WO2009108073A1 - Biomarkers for prediction of preeclampsia and/or cardiovascular disease - Google Patents

Biomarkers for prediction of preeclampsia and/or cardiovascular disease Download PDF

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WO2009108073A1
WO2009108073A1 PCT/NZ2009/000026 NZ2009000026W WO2009108073A1 WO 2009108073 A1 WO2009108073 A1 WO 2009108073A1 NZ 2009000026 W NZ2009000026 W NZ 2009000026W WO 2009108073 A1 WO2009108073 A1 WO 2009108073A1
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alpha
risk
isoform
proteins
level
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Marion Blumenstein
Robyn North
Mike Mcmaster
Michael Black
Nikola Kirilov Kasabov
Garth James Smith Cooper
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Auckland Uniservices Ltd
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Auckland Uniservices Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • PE is a disorder that occurs only during pregnancy and affects both the mother and her unborn baby.
  • the rapidly progressing condition is diagnosed when the mother develops hypertension and proteinuria, but may cause seizures, kidney failure, liver dysfunction and severe bleeding.
  • SGA small for gestational age babies
  • AGA appropriate birthweight for gestational age
  • Preeclampsia complicates 4-7% of women in their first pregnancies and it is estimated that over 8 million women are affected each year. Over 70,000 women world-wide die from PE each year. As a third of the babies are premature and there is a 3-10 fold increase in baby death, prediction of PE would be a major healthcare advance for future mothers and their babies.
  • PE pathophysiology
  • the initiating event in PE is thought to be abnormal remodelling of spiral arteries during early placentation resulting in ischemic reperfusion insults to the placenta. These events then trigger a maternal response resulting in abnormal endothelial cell function and vasoconstriction, activated inflammation and coagulation that lead to the clinical manifestations of PE.
  • PE occurs in the third trimester (28-40 weeks' gestation) of pregnancy but in severe cases, the disorder occurs in the 2nd trimester. It is very rare for the condition to occur before 22 weeks' gestation.
  • Obstetricians currently screen pregnant women for PE by serial measurements of blood pressure and protein levels in urine, combined with clinical assessment for other signs and symptoms of the syndrome.
  • CVD cardiovascular disease
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • CVD cardiovascular disease
  • certain of these proteins and their relative levels of expression may be used as markers for predicting the risk of cardiovascular disease (CVD) in a female subject. .
  • the invention provides a method for predicting the risk of a female subject developing preeclampsia, the method comprising observing the level of one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof,, and/or nucleic acids encoding the one or more proteins, subunits, proteolytic peptides, precursors and/or isoforms, wherein the one or more proteins is an HDL cargo protein.
  • the HDL cargo protein is chosen from the group consisting: phospholipid transfer protein, cholesteryl ester transfer protein, lecithin-cholesterol acyltransferase, apolipoprotein (apo) C-I , apoC-II, apoC-III, apoC-IV, paraoxonase-1 , ⁇ araoxonase-3 , serum amyloid A 4 , serum amyloid A 2 , serum amyloid A 1 , apoA-I, , apoH, apoA-IV, clusterin, apoA-II, apoL-I, apoD, apoE, apoF, apoM, angiotensinogen (AGT), alpha-2-antiplasmin, serpin peptidase inhibitor, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1 -antitrypsin, bikunin (alphal -microglobulin), kininogen,
  • the invention provides a method for predicting the risk of a female subject developing preeclampsia the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, precursors, proteolytic peptides, and/or isoforms in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids against a standard; wherein a difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing preeclampsia, and wherein the one or more
  • C4A Complement factor H-related protein 1
  • Clusterin Clusterin
  • Transthyretin Hemopexin
  • Serum amyloid P Serum amyloid A4
  • the HDL cargo protein is chosen from the group of HDL cargo proteins described herein before.
  • the precursors, isoforms, proteolytic fragments and/or subunits include: Fibrinogen alpha chain Fibrinogen beta chain Fibrinogen gamma chain
  • a fibrinogen fragment with an approximate molecular weight of 25kDa is A fibrinogen fragment with an approximate molecular weight of 25kDa
  • HKa (a fragment of HK)
  • Apolipoprotein A-I fragment with an approximately molecular weright of 17kDa Apolipoprotein A-I fragment with an approximately molecular weright of 21 kDa
  • Serum amyloid P fragment with an approximate molecular weight of 27kDa Serum amyloid P fragment with an approximate molecular weight of 27kDa
  • a higher or increased level of one or more of the following compared to a standard, is indicative of risk of developing preeclampsia:
  • Fibrinogen and/or the alpha chain, beta chain and/or gamma chain
  • Apolipoprotein A-I and/or fragments thereof with approximate molecular weights of 17,
  • Clusterin (and/or clusterin isoform I)
  • Vitronectin approximately 75 kDa
  • Angiotensinogen alpha2-HS-Glycoprotein zinc-alpha2-Glycoprotein zinc-alpha2-Glycoprotein
  • a lower or decreased level of one or more of the following compared to a standard is indicative of risk of developing preeclampsia:
  • Vitronectin approximately 65 kDa Vitronectin, approximately 75 kDa alphal -Microglobulin (bikunin)
  • a PEDF isoform with an approximate pi at 6.1 Preferably there is at least an approximately 1.3 fold increase or decrease in the level of a particular protein, precursor, isoform, proteolytic peptide, isoform and/or nucleic acid compared to the standard, more preferably at least an approximately 1.5 fold increase or decrease.
  • the invention provides a method for predicting the risk of a female subject developing preeclampsia with a small for gestational age baby (PE-SGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acid encoding the one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, proteolytic peptides, isoforms and/or nucleic acids against a standard; wherein a difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids in the sample compared to the standard is indicative of the risk of
  • the proteins, subunits, precursors, proteolytic peptides and/or isoforms are chosen from the group consisting: alpha- 1-antichymotrypsin; fibrinogen gamma chain; a fibrinogen fragment with an approximate molecular weight of 27 kDa, a fibrinogen fragment with an approximately molecular weight of 25 kDa; high molecular weight kininogen (HK); and, Vitronectin, approximately 65 kDa; Vitronectin, 75 kDa; and Clusterin.
  • a higher level of one or more of alpha- 1 -antichymotrypsin, fibrinogen gamma chain, an approximately 25 kDa fragment of fibrinogen, an approximately 27 kDa fragment of fibrinogen, an approximately 65 kDa part of the two-chain vitronectin molecule and/or clusterin in the sample compared to the standard is indicative of risk of developing PE-SGA.
  • a lower level of HK and/or vitronectin single chain with an approximate molecular weight of 75 kDa in a sample compared to the standard is indicative of risk of developing PE-SGA.
  • the invention provides a method for predicting the risk of a female subject developing PE-SGA 5 the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting apolipoprotein A-I in combination with one or more of fibrinogen, pregnancy zone protein and/or alpha-2-macroglobulin, clusterin isoform I 5 alphal-antichymotrypsin, and complement factor I and/or nucleic acids encoding said proteins in the sample; and, c) comparing the level of each of the proteins and/or nucleic acids in the sample against a standard; wherein a difference in the level of each of the proteins and/or nucleic acids in the sample compared to the standard is indicative of developing PE-SGA.
  • one or more precursors, isoforms, proteolytic peptides and/or subunits of the proteins, or nucleic acids encoding one or more thereof is detected in addition to or in lieu of detection of a particular protein.
  • the method includes detecting an approximately 17 kDa Apo-AI fragment and/or an approximately 21 kDa Apo-AI fragment and/or nucleic acids encoding same.
  • the method includes detecting fibrinogen beta chain and/or fibrinogen gamma chain, and/or nucleic acids encoding same.
  • the method includes detecting each of Apolipoprotein AI and pregnancy zone protein and/or alpha-2-macroglobulin, and/or nucleic acids encoding same.
  • a combination of a higher level of apolipoprotein A-I and a higher level of pregnancy zone protein and/or alpha-2-macroglobulin is indicative of the risk of PE-SGA.
  • the method includes detecting each of Apolipoprotein AI, pregnancy zone protein and/or alpha-2-macroglobulin, and Fibrinogen beta chain, and/or nucleic acids encoding same.
  • a combination of a higher level of apolipoprotein A-I, a higher level of fibrinogen beta chain and a higher level of pregnancy zone protein and/or alpha-2-macroglobulin is indicative of the risk of PE-SGA.
  • a higher level of apolipoprotein AI, a higher level of pregnancy zone protein and/or alpha-2-macroglobulin, a higher level of fibrinogen beta chain, a higher level of fibrinogen gamma chain, a higher level of clusterin isoform I, a higher level of alpha- 1-antichymotrypsin and a higher level of complement factor I is indicative of the risk of PE-SGA.
  • the invention provides a method for predicting the risk of a female subject developing preeclampsia with PE-SGA, the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting each of the proteins apolipoprotein A-I, serum amyloid P, and transthyretin with an approximate molecular weight of 12 kDa, and/or nucleic acids encoding same in the sample; and, c) comparing the level of each of the proteins and/or nucleic acids in the sample against a standard; wherein a difference in the level of each of the proteins and/or nucleic acids in the sample compared to the standard is indicative of developing PE-SGA.
  • the method further includes detecting fibrinogen.
  • one or more precursors, isoforms, proteolytic peptides and/or subunits of each of the proteins, or nucleic acids encoding one or more thereof is detected in addition to or in lieu of detection of a particular protein.
  • the method includes detecting an apolipoprotein A-I fragment with an approximate molecular weight of 27kDa and/or an apolipoprotein fragment with an approximate molecular weight of 17kDa, and/or nucleic acids encoding same.
  • the method includes detecting serum amyloid P with an approximate molecular weight of 27 kDa and/or a nucleic acid encoding same.
  • the invention provides a method for predicting the risk of a female subject developing preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting PEDF, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; and, c) comparing the level of the PEDF, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof against a standard; wherein a difference in the level of the PEDF, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of the risk of developing PE-AGA.
  • a higher level of PEDF, a subunit thereof, a precursor thereof, an isoform thereof, a proteolytic peptide thereof and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of risk of developing PE-AGA.
  • the isoform of PEDF is an isoform with an approximate pi of 5.4.
  • Figure 1 2D-DIGE experimental design. a Total number of spots detected by
  • DIGE as differentially expressed in 20 week pregnancy plasma from women with subsequent PE-AGA and/or PE-SGA.
  • Top7 depleted plasma B. Differentially expressed protein spots with mass spectrometry identification are annotated by spot ID numbers (see Figure 4 for protein IDs). Circled area in the upper right of panel A depicts fibrinogen which was removed from plasma by Top7 depletion (B).
  • Figure 8 Western Blot analysis of native plasma (20 weeks' gestation) with two candidate markers for the prediction of preeclampsia. Blots were incubated with polyclonal anti-human fibrinogen (left blot) and alpha- 1- antichymotrypsin antibody (right blot). Cross reacting bands were revealed by Qdot nanocrystal technology. A control blot with no primary antibody revealed no unspecific binding (far left).
  • Fibrinogen alpha, beta, and gamma chains were detected along with additional higher and lower molecular weight fibrinogen species. Densitometric analysis showed significantly higher protein expression of gamma fibrinogen in women who developed PE-SGA compared to controls, (p ⁇ 0.05). * Two fibrinogen species at approximately 27 and 25 kDa which were up-regulated in two women with PE-SGA. Western blot analysis for alpha- 1-antichymotrypsin confirmed significant up-regulation in early pregnancy plasma preceding disease, (p ⁇ 0.01).
  • Figure 11 LC-MS/MS identification of protein spots detected by 2-D DIGE as being differentially expressed in plasma at 20 weeks' gestation before the onset of preeclampsia and at the time women present with preeclampsia (36-38 weeks' gestation) compared with healthy controls.
  • Figure 12 Western Blot analysis of plasma preceding and at the time of clinical manifestation of preeclampsia reveals aberrant vitronectin processing.
  • Figure 15 PEDF 2D-DIGE Study. Maternal characteristics and pregnancy outcome in women with an abnormal uterine artery Doppler waveform who subsequently developed preeclampsia or remained healthy with an uncomplicated pregnancy. Results are mean (SD), median (IQ range) or N
  • Figure 18 Maternal Characteristics, Pregnancy Outcome and plasma pigment epithelium derived factor (PEDF) levels.
  • PE-AGA 5 preeclampsia with an appropriate birthweigth for gestational age; PE-SGA 5 preeclampsia with a small for gestational age baby. Results are mean (SD) or N (%). *P ⁇ 0.05 r P ⁇ 0.01 r P ⁇ 0.0001 compared with Controls.
  • Figure 20 Box plots showing standardized abundances of two spots (spot 427 and 579) found to be significantly (p ⁇ 0.05) up-regulated in plasma of women prior to developing preeclampsia (week 20 of gestation) in DIGE experiment III (gel picture not shown). P-value calculated by Limma t-test using false discovery rate correction comparing DIGE standardized spot volumes from women with subsequent preeclampsia versus controls.
  • the risk of developing PE can be determined by measuring specific proteins, subunits, precursors, proteolytic peptides and/or isoforms or combinations thereof in plasma, serum, urine and cervical fluid in early pregnancy, prior to the onset of disease (for example, 24 weeks of gestation or less).
  • HDL cargo proteins A significant proportion of the proteins (including particular isoforms, subunits, precursors, and/or proteolytic fragments) identified to be differentially expressed prior to preeclampsia can be grouped in a class of proteins known as high density lipoprotein (HDL) cargo proteins.
  • HDL cargo proteins any one or more HDL cargo protein may be used as a marker to determine the risk of a female subject developing PE.
  • Cardiovascular disease or “CVD” are used herein to refer to artherosclerosis and/or coronary artery disease which can manifest as angina and/or myocardial infarction, for example.
  • Risk of developing PE, CVD, or normotensive SGA is used herein to refer to the risk of developing PE , CVD or normotensive SGA at some time in the future; ie at some time following the taking of a sample from the subject for testing in accordance with the invention.
  • the subject will typically not be displaying any signs or symptoms associated with PE, CVD or normotensive SGA at the time the sample is taken.
  • the methods of the invention involve observing the levels of one or more specific proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof and/or subunits thereof in a sample taken from a female subject at a time when the female subject is pregnant.
  • the inventors contemplate the proteins to be useful as individual markers or in combinations of two or more (for example, in a classifier).
  • the inventors believe that relative expression levels of individual subunits of the proteins, as well as precursors, molecular isoforms and proteolytic peptides of the proteins are also important predictive markers.
  • detecting one or more proteins it should be taken to include reference to detecting one or more precursors, isoforms, proteolytic peptides and/or subunits of said protein(s), unless the context requires otherwise.
  • reference to detecting one or more proteins, precursors, isoforms, subunits and/or proteolytic peptides should be taken to include reference to detecting one or more nucleic acid encoding said protein(s), precursor(s), isoform(s), proteolytic peptide(s) and/or subunit(s).
  • preproteins prepeptides, preproproteins and prepropeptides including an N-terminal leader sequence.
  • methods of the invention may also include observing the levels of one or more nucleic acids (specifically transcripts or cDNA molecules based thereon) encoding the proteins, precursors and/or isoforms described herein.
  • the methods of the invention involve at least taking a sample from a female subject, detecting in the sample one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof, and/or nucleic acids encoding any one or more thereof, and comparing the level of the one or more proteins, subunits, precursors, proteolytic peptides isoforms and/or nucleic acids against the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids in a standard.
  • the difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids in the sample compared to the standard is indicative of the risk of developing PE and/or the risk of developing CVD, or the risk of developing normotensive SGA, as the case may be.
  • the terms “higher” and “lower” or “increased” and “decreased” and like terms may be used. Such terms should be taken broadly to include any change in the level of protein or nucleic acid compared to a standard. It should be appreciated that the methods of the invention may be combined with analysis of one or more other biological markers (for example, metabolites and genetic markers) or clinical observations which are known to be associated with PE and/or CVD 5 as the case may be. In a particular embodiment, methods for predicting the risk of developing CVD take into account the predicted risk of the same subject developing PE, an increased risk of developing PE being indicative of an increased risk of also developing CVD.
  • the female subject is pregnant at the time at which the sample is taken.
  • the female subject is preferably in the early stages of pregnancy, for example up to or including approximately 24 weeks, more preferably at 20 weeks of gestation or less. In one embodiment, this may also be appropriate for methods of predicting CVD.
  • exemplary precursors, isoforms, proteolytic fragments and/or subunits of use in the invention include:
  • a fibrinogen fragment with an approximate molecular weight of 27 kDa A fibrinogen fragment with an approximate molecular weight of 25kDa
  • the method includes detecting each of Apolipoprotein AI, pregnancy zone protein and/or alpha-2-macroglobulin, and Fibrinogen beta chain, and/or nucleic acids encoding same.
  • the method includes detecting fibronectm I isoform 3.
  • PEDF alone is used as a marker to assess the risk of PE-AGA.
  • an isoform of PEDF with an approximate pi of 5.4 is used as a marker.
  • the one or more proteins are chosen from the group of proteins known as HDL cargo proteins (as herein before defined).
  • the HDL cargo proteins are chosen from the group consisting of: apoM, apoE, Clusterin (apoJ), apoA-I, Serum amyloid A4, apoC-III, C3, C4A, Vitronectin,
  • the sample is preferably a blood sample, including plasma or serum samples.
  • a urine sample or a cervical fluid sample may be used.
  • samples can be taken from the patient using standard techniques known in the art.
  • high abundance proteins which have the potential to make it difficult to analyse may be removed from the sample.
  • Top6 or Top7 depletion may be used (as described herein after under the heading "Multi affinity removal system (MARS) for major abundance proteins").
  • MERS Multi affinity removal system
  • the sample may also be subject to proteolytic digestion.
  • detection of a protein, subunit, proteolytic peptide, precursor and/or isoform in accordance with the invention should be taken to include detection of any one or more fragments thereof. Fragments should be of a length sufficient to ensure specificity to the protein of interest. Such fragments will typically be at least 8 amino acids in length, more preferably at least 10, 15 or 20 amino acids in length.
  • the kit may comprise one or more antibody specific to the one or more proteins/subunits/precursors/isoforms/proteolytic peptides described herein, hi a particular embodiment, ELISA is used and the kit comprises one or more capture and/or detection antibody for one or more protein/subunit/precursor/isoform/proteolytic peptide of interest.
  • Blood was collected by venipuncture into BD EDTA- Vacutainer® collection tubes, placed on ice, centrifuged at 2400 ⁇ g for 10 min at 4 °C, and biobanked at -8O 0 C within 4 hours of collection.

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Abstract

Biomarkers and methods for use in predicting the risk of women developing preeclampsia and/or cardiovascular disease are described.

Description

BIOMARKERS FOR PREDICTION OF PREECLAMPSIA AND/OR
CARDIOVASCULAR DISEASE FIELD
The present invention relates to methods for predicting the risk of women developing preeclampsia (PE) and/or cardiovascular disease (CVD).
BACKGROUND
PE is a disorder that occurs only during pregnancy and affects both the mother and her unborn baby. The rapidly progressing condition is diagnosed when the mother develops hypertension and proteinuria, but may cause seizures, kidney failure, liver dysfunction and severe bleeding.1 A quarter of the babies born to women with preeclampsia are growth restricted (small for gestational age babies, SGA) and the remaining women have babies with an appropriate birthweight for gestational age (AGA). The only cure for PE is delivery, which results in a third of all babies born to women with PE being premature.
Preeclampsia complicates 4-7% of women in their first pregnancies and it is estimated that over 8 million women are affected each year. Over 70,000 women world-wide die from PE each year. As a third of the babies are premature and there is a 3-10 fold increase in baby death, prediction of PE would be a major healthcare advance for future mothers and their babies.
The underlying pathophysiology of PE is complex. The initiating event in PE is thought to be abnormal remodelling of spiral arteries during early placentation resulting in ischemic reperfusion insults to the placenta. These events then trigger a maternal response resulting in abnormal endothelial cell function and vasoconstriction, activated inflammation and coagulation that lead to the clinical manifestations of PE.2 Typically PE occurs in the third trimester (28-40 weeks' gestation) of pregnancy but in severe cases, the disorder occurs in the 2nd trimester. It is very rare for the condition to occur before 22 weeks' gestation. Obstetricians currently screen pregnant women for PE by serial measurements of blood pressure and protein levels in urine, combined with clinical assessment for other signs and symptoms of the syndrome.3As this 'screening' is based on detection of signs of the disease, it is too late for effective intervention to prevent its occurrence and to reduce the negative consequences of PE. Preventing the condition and the associated morbidity for the mother and child requires a method of detecting early in pregnancy which women are at high risk of later developing PE. Currently, there are no blood based screening tests available to predict PE in clinical practice. As prediction is the pre-requisite to prevention, the clinical need for a screening test for PE is widely recognised.4' 5
If women at increased risk for PE could be identified early in their pregnancies, much of the fetal and maternal morbidity and mortality could be avoided by intervention to prevent the condition and increased surveillance of their pregnancies, with timely delivery before severe maternal complications develop. Effective screening would allow women at high risk to receive targeted use of preventative treatment and intensified monitoring, whilst those at lower risk could be less intensively assessed, resulting in further savings of maternal healthcare resources.
The incidence of cardiovascular disease (CVD), including atherosclerosis and coronary heart disease, are on the rise, particularly in Western societies. Aside from the health implications, the economic costs associated with managing and treating CVD in a population are significant. Risk factors associated with CVD are numerous and include genetic predisposition, elevated total cholesterol and low density lipoprotein (LDL) cholesterol and reduced high density lipoprotein (HDL) cholesterol levels in plasma. More recently alterations in HDL cargo proteins have been associated with CVD15. PE has also been recognised as a risk factor for later development of CVD14.
Currently, there are no methods available to identify which pregnant women may later develop CVD, including CVD after an episode of PE. If women at risk of developing CVD could be identified early, the onset and progression of later CVD may be avoided or ameliorated through medical intervention.
Bibliographic details of the publications referred to herein are collected at the end of the description. OBJECT
It is an object of the present invention to provide an improved method for predicting the risk of women developing preeclampsia and/or cardiovascular disease (CVD), or at least to provide the public with a useful choice.
STATEMENT OF INVENTION
In accordance with the present invention the inventors have surprisingly identified that upregulation or downregulation of certain proteins, precursors thereof, isoforms thereof, proteolytic fragments thereof, and/or subunits thereof during early pregnancy are associated with the later development of preeclampsia and thus may provide valuable information about a female subject's risk of developing this disorder.
Further, certain of these proteins and their relative levels of expression may be used as markers for predicting the risk of cardiovascular disease (CVD) in a female subject. .
Still further, the inventors have identified a means of predicting the risk of normotensive SGA in a female subject.
In one broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia, the method comprising observing the level of one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, proteolytic peptides precursors and/or isoforms, wherein the one or more proteins is chosen from the group consisting of: Fibrinogen; Apolipoprotein A-I; Apolipoprotein C-III; Apolipoprotein E; Apolipoprotein M; Complement component 3; Complement 4A (C4A); Complement factor H-related protein 1 (FHLl); Clusterin; Transthyretin; Hemopexin; Serum amyloid P; Serum amyloid A4, alpha-2-macroglobulin; Pregnancy zone protein (PZP); alpha- 1-antichymotrypsin; alpha- 1 -antitrypsin; alpha- 1 -type I collagen; Kininogen 1 ; Vitronectin; Angiotensinogen; alpha2-HS-glycoprotein; zinc-alpha2-glycoprotein; Fibronectin 1 isoform 3; alphal -microglobulin (bikunin); Inter-alpha-trypsin inhibitor heavy chain H4; Haptoglobin-related protein, Transferrin; and Pigment epithelium-derived factor (PEDF). In another broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia, the method comprising observing the level of one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof,, and/or nucleic acids encoding the one or more proteins, subunits, proteolytic peptides, precursors and/or isoforms, wherein the one or more proteins is an HDL cargo protein.
In one embodiment the HDL cargo protein is chosen from the group consisting: phospholipid transfer protein, cholesteryl ester transfer protein, lecithin-cholesterol acyltransferase, apolipoprotein (apo) C-I , apoC-II, apoC-III, apoC-IV, paraoxonase-1 , ρaraoxonase-3 , serum amyloid A 4 , serum amyloid A 2 , serum amyloid A 1 , apoA-I, , apoH, apoA-IV, clusterin, apoA-II, apoL-I, apoD, apoE, apoF, apoM, angiotensinogen (AGT), alpha-2-antiplasmin, serpin peptidase inhibitor, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1 -antitrypsin, bikunin (alphal -microglobulin), kininogen, alpha- 1 -acid glycoprotein 2, transthyretin, inter-alpha-trypsin inhibitor H4, retinol binding protein 4, transferrin, fibrinogen, hemopexin, complement component 3 (C3), C4A, C4B, C9, and vitronectin.
In one particular embodiment, the HDL cargo protein is chosen from the group consisting: apoM, apoE, clusterin, apoA-I, serum amyloid A 4, apoC-III, C3, C4A, vitronectin, angiotensinogen, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1- antitrypsin, bikunin (alphal -microglobulin), kininogen, transthyretin, inter-alpha-trypsin inhibitor heavy chain H4, transferrin, fibrinogen, and hemopexin.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, precursors, proteolytic peptides, and/or isoforms in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids against a standard; wherein a difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing preeclampsia, and wherein the one or more proteins is chosen from the group consisting of: Fibrinogen; Apolipoprotein A-I; Apolipoprotein C-III; Apolipoprotein E; Apolipoprotein M;
Complement component 3; Complement 4 A (C4A); Complement factor H-related protein 1; Clusterin; Transthyretin; Hemopexin; Serum amyloid P; Serum amyloid A4; alpha-2- macroglobulin; Pregnancy zone protein (PZP); alpha- 1-antichymotrypsin; alpha- 1- antitrypsin; alpha- 1 -type I collagen; Kininogen I, Vitronectin; Angiotensinogen; alpha2- HS-glycoprotein; zinc-alpha2-Glycoprotein; Fibronectin 1 isoform 3; alphal- microglobulin (bikunin); Inter-alpha-trypsin inhibitor H4; Haptoglobin-related protein, Transferrin; and Pigment epithelium-derived factor.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting one or more HDL cargo proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids against a standard; wherein a difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing preeclampsia.
In one embodiment the HDL cargo protein is chosen from the group of HDL cargo proteins described herein before.
In one embodiment of the methods of the invention the precursors, isoforms, proteolytic fragments and/or subunits include: Fibrinogen alpha chain Fibrinogen beta chain Fibrinogen gamma chain
A fibrinogen fragment with an approximate molecular weight of 27 kDa
A fibrinogen fragment with an approximate molecular weight of 25kDa
Complement component 3 precursor Complement component 3 C
High Molecular Weight Kininogen (HK)
HKa (a fragment of HK)
Clusterin isoform I
Apolipoprotein A-I fragment with an approximately molecular weright of 17kDa Apolipoprotein A-I fragment with an approximately molecular weright of 21 kDa
Apolipoprotein A-I fragment with an approximately molecular weright of 3OkDa
Apolipoprotein A-I fragment with an approximately molecular weright of 25kDa
Transthyretin with an approximate molecular weight of 15kDa
Transthyretin with an approximate molecular weight of 12kDa Serum amyloid P fragment with an approximate molecular weight of 3OkDa
Serum amyloid P fragment with an approximate molecular weight of 27kDa
Vitronectin, approximately 75kDa
Virtonectin, approximately 65kDa
PEDF isoform with an approximate pi of 5.4 PEDF isoform with an approximate pi of 6.1
Preferably, a higher or increased level of one or more of the following compared to a standard, is indicative of risk of developing preeclampsia:
Fibrinogen (and/or the alpha chain, beta chain and/or gamma chain) Apolipoprotein A-I (and/or fragments thereof with approximate molecular weights of 17,
21, and/or 3OkDa)
Apolipoprotein E
Complement component 3 (and/or complement component 3 precursor and/or complement component 3 c) Complement 4a
Complement factor H-related protein I
Clusterin (and/or clusterin isoform I)
Transthyretin with an approximate molecular weight of 15 kDa Hemopexin
Serum amyloid P fragment with an approximate molecular weight of 30 kDa
Serum amyloid A4 alpha-2-macroglobulin Pregnancy zone protein (PZP) alpha- 1 - Antichymotrypsin alpha- 1 antitrypsin alpha- 1 type I Collagen
Kininogen I HKa
Vitronectin, approximately 75 kDa
Vitronectin, approximately 65kDa
Angiotensinogen alpha2-HS-Glycoprotein zinc-alpha2-Glycoprotein
Fibronectin 1 isoform 3
Transferrin
Haptoglobin-related protein
Pigment epithelium derived factor (and/or a PEDF isoform with an approximate pi of 5.4)
Preferably, a lower or decreased level of one or more of the following compared to a standard is indicative of risk of developing preeclampsia:
Apolipoprotein A-I fragment with an approximate molecular weight of 25 kDa
Apolipoprotein C-III Apolipoprotein M
Transthyretin with an approximate molecular weight of 12 kDa
Serum amyloid P fragment with an approximate molecular weight of 27 kDa
High Molecular Weight Kininogen
Vitronectin, approximately 65 kDa Vitronectin, approximately 75 kDa alphal -Microglobulin (bikunin)
Inter-alpha-trypsin inhibitor heavy chain H4
A PEDF isoform with an approximate pi at 6.1 Preferably there is at least an approximately 1.3 fold increase or decrease in the level of a particular protein, precursor, isoform, proteolytic peptide, isoform and/or nucleic acid compared to the standard, more preferably at least an approximately 1.5 fold increase or decrease.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia with a small for gestational age baby (PE-SGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acid encoding the one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, proteolytic peptides, isoforms and/or nucleic acids against a standard; wherein a difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids in the sample compared to the standard is indicative of the risk of developing PE-SGA5 and wherein the one or more proteins is chosen from the group consisting: alpha- 1- antichymotrypsin; fibrinogen; kininogen I; vitronectin, and clusterin.
In one embodiment, the proteins, subunits, precursors, proteolytic peptides and/or isoforms are chosen from the group consisting: alpha- 1-antichymotrypsin; fibrinogen gamma chain; a fibrinogen fragment with an approximate molecular weight of 27 kDa, a fibrinogen fragment with an approximately molecular weight of 25 kDa; high molecular weight kininogen (HK); and, Vitronectin, approximately 65 kDa; Vitronectin, 75 kDa; and Clusterin.
Preferably, a higher level of one or more of alpha- 1 -antichymotrypsin, fibrinogen gamma chain, an approximately 25 kDa fragment of fibrinogen, an approximately 27 kDa fragment of fibrinogen, an approximately 65 kDa part of the two-chain vitronectin molecule and/or clusterin in the sample compared to the standard is indicative of risk of developing PE-SGA.
Preferably, a lower level of HK and/or vitronectin single chain with an approximate molecular weight of 75 kDa in a sample compared to the standard is indicative of risk of developing PE-SGA.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing PE-SGA5 the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting apolipoprotein A-I in combination with one or more of fibrinogen, pregnancy zone protein and/or alpha-2-macroglobulin, clusterin isoform I5 alphal-antichymotrypsin, and complement factor I and/or nucleic acids encoding said proteins in the sample; and, c) comparing the level of each of the proteins and/or nucleic acids in the sample against a standard; wherein a difference in the level of each of the proteins and/or nucleic acids in the sample compared to the standard is indicative of developing PE-SGA.
In one embodiment, one or more precursors, isoforms, proteolytic peptides and/or subunits of the proteins, or nucleic acids encoding one or more thereof, is detected in addition to or in lieu of detection of a particular protein.
In one embodiment, the method includes detecting an approximately 17 kDa Apo-AI fragment and/or an approximately 21 kDa Apo-AI fragment and/or nucleic acids encoding same.
In one embodiment, the method includes detecting fibrinogen beta chain and/or fibrinogen gamma chain, and/or nucleic acids encoding same.
In one embodiment, the method includes detecting each of Apolipoprotein AI and pregnancy zone protein and/or alpha-2-macroglobulin, and/or nucleic acids encoding same. Preferably, a combination of a higher level of apolipoprotein A-I and a higher level of pregnancy zone protein and/or alpha-2-macroglobulin is indicative of the risk of PE-SGA.
In another embodiment, the method includes detecting each of Apolipoprotein AI, pregnancy zone protein and/or alpha-2-macroglobulin, and Fibrinogen beta chain, and/or nucleic acids encoding same. Preferably, a combination of a higher level of apolipoprotein A-I, a higher level of fibrinogen beta chain and a higher level of pregnancy zone protein and/or alpha-2-macroglobulin is indicative of the risk of PE-SGA.
In another embodiment, the method includes detecting each of Apolipoprotein AI, pregnancy zone protein and/or alpha-2-macroglobulin, fibrinogen beta chain, fibrinogen gamma chain, clusterin isoform I, alpha- 1-antichymotrypsin and complement factor I, and/or nucleic acids encoding same. Preferably a higher level of apolipoprotein AI, a higher level of pregnancy zone protein and/or alpha-2-macroglobulin, a higher level of fibrinogen beta chain, a higher level of fibrinogen gamma chain, a higher level of clusterin isoform I, a higher level of alpha- 1-antichymotrypsin and a higher level of complement factor I is indicative of the risk of PE-SGA.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia with PE-SGA, the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting each of the proteins apolipoprotein A-I, serum amyloid P, and transthyretin with an approximate molecular weight of 12 kDa, and/or nucleic acids encoding same in the sample; and, c) comparing the level of each of the proteins and/or nucleic acids in the sample against a standard; wherein a difference in the level of each of the proteins and/or nucleic acids in the sample compared to the standard is indicative of developing PE-SGA.
In one embodiment, the method further includes detecting fibrinogen. In one embodiment, one or more precursors, isoforms, proteolytic peptides and/or subunits of each of the proteins, or nucleic acids encoding one or more thereof, is detected in addition to or in lieu of detection of a particular protein.
In one embodiment, the method includes detecting an apolipoprotein A-I fragment with an approximate molecular weight of 27kDa and/or an apolipoprotein fragment with an approximate molecular weight of 17kDa, and/or nucleic acids encoding same.
In one embodiment, the method includes detecting fibrinogen beta chain and/or a nucleic acid encoding same.
In one embodiment, the method includes detecting serum amyloid P with an approximate molecular weight of 27 kDa and/or a nucleic acid encoding same.
In one embodiment, a lower level of a fragment of apolipoprotein A-I with an approximate molecular weight of 27kDa, a lower level of serum amyloid P with an approximate molecular weight of 27 kDa, a higher level of a fragment of apolipoprotein A-I with an approximate molecular weight of 17 kDa, and a lower level of a transthyretin fragment with an approximate molecular weight of 12 kDa are together indicative of risk of developing PE-SGA.
IQ another embodiment a lower level of a fragment of apolipoprotein A-I with an approximate molecular weight of 27kDa, a lower level of serum amyloid P with an approximate molecular weight of 27 kDa, a higher level of a fragment of apolipoprotein A- I with an approximate molecular weight of 17 kDa, a lower level of a transthyretin fragment with an approximate molecular weight of 12 kDa, and a higher level of fibrinogen beta chain are together indicative of risk of developing PE-SGA.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting PEDF, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; and, c) comparing the level of the PEDF, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof against a standard; wherein a difference in the level of the PEDF, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of the risk of developing PE-AGA.
Preferably, a higher level of PEDF, a subunit thereof, a precursor thereof, an isoform thereof, a proteolytic peptide thereof and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of risk of developing PE-AGA.
In one embodiment, the isoform of PEDF is an isoform with an approximate pi of 5.4.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting fibrinogen, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; and, c) comparing the level of the fibrinogen, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof against a standard; wherein a difference in the level of the fibrinogen, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of the risk of developing PE-AGA.
Preferably, a higher level of fibringoen, a subunit thereof, a precursor thereof, an isoform thereof, a proteolytic peptide thereof and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of risk of developing PE-AGA.
In one embodiment, the subunit of fibrinogen is fibrinogen beta chain. In another broad aspect the invention provides a method for predicting the risk of a female subject developing preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting Apo-AI in combination with one or more of haptoglobin-related protein( HRP), Transferrin, alpha-2-macroglobuiin, and fibronectin (FN) and/or nucleic acids encoding same in the sample; and, c) comparing the level of each of the proteins and/or nucleic acids in the sample against a standard; wherein a difference in the level of each of the proteins and/or nucleic acids in the sample compared to the standard is indicative of developing PE-AGA.
In one embodiment, one or more precursors, isoforms, proteolytic peptides and/or subunits of each of the proteins, or nucleic acids encoding one or more thereof, is detected in addition to or in lieu of detection of a particular protein.
In one embodiment, the method includes detecting an approximately 17 kDa Apo-AI fragment, an approximately 21 kDa Apo-AI fragment, and/or an approximately 30 kDa Apo-AI fragment.
In one embodiment, the method includes detecting fibronectin I isoform 3.
Preferably, a combination of a higher level of apolipoprotein AI (and/or an apolipoprotein A-I fragment of approximately 17 kDa, an apolipoprotein A-I fragment of approximately 21 kDa and/or an apolipoprotein A-I fragment of approximately 30 kDa), a higher level of HRP, a higher level of alpha 2 macroglobulin, a higher level of Fibronectin 1 isoform 3, and a higher level of transferrin are indicative of risk of developing PE-AGA.
In another aspect, the invention provides a method of predicting the risk of a female subject developing normotensive SGA, the method comprising observing the level of clusterin, a subunit thereof, a precursor thereof, a proteolytic fragment thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof. In one embodiment, the method comprises at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting clusterin, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; c) comparing the level of the clusterin, subunit, isoform, precursor, proteolytic peptide and/or nucleic acid against a standard; wherein the difference in the level of clusterin, a subunit, isoform, precursor, proteolytic peptide and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing normotensive SGA.
In one embodiment, a lower level of clusterin, a subunit thereof, a precursor thereof, an isoform thereof, proteolytic peptide and/or a nucleic acid encoding one or more thereof compared to the standard is indicative of the risk of developing normotensive SGA.
In one particular embodiment, levels of Clusterin isoform land/or nucleic acids encoding same are observed with a lower level of clusterin isoform 1 and/or nucleic acids encoding same compared to the standard is indicative of the risk of developing normotensive SGA.
In another aspect the invention provides a method of discriminating between PE-SGA and normotensive SGA the method comprising observing the level of clusterin, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoforms thereof, and/or a nucleic acid encoding one or more thereof.
In one embodiment the method comprises at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting clusterin, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; c) comparing the level of the clusterin, subunit, isoform, precursor, proteolytic peptide and/or nucleic acid against a standard; wherein the difference in the level of clusterin, subunit, isoform, precursor, proteolytic peptide and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing either PE-SGA or normotensive SGA.
In one embodiment, a lower level of clusterin, a subunit thereof, an isoform thereof, a proteolytic peptide and/or a precursor thereof, and/or a nucleic acid encoding one or more thereof compared to the standard is indicative of the risk of developing normotensive SGA and a higher level of clusterin, a subunit thereof, an isoform thereof, a proteolytic peptide thereof and/or a precursor thereof, and/or a nucleic acid encoding one or more thereof compared to the standard is indicative of the risk of developing PE-SGA.
In one particular embodiment, levels of Clusterin isoform land/or a nucleic acid encoding same are observed with a lower level of clusterin isoform 1 and/or nucleic acid encoding same compared to the standard is indicative of the risk of developing normotensive SGA and a higher level of clusterin isoform 1 and/or a nucleic acid encoding same compared to the standard is indicative of the risk of developing PE-SGA.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing CVD, the method comprising observing the level of one or more HDL cargo proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof at a time when the female subject is pregnant.
In another broad aspect the invention provides a method for predicting the risk of a female subject developing CVD the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting one or more HDL cargo proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, proteolytic peptides, isoforms and/or nucleic acids against a standard; wherein a difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing cardiovascular disease.
In one embodiment the one or more HDL cargo protein is chosen from the group consisting: phospholipid transfer protein, cholesteryl ester transfer protein, lecithin- cholesterol acyltransferase, apoC-I, apoC-II, apoC-III, apoC-IV, paraoxonase-1 , paraoxonase-3 , serum amyloid A 4 , serum amyloid A 2 , serum amyloid A 1 , apoA-I, apoH, apoA-FV, clusterin, apoA-II, apoL-I, apoD, apoE, apoF, apoM, angiotensinogen (AGT), alpha-2-antiplasmin , serpin peptidase inhibitor, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1 -antitrypsin, bikunin (alphal -microglobulin), kininogen , alpha- 1 -acid glycoprotein 2, transthyretin, inter-alpha-trypsin inhibitor heavy chain H4,Retinol Binding Protein 4, transferrin, fibrinogen, hemopexin, C3, C4A, C4B, C9, and vitronectin.
In one particular embodiment, the HDL cargo protein is chosen from the group consisting: apoM, apoE, Clusterin, apoA-I, serum amyloid A4, apoC-III, C3, C4A, vitronectin, angiotensinogen, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1- antitrypsin, bikunin (alphal -microglobulin), kininogen, transthyretin, inter-alpha-trypsin inhibitor heavy chain H4, transferrin, fibrinogen, and hemopexin.
In certain embodiments of the invention the level of the one or more proteins, a subunit thereof, an isoform thereof, a proteolytic peptide thereof and/or a precursor thereof, is determined using an immunoassay, separation based on characteristics such as molecular weight and isoelectric point, gel electrophoresis, Western Blotting or mass spectroscopy. Preferably the immunoassay is an ELISA. Preferably the gel electrophoresis is 2D gel electrophoresis or gel-free systems based on microfiuidics technologies.
Preferably the sample is a blood sample, preferably a plasma or serum sample. Alternatively the sample is urine or cervical fluid. Preferably the subject is in early pregnancy at the time the sample is taken. More preferably the subject is at or less than 24 weeks of gestation at the time the sample is taken.
In a further aspect, the invention provides a kit for use in a method of the invention, the kit comprising at least one or more reagents suitable for detection of one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding same as herein before described.
In a further aspect, there is provided the use of a method for predicting the risk of a female subject developing preeclampsia (as herein before described) in a method for predicting the risk of CVD in the subject.
In a further aspect, there is provided a method for predicting the risk of a female subject developing CVD the method comprising the step of first predicting the risk of the subject developing preeclampsia using a method as herein before described, wherein an increased risk of developing preeclampsia is indicative of an increased risk of developing cardiovascular disease.
The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
FIGURES
These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures, in which:
Figure 1 : 2D-DIGE experimental design. a Total number of spots detected by
DeCyder software after applying a 17,000 spot volume cut-off. b Matching is expressed for each gel as a ratio (mean percent ± SD) of the total number of detected spots per respective master gel.
Figure 2: Maternal and Fetal Characteristics. AGA5 appropriate birthweight for gestational age; SGA, small for gestational age. Results are mean (SD), median (IQ range) or N (%). Comparisons between the 3 groups using ANOVA. f PE-SGA versus controls PO.05 by Dunnett's t-test. tf PE-AGA versus controls and PE-SGA versus controls P<0.05 by Dunnett's t-test. * Severe Preeclampsia includes severe hypertension (BP>170/l 10 mmHg), imminent eclampsia, liver dysfunction, acute renal insufficiency or coagulopathy. §Birthweight adjusted for gestational age, fetal sex, maternal parity, ethnicity, height and weight.
Figure 3: Differentially expressed protein spots in week 20 plasma from women with subsequent preeclampsia with either an appropriate for gestational age baby
(PE-AGA) or a small for gestational age baby (PE-SGA) compared to healthy controls, a) Master spot number after visual matching across all experiments; 1C depicts spots which were also found to be a member of a disease classifier for either PE-AGA or PE-SGA by nearest shrunken centroids analysis with a bias corrected performance above 90%. b) FC, fold change between healthy versus PE-AGA or PE-SGA using median standardized abundances. FC shown only for spots significant with FDR/FWER correction. c)/?-values after applying FDR or FWER correction, d) Presence of spot in one or more 2D-DIGE experiments, statistically significant by univariate analysis (p < 0.05) with and without
FDR/FWER correction.
Figure 4: LC-MS/MS identification of plasma proteins/peptides detected by 2-D
DIGE as differentially expressed in 20 week pregnancy plasma from women with subsequent PE-AGA and/or PE-SGA. a) Master spot number after spot matching across all experiments, b) FC, fold change of protein spots by median of standardized abundances in PE-AGA or PE-SGA versus controls, c) Number of unique peptides per protein, d) Protein accession numbers as per NCBInr 2006/2007 or Mascot 20051220 databases, e) Protein name from human ref (FASTA) database, f) Member of a set of spots included in a disease classifier. ^ Spots included in a classifier but standardized abundance not significantly different in cases compared with controls after adjustment for false discovery rate
Figure 5: Plasma proteins associated with later development of preeclampsia, classified by main biological function. Differential protein expression is shown as the fold change of the median of standardized spot abundances in PE-AGA or PE-SGA versus controls. * Spots included in a classifier but standardized abundance not significantly different in cases compared with controls. Italics depict proteins which have been identified, but are unlikely to be the dominant protein in a spot based on spot location in experimental 2D gel compared to published human plasma 2D gel maps.
Figure 6: Classification models for preeclampsia with a baby of appropriate birthweight for gestational age (PE-AGA) or with a small for gestational age infant (PE-SGA). Classification (C) models were generated by nearest shrunken centroid analysis. Proteins (spot master number) included in each classifier are listed. Classification accuracy was for PE-AGA: Experiment
IH-Cl5 100%; Experiment V-Cl to C3, 90.9%; and for PE-SGA: Experiment 1-Cl to C4, 99%; Experiment IV-Cl to C2, 99.6%. HRP5 haptoglobin related protein; FN, fibronectin 1 isoform 3; A2M, alpha-2- macroglobulin; PZP, pregnancy zone protein; SAP, serum amyloid P; TTR, transthyretin.
Figure 7: Sypro Ruby stained 2-DE gel images of Top6 depleted plasma (A) and
Top7 depleted plasma (B). Differentially expressed protein spots with mass spectrometry identification are annotated by spot ID numbers (see Figure 4 for protein IDs). Circled area in the upper right of panel A depicts fibrinogen which was removed from plasma by Top7 depletion (B). Figure 8: Western Blot analysis of native plasma (20 weeks' gestation) with two candidate markers for the prediction of preeclampsia. Blots were incubated with polyclonal anti-human fibrinogen (left blot) and alpha- 1- antichymotrypsin antibody (right blot). Cross reacting bands were revealed by Qdot nanocrystal technology. A control blot with no primary antibody revealed no unspecific binding (far left). Fibrinogen alpha, beta, and gamma chains were detected along with additional higher and lower molecular weight fibrinogen species. Densitometric analysis showed significantly higher protein expression of gamma fibrinogen in women who developed PE-SGA compared to controls, (p<0.05). * Two fibrinogen species at approximately 27 and 25 kDa which were up-regulated in two women with PE-SGA. Western blot analysis for alpha- 1-antichymotrypsin confirmed significant up-regulation in early pregnancy plasma preceding disease, (p<0.01).
Figure 9: Western blot analysis for high molecular weight kininogen (HK) using plasma at week 20 of gestation from healthy controls (n=4) versus PE-SGA (n=4). HK is detected by specific anti-human mouse monoclonal antibody (R&D Systems Cat# MAB 15692) which specifically reacts with mature chain (HK) and the heavy chain of high molecular weight kininogen (HKa), but not with the light chain alone. *Two PE-SGA plasma samples show a loss of mature HK (110kDa)and over-expression of a 56 kDa proteolytic fragment of HKa heavy chain, indicating aberrant levels of HK in early pregnancy in women who later develop PE-SGA. The 'no primary (1°) antibody' control (left panel) shows no unspecific binding.
Figure 10: 2D gel of maternal plasma showing vitronectin isoforms (spot IDs 1 to 8) found to be differentially expressed prior to (at 20 weeks' gestation) and at the time of preeclampsia (at 36-38 weeks' gestation) compared with healthy controls.
Figure 11: LC-MS/MS identification of protein spots detected by 2-D DIGE as being differentially expressed in plasma at 20 weeks' gestation before the onset of preeclampsia and at the time women present with preeclampsia (36-38 weeks' gestation) compared with healthy controls.
Figure 12: Western Blot analysis of plasma preceding and at the time of clinical manifestation of preeclampsia reveals aberrant vitronectin processing.
Western Blot of maternal plasma vitronectin isoforms of women with a healthy pregnancy outcome (Cl - C3) compared to women who subsequently developed PE-AGA (PEl -PE3) at week 20 of gestation and in late pregnancy (week 36 to 37) in a longitudinal study. Each lane contained 40 ug of plasma which was separated on 4-12% BisTris gels (NUPAGE) in
MES buffer for 1 h at 200V. The no primary antibody control to the left showed no unspecific binding by the detecting antibody using nano crystal technology (Qdot conjugated anti-mouse IgG at 1:1000 from Invitrogen). Anti-human vitronectin mouse monoclonal antibody at 1 :5000 dilution (Abcam# 11591 ). M3 molecular weight marker, band size at 62 kDa. PE, preeclampsia.
Figure 13: Plasma vitronectin is altered in early pregnancy preceding the onset of preeclampsia with small for gestational age. Western Blot of maternal plasma (at 20 weeks gestation) vitronectin isoforms of women with a healthy pregnancy outcome (Cl to C4) compared to women who subsequently developed PE further complicated with SGA. Conditions were as described in Figure 12. The no primary antibody control to the left showed no unspecific binding by the detecting antibody using nano crystal technology (Invitrogen). Anti-human vitronectin mouse monoclonal Ab at
1:5000 dilution (Abcam#11591), and Qdot conjugated anti-mouse IgG at 1:1000 (Invitrogen).
Figure 14: Clusterin ELISA Study. Maternal characteristics, pregnancy outcome and plasma clusterin levels in women who subsequently develop preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA) or with a small for gestational age baby (PE-SGA) and normotensive women with a SGA baby (SGA). Results are mean (SD), median (IQ range) or N (%). § Proteinuria was defined as (spot protein: creatinine ratio >30 mg/mmol or 24 h urinary protein >0.3 g/24 h or dipstick proteinuria >2+. ss Birthweight adjusted for gestational age, fetal sex, maternal parity, ethnicity, height and weight. 1" PO.01 4PO1OOl γ P<0.0001 compared with Controls. ¥ PO.00001 compared with N-SGA
Figure 15: PEDF 2D-DIGE Study. Maternal characteristics and pregnancy outcome in women with an abnormal uterine artery Doppler waveform who subsequently developed preeclampsia or remained healthy with an uncomplicated pregnancy. Results are mean (SD), median (IQ range) or N
(%). § Birthweight adjusted for gestational age, fetal sex, maternal parity, ethnicity, height and weight. * P<0.05 f P<0.01 1 PO1OOl
Figure 16: A: 2D gel map showing protein spots containing pigment epithelium derived factor (PEDF) that are differentially regulated in week 20 plasma of women who later developed preeclampsia compared healthy controls. In this study, women with an abnormal uterine artery Doppler waveform who later developed preeclampsia were compared with women with an abnormal uterine artery Doppler waveform but remained healthy and delivered a healthy baby at term. B: Box plots showing standardized abundances of the two spots shown in Fig 15 A. P-value calculated by Limma t-test using false discovery rate correction comparing DIGE standardized spot volumes from women with subsequent preeclampsia with controls.
Figure 17: LC-MS/MS analysis of plasma proteins differentially expressed at week 20 of gestation in women prior to developing preeclampsia. 1 FD, fold difference in median standardized spot abundances in preeclampsia compared with healthy control; 2 For LC-MS/MS ion trap analysis (spot 459) Mascot (v2.1.04 Matrix Science, UK) search engine and Swiss Prot
Accession number derived from NCBInr vl2 June 2008 were used. For LTQ-FT data (spot 485) Sequest search engine and protein accession # from NCBInr version2006/2007; 3 Protein name from NCBI human FASTA data base.
Figure 18: Maternal Characteristics, Pregnancy Outcome and plasma pigment epithelium derived factor (PEDF) levels. PE-AGA5 preeclampsia with an appropriate birthweigth for gestational age; PE-SGA5 preeclampsia with a small for gestational age baby. Results are mean (SD) or N (%). *P<0.05 r P<0.01 rP<0.0001 compared with Controls.
Figure 19: LC-MS/MS analysis of plasma proteins differentially expressed in early pregnancy at week 15 and week 20 of gestation in comparison to nonpregnant control subjects. FD, fold difference of median standardized spot abundances in pregnant compared to non-pregnant control subjects. Mascot (v2.1.04 Matrix Science, UK) search engine and Swiss Prot Accession number derived from NCBInr v2006 were used.
Figure 20: Box plots showing standardized abundances of two spots (spot 427 and 579) found to be significantly (p<0.05) up-regulated in plasma of women prior to developing preeclampsia (week 20 of gestation) in DIGE experiment III (gel picture not shown). P-value calculated by Limma t-test using false discovery rate correction comparing DIGE standardized spot volumes from women with subsequent preeclampsia versus controls.
PREFERRED EMBODIMENT(S) The following is a description of the present invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the section "Examples" which provides experimental data supporting the invention and specific examples thereof.
The inventors have surprisingly discovered individual proteins, precursors thereof, isoforms thereof, proteolytic peptides or fragments thereof and/or subunits thereof as well as sets of these proteins, subunits, precursors, proteolytic peptides and/or isoforms, present in the maternal circulation early in pregnancy, are altered (for example are up-regulated or down-regulated) in women who subsequently present with PE-AGA and/or PE-SGA compared to those that do not. The inventors believe these marker proteins, subunits, precursors, isoforms, and/or proteolytic peptides and their relative blood levels in early pregnancy can be used to predict the risk of women developing PE. The risk of developing PE can be determined by measuring specific proteins, subunits, precursors, proteolytic peptides and/or isoforms or combinations thereof in plasma, serum, urine and cervical fluid in early pregnancy, prior to the onset of disease (for example, 24 weeks of gestation or less).
A significant proportion of the proteins (including particular isoforms, subunits, precursors, and/or proteolytic fragments) identified to be differentially expressed prior to preeclampsia can be grouped in a class of proteins known as high density lipoprotein (HDL) cargo proteins. Thus, the inventors contemplate that any one or more HDL cargo protein may be used as a marker to determine the risk of a female subject developing PE.
Further, the inventors' note that changes in the expression of a number of HDL cargo proteins are reported to be associated with CVD. They also note that PE is associated with increased risk of CVD. The inventors' contemplate the use of HDL cargo proteins and their relative blood levels during pregnancy as markers for predicting the risk of a female subject later developing CVD, including but not limited to predicting the risk of developing CVD associated with PE (ie, the risk of developing CVD at the time of, or subsequent to, an episode of PE).
Further, the inventors have identified that levels of the protein clusterin discriminate between PE-SGA and normotensive SGA. In particular, decreases in the level of clusterin, particularly clusterin isoform I, in maternal blood early in pregnancy, is associated with normotensive SGA, while increases in clusterin, particularly clusterin isoform I, are associated with later development of PE-SGA. Thus levels of clusterin may be used to predict the risk of a female developing normotensive SGA or to distinguish between between the risk of normotensive SGA and PE-SGA.
As used herein PE should be taken broadly to refer to conditions in which a pregnant female presents with new onset hypertension (usually developing after 20 weeks' gestation) and proteinuria or coagulopathy (thrombocytopenia or disseminated intravascular coagulation) or liver involvement (abnormal liver function specifically aspartate transaminase and/or alanine transaminase, liver rupture or subcapsular haematoma) or renal impairment or imminent eclampsia or eclampsia (generalised seizures). Unless the context requires otherwise, reference to PE should be taken to include reference to PE-AGA5 PE-SGA, HELLP syndrome (haemolysis, elevated liver enzymes, low platelets) and eclampsia.
"Cardiovascular disease" or "CVD" are used herein to refer to artherosclerosis and/or coronary artery disease which can manifest as angina and/or myocardial infarction, for example.
"Normotensive SGA" is used to refer to a condition in which the mother was normotensive during pregnancy (i.e. did not have gestational hypertension, preeclampsia or chronic hypertension) but has a baby with a birthweight <1 Oth customised birthweight centile.
"Risk of developing" PE, CVD, or normotensive SGA, as the case may be, is used herein to refer to the risk of developing PE , CVD or normotensive SGA at some time in the future; ie at some time following the taking of a sample from the subject for testing in accordance with the invention. The subject will typically not be displaying any signs or symptoms associated with PE, CVD or normotensive SGA at the time the sample is taken.
The methods of the invention involve observing the levels of one or more specific proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof and/or subunits thereof in a sample taken from a female subject at a time when the female subject is pregnant. The inventors contemplate the proteins to be useful as individual markers or in combinations of two or more (for example, in a classifier). In addition, the inventors believe that relative expression levels of individual subunits of the proteins, as well as precursors, molecular isoforms and proteolytic peptides of the proteins are also important predictive markers. Therefore, where reference is made herein to detecting one or more proteins, it should be taken to include reference to detecting one or more precursors, isoforms, proteolytic peptides and/or subunits of said protein(s), unless the context requires otherwise. Similarly, reference to detecting one or more proteins, precursors, isoforms, subunits and/or proteolytic peptides should be taken to include reference to detecting one or more nucleic acid encoding said protein(s), precursor(s), isoform(s), proteolytic peptide(s) and/or subunit(s).
As used herein, "isoforms" is intended to refer to different forms of the same protein. "Isoforms" should be taken to include, for example, those proteins which result from alternative splicing of mRNA, polymorphisms contained within a gene, and/or by posttranslational modification (for example, glycosylation).
The term "precursor(s)" should be interpreted broadly and includes for example preproteins, prepeptides, preproproteins and prepropeptides including an N-terminal leader sequence. To the extent that these proteins may be expressed at different levels at the nucleic acid level, the methods of the invention may also include observing the levels of one or more nucleic acids (specifically transcripts or cDNA molecules based thereon) encoding the proteins, precursors and/or isoforms described herein.
Preferably the methods of the invention involve at least taking a sample from a female subject, detecting in the sample one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof, and/or nucleic acids encoding any one or more thereof, and comparing the level of the one or more proteins, subunits, precursors, proteolytic peptides isoforms and/or nucleic acids against the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids in a standard. The difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids in the sample compared to the standard is indicative of the risk of developing PE and/or the risk of developing CVD, or the risk of developing normotensive SGA, as the case may be.
When referring to the level of a particular protein, subunit, precursor, isoform, proteolytic peptide or nucleic acid from a subject compared to a standard, the terms "higher" and "lower" or "increased" and "decreased" and like terms may be used. Such terms should be taken broadly to include any change in the level of protein or nucleic acid compared to a standard. It should be appreciated that the methods of the invention may be combined with analysis of one or more other biological markers (for example, metabolites and genetic markers) or clinical observations which are known to be associated with PE and/or CVD5 as the case may be. In a particular embodiment, methods for predicting the risk of developing CVD take into account the predicted risk of the same subject developing PE, an increased risk of developing PE being indicative of an increased risk of also developing CVD.
As mentioned herein before, the female subject is pregnant at the time at which the sample is taken. In embodiments of the invention relating to methods for predicting the risk of the subject developing PE and normotensive SGA5 the female subject is preferably in the early stages of pregnancy, for example up to or including approximately 24 weeks, more preferably at 20 weeks of gestation or less. In one embodiment, this may also be appropriate for methods of predicting CVD.
In one embodiment of the invention relating to methods of predicting risk of developing PE the one or more proteins include:
Fibrinogen
Apolipoprotein A-I
Apolipoprotein C-III Apolipoprotein E
Apolipoprotein M
Complement component 3
Complement 4A
Complement factor H-related protein 1 Clusterin
Transthyretin
Hemopexin
Serum amyloid P
Serum amyloid A4 alpha-2-macroglobulin
Pregnancy zone protein (PZP) alpha- 1 -antichymotrypsin alpha- 1 -antitypsin alpha-1-type I collagen Kininogen I Vitronectin Angiotensinogen alpha2-HS-glycoprotein zinc-alpha2-glycoprotein Fibronectin 1 isofortπ 3 alphal -microglobulin (bikunin) Inter-alpha-trypsin inhibitor heavy chain H4 Haptoglobin-related protein
Transferrin PEDF
In one embodiment, exemplary precursors, isoforms, proteolytic fragments and/or subunits of use in the invention include:
Fibrinogen alpha chain
Fibrinogen beta chain
Fibrinogen gamma chain
A fibrinogen fragment with an approximate molecular weight of 27 kDa A fibrinogen fragment with an approximate molecular weight of 25kDa
Complement component 3 precursor
Complement component 3 c
High Molecular Weight Kininogen (HK)
HKa Clusterin isoform I
Apolipoprotein A-I fragment with an approximately molecular weright of 17kDa
Apolipoprotein A-I fragment with an approximately molecular weright of 2IkDa
Apolipoprotein A-I fragment with an approximately molecular weright of 3OkDa
Apolipoprotein A-I fragment with an approximately molecular weright of 25kDa Transthyretin with an approximate molecular weight of 15kDa
Transthyretin with an approximate molecular weight of 12kDa
Serum amyloid P fragment with an approximate molecular weight of 3OkDa
Serum amyloid P fragment with an approximate molecular weight of 27kDa Vitronectin, single chain with an approximate molecular weight of 75kDa Virtonectin approximately 65kDa part of the double chain isoform PEDF isoform with an approximate pi of 5.4 PEDF isoform with an approximate pi of 6.1
In one embodiment, the protein is chosen from the group of proteins known as HDL cargo proteins. HDL cargo proteins include proteins complexed to HDL particles including those implicated in the biological pathways associated with lipid metabolism, complement regulation, regulation of proteolysis (for example proteinase inhibition) and acute-phase response (proteins whose plasma concentrations are altered markedly by acute infection and chronic inflammation)16, for example. By way of further example, the family of HDL cargo proteins includes the proteins phospholipid transfer protein, cholesteryl ester transfer protein, lecithin-cholesterol acyltransferase, apoC-I, apoC-II, apoC-III, apoC-IV, paraoxonase-1 , paraoxonase-3 , serum amyloid A 4 , serum amyloid A 2 , serum amyloid A l , apoA-I, , apoH, apoA-IV, clusterin, apoA-IL apoL-I, apoD, apoE, apoF, apoM, angiotensinogen (AGT), alpha-2-antiplasmin , serpin peptidase inhibitor , alpha-2-HS- glycoprotein, haptoglobin-related protein, alpha- 1 -antitrypsin, bikunin (alphal- microglobulin), kininogen (for example, kininogen I)5 alpha- 1 -acid glycoprotein 2, transthyretin, inter-alpha-trypsin inhibitor heavy chain H4, Retinol Binding Protein 4, transferrin, fibrinogen, hemopexin, C3, C4A, C4B, C9, and vitronectin.
In one particular embodiment, the HDL cargo protein is chosen from the group consisting: apoM, apoE, Clusterin, apoA-I, serum amyloid A 4, apoC-III, C3, C4A, vitronectin, angiotensinogen, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1- antitrypsin, bikunin (alpha 1- microglobulin), kininogen, transthyretin, inter-alpha-trypsin inhibitor heavy chain H4, transferrin, fibrinogen, and hemopexin.
Amino acid and/or nucleic acid sequence information for each protein of use in the invention can be deduced from the accession numbers (listed in Figure 4) via public databases, for example National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA), MSDBv2005 (Imperial College, London, UK) or IPI-human database v3.27 (European Bioinformatics Institute). These databases may be accessed via http ://ca.expasy.org. Amino acid and/or nucleic acid sequence information for proteins not listed in Figure 4 herein (such as certain HDL cargo proteins) can be obtained from the publicly available databases mentioned above, using the name of a particular protein and/or the accession numbers provided by Vaisar et al15. In addition, details of the nomenclature used for the genes relating to these proteins may be found on the website of the HUGO Gene Nomenclature Committee (www.genenames.org).
In the context of the present invention, reference to "nucleic acids encoding" a protein, subunit, precursor, proteolytic peptide or isoform thereof should be taken to be a reference to mRNA transcripts encoding said protein, subunit, precursor, proteolytic peptide and/or isoform, or cDNAs derived from said transcripts.
The inventors have identified that changes in the levels of specific proteins, and in particular clusters or sets of these proteins, can be used to predict the risk of PE-SGA. These proteins include alpha- 1-antichymotrypsin, fibrinogen (including in particular fibrinogen gamma chain, fibrinogen beta chain, two fibrinogen fragments with an approximate molecular weight of approximately 27 and approximately 25 kDa), high molecular weight kininogen, apolipoprotein A-I (including in particular apolipoprotein A-I precursor and/or fragments of apolipoprotein A-I), pregnancy zone protein, alpha-2- macroglobulin, clusterin (including in particular clusterin isoform I), alpha- 1- antichymotrypsin, serum amyloid P, transthyretin, and vitronectin (including in particular a 65kDa part of the two-chain vitronectin molecule and vitronectin single chain which an approximate molecular weight of 75kDa).
Proteins which may individually (or in combination) be of use to predict the risk of PE- SGA include alpha- 1-antichymotrypsin, fibrinogen gamma chain, fibrinogen beta chain an approximately 27kDa fragment of fibrinogen, an approximately 25 kDa fragment of fibrinogen, and high molecular weight kininogen, vitronectin (an/or including in particular vitronectin, 65 kDa part of the two chain vitronectin molecule and/or vitronectin, single chain with an approximate molecular weight of 75kDa) and clusterin.
In a particular embodiment apolipoprotein A-I (and/or including in particular fragments thereof including an approximately 17kDa Apo-AI fragment) in combination with one or ore of fibrinogen, pregnancy zone protein, alpha-2-macroglobulin, clusterin isoform I, alpha-I-aαtichymotrypsin, and complement factor I are used as markers for assessing the risk of PE-SGA.
In one embodiment, the method includes detecting an approximately 17 kDa Apo-AI fragment and/or an approximately 21 kDa Apo-AI fragment.
In one embodiment, the method includes detecting fibrinogen beta chain and/or fibrinogen gamma chain.
In one embodiment, the method includes detecting each of Apolipoprotein AI and pregnancy zone protein and/or alpha-2-macroglobulin.
In another embodiment, the method includes detecting each of Apolipoprotein AI, pregnancy zone protein and/or alpha-2-macroglobulin, and Fibrinogen beta chain, and/or nucleic acids encoding same.
In another embodiment, the method includes detecting each of Apolipoprotein AI, pregnancy zone protein and/or alpha-2-macroglobulin, fibrinogen beta chain, fibrinogen gamma chain, clusterin isoform I, alpha- 1-antichymotrypsin and complement factor I, and/or nucleic acids encoding same.
In another embodiment, apolipoprotein A-I, serum amyloid P and transthyretin with an approximate molecular weight of 12 kDa are used in combination as markers for assessing risk of PE-SGA.
In one embodiment, the method further includes detecting fibrinogen.
In one embodiment, the method includes detecting an apolipoprotein A-I fragment with an approximate molecular weight of 27kDa and/or an apolipoprotein fragment with an approximate molecular weight of 17kDa.
In one embodiment, the method includes detecting fibrinogen beta chain. In one embodiment, the method includes detecting serum amyloid P with an approximate molecular weight of 27 kDa.
The inventors have also identified that changes in the levels of PEDF and/or fibrinogen and/or particular clusters or sets of proteins can be used to predict the risk of PE-AGA.
In one embodiment, Apo-Al (and/or including in particular fragments thereof including a fragment of 17 kDa, an Apo-AI fragment of 21 kDa and/or an Apo-AI fragment of 30 kDa), hi combination with one or more of HRP, Transferrin, alpha-2-macroglobulin, and fibronectm are used as markers to assess the risk of PE-AGA.
In one embodiment, the method includes detecting fibronectm I isoform 3.
In another embodiment, PEDF alone is used as a marker to assess the risk of PE-AGA. In one embodiment, an isoform of PEDF with an approximate pi of 5.4 is used as a marker.
In another embodiment, fibrinogen alone is used as a marker to assess the risk of PE-AGA. In one embodiment, the fibrinogen beta chain is used as a marker.
In one embodiment of the invention, a higher or increased level of one or more of the following compared to a standard, is indicative of risk of developing PE:
Fibrinogen (and/or alpha, beta and/or gamma chains of fibrinogen)
Apolipoprotein A-I fragments with approximate molecular weights of 17, 21, and/or
3OkDa Apolipoprotein E
Complement component 3 precursor
Complement component 3c
Complement factor H-related protein I
Complement 4a Clusterin (and/or Clusterin isoform I)
Transthyretin with an approximate molecular weight of 15 kDa
Hemopexin
Serum amyloid P fragment with an approximate molecular weight of 30 kDa Serum amyloid A4 alpha-2-macroglobulin
Pregnancy zone protein (PZP) alpha- 1 -Antichymotrypsin alpha- 1-antitypsin alpha- 1 type I collagen
Kininogen I
HKa
Vitronectin, single chain with an approximate molecular weight of 75 kDa Vitronectin approximatly 65kDa part of the double chain isoform
Angiotensinogen alpha2-HS-glycoprotein zinc-alpha2-glycoprotein
Fibronectin 1 isoform 3 Transferrin
Haptoglobin-related protein
PEDF (and/or a PEDF isoform with an approximate pi of 5.4)
Similarly, a lower or decreased level of one or more of the following compared to a standard, is indicative of risk of developing PE:
Apolipoprotein A-I fragment with an approximate molecular weight of 25 kDa
Apolipoprotein C-III
Apolipoprotein M
Transthyretin with an approximate molecular weight of 12 kDa Serum amyloid P fragment with an approximate molecular weight of 27 kDa
High Molecular Weight Kininogen
Vitronectin, 65 kDa part of the two-chain vitronectin molecule
Vitronectin, single chain with an approximate molecular weight of 75 kDa alphal -microglobulin (bikunin) Inter-alpha-trypsin inhibitor heavy chain H4
A PEDF isoform with an approximate pi at 6.1 As used generally herein the terms "higher" and "lower" or "increased" and "decreased" and like terms, when used in relation to the level of a particular protein, subunit thereof, precursor thereof, isoform thereof, proteolytic peptide thereof and/or nucleic acid encoding said proteins, subunits, precursors, proteolytic peptides and isoforms should be taken broadly to include any change in the level of protein or nucleic acid compared to a standard. However, in a preferred embodiment, at least an approximately 1.3 -fold increase or decrease in the level of a particular protein, subunit, precursor, proteolytic peptide, isoform and/or nucleic acid compared to the standard is indicative of the risk of developing PE, CVD or normotensive SGA, as the case may be. In a more preferred embodiment, there is at least an approximately 1.5-fold change in the level of a particular protein, precursor, isoform, proteolytic peptide, subunit and/or nucleic acid. In a particular embodiment there is from an approximately 1.3 to an approximately 12.2 to 13-fold change. Specific fold-changes referred to herein are fold-changes measured by 2D-DIGE in accordance with the methods described hereinafter, unless otherwise stated.
In particular preferred embodiments one or more of the following are indicative of the risk of developing PE: an approximately 3 -fold increase in the level of Fibrinogen alpha chain, an approximately 1.5- to 6.4-fold increase of fibrinogen beta chain, an approximately 1.5- to 3.6-fold increase of fibrinogen gamma chain, an approximately 1.9- to 12.2-fold increase in the level of Apolipoprotein A-I fragments with approximate molecular weights of 17, 21, and/or 30 kDa, an approximately 1.9-fold decrease in the level of apolipoprotein C-III, an approximately 1.5- to 2.2-fold increase in the level of apolipoprotein E, an approximately 1.6-fold decrease in the level of apolipoprotein M, an approximately 1.4- to 3.2-fold increase in the level of Complement component 3 precursor and/or complement component 3c, an approximately 2.7-fold increase in the level of complement C4a, an approximately 6.4-fold increase in the level of Complement factor H-related 1, an approximately 2.0- to 3.1 -fold increase in the level of clusterin isoform I, an approximately 1.15 fold increase in the level of Clusterin (as observed by ELISA), an approximately 1.5- to 7.5-fold increase in the level of Transthyretin fragment with an approximate molecular weight of 15 kDa, an approximately 1.8- to 2.2-fold decrease in the level of Transthyretin fragment with an approximate molecular weight of 12 kDa, an approximately 6.9-fold increase in the level of Hemopexin, an approximately 1.5-fold increase in the level of Serum amyloid P fragment with an approximate molecular weight of 30 kDa, an approximately 1.6- to 2.0-fold decrease in the level of Serum amyloid P fragment with an approximate molecular weight of 27 kDa, an approximately 1.5- to 2.9- fold increase in the level of Serum amyloid A4, an approximately 1.5- to 5.5-fold increase in the level of Alpha-2-macroglobulin, an approximately 1.5- to 5.5-fold increase in the level of PZP5 an approximately 2.0- to 3.1-fold increase in the level of Alpha- 1-antichymotrypsin, an approximately 1.3- to 2.2-fold increase in the level of Alpha- 1 -antitrypsin, an approximately 3.3- to 4.6-fold increase in the level of Alpha- 1 -type I collagen, an approximately 1.3-fold increase in Angiotensinogen, an approximately 1.5- to 2.9-fold increase in alpha2-HS-glycoprotein, an approximately 1.5-fold increase in zinc-alpha2- glycoprotein, an approximately 1.5-fold increase in Fibronectin 1 isoform 3, an approximately 2-fold increase in Haptoglobin related protein, an approximately 1.5- to 2.0 fold increase in transferrin, an approximately 2-fold increase in the level of cleaved HK, namely HKa (as observed by Western Blot), an approximately 1.5 to 1.7 fold decrease in Kininogen I, an approximately 1.5- to 2.0-fold increase in the level of single chain vitronectin with a predicted molecular weight of 75 kDa, an approximately 1.5- to 1.8-fold decrease in the level of the 65 kDa part of the two-chain vitronectin, an approximately 10- fold increase in the level of the 65 kDa part of the two-chain vitronectin (as observed by Western blot), an approximately 10-fold decrease in the level of single chain vitronectin with a predicted molecular weight of 75 kDa (as observed by Western blot), decreased plasma levels of high molecular weight kininogen (for example approximately a 1.5 fold decrease), an approximately 1.6-fold increase in PEDF isoform with an approximate pi of 5.4, an approximately 1.2 -fold increase in total plasma PEDF in PE-AGA (as observed by ELISA), an approximately 1.5-fold decrease in alpha- 1 -microglobulin, an approximately 1.7- tol.9-fold decrease in inter-alpha-trypsin inhibitor heavy chain H4, and/or an approximately 1.6-fold decrease in PEDF isoform with an approximate pi at 6.1 , compared to a standard.
In a specific embodiment, a higher level of alpha- 1-antichymotrypsin, fibrinogen gamma chain, an approximately 27kDa fragment of fibrinogen, an approximately 25 kDa fragment of fibrinogen, a lower level of high molecular weight kininogen, a lower level of single chain vitronectin with an approximately molecular weight of 75kDa, a higher level of the 65 kDa part of the two-chain vitronectin and/or a higher level of clusterin compared to a standard, is indicative of risk of developing PE-SGA. In one preferred embodiment, an approximately 2.4- to 3.1 -fold higher level of clusterin, an approximately 2-fold higher level of alpha- 1-antichymotrypsin, an approximately 1.5-fold higher level of fibrinogen gamma chain, an approximately 1.5-fold higher level of a 27 kDa fragment of fibrinogen, an approximately 1.5-fold higher level of a 25 kDa fragment of fibrinogen, an approximately 10-fold higher level of the approximately 65 kDa part of the two-chain vitronectin (as observed by Western blot), an approximately 10-fold decrease in the level of single chain vitronectin with a predicted molecular weight of 75 kDa (as observed by Western blot), an approximately 1.5-fold lower level of high molecular weight kininogen and/or at least an approximately 2.4- to 3.1 -fold higher level of clusterin compared to standard, is indicative of risk of developing PE-SGA.
In a further particular embodiment a higher level of apolipoprotein A-I in combination with one or more of a higher level of fibrinogen (including in particular the beta chain), a higher level of pregnancy zone protein, a higher level of alpha-2-macroglobulin, a higher level of clusterin isoform I, a higher level of alpha 1 antichymotrypsin and a higher level of complement factor I is indicative of the risk of PE-SGA.
In one preferred embodiment there is an approximately 2.4-fold higher level of apolipoprotein A-I, an approximately 1.9-fold higher level of fibrinogen, an approximately 1.7-fold higher level of pregnancy zone protein, an approximately 1.7-fold higher level of alpha-2-macroglobulin, an approximately 2.4-fold higher level of clusterin isoform I, an approximately 2.0- to 3.1-fold increase in the level of Alpha- 1-antichymotrypsin, and an approximately 2.0- to 3.1 -fold higher level in complement factor I.
In another embodiment, a lower level of a fragment of apolipoprotein A-I5 a lower level of serum amyloid P, a higher level of another fragment of apolipoprotein A-I, and a lower level of transthyretin are together indicative of risk of developing PE-SGA. In another embodiment, additionally a higher level of fibrinogen (including in particular the beta chain) is also indicative of risk of developing PE-SGA.
In one preferred embodiment, there is an approximately 2.0-fold lower level of fragment of apolipoprotein A-I (with a molecular weight of approximately 27kDa), an approximately 2.0-fold lower level of serum amyloid P with an approximate molecular weight of 27 kDa, an approximately 2.4-fold higher level of fragments of apolipoprotein A-I with approximate molecular weights of 17, 21, and/or 30 kDa, an approximately 1.8-fold lower level of fragment of transthyretin with an approximate molecular weight of 12kDa, and an approximately 1.9-fold lower level of fibrinogen beta chain.
In another embodiment a higher level of PEDF is indicative of risk of developing PE- AGA. In one preferred embodiment, an approximately 1.6-fold increase in PEDF isoform with an approximate pi of 5.4, an approximately 1.2 -fold increase in total plasma PEDF in PE-AGA (as observed by ELISA), an approximately 1.6-fold decrease in PEDF isoform with an approximate pi at 6.1 , compared to a standard is indicative of the risk of developing PE-AGA.
In another embodiment a higher level of fibrinogen (including in particular fibrinogen beta chain) is indicative of the risk of developing PE-AGA. In one preferred embodiment, an approximately 3.0-fold to an approximately 6.4-fold increase in fibrinogen beta chain is indicative of the risk of developing PE-AGA.
In another embodiment, a higher level of apolipoprotein A-I (including in particular an apolipoprotein A-I fragment of approximately 17 kDa, an apolipoprotein A-I fragment of approximately 21 kDa, and/or an apolipoprotein A-I fragment of approximately 30 kDa), in combination with one or more of a higher level of Fibronectin 1 isoform 3, a higher level of transferrin, a higher level of HRP, and a higher level of alpha-2-macroglobulin is indicative of risk of developing PE-AGA. In one preferred embodiment there is an approximately 2.5 to 5-fold increase in the level of an apolipoprotein A-I of approximately 17 kDa, an approximately 1.9-fold increase in the level of an apolipoprotein A-I fragment of approximately 21 kDa, an approximately 2-fold increase in the level of an apolipoprotein A-I fragment of approximately 30 kDa, an approximately 1.5-fold increase in the level of Fibronectin 1 isoform 3, an approximately 1.6-fold increase in the level of transferrin, an approximately 2-fold increase in the level of HRP, and an approximately 1.5-fold increase in the level of alpha-e-macroglobulin.
As mentioned previously herein, the inventors have identified that differences in the level of clusterin (including subunits, precursors, proteolytic peptides and/or isoforms thereof) can be used to predict the risk of a woman deleveloping PE-SGA or normotensive SGA. These differences also allow one to distinguish between these disorders. Methods relating to this aspect of the invention involve observing the level of clusterin in a sample compared to a standard, as generally described elsewhere herein.
In one embodiment, a lower level of clusterin is indicative of the risk of developing normotensive SGA and a higher level of clusterin (in particular clusterin isoform I) is indicative of the risk of developing PE-SGA. In a particular embodiment, an approximately 1.2-fold decrease in clusterin (as observed by ELISA) is indicative of the risk of developing normotensive SGA. In another particular embodiment, an approximately 1.15-fold increase in clusterin (as observed by ELISA) is indicative of the risk of developing PE-SGA.
In embodiments of the invention relating to methods for predicting the risk of a female subject developing CVD the one or more proteins are chosen from the group of proteins known as HDL cargo proteins (as herein before defined). In one particular embodiment, the HDL cargo proteins are chosen from the group consisting of: apoM, apoE, Clusterin (apoJ), apoA-I, Serum amyloid A4, apoC-III, C3, C4A, Vitronectin,
Angiotensinogen, alpha-2-HS-glycoprotein, Haptoglobin-related protein, alpha-a- antitrypsin, alpha-a-Microglobulin (Bikunin), Kininogen 1, Transthyretin, Inter-alpha- 1- inhibitor heavy chain H4, Transferrin, Fibrinogen and Hemopexin.
A standard in accordance with the invention is a level of one or more of the proteins, precursors, isoforms, subunits, proteolytic peptides, or nucleic acids encoding the one or more proteins, precursors, subunits, proteolytic peptids and/or isoforms thereof, known to be associated with normal pregnancy (in the case of a test for prediction of the risk of PE or normotensive SGA), and/or associated with individuals having substantially no history and/or evidence (symptoms or signs) of cardiovascular disease. As used herein "normal pregnancy" means a pregnancy in which PE does not present. Preferably, the standard will be a control sample having a known level of one or more specific proteins, isoforms thereof, and/or nucleic acids encoding same, which is tested concurrently with the sample from a subject in accordance with the invention. However, in another embodiment, the standard could be a printed chart or electronic information or the like containing previously generated data considered to provide appropriate standards (as herein before described) and which the test samples could be compared to on the basis of colour or fluorescence levels, for example.
The proteins, including precursors, isoforms, proteolytic peptides and subunits thereof, and nucleic acids encoding same may be detected and the levels thereof compared to a standard using any one or a combination of techniques which are of use in identifying, quantifying and/or highlighting differential levels or expression of one or more proteins. Such techniques will be readily appreciated by persons of ordinary skill in the art to which the invention relates. However, by way of example protein purification methods, immunological techniques, separation of proteins based on characteristics such as molecular weight and isoelectric point including gel electrophoresis and microfluidics- based technologies as for example in gel-free protein separation techniques, and mass spectroscopy (MS) utilizing isobaric label based MS such as iTRAQ6 or label-free approaches such as multiple reaction monitoring (MRM)7 may be employed.
Appropriate immunological techniques include enzyme linked immunosorbent assay (ELISA) (sandwich ELISA, double sandwich ELISA, direct ELISA, microparticle ELISA), radioimmunoassay (RIA), immunoprecipitation, Western blotting, immunohistochemical staining, antibody arrays, or agglutination assays. Protocols for carrying out such techniques are readily available; for example, see "Antibodies a Laboratory Manual", Cold Spring Harbor Laboratory Press (1988), or the protocols described herein after under the heading "Experimental Proceedures".
Antibodies of use in such immunological techniques may be purchased commercially or produced according to standard methodology in the art having regard to the nature of the proteins to be tested. For example, polyclonal antibodies and monoclonal antibodies may be produced in accordance with the procedures described in the text "Antibodies a Laboratory Manual" (Cold Spring Harbor Laboratory Press, 1988) using an appropriate protein or fragment thereof as antigen. Preferably monoclonal antibodies are used.
In the event that one isoform, proteolytic peptide or subunit of a particular protein, for example a glycoisoform, is up-regulated and a second, related isoform, proteolytic peptide or subunit is downregulated in the disease state, then it is to be expected that monoclonal antibodies will be developed that can distinguish between the two isoforms, proteolytic peptides or subunits . In such eventualities, suitable isoform-specific, peptide-specific or subunit-specific-MAbs can be incorporated into methods of the invention, including in particular multiplex tests, for detection of said up - down-regulation, and will constitute informative aspects of the methods with respect to predicting disease.
Nucleic acid-based techniques of use in the invention include differential display procedures, Northern Blotting, and competitive PCR. Persons skilled in the art to which the invention relates will readily appreciate methodology for performing these techniques.
Nucleic acids, such as oligonucleotide probes and primers, of use in detecting expression levels of proteins in accordance with the invention (for example using Northern blotting or competitive PCR) will be readily appreciated by skilled persons having regard to the information contained herein and any published amino acid and/or nucleic acid sequence information for the proteins of relevance to the invention. The nucleic acids will be capable of hybridising specifically to a mRNA or cDNA associated with a protein of interest and in the case of primers they will be capable of priming a PCR or like reaction. While such nucleic acids will preferably have 100% complementarity to their target region of the mRNA or cDNA of the protein of interest, they may contain one or more non- complementary nucleotides at a particular position while still substantially retaining its specificity in respect of the protein or isoform of interest. By way of example, the nucleic acids may have approximately 80%, approximately 90%, approximately 95%, or approximately 99% complementarity or homology to its target. Typically, the nucleic acids will hybridise to their target nucleic acid under stringent hybridisation conditions (see for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
Nucleic acid probes and primers of use in the invention may be prepared by any number of conventional DNA synthesis methods including recombinant techniques and chemical synthesis. It will be appreciated that the usefulness of any probe or primer may be evaluated, at least notionally, using appropriate software and sequence information for the nucleic acid encoding the protein of interest. For example software packages such as PC Oligo5 (National Bioscience Inc) or Amplify (University of Wisconsin), and the PrimerSelect program (DNAStar Inc) may be used to evaluate primers.
The methods of the invention may be performed with the aid of microarray technology. For example, antibody microarrays which contain an array of antibodies specific to one or more proteins/isoforms/subunits/proteolytic peptides/precursors of use in the invention, or nucleic acid microarrays which contain an array of nucleic acid probes specific to one or more nucleic acids encoding one or more proteins/isoforms/precursors/subunits/proteolytic peptides of use in the invention, may be used.
Mass spectroscopy techniques of use in the invention are described for example in "Proteins and proteomics-A laboratory manual" (RJ Simpson, Cold Spring Harbour Laboratory Press (2002) and elsewhere herein.
In embodiments of the invention which rely on differences in the level of isoforms resulting from posttranslational modifications, such as differential glycoslyation (for example in the case of Clusterin), measurement by appropriate techniques include glycan detection methods using antibody arrays, 2D gel electrophoresis, non gel based 2D systems, mass spectrometry or any other method capable of identifying protein isoforms.
The difference in the levels of proteins, precursors, isoforms, proteolytic peptides and/or nucleic acids in a sample versus a standard may be compared using standard technology having regard to the method employed to detect the proteins, precursors, isoforms, proteolytic peptides and/or subunits. For example, colorimetric and fluorometric techniques may be used in which a detection molecule (such as an antibody or nucleic acid probe or primer) is labelled with a molecule which can be visualised by the naked eye or otherwise detected using a spectrophotometer, or fluorometer for example. Alternatively, detection molecules could be labelled with radio-isotopes. Incorporating labels into nucleic acids during PCR amplification where it is employed (as opposed to labelling a detection molecule such as a probe or primer), is also contemplated. Methods for labelling molecules and subsequently measuring the intensity of signals generated will be known to those of skill in the art to which the invention relates. However, examples are provided in the "Examples" section herein after.
It should be appreciated that in addition to the samples and standards mentioned herein before, the method may include the testing of one or more positive or negative control samples to ensure the integrity of the results. For example, one could include a sample containing no protein and one or more samples containing a known level of protein so that results can be calibrated across different runs of the method.
The sample is preferably a blood sample, including plasma or serum samples. Alternatively, a urine sample or a cervical fluid sample may be used. Such samples can be taken from the patient using standard techniques known in the art.
The sample may be processed prior to detecting the one or more proteins, subunits, proteolytic peptides, precursors and/or isoforms to facilitate analysis of the proteins or nucleic acids encoding them. Skilled persons will readily appreciate appropriate processing steps and techniques suitable for performing them.
In one embodiment, high abundance proteins which have the potential to make it difficult to analyse, such as detect and/or measure the level of proteins of interest, may be removed from the sample. For example, Top6 or Top7 depletion may be used (as described herein after under the heading "Multi affinity removal system (MARS) for major abundance proteins"). The sample may also be subject to proteolytic digestion. As such detection of a protein, subunit, proteolytic peptide, precursor and/or isoform in accordance with the invention should be taken to include detection of any one or more fragments thereof. Fragments should be of a length sufficient to ensure specificity to the protein of interest. Such fragments will typically be at least 8 amino acids in length, more preferably at least 10, 15 or 20 amino acids in length.
Processing steps for preparing the sample for analysis of nucleic acids encoding one or more protein, subunit, precursor, proteolytic peptide and/or isoform thereof, may include lysing blood cells, isolating inRNA, and generating cDNA using standard procedures such as RT-PCR as will be known in the art to which the invention relates.
Skilled persons may readily appreciate other means by which the sample may be processed for use in the invention.
The invention also relates to a kit of use in a method of the invention, the kit comprising at least one or more reagents suitable for detection of one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, sύbunits thereof, and/or nucleic acids encoding same as herein before described.
By way of example, where an immunological procedure is used in a method of the invention the kit may comprise one or more antibody specific to the one or more proteins/subunits/precursors/isoforms/proteolytic peptides described herein, hi a particular embodiment, ELISA is used and the kit comprises one or more capture and/or detection antibody for one or more protein/subunit/precursor/isoform/proteolytic peptide of interest.
By way of further example, where a method of the invention involves detection of nucleic acids encoding one or more proteins, subunits, precursors, proteolytic peptides, or isoforms described herein, it may comprise one or more nucleic acid probes and/or primers which have specificity for nucleic acids encoding specific proteins/subunits/precursors/proteolytic peptides/isoforms of interest.
Reagents of use in processing samples for analysis may also be contained in the kits of the invention. They may also comprise one or more standard and/or other controls containing known levels of one or more protein, precursor, isoform, proteolytic peptide, subunit or nucleic acid in accordance with the invention. The kit may also include one or more reagents suitable for detection of one or more additional biomarkers that may be analysed in combination with the proteins/subunits/precursors/isoforms/proteolytic peptides/nucleic acids identified herein. Further, kits of the invention can also comprise instructions for the use the components of the kit as well as printed charts or the like that could be used as standards against which results obtained from test samples could be compared. Reagents may be held in any suitable container. EXAMPLES Experimental procedures Materials Urea, Tris and IEF acrylamide were of ultrapure, MB quality and purchased from GE Healthcare, Auckland, NZ along with the minimal CyDye kit, immobiline™ 11 cm pH 4-7 DryStrips and IPG buffer (pH 4-7). Detergent C7BzO, dimethylformamide (DMF), thiourea (ACS reagent) were purchased from SIGMA- Aldrich (Auckland, NZ), CHAPS from AppliChem, (Darmstadt, Germany), ultrapure DDT and TrisGlycineSDS gel running buffer from Invitrogen (Auckland, NZ), TrisHCl 8-16% Criterion gradient gels from BIORAD (Auckland, NZ).
Study group and plasma collection
A series of case-control studies were conducted to identify changes in the proteome in early pregnancy plasma, obtained at 20 weeks' gestation, prior to the onset of preeclampsia. Seven experiments were conducted comparing women who developed preeclampsia with a baby with an appropriate birthweight for gestational age (PE-AGA) or women developed preeclampsia and a small for gestational age baby (PE-SGA) with healthy pregnant controls, Figure 1. All women were participants in the SCOPE study (SCreening fOr Pregnancy Endpoints, Australian Clinical Trials Registry
ACTRN12607000551493), a prospective screening study of nulliparous women. The study protocol was approved by the Auckland Ethics Committee. Written informed consent was obtained from each participant.
Different women were included in the experiments except in the following. The same subjects with PE-SGA were used in experiments IV and VI, with a subset of these in experiment I and II. Experiment I and II used the same set of eight controls and experiments III and TV used the same set of 12 controls. EDTA-plasma from women at 20 ± 1 weeks gestation who had uncomplicated pregnancies (controls, n=57) or subsequently developed PE-AGA (n=27) and PE-SGA (n=12) were obtained from the SCOPE biobank (Auckland, NZ). Blood was collected by venipuncture into BD EDTA- Vacutainer® collection tubes, placed on ice, centrifuged at 2400χ g for 10 min at 4 °C, and biobanked at -8O0C within 4 hours of collection. Preeclampsia was defined systolic blood pressure >140 mm Hg and/or diastolic blood pressure >90 mm Hg on two or more occasions after 20 weeks gestation but prior to the onset of labour or postpartum systolic BP >140 mmHg and/or diastolic BP >90mmHg postpartum on at least 2 occasions 4 hours apart, combined with either proteinuria (spot protein creatinine ratio >30 mg/mmol or 24 h urinary protein >0.3 g/24 h or dipstick proteinuria>2+) or any multi-organ complication. Severe preeclampsia was defined as having any of the following: persistent severe hypertension (systolic hypertension >170 mmHg or diastolic hypertension >110 mmHg), coagulopathy (thrombocytopenia with platelets <100 x 109/L or disseminated intravascular coagulation), microangiopathic haemolysis, liver dysfunction with aspartate transaminase and/or alanine transaminase >45 JXJfL, imminent eclampsia, eclampsia or acute renal insufficiency.
Small for gestational age (SGA) was defined as a birthweight less than the 10th centile adjusted for infant gender and maternal parity, ethnicity, height and weight, as determined by the customised centile calculator at http://www.gestation.net. Gestational age was based on the last menstrual period if these dates were certain and the expected delivery date (EDD) concurred within 7 days of the EDD calculated from a scan performed before 16 weeks' gestation or 10 days of the EDD determined from a scan at 19-21 weeks. If the date for the last menstrual period was uncertain or the EDD based on the last menstrual period differed by more than 7 days from a scan performed before 16 weeks or more than 10 days from a scan performed at 19-21 week, then gestation was based on data from the earliest scan.
MuMi affinity removal system (MARS) for major abundance proteins
High abundance plasma proteins were removed by either Top6 (albumin, transferrin, IgG, IgA, haptoglobin, alpha- 1 -antitrypsin) or Top7 (six most abundant proteins plus fibrinogen) depletion using the multi affinity removal system (MARS) from Agilent Technologies.
Top6 depletion was carried out with a high capacity 4.6 x 100 mm Hu-6HC column (Agilent Technologies Mfg,Waghaeusel-Wiesenthal, Germany) with a binding capacity for 90 μL of human plasma. Binding capacity of the 4.6 x 100 mm Top7 column was 60 μL of human plasma. Chromatographic separation of abundance proteins by MARS column was performed with buffer A and buffer B (Agilent) in the mobile phase according to a standard LC protocol supplied by the manufacturer. In brief, 150 μL aliquots of crude human plasma samples were removed from the biobank (-800C)5 allowed to thaw on ice and diluted five times for Top6 and four times for Top7 depletion with buffer A containing a protease inhibitor cocktail (EDTA-free complete™, Roche, Auckland, NZ). The final concentration of protease inhibitors in diluted plasma samples was one times, diluted from a stock solution of one complete™ tablet per ImL of buffer A. Only one specimen at a time was thawed and immediately depleted by MARS to minimise proteolytic degradation of plasma proteins. Diluted plasma was filtered (0.22 μm Spin-X® filters from Corning, NY, USA) by centrifugation at 16 000 x g at room temperature for 2 min. The filtered samples were kept on ice until injected onto the MARS column. Flow-through fractions were collected and immediately stored at -8O0C until used for Vivaspin concentration and subsequent 2D proteomics analysis of depleted plasma proteins.
For protein concentration and buffer exchange of depleted plasma fractions, frozen fractions of Top6 (4 mL each) and Top7 (2 mL each) were thawed at room temperature. Each fraction was placed in a 5,000 molecular weight cutoff Vivaspin4 filter (Vivascience, Hannover, Germany) and centrifuged at 3,700 x g at 18°C for 30 min. The filtrate was discarded. To remove excess salt from the depleted plasma fractions, each retentate was washed three times with 3-4 mL of 0.1 % trifluoroacetic acid (TFA, protein sequencer grade from Applied Biosystems, Warrington, UK ) followed by centrifugation as above. A buffer exchange was then carried out by centrifugation of the retentate with 7 M urea, 2 M thiourea, 2% CHAPS by centrifugation at 3,700 x g at 18°C for 60 min, which was repeated twice. Once the volume was reduced to approximately 250 μL, the sample was removed from the filter and stored at -800C until used for 2D-DIGE analysis. Protein concentrations of the resulting concentrates were determined by 2D Quant protein assay (GE Healthcare) ranging from 1.75 to 6.9 μgper μL. The average coefficient of variation (CV) of the 2D Quant kit was less than 10%.
2D-DIGE protocol
Depleted plasma samples stored at -8O0C in 7 M urea, 2 M thiourea, 2 % CHAPS were thawed on ice and labeled with 200 pmol per 50 μg of protein each with CyDye Fluor minimal dyes (GE Healthcare) according to the manufacturers protocol. In order to adjust the pH of the sample to the recommended range of pH 8 to 9, 10OmM Tris (w/v) was added to each sample to a final concentration of 10 mM prior to CyDye labeling. For the pooled internal standard, either an equal amount of each sample included in the experimental sample set was combined or equal amounts of representative specimens (n = 4 - 6 per experimental group) were used. Samples were labeled with respective CyDyes: the internal standard was labeled with Cy2 and controls and cases with Cy3 and Cy5, whereby each case and control group was dye balanced i.e. half of controls and half the cases were labeled with Cy3 and the other half of each group labeled with Cy5.
Each 2D gel contained a case, control and pooled internal standard (see Figure 1 for experimental design) whereby 50 μg of Cy2 labeled internal standard, a Cy3 or Cy5 labeled control (50 μg) and a Cy5 or Cy3 labeled preeclamptic sample (50 μg) were combined and diluted with an equal volume of two times rehydration buffer consisting of 7 M urea, 2 M thiourea, 2% C7BzO (w/v), 2% IPG pH 4 -7 buffer (v/v), 130 mM DTT, trace of bromophenol blue. After 10 min incubation on ice, the volume of the Cy2/Cy3/Cy5 multiplexed sample was adjusted to 200 μL with one times rehydration buffer as above, except for 65 mM DTT and 1% each of C7BzO and IPGs pH 4-7. Samples were centrifuged at 20,000 x g at 18°C for 10 min to remove any particulates prior to overnight passive rehydration of 11 cm pH 4-7 IPG strips using 150 μg of protein per gel.
First dimension focusing was performed on a Multiphor II flatbed (GE Healthcare) at 20°C as follows: 500V for 5 h (step), 3500V for 5 h (gradient) followed by 3500V for 12 h (step) to a total applied 54 500 volt hours. Focused strips were used for 2D gel electrophoresis. For some experiments, such as experiment VII involving 24 gels, all strips were stored frozen (-800C) until used. Equilibration of IEF strips was carried out for 15 min (in buffer containing 6 M urea, 20% glycerol (v/v), 2% SDS (w/v), 20 mM TrisHCl pH 8.8, 65 mM DTT) and alkylated for another 15 min in the same buffer where DTT was substituted with 2.5 % acrylamide (w/v). IEF strips were then rinsed with TrisGlySDS buffer and second dimension SDS-PAGE was performed on 8-16% criterion gels for 15 mA per gel for 30 min followed by 30 mA per gel for 90 min using a Dodeca cell (BIORAD), running twelve gels at a time. GeIs were scanned on a Typhoon 9400 (GE Healthcare) at appropriate excitation and emission wavelengths for each CyDye (Cy2, Cy3, and CyS)9 100 μm, PMT voltage ranging from 440 to 500, generating three images per gel. Gel images were cropped in ImageQuant (GE Healthcare) and imported into DeCyder-2D Differential Analysis software v6.5 (GE Healthcare) for analysis. Spot detection and matching of all spots to an automatically chosen master gel was performed in the batch processor, setting the estimated number of spots to 10,000 (maximum) and applying a spot volume filter to greater than 17,000. The biological variance analysis module (BVA) was then used to check spot matches and re-match the quantified spots of all gels using a manually chosen master gel if the automatic master (batch processor) exhibited a high spot number due to technical artefacts, as for example in gel streaking.
DeCyder software calculates a match quality score (MQS) for each individual spot which is expected to be in the range of 1 to 15, with values approaching zero as a measure of good spot matching. In the present study global match quality scores (mean MQS) were reproducible over all gel images under investigation ranging from 0.8 to 1.8 indicating consistent spot matching over several different experiments. The total number of detected spots, after applying a 17,000 spot volume filter, ranged from 823 to 1094 per master gel with an overall average matching rate of 80.2 ± SD 2.56 % (Figure 1).
The standardized abundance data (spot volume ratios relative to the internal standard,
Cy3/Cy2 and Cy5/Cy2) for each gel was exported from DeCyder in a tab delimited file, constructed by the XML toolbox of DeCyder software.
Bioinformatics analysis of 2D-DIGE data
Clinical Data
Continuous clinical data from PE-AGA, PE-SGA and healthy pregnant women were compared using ANOVA with post hoc analysis using the Dunnett's test, SAS version 9.1 (Gary, USA). Categorical data were analyzed using Chi-Squared or Fisher's Exact Test. A p-value less than 0.05 was considered to be statistically significant.
Data transformation Standardized abundances of down-regulated spots were transformed from negative foldchanges to values ranging from 0 to 1. Log-transformation (base 2) was then applied, and the data from each gel was normalized by zero-centering through median subtraction. Box plots were used to assess outlying gels and two gels in experiment III were removed from analysis. The logged, median-centered relative abundances were used for both univariate analyses to identify differentially expressed proteins, and for nearest shrunken centroids (NSC) analysis for constructing disease classifiers.
Univariate statistical tests for differential protein expression Mann Whitney tests and the moderated t-test implemented in Limma (Linear Models for Microarray Data9) were carried out independently using the log-transformed standardized abundance of all gel spots. False discovery rate (FDR) correction was applied to correct for multiple testing, except for experiment III and IV where a large number of spots remained statistical significant using FDR correction10. Here the more stringent family- wise error rate (FWER) was used, effectively decreasing the likelihood of false positive results. An adjusted p-valuQ of less than 0.05 was considered statistically significant
Nearest shrunken centroids for disease classification DIGE data were analyzed by the NSC method for identification of disease classifiers (preeclamptic versus healthy) after imputing the spot volume for missing values from the median for all cases and controls in that experiment.11 The scores for each spot were shrunk towards zero using an approach designed to eliminate non-informative spots from the analysis and determine sets of spots that best discriminate disease classes. The generalizable performance of this classifier was then estimated by assessing the unbiased performance of this model selection procedure.12 The parametric confidence interval around this estimate was calculated based on a binomial likelihood function with n = 24 and the number correctly classified equal to 24 multiplied by the bias-corrected misclassification rate.
Selection criteria for protein spots of interest
Spots were defined as 'proteins of interest' from replicate proteome comparisons between controls versus PE-AGA or PE-SGA based on the following criteria: 1) protein spots that were significantly 'up-regulated' or 'down-regulated' defined asj9<0.05 with FDR correction for experiments I, H5 V5 and VI or/?<0.05 with FWER correction for experiments HI and IV by Limma or Mann Whitney tests and also significant in at least one other experiment (with or without/?- value correction) or 2) identified in a set of proteins as a classifier by NSC analysis with a bias-corrected classification performance > 90 % .
Identification of proteins by LC-MS/MS
Spots identified as 'proteins of interest' were excised from either several DIGE or preparative gels and identified by mass spectrometry To enhance spot visibility for picking, gels were first stained overnight with Sypro Ruby (Tnvitrogen), scanned on a Fuji- FLA2000 scanner, then over-stained with colloidal Coomassie blue (SERV A-Blue G). Very low abundant spots unable to be visualized by colloidal Coomassie staining were excluded for excision from the gel even if statistically significant. Sypro Ruby digitized images were used as protein maps to aid spot excision using OneTouch (The gel Company, San Francisco, USA) spot pickers. Excised protein plugs were destained, digested with Trypsin Gold (Promega) according to an online published method at the University of California San Francisco (www.ucsf.edu/brc). and submitted for LC-MS/MS analysis on a QSTAR XL ESI-qTOF (Applied Biosystems) at the Maurice Wilkins Centre, University of Auckland or by nanospray LC-MS/MS at HortResearch, Ruakura, New Zealand, using a LTQ linear ion trap mass spectrometer. Separations using the QSTAR were carried out on an Ultimate capillary LC system (LC Packings, San Francisco, CA, USA) fitted with a Zorbax 300 SB Cl 8 column (Agilent Technologies). Mass spectrometry was performed in positive ion mode with a mass range of 300-1600 (m/z) with the 2 most abundant peptides selected for fragmentation.
For LTQ LC-MS/MS analysis of tryptic peptides, the instrument was fitted with a nanospray ESI interface (ThermoQuest, Finnigan, San Jose, CA, USA) and coupled to an EttanTM MDLC (GE Healthcare). The MDLC was used in the high-throughput configuration with two RPC trap columns (Zorbax 300-SB C 18) for on-line desalting and sample clean-up, followed by two nanoscale RPC analytical columns (LC Packings) for high-resolution separation. One set of RPC trap/analytical columns was equilibrated while the second set separated the sample. The mobile phases were A: 0.1% aqueous formic acid and B: 84% acetonitrile + 0.1% formic acid. 2 μL of the digested protein sample was injected and tryptic peptides were separated at a flow rate of 280nL/min with a linear gradient from 0 to 60% B over 50 mins. The mass spectrometer was operated in the positive ion mode and the mass range acquired was m/z 300-1800. Data was acquired using a top 3 experiment in data-dependent mode with dynamic exclusion enabled.
Database Searching and Data Interpretation
QSTAR MS/MS data were extracted from raw spectra using Mascot Search vl.6bl3 (Matrix Science, London, UK) and searched against a mammals subset of MSDB v2005 (Imperial College, London, UK) or IPI-human database v3.27 (European Bioinformatics Institute) using Mascot search engine v2.0.05 (Matrix Science) with the following parameters: semi-trypsin cleavage with 1 missed cleavage allowed; fixed modification: propionamide; variable modifications: oxidation (M), deamidation (N and Q), pyroglutamic acid conversion (N-terminal Q & E), acetylation; peptide tolerance: ±0.1 Da; MS/MS tolerance ±0.1 Da; peptide charge 2+ and 3+.
LTQ nanospray LC-MS/MS data were extracted using SEQUEST.13 Spectra were searched against a human subdata base from the latest version of the public non-redundant protein database (NR) of the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA) with the folio whig search parameters: mass type: monoisotopic precursor and fragments; enzyme and enzyme limits: trypsin (KR), fully enzymatic cleaving at both ends allowing up to 2 missed cleavages; peptide tolerance: 1.4000 atomic mass units (AMU); fragment ion tolerance: 1.0 AMU; number of results scored: 250; ion and ion series calculated: B and Y ions; static modifications: C-cysteine (propionamide); differential modifications: N term peptide (acetylation), methionine (oxidation).
For positive peptide identification the following filter criteria were set in Bio works v3.3 (Thermo Electron Corporation): single, double and triple charge peptides with a correlation factor (XCorr) greater than 1.30, 1.80, and 2.30, respectively; delta cross-correlation factor (dCn) greater than 0.08 indicating a significant difference between the best match reported and the next best match; (Sp) preliminary score greater than 250; protein probability Ie- 002; different (unique) peptides only. All matched peptides were confirmed by visual examination of the spectra. Western Blot analysis
Six differentially expressed proteins found by proteomics analysis were further evaluated by Western blot. Native plasma (20 β L) from week 20 of gestation was separated on 4- 12% NuPAGE gradient gels (Invitrogen) according to the manufacturer's instructions. Proteins were transferred to Immobilon FL transfer membrane (Millipore Corporation) and blocked over night at 40C in 5% BSA. Detection of alpha- 1-antichymotrypsin (DakoCytomation, Glostrup, Denmark, #A0022 at 1:5000), fibrinogen (Dako #A0080 at 1:10,000), complement 3c (Dako #A0062 at 1:10,000), gelsolin (Dako at 1:10,000), clusterin isoform l(Alexis Biochemicals Inc., #ALX-210-451) and serum amyloid A4 (R&D Systems Inc., #AF3856) was accomplished with the respective quantum dots Q655 Qdot conjugated IgGs (Invitrogen) at 1:1000 dilution. A FujiLAS-3000 CCD camera (FujiFilm) was used for signal detection (UV light excitation with a 655nmWB20 filter).
Results
Subject characteristics
Among the 2065 women recruited into SCOPE in Auckland, 31 (1.5%) were lost to follow-up or had a miscarriage or termination before 20 weeks. Of the 2034 women with pregnancies continuing after 20 weeks' gestation, 85 (4.2%) developed preeclampsia of whom a quarter had an SGA infant (n=21). In a series of 2D-DIGE experiments the inventor's analyzed the differential expression of plasma proteins in women who subsequently developed preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA) or PE associated with delivery of a small for gestational age infant (PE- SGA) compared to those who remained healthy. In total, they studied 27 women who developed PE-AGA, 12 who developed PE-SGA and 57 healthy controls. Figure 1 shows the number of plasma samples analyzed in the disease and control groups for each of the seven experiments conducted (I- VII).
The clinical characteristics of the women and the maternal and neonatal outcomes in each group are shown in Figure 2. PE was diagnosed according to strict criteria described herein. There were no perinatal or maternal deaths.
High-abundance protein depletion In order to achieve broader proteome coverage and detect lower abundance proteins, plasma samples were depleted of high abundance proteins using multiple irnmunoaffinity columns prior to 2D DIGE analysis. In two initial experiments (I and II), removal of the six highest abundance plasma proteins (albumin, IgG5 IgA, transferrin, haptoglobin, and antitrypsin) was accomplished using a Top6 MARS column (Agilent). For subsequent experiments (III- VII), a Top7 MARS column, which also removes fibrinogen, was used. Representative gel images for Top6- and Top7 -depleted plasma samples are shown in Figures 7A and 7B respectively. Similar numbers of spots were detected using both depletion schemes (Figure 1) and the overall 2-DE gel patterns observed were the same (Figures 7 A and 7B) except for the removal of fibrinogen spots in Top7-depleted gels (circled area in Fig 7A). Routinely, the protein yield of Top7 depleted plasma from the flow-through fraction was around 20 percent of the initial injected amount with reproducible and consistent protein recoveries across experiments. The coefficient of variation (C V%) for protein recovery ranged from 5 to 11% per experiment in both immunodepletion schemes.
Detection of plasma proteins differentially expressed in women who developed preeclampsia
A DIGE proteomics approach reduces inter-gel variability by simultaneously analyzing plasma from preeclamptic women and healthy controls in relation to a pooled Cy2-labeled internal standard16 We determined that at 80% power, three quarters of protein expression levels above a 2-fold difference between cases and controls would be detected in a sample size of 12 women per group using DIGE.
Comparison of plasma protein expression levels by univariate analyses identified a total of 131 protein spots, found to be significantly up- or down-regulated in the PE-AGA or PE- SGA samples with FDR (experiments I5 II, V5 VI5 and VII) or FWER (experiments III and IV) correction (p < 0.05). These differentially expressed spots were visually inspected in the gel images and technical artifacts excluded. Spots of interest were then selected for further analysis if they were significant in repeated experiments or members of a classifier group of spots as determined by multivariate analyses. By these inclusion criteria, forty- nine spots were identified; 19 spots were differentially expressed in PE-AGA samples, 13 in PE-SGA samples and 17 spots were up- or down-regulated in both conditions (Figure 3). Of these 49 differentially expressed protein spots, 22 spots were also included in a classification model generated by nearest shrunken centroids. It was notable that 9 of the 13 protein spots associated with PE-SGA alone were down-regulated (2.6 to 1.3 fold) compared to healthy controls. This contrasts with findings in the PE-AGA group where 18 of the 19 differentially expressed protein spots were up-regulated (1.5 to 7.8 fold) compared to healthy controls. Of the 17 differentially expressed spots in both PE-AGA and PE-SGA, the majority (13 out of the 17) were significantly increased 1.5 to 12.2 fold compared to healthy controls. Four proteins overlapping both PE subgroups were found to be decreased, with 1.6 to 2.2-fold lower levels compared to healthy controls.
Identification of proteins of interest by MS/MS
Protein spots of interest were excised from preparative gels and subjected to in-gel trypsin digestion and LC-MS/MS for identification. Protein IDs were unable to be obtained for 10 spots due to being very low abundance. The gel locations of 36 differentially expressed protein spots and a further 3 spots that were part of a disease classifier only identified by LC-MS/MS (2 or more peptides per protein) are shown in 2DE-maps of Top6- and Top7- depleted pregnancy plasma in Figure 7. The protein accession numbers for public databases MSDB v2005 (Imperial College, London, UK), IPI-hurnan database v3.27 (European Bioinformatics Institute), and the public non-redundant protein database (NR) of the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA) NCBI are listed in Figure 4.
LC-MS/MS analysis of tryptic digests from spots excised from 2D gels of plasma often result in the identification of more than one protein per spot. The majority of these proteins can be assigned to coagulation, lipid metabolism, inflammation or immune response, haemolysis, vasoactive/angiogenesis, protease inhibitors, extracellular matrix remodelling protein carriers and acute-phase responses according to their proposed biological function (Figure 5).
Classification of PE-AGA and PE-SGA patients by protein clusters
The inventors performed multivariate analysis in order to identify groups of markers that are co-regulated in plasma samples from women who developed either PE-AGA or PE- SGA compared to controls. Using the nearest shrunken centroids approach three different models with a bias corrected performance above 90% (classification accuracy) were identified for the PE-AGA group and six models for the PE-SGA group, Figure 6. In PE- SGA, various combinations of three key proteins (fragments of apoA-I with estimated molecular weights of 17 kDa and 21 kDa , pregnancy zone protein ( PZP) or alpha-2- macroglobulin, fibrinogen), performed equally well as classifiers in experiment I with a classification accuracy of 99%. Classifier I-C4 also included clusterin isoform I, alpha- 1- antichymotrypsin and complement factor I. In experiment IV, the same fragments of fibrinogen (spot 11) and apoA-1 (spot 41) were again part of the classifier for PE-SGA, along with serum amyloid P precursor (spot 30) and transthyretin with an approximate molecular weight of 12 kDa (spot 47) .
Western blot analysis of candidate markers
Six of the potential preeclampsia markers were selected for immunodetection in native
(undepleted) early pregnancy plasma. Using specific antibodies to clusterin isoform I, fibrinogen, complement 3 c, alpha- 1-antichymotrypsin, human serum amyloid A4 (constitutive) and plasma gelsolin, the inventor's investigated whether these proteins revealed by 2D-DIGE as differentially expressed could be identified by immunoblotting. Plasma samples taken at week 20 of gestation from women who subsequently developed PE-SGA (n = 4-5) were used for Western blotting. Western blot analysis confirmed differential plasma levels for fibrinogen and alpha- 1-antichymotrypsin consistent with our 2D-DIGE findings (Figure 8). Two proteolytic fragments of fibrinogen corresponding to 27 and 25 kDa appear to be highly up-regulated in two of the four PE-SGA patients (Figure 8). Densitometric analysis of the three main fibrinogen chains alpha, beta and gamma confirmed the up-regulation of fibrinogen gamma in the PE-SGA group (274 ± 75 SD, mean intensity units xl O3) versus controls (181 ± 19 SD), p<0.05 (Student' s T-test, one-tailed). Immunoblot analysis showed increased levels of alpha- 1-antichymotrypsin in plasma of women who later developed PE-SGA with a mean band intensity of 309 ± 42 SD in the PE-SGA group versus controls (218 ± 21 SD),/?<0.01.
No differences could be detected by immunoblot in the levels of plasma gelsolin, serum amyloid A4, complement 3 c, and clusterin isoform 1 (data not shown). This may be due to being unable to detect differential expression of isoforms in ID gels or a lack of sensitivity to detect differences between cases and controls by Western blot. High molecular weight kininogen (HK) was detected by immuuoblot in native 20 weeks' gestation plasma from healthy women with an uncomplicated pregnancy. This finding contrasts with the findings in plasma obtained at 20 weeks' gestation from women with subsequent PE-SGA, whereby prior to the development of PE-SGA, two of the four women had markedly diminished levels of HK (Figure 9).
Vitronectin
In a separate study of six women who developed PE-AGA and six healthy pregnant controls, 2D-DIGE analysis of plasma was performed using blood obtained at 20 weeks' gestation and at 36-38 weeks gestation from the same women. The standardised abundance of protein spots were significantly different (p<0.05, two-way ANOVA with False Discovery Rate Correction) in plasma from women prior to and with established preeclampsia (n=6) compared to healthy controls (n=6) (Figure 10).
The five proteins were present in two distinct protein spot trains, indicating the presence of different isoforms of the same protein along each chain possibly due to differential glycosylation. The two protein spots in the upper chain (spots 1 and 2) showed an increase in expression in women who developed preeclampsia and the three protein spots in the lower chain (spots5 to 7) showed a decrease in expression when compared to healthy controls (Figure 10). All five spots were identified as vitronectin (Figure 11 for protein accession numbers).
The above 2D gel electrophoresis results were confirmed by Western Blot (Figure 12). Compared to healthy controls, women who later developed PE, over-expressed the single chain 75 kDa vitronectin molecule at 20 weeks of gestation and also at the time the condition was manifest clinically in two of the three PE cases. The results indicate that 75 kDa vitronectin isoforms are up-regulated in PE-AGA both in early pregnancy before onset of disease and after clinical manifestation, whereas the double-chain vitronectin (65+10 kDa) is down-regulated in PE-AGA.
In a further study the inventors investigated plasma at week 20 of gestation of women with a healthy pregnancy outcome (Cl to C4) compared to women who subsequently developed PE-SGA (PS) (Figure 13). Plasma of women with a healthy pregnancy outcome was found to contain mainly single chain vitronectin with a molecular weight of 75 kDa. Women who subsequently developed PE-SGA not only exhibited lower levels of the 75 kDa vitronectin but expressed under reducing conditions predominantly the 65 kDa fragment of the two-chain vitronectin molecule (65+10 kDa) (3 out of 4 cases). These results indicate down-regulation of single chain vitronectin at 75kDa and up-regulation of the two-chain isoform of vitronectin in PE-SGA.
Clusterin In a separate case control study the inventors determined plasma levels of clusterin, measured by ELISA5 prior to the onset of preeclampsia and SGA pregnancies. The pregnant women were participants in the SCOPE (Screening for Pregnancy Endpoints) study in Auckland, New Zealand, (Australian Clinical Trials Registry ACTRN12607000551493). The three case groups were 1) preeclampsia with an appropriately grown baby for gestational age baby ( PE-AGA, n=20); 2) preeclampsia with a small for gestational age baby (PE-SGA, n=20) and 3) normotensive women with an SGA baby (N-SGA n=20). Controls comprised of women who had an uncomplicated pregnancy defined as a pregnancy with no antenatal obstetric or medical complications who delivered an appropriately grown, healthy baby at 37 or greater weeks' gestation (N=40).
Clusterin ELISA
Clusterin was measured in EDTA plasma collected at 20±l weeks' gestation using a human clusterin ELISA kit (Bio Vendor, Modrice, Czech Republic) according to the manufacturer's protocol. Student's t- test was used to compare continuous data between groups and Chi-square or Fisher's exact tests, as appropriate, were used to compare categorical variables, (SAS 9.1 ®). Statistical significance was defined as P O.05.
Of the 2034 women with pregnancies continuing after 20 weeks' gestation, 85 (4.2%) developed preeclampsia of whom a quarter had an SGA infant (n=21). Ten percent of women had an SGA baby (n=203)5 of whom 159 (78.3%) were born to women who were normotensive. Maternal characteristics and pregnancy outcome in cases and controls are shown in Figure 14. Nineteen of the 20 women in PE-AGA and in PE-SGA groups had proteinuria, with the last woman in each group diagnosed as she developed multi-organ complications of preeclampsia. Plasma clusterin values were elevated at 20 weeks' gestation in women who later developed PE-SGA (81.5 SD 14.8 μg/ml; PO.01) and lower in women who subsequently had an SGA baby but remained normotensive (58.3SD 11.7 μg/ml, P<0.001) and compared with healthy controls who had an uncomplicated pregnancy (70.89, SD12.06 μg/ml), (Figure 14). The PE-AGA group had similar clusterin values to controls, P=0.92.
In summary, clusterin was identified as being elevated prior to PE-AGA and PE-SGA in 2D-DIGE experiments. However, plasma clusterin was not found to be elevated in PE- SGA (n=4) using semi-quantitative immunoblots, but was found to be elevated in PE-SGA by ELISA (n-=20). These contrasting results are likely to be due to small sample size and semi-quantitative nature of measurements in the Western blots. The inventors consider the more precise, larger ELISA study confirms the 2D-DIGE finding that plasma clusterin is elevated prior to the onset of PE-SGA. Plasma clusterin, as determined by ELISA measurement (Bio Vendor, Czech Republic), was not found to be increased in women who later developed PE-AGA. This does not negate the 2D-DIGE results, as this ELISA would not detect elevation of specific isoforms of clusterin detected in 2D-DIGE due to the use of a total clusterin antibody with unknown epitope included in the ELISA detection kit used.
In summary, the inventors would like to raise the possibility that beyond total levels of circulating clusterin, the differential expression of clusterin isoforms due to posttranslational modifications could have potential value as preeclampsia biomarkers. A previous example of the utility of specific clusterin isoforms rather than total plasma/serum levels was demonstrated for patients with colorectal cancers.17
Pigment Epithelium Derived Factor (PEDF)
2D-DIGE identified PEDF
In separate 2D-DIGE study, the inventors investigated a group of pregnant women who had an abnormal utero-placental blood supply (determined by uterine artery Doppler ultrasound) and compared women who subsequently developed preeclampsia (N= 12) with those who had uncomplicated pregnancies (N=I 2). Plasma specimens were collected at 20+1 weeks' gestation from participants in the SCOPE study in Auckland, New Zealand, (Australian Clinical Trials Registry ACTRN12607000551493). Blood was collected by venipuncture into BD EDTA- Vacutainer®, placed on ice, centrifuged at 240Ox g for 10 min at 4 °C and stored at -80°C. The mean time between collection and storage was 2.2 (SD 1.0) hours. Participants had uterine artery Doppler ultrasound examinations at 20 and 24 weeks of gestation. An abnormal uterine artery Doppler waveform was defined as a resistance index (RI) at 24 weeks > 90thC (0.62) or a RI at 20 weeks > 99thC (0.80).
Maternal characteristics and pregnancy outcome in the PEDF 2D-DIGE study are shown in Figure 15. All women had an abnormal uterine artery Doppler ultrasound and women who later developed preeclampsia were compared with those who remained healthy and had an uncomplicated pregnancy. Eleven of the 12 women with preeclampsia had proteinuria and the last was diagnosed as she developed multi-organ complications of preeclampsia. None of the women smoked. A third of the babies in the PE group were SGA (n=4) and half were premature requiring admission to a neonatal unit (n=6).
Pigment epithelium derived factor (PEDF) was identified by mass spectroscopy in two differentially expressed spots in the preeclampsia group (PO.01, false discovery rate corrected, (Figures 16 and 17). One isoform of PEDF (spot ID 459, pi 5.4) was increased, and the other (spot ID 485) at pi 6.1 was reduced in preeclampsia. Here, we investigated total unbound PEDF in plasma of women at 20 weeks of gestation prior to the onset of preeclampsia by ELISA.
In summary, the inventors would like to raise the possibility that beyond total levels of circulating PEDF, that different PEDF isoforms due for example to posttranslational modifications (e.g. glycoisoforms) have potential value as biomarkers for the prediction of preeclampsia.
Plasma PEDF levels by ELISA prior to preeclampsia
In a separate case control study, the inventors investigated PEDF measured by ELISA in plasma at 20±l weeks' gestation prior to the onset of PE-AGA and PE-SGA pregnancies. The pregnant women were participants in the SCOPE study, Auckland, New Zealand, as described above for Clusterin. The two case groups comprised PE-AGA (n=20) and PE- SGA (n=20) and controls comprised of healthy women who had an uncomplicated pregnancy and delivered an appropriately grown, healthy baby at term (N=20, 20). PEDF was measured by ELISA using the commercially available ChemiKine™ human PEDF Sandwich ELISA kit (Millipore Australia Pty Ltd, North Ryde, NSW). Plasma samples were treated with urea (7 M final concentration) according to the manufacturer's protocol prior to ELISA procedure to detect total unbound PEDF. Student's t- test was used to compare continuous data between groups and Chi-square or Fisher's exact tests, as appropriate, were used to compare categorical variables (SAS 9.1 ®). Statistical significance was defined as P <0.05.
Maternal characteristics and pregnancy outcome in the PEDF ELISA study are shown in Figure 18. Thirteen (65%) women in each preeclampsia group had severe disease. Of the PE-AGA and PE-SGA groups, 7 (35%) and 12 (60%) babies, respectively, were born premature. Plasma PEDF levels were elevated in women who later developed PE-AGA (14.5 SD 2.6 μg/ml; P=0.02) compared with healthy controls who had an uncomplicated pregnancy (12.2 SD 3.4 μg/ml). Women who later developed PE- SGA had similar PEDF values to controls, (12.0 SD 4.0 versus 11.7 SD2.5; P=0.73).
Complement factor H-related 1 (FHL-I) Protein
Increased expression of complement factor H-related 1 (FHL-I) was initially discovered in a small longitudinal 2D DIGE study including plasma samples from two women at week 15 and two women at week 20 of their of pregnancy who subsequently had a normal pregnancy outcome in comparison to non-pregnant control subjects (n=2). Two protein spots (spots 517 and 520) were found to be increased 10- and 23 -fold, respectively, in pregnant women compared to non-pregnant controls. Those two spots were identified by LC-MS/MS mainly as complement factor H-related 1 (FHL-I) protein (Figure 19). Moreover, FHL-I was also found as part of two spots in another 2D DIGE experiment (Experiment III) investigating plasma samples from women in early pregnancy (week 20 of gestation) prior to developing preeclampsia in comparison to gestation matched controls. These two spots (spots 427 and 579) were 3- and 4.8-fold up-regulated, respectively, in women with subsequent preeclampsia (FDR corrected P- valueO .01; Figure 20). FHL-I is derived from complement factor H, a key protein in the alternative pathway of complement activation, by means of alternative rnRNA splicing. Detection of FHL-I in pregnancy plasma from women with PE-SGA was unsuccessful, possibly due to 10-fold lower levels of FHL-I present in plasma compared to complement factor H (30 versus 400 ug/mL).
The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. Titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.
The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country in the world.
Throughout this specification and any claims which follow, unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to".
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Claims

1. A method for predicting the risk of a female subject developing preeclampsia, the method comprising observing the level of one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, proteolytic peptides precursors and/or isoforms, wherein the one or more proteins is chosen from the group consisting of: Fibrinogen; Apolipoprotein A-I; Apolipoprotein C-III; Apolipoprotein E; Apolipoprotein M; Complement component 3; Complement 4A (C4A); Complement factor H-related protein 1 (FHLl); Clusterin; Transthyretin;
Hemopexin; Serum amyloid P; Serum amyloid A4, alpha-2-macroglobulin; Pregnancy zone protein (PZP); alpha- 1-antichymotrypsin; alpha- 1 -antitrypsin; alpha- 1 -type I collagen; Kininogen 1; Vitronectin; Angiotensinogen; alpha2-HS- glycoprotein; zinc-alpha2-glycoprotein; Fibronectin 1 isoform 3; alphal- microglobulin (bikunin); Inter-alpha-trypsin inhibitor heavy chain H4;
Haptoglobin-related protein, Transferrin; and Pigment epithelium-derived factor (PEDF).
2. A method for predicting the risk of a female subject developing preeclampsia, the method comprising observing the level of one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof,, and/or nucleic acids encoding the one or more proteins, subunits, proteolytic peptides, precursors and/or isoforms, wherein the one or more proteins is an HDL cargo protein.
3. A method as claimed in claim 2 wherein the HDL cargo protein is chosen from the group consisting: phospholipid transfer protein, cholesteryl ester transfer protein, lecithin-cholesterol acyltransferase, apolipoprotein (apo) C-I , apoC-II, apoC-III, apoC-IV, paraoxonase-1 , paraoxonase-3 , serum amyloid A 4 , serum amyloid A 2, serum amyloid A 1 , apo A-I, , apoH, apoA-IV, clusterin, apoA-IL apoL-I, apoD, apoE, apoF, apoM, angiotensinogen (AGT), alpha-2-antiplasmin, serpin peptidase inhibitor, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1- antitrypsin, bikunin (alphal -microglobulin), kininogen, alpha- 1 -acid glycoprotein 2, transthyretin, inter-alpha-trypsin inhibitor H4, retinol binding protein 4, transfeπin, fibrinogen, hemopexin, complement component 3 (C3), C4A, C4B, C9, and vitronectin.
4. A method as claimed in claim 3 wherein the HDL cargo protein is chosen from the group consisting: apoM, apoE, clusterin. apoA-I, serum amyloid A 4, apoC-III, C3, C4A, vitronectin, angiotensinogen, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1- antitrypsin, bikunin (alphal -microglobulin), kininogen, transthyretin, inter-alpha- trypsin inhibitor heavy chain H4, transferrin, fibrinogen, and hemopexin.
5. A method as claimed in any one of claims 1 to 4 comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting the one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, precursors, proteolytic peptides, and/or isoforms in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids against a standard; wherein a difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing preeclampsia.
7. A method as claimed in any one of claims 1 to 6 wherein the precursors, isoforms, proteolytic fragments and/or subunits include: Fibrinogen alpha chain, Fibrinogen beta chain, Fibrinogen gamma chain, A fibrinogen fragment with an approximate molecular weight of 27 kDa, A fibrinogen fragment with an approximate molecular weight of 25kDa, Complement component 3 precursor, Complement component 3 C, High Molecular Weight Kininogen (HK), HKa (a fragment of HK), Clusterin isoform I, Apolipoprotein A-I fragment with an approximately molecular weright of 17kDa, Apolipoprotein A-I fragment with an approximately molecular weright of 2IkDa, Apolipoprotein A-I fragment with an approximately molecular weright of 3OkDa, Apolipoprotein A-I fragment with an approximately molecular weright of 25kDa, Transthyretin with an approximate molecular weight of 15kDa, Transthyretin with an approximate molecular weight of 12kDa, Serum amyloid P fragment with an approximate molecular weight of 3OkDa, Serum amyloid P fragment with an approximate molecular weight of 27kDa, Vitronectin (approximately 75kDa), Vitronectin (approximately 65kDa), PEDF isoform with an approximate pi of 5.4, and PEDF isoform with an approximate pi of 6.1.
8. A method as claimed in any one of claims 1 to 7 wherein a higher or increased level of one or more of the following compared to a standard, is indicative of risk of developing preeclampsia: Fibrinogen (and/or the alpha chain, beta chain and/or gamma chain), Apolipoprotein A-I (and/or fragments thereof with approximate molecular weights of 17, 21, and/or 3OkDa), Apolipoprotein E, Complement component 3 (and/or complement component 3 precursor and/or complement component 3c)5 Complement 4a, Complement factor H-related protein I, Clusterin (and/or clusterin isoform I), Transthyretin with an approximate molecular weight of
15 kDa, Hemopexin, Serum amyloid P fragment with an approximate molecular weight of 30 kDa, Serum amyloid A4, alpha-2-macroglobulin, Pregnancy zone protein (PZP), alpha- 1-Antichymotrypsin, alpha- 1 antitrypsin, alpha- 1 type I Collagen, Kininogen I, HKa, Vitronectin (approximately 75 kDa), Vitronectin (approximately 65kDa), Angiotensinogen, alpha2-HS-Glycoprotein, zmc-alpha2-
Glycoprotein, Fibronectin 1 isoform 3, Transferrin, Haptoglobin-related protein and Pigment epithelium derived factor (and/or a PEDF isoform with an approximate pi of 5.4).
9. A method as claimed in any one of claims 1 to 7 wherein a lower or decreased level of one or more of the following compared to a standard is indicative of risk of developing preeclampsia: Apolipoprotein A-I fragment with an approximate molecular weight of 25 kDa, Apolipoprotein C-III, Apolipoprotein M, Transthyretin with an approximate molecular weight of 12 kDa, Serum amyloid P fragment with an approximate molecular weight of 27 kDa, High Molecular
Weight Kininogen, Vitronectin (approximately 65 kDa), Vitronectin (approximately 75 kDa), alphal -Microglobulin (bikunin), Inter-alpha-trypsin inhibitor heavy chain H4 and A PEDF isoform with an approximate pi at 6.1.
10. A method as claimed in claims 8 or 9 wherein there is at least an approximately 1.3 fold increase or decrease in the level of a particular protein, precursor, isoform, proteolytic peptide, isoform and/or nucleic acid compared to the standard, more preferably at least an approximately 1.5 fold increase or decrease.
11. A method for predicting the risk of a female subject developing preeclampsia with a small for gestational age baby (PE-SGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting one or more proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acid encoding the one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, proteolytic peptides, isoforms and/or nucleic acids against a standard; wherein a difference hi the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acids in the sample compared to the standard is indicative of the risk of developing PE-SGA, and wherein the one or more proteins is chosen from the group consisting: alpha- 1- antichymotrypsm; fibrinogen; kininogen I; vitronectin, and clusterin.
12. A method as claimed in claim 11 wherein the proteins, subunits, precursors, proteolytic peptides and/or isoforms are chosen from the group consisting: alpha- 1- antichymotrypsin; fibrinogen gamma chain; a fibrinogen fragment with an approximate molecular weight of 27 kDa, a fibrinogen fragment with an approximately molecular weight of 25 kDa; high molecular weight kininogen (HK); and, Vitronectin, approximately 65 kDa; Vitronectin, 75 kDa; and Clusterin.
13. A method as claimed in claims 11 or 12 wherein a higher level of one or more of alpha- 1 -antichymotrypsin, fibrinogen gamma chain, an approximately 25 kDa fragment of fibrinogen, an approximately 27 kDa fragment of fibrinogen, an approximately 65 kDa part of the two-chain vitronectin molecule and/or clusterin in the sample compared to the standard is indicative of risk of developing PE-SGA.
14. A method as claimed in claims 11 or 12 wherein a lower level of HK and/or vitronectin single chain with an approximate molecular weight of 75 kDa in a sample compared to the standard is indicative of risk of developing PE-SGA.
15. A method for predicting the risk of a female subject developing PE-SGA, the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting apolipoproteixi A-I in combination with one or more of fibrinogen, pregnancy zone protein and/or alpha-2-macroglobulin, clusterin isoform I, alphal-antichymotrypsin, and complement factor I and/or nucleic acids encoding said proteins in the sample; and, c) comparing the level of each of the proteins and/or nucleic acids in the sample against a standard; wherein a difference in the level of each of the proteins and/or nucleic acids in the sample compared to the standard is indicative of developing PE-SGA.
16. A method as claimed in claim 15 wherein one or more precursors, isoforms, proteolytic peptides and/or subunits of the proteins, or nucleic acids encoding one or more thereof, is detected in addition to or in lieu of detection of a particular protein.
17. A method as claimed in claim 16 wherein the one or more precursors, isoforms, proteolytic peptides and/or subunits includes an approximately 17 kDa Apo-AI fragment, an approximately 21 kDa Apo-AI fragment, fibrinogen beta chain, and fibrinogen gamma chain.
18. A method as claimed in any one of claims 15 to 17 wherein the method includes detecting each of Apolipoprotein AI and pregnancy zone protein and/or alpha-2- macroglobulin, and/or nucleic acids encoding same.
19. A method as claimed in claim 18 wherein a combination of a higher level of apolipoprotein A-I and a higher level of pregnancy zone protein and/or alpha-2- macroglobulin is indicative of the risk of PE-SGA.
20. A method as claimed in any one of claims 15 to 17 wherein the method includes detecting each of Apolipoprotein AI, pregnancy zone protein and/or alpha-2- macroglobulin, and Fibrinogen beta chain, and/or nucleic acids encoding same.
21. A method as claimed in claim 20 wherein a combination of a higher level of apolipoprotein A-I, a higher level of fibrinogen beta chain and a higher level of pregnancy zone protein and/or alpha-2-macroglobulin is indicative of the risk of PE-SGA.
22. A method as claimed in any one of claims 15 to 17 wherein the method includes detecting each of Apolipoprotein AI, pregnancy zone protein and/or alpha-2- macroglobulin, fibrinogen beta chain, fibrinogen gamma chain, clusterin isoform I, alpha- 1-antichymotrypsin and complement factor I5 and/or nucleic acids encoding same.
23. A method as claimed in claim 23 wherein a higher level of apolipoprotein AI, a higher level of pregnancy zone protein and/or alpha-2-macroglobulin, a higher level of fibrinogen beta chain, a higher level of fibrinogen gamma chain, a higher level of clusterin isoform I, a higher level of alpha- 1-antichymotrypsin and a higher level of complement factor I is indicative of the risk of PE-SGA.
24. A method for predicting the risk of a female subject developing preeclampsia with PE-SGA, the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting each of the proteins apolipoprotein A-I, serum amyloid P, and transthyretin with an approximate molecular weight of 12 kDa, and/or nucleic acids encoding same in the sample; and, c) comparing the level of each of the proteins and/or nucleic acids in the sample against a standard; wherein a difference in the level of each of the proteins and/or nucleic acids in the sample compared to the standard is indicative of developing PE-SGA.
25. A method as claimed in claim 24 wherein the method further includes detecting fibrinogen.
26. A method as claimed in claims 24 or 25 wherein one or more precursors, isoforms, proteolytic peptides and/or subunits of each of the proteins, or nucleic acids encoding one or more thereof, is detected in addition to or in lieu of detection of a particular protein.
27. A method as claimed in claim 26 wherein the one or more precursors, isoforms, proteolytic peptides and/or subunits includes an apolipoprotein A-I fragment with an approximate molecular weight of 27kDa, an apolipoprotein fragment with an approximate molecular weight of 17kDa, fibrinogen beta chain, and serum amyloid
P with an approximate molecular weight of 27 kDa.
28. A method as claimed in any one of claims 24 to 27 wherein a lower level of a fragment of apolipoprotein A-I with an approximate molecular weight of 27kDa, a lower level of serum amyloid P with an approximate molecular weight of 27 kDa, a higher level of a fragment of apolipoprotein A-I with an approximate molecular weight of 17 kDa, and a lower level of a transthyretin fragment with an approximate molecular weight of 12 kDa are together indicative of risk of developing PE-SGA.
29. A method as claimed hi any one of claims 24 to 27 wherein a lower level of a fragment of apolipoprotein A-I with an approximate molecular weight of 27kDa, a lower level of serum amyloid P with an approximate molecular weight of 27 kDa, a higher level of a fragment of apolipoprotein A-I with an approximate molecular weight of 17 kDa, a lower level of a transthyretin fragment with an approximate molecular weight of 12 kDa, and a higher level of fibrinogen beta chain are together indicative of risk of developing PE-SGA.
30. A method for predicting the risk of a female subject developing preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting PEDF, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; and, c) comparing the level of the PEDF, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof against a standard; wherein a difference in the level of the PEDF, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of the risk of developing PE-AGA.
31. A method as claimed in claim 30 wherein a higher level of PEDF, a subunit thereof, a precursor thereof, an isoform thereof, a proteolytic peptide thereof and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of risk of developing PE-AGA.
32. A method as claimed in claim 30 or 31 wherein the isoform of PEDF is an isoform with an approximate pi of 5.4.
33. A method for predicting the risk of a female subject developing preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA), the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting fibrinogen, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; and, c) comparing the level of the fibrinogen, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof against a standard; wherein a difference in the level of the fibrinogen, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid encoding one or more thereof in the sample compared to the standard is indicative of the risk of developing PE-AGA.
34. A method as claimed in claim 33 wherein a higher level of the fibrinogen, subunit, precursor, isoform, proteolytic peptide and/or nucleic acid is indicative of the risk of developing PE-AGA.
35. A method as claimed in claim 33 or 34 wherein the subunit of fibrinogen is fϊbriogen beta chain.
36. A method for predicting the risk of a female subj ect developing preeclampsia with an appropriate birthweight for gestational age baby (PE-AGA), the method comprising at least the steps of: a) taking a sample from the subj ect at a time when the subj ect is pregnant; b) detecting Apo-AI in combination with one or more of HRP, Transferrin, alpha- 2-macroglobulin, and fibronectin and/or nucleic acids encoding same in the sample; and, c) comparing the level of each of the proteins and/or nucleic acids in the sample against a standard; wherein a difference in the level of each of the proteins and/or nucleic acids in the sample compared to the standard is indicative of developing PE-AGA.
37. A method as claimed in claim 36 wherein one or more precursors, isoforms, proteolytic peptides and/or subunits of each of the proteins, or nucleic acids encoding one or more thereof, is detected in addition to or in lieu of detection of a particular protein.
38. A method as claimed in claim 37 wherein the one or more precursors, isoforms, proteolytic peptides and/or subunits includes an approximately 17 kDa Apo-AI fragment, an approximately 21 kDa Apo-AI fragment, an approximately 30 kDa Apo-AI fragment and fibronectin I isoform 3.
39. A method as claimed in any one of claims 36 to 38 wherein a combination of a higher level of apolipoprotein AI (and/or an apolipoprotein A-I fragment of approximately 17 kDa, an apolipoprotein A-I fragment of approximately 21 kDa and/or an apolipoprotein A-I fragment of approximately 30 kDa), a higher level of HRP, a higher level of alpha 2 macroglobulin a higher level of Fibronectin 1 isoform 3, and a higher level of transferrin are indicative of risk of developing PE- AGA.
40. A method of predicting the risk of a female subject developing normotensive SGA, the method comprising observing the level of clusterin, a subunit thereof, a precursor thereof, a proteolytic fragment thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof.
41. A method as claimed in claim 40 wherein the method comprises at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting clusterin, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; c) comparing the level of the clusterin, subunit, isoform, precursor, proteolytic peptide and/or nucleic acid against a standard; wherein the difference in the level of clusterin, a subunit, isoform, precursor, proteolytic peptide and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing normotensive SGA.
42. A method as claimed in claims 40 or 41 wherein a lower level of clusterin, a subunit thereof, a precursor thereof, an isoform thereof, proteolytic peptide and/or a nucleic acid encoding one or more thereof compared to the standard is indicative of the risk of developing normotensive SGA.
43. A method as claimed in any one of claims 40 to 42 wherein the isoform is Clusterin isoform 1 and a lower level of clusterin isoform 1 compared to the standard is indicative of the risk of developing normotensive SGA.
44. A method of discriminating between PE-SGA and normotensive SGA the method comprising observing the level of clusterin, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoforms thereof, and/or a nucleic acid encoding one or more thereof.
45. A method as claimed in claim 44 wherein the method comprises at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting clusterin, a subunit thereof, a precursor thereof, a proteolytic peptide thereof and/or an isoform thereof, and/or a nucleic acid encoding one or more thereof in the sample; c) comparing the level of the clusterin, subunit, isoform, precursor, proteolytic peptide and/or nucleic acid against a standard; wherein the difference in the level of clusterin, subunit, isoform, precursor, proteolytic peptide and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing either PE-SGA or normotensive SGA.
46. A method as claimed in claims 44 or 45 wherein a lower level of clusterin, a subunit thereof, an isoform thereof, a proteolytic peptide and/or a precursor thereof, and/or a nucleic acid encoding one or more thereof compared to the standard is indicative of the risk of developing normotensive SGA and a higher level of clusterin, a subunit thereof, an isoform thereof, a proteolytic peptide thereof and/or a precursor thereof, and/or a nucleic acid encoding one or more thereof compared to the standard is indicative of the risk of developing PE-SGA.
47. A method as claimed in any one of claims 44 to 46 wherein the isoform of clusterin is Clusterin isoform 1 and a lower level of clusterin isoform 1 and/or nucleic acid encoding same compared to the standard is indicative of the risk of developing normotensive SGA and a higher level of clusterin isoform 1 and/or a nucleic acid encoding same compared to the standard is indicative of the risk of developing PE- SGA.
48. A method for predicting the risk of a female subject developing CVD, the method comprising observing the level of one or more HDL cargo proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof at a time when the female subject is pregnant.
49. A method as claimed in claim 48 the method comprising at least the steps of: a) taking a sample from the subject at a time when the subject is pregnant; b) detecting one or more HDL cargo proteins, precursors thereof, isoforms thereof, proteolytic peptides thereof, and/or subunits thereof, and/or nucleic acids encoding the one or more proteins, subunits, precursors, proteolytic peptides and/or isoforms thereof in the sample; and, c) comparing the level of the one or more proteins, subunits, precursors, proteolytic peptides, isoforms and/or nucleic acids against a standard; wherein a difference in the level of the one or more proteins, subunits, precursors, isoforms, proteolytic peptides and/or nucleic acid in the sample compared to the standard is indicative of the risk of developing cardiovascular disease.
50. A method as claimed in claims 48 or 49 wherein the one or more HDL cargo protein is chosen from the group consisting: phospholipid transfer protein, cholesteryl ester transfer protein, lecithin-cholesterol acyltransferase, apoC-I, apoC-II, apoC-III, apoC-IV, paraoxonase-1 , paraoxonase-3 , serum amyloid A 4 , serum amyloid A 2 , serum amyloid A 1 , apoA-I, apoH, apoA-IV, clusterin, apoA- II, apoL-I, apoD, apoE, apoF, apoM, angiotensinogen (AGT)5 alpha-2-antiplasmin , serpin peptidase inhibitor, alpha-2-HS-glycoprotein, haptoglobin-related protein, alpha- 1 -antitrypsin, bikunin (alphal -microglobulin), kininogen , alpha- 1 -acid glycoprotein 2, transthyretin, inter-alpha-trypsin inhibitor heavy chain H4,Retinol Binding Protein 4, transferrin, fibrinogen, hemopexin, C3, C4A, C4B, C9, and vitronectin.
51. A method as claimed in claim 50 wherein the HDL cargo protein is chosen from the group consisting: apoM, apoE, Clusterin, apoA-I, serum amyloid A4, apoC-III, C3, C4A, vitronectin, angiotensinogen, alpha-2-HS-glycoprotein, haptoglobin- related protein, alpha- 1 -antitrypsin, bikunin (alphal -microglobulin), kininogen, transthyretin, inter-alpha-trypsin inhibitor heavy chain H4, transferrin, fibrinogen, and hemopexin.
52. The use of a method for predicting the risk of a female subject developing preeclampsia as claimed in any one of claims 1 to 47 in a method for predicting the risk of CVD in the subject.
53. A method for predicting the risk of a female subj ect developing CVD the method comprising the step of first predicting the risk of the subject developing preeclampsia using a method as claimed in any one of claims 1 to 44, wherein an increased risk of developing preeclampsia is indicative of an increased risk of developing cardiovascular disease.
54. A method as claimed in any one of claims 1 to 53 wehrein the level of the one or more proteins, a subunit thereof, an isofoπn thereof, a proteolytic peptide thereof and/or a precursor thereof, is determined using an immunoassay, separation based on characteristics such as molecular weight and isoelectric point, gel electrophoresis, Western Blotting or mass spectroscopy. Preferably the immunoassay is an ELISA. Preferably the gel electrophoresis is 2D gel electrophoresis or gel-free systems based on microfluidics technologies.
55. A method as claimed in any one of claims 1 to 54 wherein the sample is a blood sample, plasma sample, serum sample, urine sample or cervical fluid sample.
56. A method as claimed in any one of claims 1 to 55 wherein the subject is in early pregnancy at the time the sample is taken, preferably at or less than 24 weeks of gestation at the time the sample is taken.
57. A kit for use in a method as claimed in any one of claims 1 to 56, the kit comprising at least one or more reagents suitable for detection of one or more proteins, and/or subunits thereof, and/or precursors thereof, and/or isoforms thereof, and/or proteolytic peptides thereof and/or nucleic acids encoding any one or more thereof as defined in any one of the preceding claims.
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