Proteomic Analysis of Amniotic Fluid in Pregnancies with Turner Syndrome Fetuses Ariadni Mavrou,† Athanasios K. Anagnostopoulos,†,‡ Aggeliki Kolialexi,† Konstantinos Vougas,‡ Nikos Papantoniou,§ Aris Antsaklis,§ Michael Fountoulakis,‡ and George Th. Tsangaris*,‡ Medical Genetics, Athens University School of Medicine, Athens, Greece, Proteomics Research Unit, Centre of Basic Research II, Biomedical Research Foundation, Academy of Athens, Athens, Greece, and 1st Department of Obstetrics & Gynaecology, Athens University School of Medicine, Athens, Greece Received September 10, 2007
Turner syndrome, occurring in 1:2500 female births, is caused by the complete or partial absence of one X chromosome. Amniotic fluid supernatant proteins from five second trimester pregnancies with Turner syndrome fetuses and five normal ones were analyzed by 2DE, MALDI-TOF-MS, and Western blot. Serotransferin, lumican, plasma retinol-binding protein, and apolipoprotein A-I were increased in Turner syndrome, while kininogen, prothrombin, and apolipoprotein A-IV were decreased. Since differentially expressed proteins are likely to cross the placenta barrier and be detected in maternal plasma, proteomic analysis may enhance research for noninvasive prenatal diagnosis of Turner syndrome. Keywords: Amniotic fluid • Amniotic fluid supernatant • Biomarkers • Turner syndrome • Prenatal diagnosis • Proteomics • MALDI TOF-MS • Mass spectrometry • Two-dimensional electrophoresis
Introduction Turner Syndrome, occurring in approximately 1:2500 female births, is caused by a sex chromosome abnormality involving the presence of only one normal X chromosome and the complete or partial absence of the second.1 Short stature and/ or ovarian failure are the most common clinical presentations and the majority of women never produce ovarian hormones. Typical congenital malformations are pterygium colli, horse shoe kidney, and coarctation of the aorta, as well as less severe heart defects, especially associated with the 45, XO karyotype. Osteoporosis, hypothyroidism, and type 2 diabetes are often seen in TS. There is epidemiological evidence for increased morbidity due to ischemic heart disease, hypertension, and stroke.1 Progress made in proteomics over the past few years was rendered possible through the development of protein separation approaches and mass spectrometry (MS). Two-dimensional electrophoresis (2DE) is an excellent tool for proteomic analysis and can be used to compare patterns of protein expression in various biological materials under physiological and pathological conditions. MS, in association with bioinformatics, provides a powerful tool for the rapid identification of protein spots on 2DE.2 * Corresponding author: George Th. Tsangaris Ph.D., Proteomics Research Unit, Centre of Basic Research II, Biomedical Research Foundation, Academy of Athens, Soranou Efesiou 4, 115 27 Athens, Greece. Tel.: ++ 210 6597075. Fax: ++ 210 6597545. E-mail:
[email protected]. † Medical Genetics, Athens University School of Medicine. ‡ Biomedical Research Foundation. § 1st Department of Obstetrics & Gynaecology, Athens University School of Medicine.
1862 Journal of Proteome Research 2008, 7, 1862–1866 Published on Web 03/26/2008
Amniotic fluid, routinely used for prenatal diagnosis, contains large amounts of proteins produced by the amnion epithelial cells, fetal tissues, fetal excretions, and placental tissues. Additionally, molecules originating from maternal circulation are released in the amniotic cavity. The biochemical composition of AF is modified throughout pregnancy and its protein profile reflects both physiological and pathological changes affecting the fetus and the mother.3,4 Identification of changes in the balance of proteins, therefore, may be used to detect a particular type of pathology, or to ascertain a specific genetic disorder. Potential biomarkers in the AF have already been identified in cases of inflammation and premature rupture of fetal membranes.5–7 Witht he use of surface-enhanced laser desorption/ionization time-of-flight MS (SELDI-TOF-MS) distinct proteomic profiles in AF were detected in pregnancies with normal fetuses and fetuses with abnormal karyotypes, such as trisomy 21 (Down syndrome) and trisomy 18.8 We have previously reported on a set of 8 proteins which were differentially expressed in the AF of Down syndrome.9 Turner syndrome is the second in frequency, after Down syndrome, of chromosomal abnormalities identified prenatally, and although the phenotypic abnormalities of this disorder are not as severe, multi-center studies indicate that a majority of Turner syndrome fetuses diagnosed prenatally are legally aborted.10 Ultrasonography can identify fetuses at risk for Turner syndrome, but currently, there are no biochemical screening tests for the identification of pregnancies at risk.
Materials and Methods Materials. In the present study, we used a combined approach, based on 2DE and MS, in order to compare the 10.1021/pr700588u CCC: $40.75
2008 American Chemical Society
research articles
Proteomic Analysis of Amniotic Fluid in Turner Syndrome protein composition of AFS coming from pregnancies with normal and TS fetuses. Twenty milliliters of AF samples were obtained, after written informed consent, from women undergoing amniocentesis in the 16-18th week of gestation (mean gestational age: 16.8 weeks). Following centrifugation for the collection of amniocytes for cytogenetic analysis, supernatants were centrifuged again at 12 000g at 4 °C for 30 min for the removal of insoluble components, aliquoted, and frozen at -80 °C. Ten 2-mL aliquots were chosen for proteomic analysis, 5 from pregnancies that according to conventional cytogenetics carried fetuses with TS (karyotype 45, XO) and 5 from pregnancies, matched for gestational age, with chromosomally normal female fetuses. Samples were not pooled but analyzed independently. Women with gestational diseases or pregnancy complications were excluded from the study and all women used as controls had normal uneventful deliveries at term. The protocol was approved by the Institutional Ethics Board. Methods. 1. Two-Dimensional Gel Electrophoresis (2DE). After precipitation, the protein content of AFS was determined using the EXPERION Automated Electrophoresis Station in combination with the Protein 260 Analysis Kit (Bio-Rad Laboratories, Hercules, CA) as previously described.9 2DE was performed on 17 cm immobilized pH 3–10 nonlinear gradient IPG strips (Bio-Rad). For the second-dimensional electrophoresis 12% SDS polyacrylamide gels were used in a Proteiner apparatus (Bio-Rad). Gels were stained with colloidal Coomassie Blue (Novex, San Diego, CA), scanned in a GS-800 Calibrated Densitometer (Bio-Rad), and analyzed using the PDQuest image processing software (Bio-Rad). Expression levels >1 indicated overexpression and 55 indicate identity or extensive homology at the p < 0.05 level.
protein A-I (ApoA1) were significantly increased in TS cases, whereas kininogen (KNG1), prothrombin (THBR), and apolipoprotein A-IV (ApoA4) were decreased. From the identified proteins, RETBP, LUM, and KNG1, were selected for Western blot analysis in order to further validate the quantitative differences by the PDQuest software: one highly increased (RETBP), one slightly increased (LUM), and one decreased (KNG1) in TS, as compared to normal. Western blotting confirmed the reduction of KNG1 levels and the 1864
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increase of LUM and RETBP in AFS obtained from cases with TS (Figure 2A). Densitometric analysis showed that the AFS coming from women with TS fetuses contained 40% less KNG1, while LUM and RETBP were increased 2.4 and 14.9 times, respectively (Figure 2B).
Discussion The present study identified a set of 7 proteins differentially expressed in AFS coming from fetuses with TS and a 45, XO
Proteomic Analysis of Amniotic Fluid in Turner Syndrome a
Table 2. Expression Levels of the Identified Proteins 45, XO
46, XX
protein symbol
% total density (×1000)
% total density (×1000)
expression level
TRFE LUM RETBP ApoA1 KNG THRB ApoA4
42.8 ( 10 173 ( 30 150 ( 19 185 ( 30 23 ( 12 17 ( 7 29 ( 8
21.1 ( 11 50 ( 25 20 ( 10 25 ( 11 60 ( 22 36 ( 10 62 ( 10
2.02* 3.46* 7.7** 7.4** 0.38* 0.47* 0.46*
a The density of each spot in each sample was measured by the PDQuest software and expressed as percentage to the total optical density. The total level of each protein was calculated as the sum of percentages of all spots containing the same protein (*p < 0.05, **p < 0.005).
Figure 2. Western blot analysis of RETBP, LUM, and KNG1 in normal (46, XX) and TS (45, X0) AFS. (A) Equal protein amounts of AFS from women carrying normal and TS fetuses were separated by gel electrophoresis and immunoblotted with the appropriate dilution of antibody. One membrane out of the three independent replicates is shown. The nonspecific bindings of RETBP polyclonal antibody and human IgG were used as internal loading controls. (B) Quantification of protein content of RETBP, LUM, and KNG1 using scanning densitometry. Each bar represents the mean ( SD of three independent experiments. Dark stripped bars, 45, X0; light bars, 46, XX; *p < 0.05, **p < 0.005.
karyotype. Four of them (TRFE, RETBP, LUM, ApoA1) were upregulated and three (ApoA4, THBR, KNG1) down-regulated.
research articles Since quantitative changes are reported in the relative amount of several proteins in AF between the first and second trimester, only gestational age matched controls were analyzed.12 Database search showed that genes encoding for these proteins are located on chromosomes other than chromosome X, but their expression is possibly regulated by transcription factors located on the X chromosome. A similar assumption was made regarding the contribution to Down syndrome phenotype of genes located on chromosome 21.9,13 All 7 proteins differentially expressed in AFS coming from fetuses with TS are mainly involved in fetal growth and development and include nutrient/ cofactors and hormone carriers. TRFE and RETBP, both carrier proteins, were increased in TS AFS. TRFE binds and transports iron, while RETBP belongs to a group of proteins that bind to and transport thyroid hormones and is indirectly implicated in the transport of vitamin A. Retinols are essential for normal reproduction and play an important role in ovarian steroidogenesis, oocyte maturation, and early embryonic development.14,15 LUM levels were increased by 2.4 times in AFS coming from TS fetuses. LUM is a keratan sulfate proteoglycan that belongs to the family of small leucine-rich repeat proteoglycans. Although this protein was first described in the cornea, it is present in the extracellular matrix of many tissues such as cartilage, aorta, liver skin, muscle intestine, and human amniotic membranes. LUM is known to interact with fibrillar collagen and may influence the interaction of collagen fibrils with other components of the extracellular matrix, participating in its maintenance.16–20 Changes in the expression of ApoA1 and ApoA4 levels were noted in TS AFS. The relative amount of the HDL associated ApoA4 was decreased in TS. ApoA4 is known to possess antiatherogenic properties and the decrease observed is possibly related to the risk for atherosclerosis and coronary artery disease associated with TS.21 On the other hand, ApoA1 was increased. ApoA1 is also reported increased in the peripheral blood of premenarcheal females.22 In ovariectomized baboons, administration of estrogen increases ApoA1 production22 and the same is seen in premenopausal women,23 probably because of increased degradation of HDL. A decrease in THRB levels, an important coagulation factor produced by the liver, was noted. Hepatic abnormalities are relatively frequent in TS and surveillance of liver function is included in the management of these patients.24 KNG1, which also has an important role in blood coagulation, was decreased by 40%. KNG1, a protein with multifunctional domains, serves as the precursor of potent vasoactive kinin peptides and functions as a cysteine proteinase inhibitor.25 During coagulation, high molecular weight kininogen helps to position optimally prekallikrein and factor XI next to factor XII and inhibits the thrombin-plasmin induced aggregation of thrombocytes. Northern and dot blot analysis in the liver of female rats showed a 4-fold increase in kininogen mRNA levels as compared to males.26 Furthermore, ovariectomy is known to reduce kininogen transcripts in the liver, while estradiol replacement of the ovariectomized rats increases kininogen mRNA levels. In contrast, progesterone treatment of the ovariectomized rats results in an increase in renal kallikrein mRNA levels, while it reduces kininogen gene expression as compared to vehicle-treated ovariectomized animals.27 Immunoreactive kininogen levels in the serum are increased by estradiol, but are slightly decreased by progesterone treatment. Journal of Proteome Research • Vol. 7, No. 5, 2008 1865
research articles The differentially expressed proteins identified in TS AFS could be related to the phenotype of the syndrome, and if they cross the placenta barrier, they may represent potential biomarkers in maternal blood. Since, for the time being, the utility of fetal cells and free fetal DNA for noninvasive prenatal diagnosis is limited by their small quantity and difficulty to distinguish them from maternal material, some of these proteins may represent useful potential biomarkers for noninvasive prenatal diagnosis. Nevertheless, follow-up experiments are necessary in order to verify and evaluate the relevance of our findings. Abbreviations: TS, Turner syndrome; MS, mass spectrometry; 2DE, two-dimensional electrophoresis; AF, amniotic fluid; AFS, amniotic fluid supernatant; MALDI TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Acknowledgment. The first two authors contributed equally to the study and should be both considered as first author. Funding from the European Commission for the Special Non-Invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence (LSHB-CT-2004503243) for which this study was funded is gratefully acknowledged. References (1) Gravholt, C. H. Nat. Clin. Pract. Endocrinol. Metab. 2005, 1, 41– 52. (2) Duncan, M. W.; Hunsucker, S. W. Exp. Biol. Med. 2005, 230, 808– 817. (3) Jauniaux, E.; Jurkovic, D.; Gulbis, B.; Collins, W. P.; Zaidi, J.; Campbell, S. Am. J. Obstet. Gynecol. 1994, 170, 1365–1369. (4) Kolialexi, A.; Mavrou, A.; Tsangaris, G. T. Prot. Clin. Applic. 2007, 1, 853–860. (5) Brenner, B. Thromb. Res. 2004, 114, 409–414. (6) Vuadens, F.; Benay, C.; Crettaz, D.; Gallot, D.; Sapin, V.; Schneider, P.; Bienvenut, W. V.; Lemery, D.; Quadroni, M.; Dastugue, B.; Tissot, J. D. Proteomics 2003, 3, 1521–1525. (7) Buhimschi, C. S.; Weiner, C. P.; Buhimschi, I. A. Obstet. Gynecol. Surv. 2006, 61, 481–486.
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