18] [0.27–1.95] [0.37–4.08]
a
block 1: rs1049174/rs2255336/rs2617160, block 2: rs2246809/rs2617169/rs2617170/rs2617171 haplotypes. aOR = adjusted odds ratio, adjusted for age and BMI. c Pc = corrected P according to Bonferroni correction. Underlined characters indicate minor allele. Bold values indicate significant association. b
a potential cytotoxic attack by maternal NK was the down-regulation of the activating NK-cell receptor NKG2D by the soluble MIC molecules. The soluble isoform of MICA (sMICA) results from the proteolytic shedding of the membrane-bound molecules by placental exosomes.16–18 The MICA-129 genotype did not influence sMICA levels.15 Several studies reported that NKG2D plays an important role through cytotoxicity and cytokine release in the pathogenesis of infections, autoimmune diseases, and GVHD.19–21 In our study, only NKG2D 11 (rs2617170) showed a significant association with RM (P = 0.043) under the additive but not dominant and recessive models, which remained significant after adjustment for the age and BMI. This association translate a low protective effect against RM [OR (95%) = 0.63 (0.40–0.98)] for the T/T genotype. The latter genotype may contribute to successful implantation in the Tunisian population. Hayashi et al.8 succeeded in identifying a haplotype constructed from five SNPs located in the NKG2D gene (rs1049174, rs2617160, rs2617170, rs2617171, and rs1983526) and which was associated with the natural cytotoxic activity. The T/T genotype in SNP rs2617170 was associated with a higher cytotoxic activity and a decreased risk of cancer development, while it is associated with successful pregnancy in this cohort. American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
The role of NK cells in allograft tolerance has been established suggesting their inability to kill the semiallogenic fetal cells.22,23 Our findings suggest that the variant NKG2D 11 (rs2617170) may influence the successful pregnancy outcome. The NK-cell cytotoxicity against trophoblast cells is tightly regulated by specific inhibitory and activating receptors contributing to immunomodulation at the maternal–fetal interface.24,25 Many studies reported associations between genetic polymorphisms in NKG2D gene and viral defense and autoimmune diseases. Both GG genotype in rs2255336 and TT genotype in rs2617160 conferred a high cytotoxic activity. They were associated, respectively, with an increased risk of SLE and chronic hepatitis B.20,26 In this current study, we identified two haplotype blocks from seven SNPs located in the NKG2D gene on the prevalence of individual SNPs and LD between them (Table IV). After adjustment for age and BMI, differences between patients and controls remain significant for CAT and GGA haplotypes suggesting their susceptibility contribution on the RM risk (aOR = 2.13 [1.24–3.68], aOR = 2.02 [1.03– 3.96], respectively. While, CAA haplotype seems to be protective against RM (aOR = 0.72 [0.54–0.92]). In block2, significant difference between GATG and ATTC haplotypes was seen, thus conferring disease 7
HIZEM ET AL.
susceptibility and protective nature to these haplotypes, respectively (Table V). Our results are different from those of Tomonori Hayashi et al. and J. Luis Espinoza et al., who found a correlation between NKG2D haplotype and phenotype (high or low natural cytotoxic activity) in the occurrence of cancer and the transplants outcomes. The HNK1 haplotype constructed from five SNPs (rs1049174, rs2617160, rs2617170, rs2617171, rs1983526) was significantly associated with reduced risk of cancer and better clinical outcome after transplantation.8,27 Discordance in results may be due to differences in NK cells functions in these different diseases. In pregnancy, other combined alleles and haplotypes seem to affect the cytotoxic activity of NK cells which contributes to fetal reject. On other hand, cytotoxicity of the maternal NK cells has also a positive role in placental formation by limiting excessive trophoblast invasion thus contributing in successful pregnancy.5,6 Kusakabe et al.28 reported that in the process of spontaneous abortion, complement C3 appears to be influential while the cytotoxicity of uterine NK cells is inhibited suggesting the benefic role of NK cells cytotoxicity in successful pregnancy. Conclusion In conclusion, our data support an effect of the NKG2D 11(rs2617170) variant on pregnancy loss risk in the Tunisian women, whereas the other variants tested were not found to play a major role in RM. The strength of our study lies in the large sample population (312 patients and 334 controls), the homogeneity of the ethnic backup (only Tunisian Arab women were included) and in the careful selection of controls, so as to avoid the contribution of confounding factors. It is noteworthy that the association of NKG2D genetic variants to RM risk should be regarded in the context of the cytokine environment in pregnancy, as well as likely participation of other polymorphisms in NKG2D and other modifying genes. Further studies, on other ethnic groups are needed to confirm the current findings and to reveal the mechanism by which NKG2D polymorphisms influence overall RM risk. Acknowledgments We especially thank all of the participants of this project who donated blood for this study. We also 8
especially thank the medical and non-medical staff from the Obstetrics and Gynecology Department of Hospital Farhat Hached in Sousse for collecting the samples involved in this work. Funding This study was supported by the Ministry of Education and Scientific Research in Tunisia and by INSERM, U940, Saint Louis Hospital, Paris, F75010, France. Conflict of interest None declared. References 1 Aruna M, Sudheer PS, Andal S, Tarakeswari S, Reddy AG, Thangaraj K, Singh L, Reddy BM: HLA-G polymorphism patterns show lack of detectable association with recurrent spontaneous abortion. Tissue Antigens 2010; 76:216–222. 2 Kwak-Kim J, Yang KM, Gilman-Sachs A: Recurrent pregnancy loss: a disease of inflammation and coagulation. J Obstet Gynaecol Res 2009; 35:609–622. 3 Branch DW, Gibson M, Silver RM: Clinical practice. Recurrent miscarriage. N Engl J Med 2010; 363:1740–1747. 4 Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V, Benharroch D, Porgador A, Keshet E, Yagel S, Mandelboim O: Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med 2006; 12:1065–1074. 5 Dosiou C, Giudice LC: Natural killer cells in pregnancy and recurrent pregnancy loss: endocrine and immunologic perspectives. Endocr Rev 2005; 26:44–62. 6 Saito S, Nakashima A, Myojo-Higuma S, Shiozaki A: The balance between cytotoxic NK cells and regulatory NK cells in human pregnancy. J Reprod Immunol 2008; 77:14–22. 7 Mincheva-Nilsson L, Nagaeva O, Chen T, Stendahl U, Antsiferova J, Mogren I, Hernestal J, Baranov V: Placenta-derived soluble MHC class I chain-related molecules down-regulate NKG2D receptor on peripheral blood mononuclear cells during human pregnancy: a possible novel immune escape mechanism for fetal survival. J Immunol 2006; 176:3585–3592. 8 Hayashi T, Kazue Imai K, Yukari Morishita Y, Kusunoki Y, Nakachi K: Identification of the NKG2D haplotypes associated with natural cytotoxic activity of peripheral blood lymphocytes and cancer immunosurveillance. Cancer Res 2006; 66:563–570. 9 Beaman KD, Ntrivalas E, Mallers TM, Jaiswal MK, Kwak-Kim J, Gilman-Sachs A: Immune etiology of recurrent pregnancy loss and its diagnosis. Kenneth D. Beaman, Evangelos Ntrivalas. Am J Reprod Immunol 2012; 67:319–325. 10 Dimasi N, Moretta L, Biassoni R: Structure of the Ly49 family of natural killer (NK) cell receptors and their interaction with MHC class I molecules. Immunol Res 2004; 30:95–104. 11 Eagle RA, Trowsdale J: Promiscuity and the single receptor: NKG2D. Nat Rev Immunol 2007; 7:737–744.
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12 Hedlund M, Stenqvist AC, Nagaeva O, Kjellberg L, Wulff M, Baranov V, Mincheva-Nilsson L: Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function. J Immunol 2009; 183:340–351. 13 Amroun H, Djoudi H, Busson M, Allat R, El Sherbini S, Sloma I, Ramasawmy R, Brun M, Dulphy N, Krishnamoorthy R, Toubert A, Charron D, Abbadi MC, Tamouza R: Early-onset ankylosing spondylitis is associated with a functional MICA polymorphism. Hum Immunol 2005; 66:1057–1061. 14 Douik H, Ben Chaaben A, Attia Romdhane N, Romdhane HB, Mamoghli T, Fortier C, Boukouaci W, Harzallah L, Ghanem A, Gritli S, Makni M, Charron D, Krishnamoorthy R, Guemira F, Tamouza R: Association of MICA-129 polymorphism with nasopharyngeal cancer risk in a Tunisian population. Hum Immunol 2009; 70:45–48. 15 Boukouaci W, Marc Busson M, Regis Peffault de Latour P, Rocha V, Suberbielle C, Bengoufa D, Dulphy N, Haas P, Scieux C, Amroun H, Gluckman E, Krishnamoorthy R, Toubert A, Charron D, Socie G, Tamouza R: MICA-129 genotype, soluble MICA, and anti-MICA antibodies as biomarkers of chronic graft-versus-host disease. Blood 2009; 114:5216–5224. 16 Porcu-Buisson G, Lambert M, Lyonnet L, Loundou A, Gamerre M, Camoin-Jau L, Dignat-George F, Caillat-Zucman S, Pascale Paul P: Soluble MHC Class I chain-related molecule serum levels are predictive markers of implantation failure and successful term pregnancies following IVF. Hum Reprod 2007; 22:2261–2266. 17 Choy MK, Phipps ME: MICA polymorphism: biology and importance in immunity and disease. Trends Mol Med 2010; 16:97–106. 18 Hang SY, Chang CH, Chen FP, Yu CL: The alteration of placentalderived soluble MHC class I chain-related protein A and B during pregnancy. Acta Obstet Gynecol Scand 2011; 90:802–807. 19 Park KS, Park JH, Song YW: Inhibitory NKG2A and activating NKG2D and NKG2C natural killer cell receptor genes: susceptibility for rheumatoid arthritis. Tissue Antigens 2008; 72:342–346.
American Journal of Reproductive Immunology (2014) ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
20 Kabalak G, Thomas RM, Martin J, Ortego-Centeno N, JimenezAlonso J, de Ram on E, Buyny S, Hamsen S, Gross WL, Schnarr S, Zeidler H, Gromnica-Ihle E, Schmidt RE, Witte T: Association of an NKG2D gene variant with systemic lupus erythematosus in two populations. Hum Immunol 2010; 71:74–78. 21 Zingoni A, Ardolino M, Santoni A, Cerboni C: NKG2D and DNAM1activating receptors and their ligands in NK–Tcell interactions: role in the NK cell mediated negative regulation of Tcell responses. Front Immunol 2013; 3:408. 22 Beilke JN, Kuhl NR, Van Kaer L, Gill RG: NK cells promote is let allograft tolerance via a perforin-dependent mechanism. Nat Med 2005; 11:1059–1065. 23 Yu G, Xu X, Vu MD, Kilpatrick ED, Li XC: NK cells promote transplant tolerance by killing donor antigen-presenting cells. J Exp Med 2006; 203:1851–1858. 24 Moffett-King A: Natural killer cells and pregnancy. Nat Rev Immunol 2002; 2:656–663. 25 Lash GE, Bulmer JN: Do uterine natural killer (uNK) cells contribute to female reproductive disorders? J Reprod Immunol 2011; 88:156–164. 26 Ma J, Guo X, Wu X, Li J, Zhu X, Li Z, Li J, Pan L, Li T, Li H, Liu Y: Association of NKG2D genetic polymorphism with susceptibility to chronic hepatitis B in a Han Chinese population. Med Virol 2010; 82:1501–1507. 27 Espinoza JL, Takami A, Onizuka A, Sao H, Akiyama H, Miyamura K, Okamoto S, Inoue M, Kanda Y, Ohtake S, Fukuda T, Morishima Y, Kodera Y, Shinji Nakao S: NKG2D gene polymorphism has a significant impact on transplant outcomes after HLA-fully-matched unrelated bone marrow transplantation for standard risk hematologic malignancies. Haematol 2009; 94:1427–1434. 28 Kusakabe K, Naka M, Yuko Ito Y, Eid N, Otsuki Y: Regulation of natural-killer cell cytotoxicity and enhancement of complement factors in the spontaneously aborted mouse placenta. Fertil Steril 2008; 90:1451–1459.
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