A METHOD OF DIAGNOSING AND/OR PROGNOSING PREECLAMPSIA

Abstract

The present invention relates to a method of diagnosing and/or prognosing preeclampsia. Specifically, the method involves determining the quantitative level of one or more biomarkers in a biological sample from the subject and either diagnosing preeclampsia; prognosing unstable moderate early-onset preeclampsia; and/or diagnosing preeclampsia and prognosing unstable moderate early-onset preeclampsia in the subject.

Claims

1. A method of diagnosing preeclampsia and/or prognosing unstable moderate early-onset preeclampsia, in a subject; the method comprising the steps of: (a) determining the quantitative level of one or more biomarkers in a biological sample from the subject; and (b) diagnosing preeclampsia and/or prognosing unstable moderate early-onset preeclampsia in the subject based on the quantitative level of the or each biomarker in the biological sample; wherein the or each biomarker is selected from FN1, CPB2, ORM2, IGLC2, C5, C9, ENDOD1, FGA, HBD, PSG1, STOM, EHD1 and DNM1L.

2. The method according to claim 1 wherein the method is a method of diagnosing preeclampsia, and the or each biomarker is selected from FN1, CPB2, ENDOD1 and EHD1.

3. The method according to claim 2 wherein the or each biomarker is further selected from C5, PSG1, DNM1L, PRDX2, IGLC2, ORM2, FBLN1, HBD, STOM, FGA, AMBP, C3, GUSB, C9, CCT7, PSG2, CFB, HBB, SERPINA1, APOC3, FGG and IGHG2.

4. The method according to claim 1 wherein the method is a method of prognosing unstable moderate early-onset preeclampsia, and the or each biomarker is selected from FN1, CPB2, C5, PSG1, DNM1 L, IGLC2, HBD and ORM2.

5. The method according to claim 4 wherein the or each biomarker is further selected from ENDOD1, EHD1, STOM, FGA, COL4A2, APOE, PSG9, C9, ALB, PRKAR2B, LBP, HPSE, PSG3, SHBG, SVEP1, UBE2L3 and SERPIND1.

6. The method according to claim 1 wherein the or each biomarker for diagnosing preeclampsia and/or prognosing unstable moderate early-onset preeclampsia is a peptide having an amino acid sequence selected from any one or more of FGFCPMAAHEEICTTNEGVMYR, YSFCTDHTVLVQTR, VDVIPVNLPGEHGQR, EATIPGHLNSYTIK, IYLYTLNDNAR, TYLGNALVCTCYGGSR, ESKPLTAQQTTK, IHIGSSFEK, NWGLSFYADKPETTK, EQLGEFYEALDCLCIPR, SYSCQVTHEGSTVEK, ATLVCLISDFYPGAVTVAWK, GGSASTWLTAFALR, MVETTAYALLTSLNLK, LSPIYNLVPVK, VVEESELAR, RPWNVASLIYETK, ALTPQCGSGEDLYILTGTVPSDYR, HPDEAAFFDTASTGK, VQHIQLLQK, GLIDEVNQDFTNR, VNVDAVGGEALGR, GTFSQLSELHCDK, FTFTLHLETPKPSISSSNLNPR, LPKPYITINNLNPR, EASMVITESPAALQLR, VIAAEGEMNASR, VYGALMWSLGK and IFSPNVVNLTLVDLPGMTK.

7. The method according to claim 2 wherein the or each biomarker is a peptide having an amino acid sequence selected from any one or more of FGFCPMAAHEEICTTNEGVMYR, YSFCTDHTVLVQTR, VDVIPVNLPGEHGQR, EATIPGHLNSYTIK, IYLYTLNDNAR, TYLGNALVCTCYGGSR, IHIGSSFEK, ALTPQCGSGEDLYILTGTVPSDYR and VYGALMWSLGK.

8. The method according to claim 3 wherein the or each biomarker is a peptide having an amino acid sequence selected from any one or more of GGSASTWLTAFALR, FTFTLHLETPKPSISSSNLNPR, IFSPNVVNLTLVDLPGMTK, LSEDYGVLK, EGGLGPLNIPLLADVTR, SYSCQVTHEGSTVEK, NWGLSFYADKPETTK, ATLVCLISDFYPGAVTVAWK, AITPPHPASQANIIFDITEGNLR, VNVDAVGGEALGR, EASMVITESPAALQLR, HPDEAAFFDTASTGK, ETLLQDFR, AFIQLWAFDAVK, SSLSVPYVIVPLK, IPIEDGSGEVVLSR, SLLEQYHLGLDQK, LSPIYNLVPVK, SLHDAIMIVR, NSATGEESSTSLTVK, SDPVTLNLLHGPDLPR, DFHINLFQVLPWLK, GTFATLSELHCDK, LYHSEAFTVNFGDTEEAK, GWVTDGFSSLK, IHLISTQSAIPYALR and NQVSLTCLVK.

9. The method according to claim 4 wherein the biomarker is a peptide having an amino acid sequence selected from any one or more of ESKPLTAQQTTK, IHIGSSFEK, GGSASTWLTAFALR, MVETTAYALLTSLNLK, FTFTLHLETPKPSISSSNLNPR, LPKPYITINNLNPR, IFSPNVVNLTLVDLPGMTK, SYSCQVTHEGSTVEK, GTFSQLSELHCDK and EQLGEFYEALDCLCIPR.

10. The method according to claim 5 wherein the or each biomarker is a peptide having an amino acid sequence selected from any one or more of ALTPQCGSGEDLYILTGTVPSDYR, VYGALMWSLGK, VIAAEGEMNASR, VQHIQLLQK, SVSIGYLLVK, VQAAVGTSAAPVPSDNH, SNPVILNVLYGPDLPR, GLIDEVNQDFTNR, VVEESELAR, RPWNVASLIYETK, DVFLGMFLYEYAR, LVRPEVDVMCTAFHDNEETFLK, AATITATSPGALWGLDR, ITLPDFTGDLR, TDFLIFDPK, LFIPQITTK, DIPQPHAEPWAFSLDLGLK, LLSDFPVVPTATR, IEINFPAEYPFKPPK and FPVEMTHNHNFR.

11. The method according to claim 8 wherein the determining step (a) further comprises determining the quantitative level in a first set of biomarkers, wherein the quantitative level of the first set of biomarkers greater than the quantitative level of the respective biomarkers in a normal sample is indicative of preeclampsia, wherein the first set of biomarkers is selected from any one or more of FGFCPMAAHEEICTTNEGVMYR, YSFCTDHTVLVQTR, VDVIPVNLPGEHGQR, EATIPGHLNSYTIK, IYLYTLNDNAR, TYLGNALVCTCYGGSR, VYGALMWSLGK, GGSASTWLTAFALR, LSEDYGVLK, EGGLGPLNIPLLADVTR, NWGLSFYADKPETTK, AITPPHPASQANIIFDITEGNLR, VNVDAVGGEALGR, EASMVITESPAALQLR, HPDEAAFFDTASTGK, ETLLQDFR, AFIQLWAFDAVK, SSLSVPYVIVPLK, SLHDAIMIVR, NSATGEESSTSLTVK, GTFATLSELHCDK, LYHSEAFTVNFGDTEEAK, GWVTDGFSSLK and IHLISTQSAIPYALR.

12. The method according to claim 8 wherein the determining step (a) further comprises determining the quantitative level in a second set of biomarkers, wherein the quantitative level of the second set of biomarkers less than the quantitative level of the respective biomarkers in a normal sample is indicative of preeclampsia, wherein the second set of biomarkers is selected from any one or more of IHIGSSFEK, ALTPQCGSGEDLYILTGTVPSDYR, FTFTLHLETPKPSISSSNLNPR, IFSPNVVNLTLVDLPGMTK, SYSCQVTHEGSTVEK, ATLVCLISDFYPGAVTVAWK, IPIEDGSGEVVLSR, SLLEQYHLGLDQK, LSPIYNLVPVK, SDPVTLNLLHGPDLPR, DFHINLFQVLPWLK and NQVSLTCLVK.

13. The method according to claim 9 wherein the determining step (a) further comprises determining the quantitative level in a third set of biomarkers, wherein the quantitative level of the third set of biomarkers greater than the quantitative level of the respective biomarkers in a normal sample is indicative of unstable moderate early-onset preeclampsia, wherein the third set of biomarkers is selected from any one or more of GGSASTWLTAFALR, MVETTAYALLTSLNLK, FTFTLHLETPKPSISSSNLNPR, LPKPYITINNLNPR, SYSCQVTHEGSTVEK, EQLGEFYEALDCLCIPR, VYGALMWSLGK, SVSIGYLLVK, VQAAVGTSAAPVPSDNH, SNPVILNVLYGPDLPR, RPWNVASLIYETK, DVFLGMFLYEYAR, LVRPEVDVMCTAFHDNEETFLK, AATITATSPGALWGLDR, ITLPDFTGDLR and FPVEMTHNHNFR.

14. The method according to claim 9 wherein the determining step (a) further comprises determining the quantitative level in a fourth set of biomarkers, wherein the quantitative level of the fourth set of biomarkers less than the quantitative level of the respective biomarkers in a normal sample is indicative of unstable moderate early-onset preeclampsia, wherein the fourth set of biomarkers is selected from any one or more of ESKPLTAQQTTK, IHIGSSFEK, IFSPNVVNLTLVDLPGMTK, GTFSQLSELHCDK, ALTPQCGSGEDLYILTGTVPSDYR, VIAAEGEMNASR, VQHIQLLQK, GLIDEVNQDFTNR, VVEESELAR, TDFLIFDPK, LFIPQITTK, DIPQPHAEPWAFSLDLGLK, LLSDFPVVPTATR and IEINFPAEYPFKPPK.

15. The method according to any one of claims 1-3, 6-8 and 11-12 wherein the method of diagnosing preeclampsia is a method of diagnosing early-onset preeclampsia in a subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Embodiments of the present invention will now be described with reference to the following non-limiting examples and the accompanying drawings, in which:

[0143] FIG. 1 illustrates mass spectrometry analysis of pregnancy and early-onset preeclampsia (EOP) platelet releasate;

[0144] FIG. 2 illustrates that 36 peptides are differentially expressed in EOP patients;

[0145] FIG. 3 illustrates that 36 differentially expressed peptides are capable of 100% separation of patients into healthy pregnancy or EOP group;

[0146] FIG. 4 illustrates that 30 peptides are differentially expressed in UM EOP patients;

[0147] FIG. 5 illustrates that 30 differentially expressed peptides are capable of 100% separation of patients into SM EOP or UM EOP; and FIG. 6 illustrates plasma expression of the PR biomarkers, wherein the top biomarkers were chosen to confirm their presence in the plasma of EOP patients, wherein both C5 and CPB2 were quantified in the plasma of healthy pregnant and all EOP patients including stable moderate, unstable moderate and severe EOP patients.

[0148] Materials & Methods

[0149] Patient Recruitment

[0150] Following an informed consent, patients were recruited from the Rotunda Hospital in Dublin. Early-onset preeclampsia (EOP) was diagnosed before 34 weeks of gestation with a new-onset hypertension above 140/90 mmHg measured on two separate occasions, proteinuria 0.3 g/24 hrs or 1+ on a dipstick test, low platelet count, IUGR presence and the evidence of abnormal blood flow. Patients were classified as severe EOP if blood pressure exceeded 160/110 mmHg with proteinuria 1.5 g/24 hrs (or 2+ on a dipstick test). Patients were classified into 3 groups based on their disease progression: [0151] 1. patients with moderate preeclampsia at the time of admission and at delivery but excluding those with diastolic blood pressure (DBP) 90-99 mmHg and systolic blood pressure (SBP) 140-149 mmHg); termed stable moderate early onset preeclampsia (SM EOP); [0152] 2. patients with severe preeclampsia at the time of admission and delivery; termed stable severe preeclampsia (SS EOP); and [0153] 3. patients with moderate preeclampsia at the time of admission, whose condition worsened and ultimately experienced severe PE at the time of delivery (excluding those with DBP 90-99 mmHg and SBP 140-149 mmHg on admission); termed unstable moderate preeclampsia (UM EOP).

[0154] Platelet Releasate Isolation

[0155] Human platelets were obtained from 18 healthy pregnant and 22 early-onset preeclampsia patients in accordance with approved guidelines from University College Dublin and the Rotunda Hospital,

[0156] Dublin. Human platelet releasate was isolated from washed platelets. Blood was drawn from human volunteers free from medication into 0.15% v/v acid/citrate/dextrose (ACD) anticoagulant (38 mM anhydrous citric acid, 75 mM sodium citrate, 124 mM D-glucose). Blood was centrifuged at 150 g for 10 minutes at room temperature and platelet rich plasma (PRP) aspirated. Platelets were pelleted from PRP by centrifugation at 720 g for 10 min at room temperature and resuspended in a modified Tyrodes buffer (JNL) (130 mM NaCl, 10 mM trisodium citrate, 9 mM NaHCO.sub.3, 6 mM dextrose, 0.9 mM MgCl.sub.2, 0.81 mM KH.sub.2PO.sub.4, 10 mM Tris pH 7.4) and supplemented with 1.8 mM CaCl.sub.2. Platelet counts were adjusted to 2×10.sup.8/mL using a Sysmex™ haematology analyser (TOA Medical Electronics, Kobe, Japan). Platelets were activated with 1 U/ml thrombin (Roche, Basel, Switzerland) under constant stirring (1000 rpm) for 5 mins using a Chronolog-700 platelet aggregometer (Chronolog Cor, Manchester, UK). Platelet aggregates were removed by centrifugation by centrifugation three times at 10,000×g for 10 minutes. Harvested platelet releasates were stored at −80° C. until subsequent use.

[0157] Sample Preparation and Mass Spectrometry

[0158] PR samples were solubilised in RIPA buffer and proteins precipitated overnight with 95% acetone (4:1 acetone: sample volume) at −20° C. Dried protein pellets were resuspended in 8M urea/24 mM Tris-HCL, pH 8.2, at 37° C. for one hour. Disulphide bonds were reduced with 5 mM DTT and protected with 15 mM iodoacetamide. PR samples were digested with Lys-C(1:100; Promega, Madison, Wis.) followed by digestion with trypsin (1:100; Promega). Peptides were purified using ZipTipC.sub.18 pipette tips (Millipore, Billerica, Mass., USA) and resuspended in 1% formic acid. For data-dependent acquisition (DDA), samples were analysed using a Thermo-Scientific Q-Exactive mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) liquid chromatography (LC) system. In brief, each 5 μg sample was loaded onto a fused silica emitter (75 μm ID), pulled using a laser puller (Sutter Instruments P2000, Novato, Calif., USA), packed with Reprocil Pur (Dr Maisch, Ammerbuch-Entringen, Germany) C18 (1.9 μm; 12 cm in length) reverse-phase media and separated by an increasing acetonitrile gradient over 47 minutes (flow rate=250 nUmin) direct into a Q-Exactive MS. The MS was operated in positive ion mode with a capillary temperature of 320° C., and with a potential of 2300 V applied to the frit. All data was acquired while operating in automatic data-dependent switching mode. A high resolution (70,000) MS scan (300-1600 m/z) was performed using the Q Exactive to select the 12 most intense ions prior to MS/MS analysis using high-energy collision dissociation (HCD).

[0159] The data-independent acquisition (DIA) isolation scheme and multiplexing strategy was based on that from Egertson et al., 2013 in which five 4-m/z isolation windows are analysed per scan. DIA data were acquired for the psychotic experiences group (see Table 1; 40Cases/66 Controls). Samples were run on the Thermo Scientific Q Exactive mass spectrometer in DIA mode. Each DIA cycle contained one full MS-SIM scan and 20 DIA scans covering a mass range of 490-910.sup.Th with the following settings: the SIM full scan resolution was 35,000; AGC 1e6; Max IT 55 ms; profile mode; DIA scans were set at a resolution of 17,000; AGC target 1e5; Max IT 20 ms; loop count 10; MSX count 5; 4.0 m/z isolation windows; centroid mode (Egertson et al., 2013). The cycle time was 2s, which resulted in at least ten scans across the precursor peak. For the library, QC samples were injected in DDA mode at the beginning of the run, and after every ten injections throughout the run. The relative fragment-ion intensities, peptide-precursor isotope peaks and retention time of the extracted ion chromatograms from the DIA files were used to confirm the identity of the target molecular species.

[0160] Protein Identification and Quantification

[0161] Raw DDA MS files were analysed by MaxQuant (MQ) version 1.5.0.30. MS/MS spectra were searched by the Andromeda search engine against a human FASTA (August 2016) obtained from UniProt. MQ analysis included an initial search with a precursor mass tolerance of 20 ppm the results of which were used for mass recalibration. In the main Andromeda search precursor mass and fragment mass had an initial mass tolerance of 6 ppm and 20 ppm, respectively. The search included fixed modification of carbamidomethyl cysteine. Minimal peptide length was set to 7 amino acids and a maximum of 2 miscleavages was allowed. The false discovery rate (FDR) was set to 0.01 for peptide/protein identification. For quantitative comparison between samples we used label-free quantification (LFQ) with a minimum of two ratio counts to determine the normalized protein intensity. LFQ intensities were assigned to identified proteins by comparing the area under the curve of the signal intensity for any given peptide. The total protein approach (TPA) was used for quantitative comparison, to determine protein abundance as a fraction of total protein.

[0162] All DIA data were processed in the open-source Skyline software tool (open-source Skyline software tool (https://skyline.gs.washington.edu). This tool provided the interface for visual confirmation of protein biomarkers in the samples profiled, without any file conversion. The library was constructed from previously described MaxQuant analysis. As detailed in the online tutorials and publications by the Skyline team, the msms.txt file resulting from the MaxQuant search was used to build the library in Skyline. For our peptide targets, mass chromatograms were extracted for +2 and +3 precursor charge states and their associated fragment ions. Based on our discovery results, we analysed 89 protein candidates. For our dataset, the m/z tolerance was <10 ppm and the average retention time window was 2 minutes. All parent and fragment level data was visually confirmed across the samples run, and peak editing was undertaken where necessary, using the peptide Retention Time (RT), dotproduct (idop), mass accuracy (<10 ppm), and a confirmed library match to reliably identify and quantify peptides across the DIA runs. For statistical analysis, peak areas of the fragment level data was filtered from the Skyline document grid for analysis in mapDIA, an open source bioinformatics tool for pre-processing and quantitative analysis of DIA data. Retention time normalisation procedure was applied, followed by peptide fragment selection using 2 standard deviation threshold for outlier detection, in the independent sample setup. Differential expression analysis was analysed in Perseus software.

[0163] Data were processed in the Perseus open framework (http://www.perseus-framework.org). Protein IDs were filtered to eliminate identifications from the reverse database, proteins only identified by site, and common contaminants. TPA values were Log.sub.2 transformed. A protein was included if it was identified in at least 50% of samples in at least one group. Random forest analysis with either 80:20% or 60:40% data split was performed using Waikato Environment for Knowledge Analysis (WEKA).

EXAMPLE 1

[0164] Mass Spectrometry Analysis of Pregnancy and Early-Onset Preeclampsia (EOP) Platelet Releasate Exhibits Very Good Correlation

[0165] Referring to FIG. 1(A), strong correlations (>0.8) were found in the 18 healthy pregnancies and 22 EOP platelet releasate (PR) samples. FIG. 1(B) shows a table listing average, maximum and minimum correlations as well as standard deviation for 18 healthy pregnancy and 22 EOP PR samples. FIG. 1(C) illustrates pregnancy and EOP platelet releasate proteomes show little variation with an average coefficient of variation (CV) 5.8%±2.5% and 5.9%±2.6% respectively. The maximum variance was lower for the EOP PR (CV=21.9%) when compared to pregnancy PR (CV=24.6%).

[0166] From our discovery (DDA) mass spectrometry analysis we have uncovered 89 protein candidates to analyse through DIA. For 9 of 89 proteins we were not able to identify MS1 or MS2 spectra through our DIA methodology and further 2 did not pass DIA inclusion criteria, therefore these were removed from further analysis. Table 1 contains the complete list (78) of the proteins analysed.

TABLE-US-00001 TABLE 1 A list of proteins analysed through Skyline software following data-independent acquisition method Gene name Protein name UniProt ID FN1 Fibronectin P02751 C5 Complement C5 P01031 PSG1 Pregnancy-specific beta-1-glycoprotein 1 P11464 PRDX2 Peroxiredoxin-2 P32119 DNM1L Dynamin-1-like protein O00429 ENDOD1 Endonuclease domain-containing 1 protein O94919 CFHR1 Complement factor H-related protein 1 Q03591 ORM2 Alpha-1-acid glycoprotein 2 P19652 IGLC2 Immunoglobulin lambda constant 2 P0DOY2 FBLN1 Fibulin-1 P23142 COL4A2 Collagen alpha-2 P08572 HBD Hemoglobin subunit delta P02042 APOE Apolipoprotein E P02649 STOM Erythrocyte band 7 integral membrane protein P27105 AMBP Protein AMBP [Cleaved into: Alpha-1-microglobulin P02760 PSG9 Pregnancy-specific beta-1-glycoprotein 9 Q00887 EHD1 EH domain-containing protein 1 Q9H4M9 FGA Fibrinogen alpha chain [Cleaved into: Fibrinopeptide P02671 A; Fibrinogen alpha chain] C3 Complement C3 P01024 C7 Complement component C7 P10643 CPB2 Carboxypeptidase B2 Q96IY4 GUSB Beta-glucuronidase P08236 CCT7 T-complex protein 1 subunit eta Q99832 IGHV5-10-1 Immunoglobulin heavy variable 5-10-1 A0A0J9YXX1 PSG2 Pregnancy-specific beta-1-glycoprotein 2 P11465 CFB cDNA FLJ55673, highly similar to Complement B4E1Z4 factor B ALB Serum albumin P02768 HBB Hemoglobin subunit beta P68871 PRKAR2B cAMP-dependent protein kinase type II-beta P31323 regulatory subunit LBP Lipopolysaccharide-binding protein P18428 C9 Complement component C9 [Cleaved into: P02748 Complement component C9a; Complement component C9b] HBA1 Hemoglobin subunit alpha P69905 SERPINA1 Alpha-1-antitrypsin P01009 HPSE Heparanase Q9Y251 PSG3 Pregnancy-specific beta-1-glycoprotein 3 Q16557 TGFBI Transforming growth factor-beta-induced protein ig- Q15582 h3 SHBG Sex hormone-binding globulin P04278 IGHV3-33 Immunoglobulin heavy variable 3-33 P01772 SVEP1 Sushi, von Willebrand factor type A, EGF and Q4LDE5 pentraxin domain-containing protein 1 CP Ceruloplasmin P00450 CLEC3B Tetranectin P05452 UBE2L3 Ubiquitin-conjugating enzyme E2 L3 P68036 SERPIND1 Heparin cofactor 2 P05546 APOC3 Apolipoprotein C-III P02656 NUTF2 Nuclear transport factor 2 P61970 CTSC Dipeptidyl peptidase 1 P53634 AGT Angiotensinogen P01019 AHSG Alpha-2-HS-glycoprotein P02765 FGG Fibrinogen gamma chain P02679 PRG2 Bone marrow proteoglycan P13727 PDCD6IP Programmed cell death 6-interacting protein Q8WUM4 IGHG2 Immunoglobulin heavy constant gamma 2 P01859 MMRN1 Multimerin-1 Q13201 ST6GAL1 Beta-galactoside alpha-2,6-sialyltransferase 1 P15907 VTN Vitronectin P04004 APOC2 Apolipoprotein C-II P02655 C1S Complement C1s subcomponent P09871 CAND1 Cullin-associated NEDD8-dissociated protein 1 Q86VP6 CD84 SLAM family member 5 Q9UIB8 CSH2 Chorionic somatomammotropin hormone 2 P0DML3 DMTN Dematin Q08495 EEF1A1P5 Putative elongation factor 1-alpha-like 3 Q5VTE0 F12 Coagulation factor XII P00748 FETUB Fetuin-B Q9UGM5 GANAB Neutral alpha-glucosidase AB Q14697 HINT1 Histidine triad nucleotide-binding protein 1 P49773 HRG Histidine-rich glycoprotein P04196 IGKV2-24 Immunoglobulin kappa variable 2-24 A0A0C4DH68 ITIH3 Inter-alpha-trypsin inhibitor heavy chain H3 Q06033 KNG1 Kininogen-1 P01042 MMP1 Interstitial collagenase P03956 ORM1 Alpha-1-acid glycoprotein 1 P02763 PF4 Platelet factor 4 P02776 PLG Plasminogen P00747 PSG7 Putative pregnancy-specific beta-1-glycoprotein 7 Q13046 PZP Pregnancy zone protein P20742 SERPINA7 Thyroxine-binding globulin P05543 UGP2 UTP--glucose-1-phosphate uridylyltransferase Q16851

[0167] Skyline software allows for an analysis of individual peptides identified for each of the target proteins. Utilising mapDIA software, the area under the peak for each peptide fragments was used to assign quantitative value to a corresponding peptide. Furthermore, the quantitative data was normalised to retention time. Missing values were handled by imputation of a constant number equal to the lowest value ×0.9 for any given peptide. The quantitative peptide data was subjected to statistical analysis in Perseus software.

EXAMPLE 2

[0168] Peptide Expression in EOP Patients

[0169] A peptide level analysis was carried out to assess the diagnosis of EOP further. 207 peptides from 60 proteins were quantified by mapDIA and were subjected to statistical analysis. Student's t-test analysis with an FDR of 5% and S.sub.0 of 0.1 (denoted by the black hyperbolic lines, FIG. 2) was performed on the 71 peptides meeting the minimal difference criteria (>0.5/<−0.5). 36 peptides were found differentially expressed in EOP samples, with 12 peptides decreased (right; IHIGSSFEK, ALTPQCGSGEDLYILTGTVPSDYR, FTFTLHLETPKPSISSSNLNPR, IFSPNVVNLTLVDLPGMTK, SYSCQVTHEGSTVEK, ATLVCLISDFYPGAVTVAWK, IPIEDGSGEVVLSR, SLLEQYHLGLDQK, LSPIYNLVPVK, SDPVTLNLLHGPDLPR, DFHINLFQVLPWLK, NQVSLTCLVK) and 24 peptides increased (left; FGFCPMAAHEEICTTNEGVMYR, YSFCTDHTVLVQTR, VDVIPVNLPGEHGQR, EATIPGHLNSYTIK, IYLYTLNDNAR, TYLGNALVCTCYGGSR, VYGALMWSLGK, GGSASTWLTAFALR, LSEDYGVLK, EGGLGPLNIPLLADVTR, NWGLSFYADKPETTK, AITPPHPASQANIIFDITEGNLR, VNVDAVGGEALGR, EASMVITESPAALQLR, HPDEAAFFDTASTGK, ETLLQDFR, AFIQLWAFDAVK, SSLSVPYVIVPLK, SLHDAIMIVR, NSATGEESSTSLTVK, GTFATLSELHCDK, LYHSEAFTVNFGDTEEAK, GWVTDGFSSLK, IHLISTQSAIPYALR) in EOP.

[0170] FIG. 2 shows a volcano plot of healthy pregnancy vs EOP PR proteomes representing the peptides significantly altered in EOP patients. The black hyperbolic curved lines show the threshold for statistical significance, with an FDR of 0.05 and a minimal difference of 0.1. Our analysis revealed that the secretion levels of 24 peptides were significantly increased in EOP PR (circle) whereas 12 peptides were significantly decreased in EOP PR (triangle).

EXAMPLE 3

[0171] Peptide separation of healthy pregnant and EOP patients

[0172] The hierarchical clustering and principal component analysis of the 36 differential proteins illustrate a complete separation of healthy pregnant and EOP patients (FIG. 3).

[0173] FIG. 3(A) illustrates euclidian distance based two-way hierarchical clustering of the log.sup.2 transformed quantitative values of the 36 peptides (24 increased and 12 decreased) differentially released in EOP PR completely separates the samples into pregnancy and EOP clusters. FIG. 3(B) illustrates principal component analysis of log.sup.2 transformed quantitative values of the 36 differential peptides. Combination of component 1 and 2 enables complete separation of healthy pregnancy (triangle) and EOP (circle) PR samples.

[0174] The peptides deemed differentially expressed in EOP samples were furthermore subjected to machine learning analysis with the WEKA software. The peptides were subjected to InfoGain attribute selection and ranked according to their ability to separate the groups (Table 2).

TABLE-US-00002 TABLE 2 Attribute selection ranking for the 36 differential EOP peptides Rank Gene names Peptide SEQ ID  1 FN1 FGFCPMAAHEEICTTNEGVMYR SEQ ID NO. 1  1 FN1 YSFCTDHTVLVQTR SEQ ID NO. 2  1 FN1 VDVIPVNLPGEHGQR SEQ ID NO. 3  1 FN1 EATIPGHLNSYTIK SEQ ID NO. 4  1 FN1 IYLYTLNDNAR SEQ ID NO. 5  1 FN1 TYLGNALVCTCYGGSR SEQ ID NO. 6  7 CPB2 IHIGSSFEK SEQ ID NO. 7  8 ENDOD1 ALTPQCGSGEDLYILTGTVPSDYR SEQ ID NO. 8  9 EHD1 VYGALMWSLGK SEQ ID NO. 9 10 C5 GGSASTWLTAFALR SEQ ID NO. 10 11 PSG1 FTFTLHLETPKPSISSSNLNPR SEQ ID NO. 11 12 DNM1L IFSPNVVNLTLVDLPGMTK SEQ ID NO. 12 13 PRDX2 LSEDYGVLK SEQ ID NO. 13 14 PRDX2 EGGLGPLNIPLLADVTR SEQ ID NO. 14 15 IGLC2 SYSCQVTHEGSTVEK SEQ ID NO. 15 16 ORM2 NWGLSFYADKPETTK SEQ ID NO. 16 17 IGLC2 ATLVCLISDFYPGAVTVAWK SEQ ID NO. 17 18 FBLN1 AITPPHPASQANIIFDITEGNLR SEQ ID NO. 18 19 HBD VNVDAVGGEALGR SEQ ID NO. 19 20 STOM EASMVITESPAALQLR SEQ ID NO. 20 21 FGA HPDEAAFFDTASTGK SEQ ID NO. 21 22 AMBP ETLLQDFR SEQ ID NO. 22 23 AMBP IHIGSSFEK SEQ ID NO. 23 24 C3 SSLSVPYVIVPLK SEQ ID NO. 24 25 C3 IPIEDGSGEVVLSR SEQ ID NO. 25 26 GUSB SLLEQYHLGLDQK SEQ ID NO. 26 27 C9 LSPIYNLVPVK SEQ ID NO. 27 28 CCT7 SLHDAIMIVR SEQ ID NO. 28 29 PSG2 NSATGEESSTSLTVK SEQ ID NO. 28 30 PSG2 SDPVTLNLLHGPDLPR SEQ ID NO. 30 31 CFB DFHINLFQVLPWLK SEQ ID NO. 31 32 HBB GTFATLSELHCDK SEQ ID NO. 32 33 SERPINA1 LYHSEAFTVNFGDTEEAK SEQ ID NO. 33 34 APOC3 GWVTDGFSSLK SEQ ID NO. 34 35 FGG IHLISTQSAIPYALR SEQ ID NO. 35 36 IGHG2 NQVSLTCLVK SEQ ID NO. 36 The top 9 peptides (Table 3) were then used in a Random forest analysis where 80% of the data (EOP n = 18, PC n = 14) was used for training and 20% of the data (EOP n = 4, PC n = 4) was used for validation resulted in ROC AUC = 1, Specificity = 100% and Sensitivity = 100%. The addition of further peptides did not improve classification.

TABLE-US-00003 TABLE 3 Random forest classification of the healthy pregnant and EOP patients based on the 9 top peptides Classified as: Healthy pregnant EOP Healthy pregnant 4 0 EOP 0 4

EXAMPLE 4

[0175] Peptide Expression in UM EOP Patients

[0176] A peptide level analysis was carried out to assess the risk stratification for UM EOP further. 204 peptides from 63 proteins were quantified by mapDIA and were subjected to statistical analysis. Student's t-test analysis with an FDR of 5% and S0 of 0.1 (denoted by the black hyperbolic lines, (FIG. 4) was performed on the 54 peptides meeting the minimal difference criteria (>1/<−1). 30 peptides were found differentially expressed in UM EOP samples, with 14 peptides decreased (right; ESKPLTAQQTTK, IHIGSSFEK, IFSPNVVNLTLVDLPGMTK, GTFSQLSELHCDK, ALTPQCGSGEDLYILTGTVPSDYR, VIAAEGEMNASR, VQHIQLLQK, GLIDEVNQDFTNR, VVEESELAR, TDFLIFDPK, LFIPQITTK, DIPQPHAEPWAFSLDLGLK, LLSDFPVVPTATR, IEINFPAEYPFKPPK) and 16 peptides increased (left; GGSASTWLTAFALR, MVETTAYALLTSLNLK, FTFTLHLETPKPSISSSNLNPR, LPKPYITINNLNPR, SYSCQVTHEGSTVEK, EQLGEFYEALDCLCIPR, VYGALMWSLGK, SVSIGYLLVK, VQAAVGTSAAPVPSDNH, SNPVILNVLYGPDLPR, RPWNVASLIYETK, DVFLGMFLYEYAR, LVRPEVDVMCTAFHDNEETFLK, AATITATSPGALWGLDR, ITLPDFTGDLR, FPVEMTHNHNFR) in UM EOP compared to SM EOP.

[0177] FIG. 4 illustrates a volcano plot representing the peptides significantly altered in UM EOP patients. The black hyperbolic curved lines show the threshold for statistical significance, with an FDR of 0.05 and a minimal difference of 0.1. Our analysis revealed that the secretion levels of 16 peptides were significantly increased in UM EOP PR (circle) whereas 14 peptides were significantly decreased in UM EOP PR (triangle).

EXAMPLE 5

[0178] Peptide Separation of Patients into SM EOP or UM EOP

[0179] The hierarchical clustering and principal component analysis of the 30 differential peptides illustrate a complete separation of SM EOP and UM EOP patients (FIG. 5).

[0180] FIG. 5(A) illustrates euclidian distance based two-way hierarchical clustering of the log.sup.2 transformed quantitative values of the 30 peptides (16 increased and 14 decreased) differentially released in UM EOP PR completely separates the samples into SM EOP and UM EOP clusters. FIG. 5(B) illustrates principal component analysis of log.sup.2 transformed quantitative values of the 30 differential peptides. Combination of component 1 and 2 enables complete separation of SM EOP (triangle) and UM EOP (circle) PR samples.

[0181] The peptides deemed differentially expressed in SM and UM EOP samples were furthermore subjected to machine learning analysis with the WEKA software. The peptides were subjected to InfoGain attribute selection and ranked according to their ability to separate the groups (Table 4).

TABLE-US-00004 TABLE 4 Attribute selection ranking for the 30 differential UM EOP peptides Rank Gene name Peptide SEQID  1 FN1 ESKPLTAQQTTK SEQ ID NO. 37  2 CPB2 IHIGSSFEK SEQ ID NO. 7  3 C5 GGSASTWLTAFALR SEQ ID NO. 10  4 C5 MVETTAYALLTSLNLK SEQ ID NO. 38  5 PSG1 FTFTLHLETPKPSISSSNLNPR SEQ ID NO. 11  6 DNM1L IFSPNVVNLTLVDLPGMTK SEQ ID NO. 12  7 PSG1 LPKPYITINNLNPR SEQ ID NO. 39  8 IGLC2 SYSCQVTHEGSTVEK SEQ ID NO. 15  9 HBD GTFSQLSELHCDK SEQ ID NO. 40 10 ORM2 EQLGEFYEALDCLCIPR SEQ ID NO. 41 11 ENDOD1 ALTPQCGSGEDLYILTGTVPSDYR SEQ ID NO. 8 12 EHD1 VYGALMWSLGK SEQ ID NO. 9 13 STOM VIAAEGEMNASR SEQ ID NO. 42 14 FGA VQHIQLLQK SEQ ID NO. 43 15 COL4A2 SVSIGYLLVK SEQ ID NO. 44 16 APOE VQAAVGTSAAPVPSDNH SEQ ID NO. 45 17 PSG9 SNPVILNVLYGPDLPR SEQ ID NO. 46 18 FGA GLIDEVNQDFTNR SEQ ID NO. 47 19 C9 VVEESELAR SEQ ID NO. 48 20 C9 RPWNVASLIYETK SEQ ID NO. 49 21 ALB DVFLGMFLYEYAR SEQ ID NO. 50 22 ALB LVRPEVDVMCTAFHDNEETFLK SEQ ID NO. 51 23 PRKAR2B AATITATSPGALWGLDR SEQ ID NO. 52 24 LBP ITLPDFTGDLR SEQ ID NO. 53 25 HPSE TDFLIFDPK SEQ ID NO. 54 26 PSG3 LFIPQITTK SEQ ID NO. 55 27 SHBG DIPQPHAEPWAFSLDLGLK SEQ ID NO. 56 28 SVEP1 LLSDFPVVPTATR SEQ ID NO. 57 29 UBE2L3 IEINFPAEYPFKPPK SEQ ID NO. 58 30 SERPIND1 FPVEMTHNHNFR SEQ ID NO. 59 The top 10 peptides (Table 5) were then used in a Random forest analysis where 60% of the data (SM EOP n = 2, UM EOP n = 5) was used for training and 40% of the data (SM EOP n = 3, UM EOP n = 1) was used for validation resulted in ROC AUC = 1, Specificity = 100% and Sensitivity = 100% The addition of further peptides did not improve classification.

TABLE-US-00005 TABLE 5 Random forest classification of the SM EOP and UM EOP patients based on the 11 top peptides Classified as: SM EOP UM EOP SM EOP 3 0 UM EOP 0 1

TABLE-US-00006 TABLE 6 Overlapping proteins and peptides that diagnose preeclampsia; and/or prognose unstable moderate early-onset preeclampsia Peptide in Gene Peptide in prognostic Name diagnostic panel panel Protein PE v Normal UM EOP v SM EOP ORM2 NWGLSFYADKPETTK EQLGEFYEAL Alpha-1- a b ←classified as a b ←classified as (SEQ ID. 16) DCLCIPR (SEQ acid 31|a = PC 31|a = PC ID. 41) glycoprotein 04|b = PE 04|b = PE 2 IGLC2 SYSCQVTHEGSTVEK SYSCQVTHEG Immuno- a b ←classified as a b ←classified as (SEQ ID. 15); STVEK (SEQ globulin 31|a = PC 31|a = PC ATLVCLISDFYPGAVTV ID. 15) lambda 13|b = PE 04|b = PE AWK (SEQ ID. 17) constant 2 C5 GGSASTWLTAFALR GGSASTWLTA Complement a b ←classified as a b ←classified as (SEQ ID. 10) FALR (SEQ C5 31|a = PC 31|a = PC ID. 10); 04|b = PE 04|b = PE MVETTAYALLT SLNLK (SEQ ID. 38) C9 LSPIYNLVPVK (SEQ VVEESELAR Complement a b ←classified as a b ←classified as ID. 27) (SEQ ID. 48); component 13|a = PC 31|a = PC RPWNVASLIY C9 13|b = PE 13|b = PE ETK (SEQ ID. 49) CPB2 IHIGSSFEK IHIGSSFEK Carboxy- a b ←classified as a b ←classified as (SEQ ID. 7) (SEQ ID. 7) peptidase B2 40|a = PC 31|a = PC 04|b = PE 04|b = PE ENDO ALTPQCGSGEDLYILTG ALTPQCGSGE Endo- a b ←classified as a b ←classified as D1 TVPSDYR (SEQ ID. 8) DLYILTGTVPS nuclease 40|a = PC 31|a = PC DYR (SEQ domain- 04|b = PE 04|b = PE ID. 8) containing 1 protein FGA HPDEAAFFDTASTGK VQHIQLLQK Fibrinogen a b ←classified as a b ←classified as (SEQ ID.21) (SEQ ID. 43); alpha chain 22|a = PC 13|a = PC GLIDEVNQDFT 04|b = PE 13|b = PE NR (SEQ ID. 47) FN1 FGFCPMAAHEEICTTN ESKPLTAQQT Fibronectin a b ←classified as a b ←classified as EGVMYR (SEQ ID. 1); TK (SEQ ID. 37) 40|a = PC 31|a = PC YSFCTDHTVLVQTR 04|b = PE 04|b = PE (SEQ ID. 2); VDVIPVNLPGEHGQR (SEQ ID. 3); EATIPGHLNSYTIK (SEQ ID. 4); IYLYTLNDNAR (SEQ ID. 5); TYLGNALVCTCYGGSR (SEQ ID. 6) HBD VNVDAVGGEALGR GTFSQLSELH Hemoglobin a b ←classified as a b ←classified as (SEQ ID. 19) CDK (SEQ subunit 13|a = PC 21|a = PEMM ID. 40) delta 04|b = PE 01|b = PEMS PSG1 FTFTLHLETPKPSISSS FTFTLHLETPK Pregnancy- a b ←classified as a b ←classified as NLNPR (SEQ ID. 11) PSISSSNLNPR specific 31|a = PC 31|a = PC (SEQ ID. 11); beta-1- 22|b = PE 04|b = PE LPKPYITINNLN glycoprotein PR (SEQ ID. 39) 1 STOM EASMVITESPAALQLR VIAAEGEMNA Erythrocyte a b ←classified as a b ←classified as (SEQ ID. 20) SR (SEQ ID. 42) band 7 22|a = PC 40|a = PC integral 13|b = PE 04|b = PE membrane protein EHD1 VYGALMWSLGK (SEQ VYGALMWSLG EH a b ←classified as a b ←classified as ID. 9) K (SEQ ID. 9) domain- 40|a = PC 21|a = PEMM containing 04|b = PE 10|b = PEMS protein 1 DNM1L IFSPNVVNLTLVDLPGM IFSPNVVNLTL Dynamin 1 a b ←classified as a b ←classified as TK (SEQ ID. 12) VDLPGMTK Like protein 31|a = PC 30|a = PEMM (SEQ ID. 12) 04|b = PE 01|b = PEMS

[0182] Table 6 illustrates the proteins and peptides that diagnose preeclampsia, and/or prognose unstable moderate early-onset preeclampsia.

[0183] CPB2, ENDOD1 and FN1 diagnose preeclampsia with 100% specificity.

[0184] ORM2, IGLC2, C5, CBP2, FN1, HBD and PSG1 progonse UM EOP with 100% specificity.

EXAMPLE 6

[0185] Peptide Expression in Plasma Samples

[0186] To investigate if platelet-releasate-uncovered biomarkers can be found in plasma of EOP women, two top biomarkers were selected to confirm that these PR effectors can be found in plasma of early-onset preeclampsia (EOP) women. These biomarkers were quantified using an antibody-based test.

[0187] In brief, plasma from 22 early-onset preeclampsia patients and 18 healthy pregnant controls were diluted with 3% BSA in TBS and 100 μI (for CPB2 detection), or diluted with a diluent provided with the antibody and 50 μl (for C5 detection), was incubated in a 96-well plate coated with a corresponding primary antibody (Abcam plc) for 2h for C5 and 30 min for CPB2 according to the manufacturer's instructions to facilitate protein binding. Following binding of the protein to the antibody, excess plasma was removed and any unbound proteins washed off according to the manufacturer's instructions. Samples were then incubated with a complementary antibody (Abcam plc) for 1 h (C5) or 30 min (CPB2). For the detection of C5, excess of complementary antibody was washed off and samples were incubated further with conjugate antibody (Abcam plc) according to the manufacturer's instructions. The excess of the antibody was washed off again, and a chemiluminescent substrate was added to the samples for 10 min to allow for reaction to occur according to the manufacturer's instructions. The reaction was stopped, and the absorbance reading obtained at 450 nm. Protein concentration was calculated using standards of known concentrations.

[0188] The top biomarkers (C5 and CPB2) were found and quantified in the plasma of EOP patients (including unstable moderate and stable moderate EOP patients) and healthy pregnant controls (see FIG. 6.

[0189] Results

[0190] Pearson correlation coefficient (r) and coefficient of variation (CV) analysis was performed to assess biological variability in protein abundances. We found strong inter-donor reproducibility across our PR samples, averaging at r=0.932±0.034 for healthy pregnant controls (see FIGS. 1 (A&B)) and r =0.926±0.033 for EOP patients (including stable moderate (SM), unstable moderate (UM) and stable severe (SS) EOP patients) (see FIGS. 1 (A&B)). The average variance in protein quantitation was very low both in pregnant control (average CV=5.8%±2.5%) and EOP PR (average CV=5.9%±2.6%), indicating that PR contents were highly reproducible across donors. Moreover, maximum variance was low in both pregnant control and EOP PR (24.6% and 21.9% respectively; see FIG. 1 (C)). Collectively, these data suggest that the PR is a stable proteome in healthy pregnant and EOP women with vast majority of proteins exhibiting <20% associated variability.