Potential Biomarkers for Turner in Maternal Plasma: Possibility for Noninvasive Prenatal Diagnosis Aggeliki Kolialexi,†,‡ Athanasios K. Anagnostopoulos,†,‡,§ Nikos Papantoniou,| Konstantinos Vougas,§ Aris Antsaklis,| Michael Fountoulakis,§ Ariadni Mavrou,‡ 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 May 14, 2010
Turner syndrome (TS) is the most common sex chromosome abnormality in females, caused by the complete or partial absence of one X chromosome. To identify biomarkers for TS, we compared the protein composition of maternal plasma samples from pregnant women with normal and TS fetuses, using a proteomic approach consisting of 2D-E separation and MS analysis for the identification of the differentially expressed proteins. Samples were routinely obtained in the second trimester of pregnancy, stored, and used after prenatal determination of the fetal karyotype. Nine proteins (C1S, CO3, CLUS, AFAM, HABP2, IGHA1, HPT, SHBG, and CD5L) were significantly increased in the plasma of women carrying TS fetuses, whereas KNG1, IGJ, and TTHY were decreased. Identified proteins were further evaluated by immunoblot analysis while functional network association was carried out to asses significance. The identification of specific biomarkers may facilitate the development of noninvasive prenatal diagnosis and improve our understanding of the pathology of TS. Nevertheless, testing a larger cohort of pregnant women is necessary to evaluate the relevance of the reported findings. Keywords: maternal plasma • pregnancy • biomarkers • Turner syndrome • prenatal diagnosis • noninvasive prenatal diagnosis • proteomics • mass spectrometry
Introduction Turner syndrome (TS) is the most common sex chromosome abnormality in females, affecting an estimated 3% of all females conceived. The frequency among liveborn females is 1/2.5001 and as many as 15% of spontaneous miscarriages have a 45,XO karyotype.2 It has been estimated that only 1 in 100 embryos with a 45,XO karyotype survive to term.3 The syndrome is typically associated with the absence of one sex chromosome (45,XO), although mosaicism or structural abnormalities in one sex chromosome may also be responsible for the condition. There are wide variations in the clinical features of the syndrome. TS patients usually have short stature, gonadal dysgenesis, lymphedema and characteristic dysmorphisms. They are at risk of congenital heart defects (e.g., coarctation of aorta, bicuspid aortic valve) and may have progressive aortic root dilatation or dissection. They may also display congenital lymphedema, renal malformations, sensorineural hearing loss, * To whom correspondence should be addressed. 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: gthtsangaris@ bioacademy.gr. † These authors contributed equally to this manuscript. ‡ Medical Genetics, Athens University School of Medicine. § Academy of Athens. | Athens University School of Medicine.
5164 Journal of Proteome Research 2010, 9, 5164–5170 Published on Web 08/25/2010
osteoporosis, obesity, diabetes, and atherogenic lipid profile. Patients usually have normal intelligence but may have problems with nonverbal, social, and psychomotor skills. Advanced maternal age is not associated with an increased incidence of the condition. Most prenatally detected cases of TS are diagnosed in cytogenetic studies performed for advanced maternal age, congenital anomalies or biochemical screening. Multiple marker screening such as high hCG and low estriol levels also identify TS.4 Using alpha-fetoprotein, free hCG-b and inhibin A, a 53% detection rate for TS is obtained taking into account a cutoff of 1/270.5 The most useful tool in prenatal diagnosis is ultrasonography. Typical findings in Turner syndrome are increased nuchal translucency, cystic hygroma and renal and cardiac defects. When 45,XO or another karyotype-causing TS is diagnosed in the fetus, the parents are faced with the difficult question of whether or not to continue the pregnancy. Genetic counselling of parents should include a detailed discussion of the variability of somatic abnormalities and the very high likelihood of short stature and ovarian failure. Although TS patients are well integrated at almost all levels, 78.6% of the couples decide to terminate the pregnancy.6,7 Prenatal detection therefore of TS is important and necessary and a noninvasive approach will be ideal. In a series of case-control studies, a 2D-E based proteomic analysis was employed to identify changes in maternal plasma 10.1021/pr100459q
2010 American Chemical Society
Potential Biomarkers for Turner in Maternal Plasma
research articles
Methods
were scanned in a GS-800 Calibrated Densitometer (Bio-Rad Laboratories, Hercules, CA) using the scanning application/ tool of the PD-Quest v8.0 software (Bio-Rad, Hercules, CA). Protein spots of all gels contained in the analysis, were detected, aligned, matched and quantified using the PD-Quest v8.0 image processing software, according to the manufacturer’s instructions. Manual inspection of the spots was used to verify the accuracy of matching. Spot volume was used as the analysis parameter to quantify protein expression. Normalization of each individual spot was performed according to the total quantity of the valid spots in each gel, after subtraction of the background values. Optical Density (O.D.) level (%) of each protein from the control or TS group was determined separately and calculated as the sum of the volume % of all spots from all gels containing the same protein. Selection of protein spots or entire gel regions for MS analysis was based upon O.D. alteration between the two groups analyzed. A minimum of 2.0 fold change in the expression level was used as selection criterion, at the p < 0.05 level.
Sample Preparation. Plasma samples were thawed and centrifuged at 4.000× g for 30 min for the removal of insoluble components. The protein content was determined using the Bionalyzer Automated Electrophoresis Station (Agilent Technologies Inc., Waldbronn, Germany) combined with the Protein 200 plus kit (Agilent Technologies, Inc., Waldbronn, Germany) as previously described.8 Two Dimensional Gel Electrophoresis (2D-E). The experimental procedure was carried as already described.19 In brief, for the isoelectric focusing (IEF), 750 µg of total protein from each sample was diluted in 250 µL buffer consisting of 7 M urea, 50 mM Tris-HCL (pH 8.5), 2 M thiurea, 2%CHAPS, 0.4% dithioerythritol (DTE), 0.2% IPG buffer pH 3-10 (Amersham Biosciences) and 10 µL of protease inhibitors mixture (Roche Diagnostics, Basel, Swiss). After thorough mixing, diluted samples were left at room temperature for 2 h. Samples were next applied on 18 cm, pH 3-10NL, IPG strips (Bio-Rad Lab, Hercules, CA), at their basic and acidic ends, using sample cups. IPG strips had been prepared for IEF by 20 h rehydration in a buffer (8 M urea, 2% CHAPS and 0.4% DTE). To ensure maximal reproducibility in 2D-E experiments and prevent variations due to technical reasons, all 2D gel experiments were carried out simultaneously, under the same electrophoretic conditions (i.e., plasma proteins from TS embryo pregnancies were assayed, extracted and ran always simultaneously with their matched control samples). Fist dimensional electrophoresis focusing started at 250 V and voltage was gradually increased to 5000 V, with 3 V/min, where it was kept constant for 25 h (approximately 85 000 Vh totally). IEF was conducted in a PROTEAN IEF Cell, Bio-Rad apparatus. After focusing, IPG strips were equilibrated first in 6 M urea, 50 mM Tris-HCL (pH 8.8), 2% (w/v) SDS, 30% (v/v) glycerol and 0.5% (w/v) DTE for 15 min then in the same buffer containing 4% (w/v) iodoacetamide instead of DTE, for 15 more minutes. Second dimensional electrophoresis was performed on 12% SDS-polyacrylamide gels (180 × 200 × 1.5 mm3) with a run of 40 mA/gel, in PROTEIN-II multicell apparatuses (BioRad, Hercules, CA). Protein Visualization and Image Analysis. After vertical electrophoresis, gels were fixed in 50% methanol containing 5% phosphoric acid for 2 h. The fixative solution was washed off by agitation in distilled water for 45 min. Protein spots were visualized by application of Coomassie Blue G-250 staining solution (Novex, San Diego, CA) on 2D gels for 12 h. Gel images
Matrix-Assisted Laser Desorption Tandem TOF Mass Spectrometer (MALDI-TOF-MS). Protein spots of interest were annotated manually using the Melanie 4.02 software and excised from 2D gels using Proteiner SPII (Bruker Daltonics, Bremen, Germany). Gel pieces were then placed into 96-well microtitter plates, destained with 150 µL of 30% ACN in 50 mM ammonium bicarbonate and dried in a speed vacuum concentrator (MaxiDry Plus, Heto, Allered, Denmark). In-gel digestion was performed with 0.01 µg/µL trypsin (Roche Diagnostics) for 16 h at room temperature. Next 10 µL of 50% ACN containing 0.3% TFA, was added to each dried gel piece and digested peptides were extracted. Tryptic peptide mixtures (1.5 µL) were applied on an anchor chip MALDI plate with 1 µL of matrix solution, consisting of 0.08% CHCA (Sigma) and the internal standard peptides des-Arg-bradykinin (Sigma, 904.4681 Da) and adrenocorticotropic hormone fragment 18-39 (Sigma, 2465.1989 Da) in 65% ethanol, 50% ACN and 0.1% TFA. Peptide mixtures were analyzed in a MALDI-ToF mass spectrometer (Ultraflex, Bruker Daltonics). Laser shots (n ) 400) of intensity between 40% and 60% were collected and summarized and the peak list was created using the Flexanalysis v2.2 software (Bruker). Smoothing was performed with the Savitzky-Golay algorithm (width 0.2 m/z, cycle number 1). S/N was calculated by SNAP algorithm and a threshold ratio of 2.5 was allowed. Peptide matching and protein searches were performed automatically with use of MASCOT Server 2 (Matrix Science). Peptide masses were compared with the theoretical peptide masses of all available proteins from Homo sapiens in the SWISS-PROT and TREmBL databases. Stringent criteria were used for protein identification with a maximum allowed mass error of 25 ppm and a minimum of four matching peptides. Probability score with p < 0.05 was used as the criterion for affirmative protein identification. Monoisotopic masses were used, and one missed trypsin cleavage site was calculated for proteolytic products. Search parameters included potential residue mass modification for carbamidomethylation and oxidation. Redundancy of proteins that appeared in the database under different names and accession numbers was eliminated. If more than one protein was identified under one spot, the single protein member with the highest protein score was singled out from the multiprotein family. Western Blot Analysis. Ten µg total protein (2 µg in the case of immunoblot for Kininogen) of control and TS embryo samples were separated by 10% SDS-PAGE under reducing
proteome in early pregnancy, in women known to carry fetuses with TS as compared to healthy pregnant women carrying chromosomally normal females.
Study Group and Plasma Collection Maternal peripheral blood was collected from women with uncomplicated pregnancies, undergoing prenatal diagnosis at 16-18 wk gestation. Blood was collected by venipuncture into EDTA-Vacutainers, placed on ice and centrifuged at 2500× g for 10 min at 4 °C. Plasma was stored at -80 °C within 4 h of collection. Upon completion of the fetal karyotype, plasma from 10 women carrying a TS fetus and from 10 women matched for gestational and maternal age with normal fetuses, were chosen for proteomic studies. Written informed consent was obtained from each participant. The protocol was approved by the Athens University Ethics Board.
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Kolialexi et al.
Figure 1. 2D-E master gel of plasma proteins and modified 2D patterns among pregnant women carrying (A) Turner s. fetuses and (B) Normal fetuses. Panels I-X designate in detail 2D gel areas in which spots with altered expression are contained. Numbered arrows indicate all differentially expressed proteins identified by MS, as reported in Table 1.
conditions and electroblotted to Hybond_ECL NC membranes (Amersham Biosciences, Upsala, Sweden). After blocking with 5% nonfat dried milk in TBST solution (20 mM Tris/pH 7.6, 137 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, membranes were washed with TBST and incubated overnight at 4 °C with the appropriate primary antibodies against Clusterin (dilution 1:200), (sc-56079), Kininogen (dilution 1:1000) (sc-23914) and Afamin (dilution 1:200) (sc-74311). Next, membranes were washed with TBST and incubated with antimouse HRP-conjugated secondary antibody (1:5000). After a final wash with TBST solution proteins were detected by the ECL, west pico (Thermo scientific) detection system. Western blots were scanned with a GS-800 calibrated densitometer (Bio-Rad Lab). Band quantification was performed with the Quantity One image processing software (Bio-Rad Lab). Human IgG protein was used as internal control to ensure equal sample loading. All antibodies were purchased by Santa Cruz Biotechnology (CA). Statistical Analysis. To ensure confidence in our experimental approach we employed a design which involved duplicate 2D gels per sample (i.e., to determine analytical variation) and separate preparations for each replicate sample per experiment (i.e., to determine biological variation), summing up to 40 2D gels in total. 5166
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Mean densitometry values of all spots corresponding to a specific protein from each group were first checked for normal distribution using the Kolmogorov-Smirnov/Lilliefor test (StatPlus 2007 software, AnalystSoft, Vancouver, Canada). Data with normally distributed densitometric values were exported to Microsoft Excel 2007 software and compared with the two pair t-test assuming unequal variances. Means of spot intensities for proteins with not normally distributed values were compared for statistical significance with the Mann-Whitney nonparametric test (GraphPad Instat 3 software, GraphPad software Inc., La Jolla, CA). Statistical significance (a-level) was defined as p < 0.05. To control the False Discovery Rate (FDR), individual a-levels for each spot were adjusted following the FDR correction procedure.9 In Western Blot experiments, mean protein quantification was performed by three independent experiments for each protein analyzed, designed to contain per blot 4 out of 10 plasma samples from cases known to carry chromosomally normal fetuses randomly selected and four samples from pregnant women with TS fetuses. Optical density means of the bands for each protein of the control and TS groups were compared with two sample t-test assuming unequal variances of the Microsoft Excel 2007 software. A p-value < 0.01 was considered statistically significant.
research articles
Potential Biomarkers for Turner in Maternal Plasma
Table 1. Proteins Differentially Present in the 2nd Trimester Maternal Plasma of Women Carrying Turner Syndrome Fetusesa
spot no.
accession no.
1-5
P09871
6-13
P01042
14-21
P01024
22-34
P10909
35-39
P01591
40-44
P43652
45-47
Q14520
48-56
P01876
57,63,64,65,69
P00738
58,59,66,67
P04278
60,62,68
O43866
70-73
P02766
protein entry name/identity
C1S_HUMAN Complement C1s subcomponent precursor KNG1_HUMAN Kininogen-1 precursor CO3_HUMAN Complement C3 precursor CLUS_HUMAN Clusterin precursor IGJ_HUMAN Immunoglobulin J chain AFAM_HUMAN Afamin precursor HABP2_HUMAN Hyaluronan-binding protein 2 precursor IGHA1_HUMAN Ig alpha-1 chain C region HPT_HUMAN Haptoglobin precursor [Contains: Haptoglobin alpha chain] SHBG_HUMAN Sex hormone-binding globulin precursor CD5L_HUMAN CD5 antigen-like precursor TTHY_HUMAN Transthyretin precursor
MW (Da)
pI
Mascot matched coverage score peptides (%)
mean density% ( SD (× 1000) 45,X0
46,XX
expression level
78,174 6.02
163
32/70
20
13 ( 3.21 3.53 ( 0.75
4.33b
72,996 6.4
104
19/88
27
31 ( 2.9
0.46b
134
32/70
20
33.5 ( 5.9
11 ( 0.07
3.04c
53,031 5.9
126
18/72
38
59.2 ( 6,1
25 ( 3.6
2.36c
16,041 4.4
62
4/23
32
5.7 ( 2.17 13.7 ( 1.69
0.41c
70,963 5.6
63
8/61
16
9.2 ( 2.6
3.4 ( 0.74
2.69c
64,740 6.1
69
13/83
23
26.2 ( 3.6
11.8 ( 1.69
2.22b
68,486 6.08
61
6/86
41
49.5 ( 6.2
16.4 ( 2.33
3.01c
45,861 6.1
122
16/57
33
6.2 ( 0.7
2.89 ( 0.2
2.17b
43,980 6.2
108
15/74
50
56.8 ( 2.7
25.8 ( 0.9
2.2b
39,603 5.17
72
11/49
45
15,991 5.4
76
6/32
48
188,569 6
8.8 ( 1.38 13.5 ( 2.2
67.2 ( 5.2
4.3 ( 0.35 35.4 ( 4.1
2.04c 0.38b
a Proteins are presented with their symbol and spot numbers as indicated in Figure 1. Theoretical pI, and molecular weight calculated by the CalPI/MW available on the Swiss-Prot Web site. Score is -10 Log (p), where p is the probability that the observed match is a random event. Scores >55 indicate identity or extensive homology at the p < 0.05 level. Quantification of the differentially expressed proteins (%Total density) spot density/densities of all spots resolved in the gel), is presented in the last three columns. Protein expression >1 states over-expression, while
