Patent application title: METHOD FOR THE DIAGNOSIS OF PREECLAMPSIA
Won Sun Park (Busan, KR)
Na Ri Kim (Busan, KR)
Mohamad Warda (Busan, KR)
Jin Han (Busan, KR)
Jin Han (Busan, KR)
IPC8 Class: AC12Q168FI
Class name: Measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid with significant amplification step (e.g., polymerase chain reaction (pcr), etc.)
Publication date: 2012-12-06
Patent application number: 20120309009
The present invention relates to a biomarker and a composition for
diagnosis of preeclampsia. In accordance with one aspect of the present
invention, there is provided a biomarker for diagnosis of preeclampsia
using an enzyme selected from the group consisting of placental
chondroitin 4-O-sulfotransferase 1 (C4ST), chondroitin 6-sulfotransferase
(C6S), heparan sulfate 6-O-sulfotransferase 1 (HS6S), and
dermatan/chondroitin sulfate 2-sulfotransferase (CS-2OST), or uronic
5. A method for identifying preeclampsia of pregnancy in a pregnant woman, the method comprising: isolating a placental tissue from the pregnant woman; performing RT-PCR assays to detect the level of chondroitin 6-sulfotransferase (C6S) in the isolated placental tissue of the pregnant woman; measuring an amount of mRNA of the C6S by Quantitative Real-Time PCR (qRT-PCR); and identifying preeclampsia of pregnancy in the pregnant woman based on the amount of mRNA of the C6S from the isolated placental tissue of the pregnant woman, wherein an amount of mRNA of the C6S in a placental tissue of a pregnant woman having preeclampsia is lower than an amount of mRNA of the C6S in a placental tissue of a pregnant woman who does not have preeclampsia.
6. The method of claim 5, further comprising: comparing the amount of mRNA of the C6S in the isolated placental tissue of the pregnant woman with the amount of mRNA of the C6S in the placental tissue of the pregnant woman who does not have preeclampsia.
CROSS-REFERENCE TO RELATED APPLICATION
 This application claims priority to and the benefit of Korean Patent Application No. 10-2007-0069002 filed in the Korean Intellectual Property Office on Jul. 10, 2007, the entire content of which is incorporated herein by reference.
FIELD OF THE INVENTION
 The present invention relates to a biomarker and a composition for diagnosis of preeclampsia.
BACKGROUND OF THE INVENTION
 Preeclampsia in pregnancy can be a very serious health problem. It can cause fetal growth restriction, fetal death and morbidity, premature deliveries, and death of the mother. The exact cause of preeclampsia is not known, and treatments for efficiently curing or preventing preeclampsia are not also available yet. Preeclampsia is known to cause several problems at the same time, such as high blood pressure (hypertension), pathological edema and leakage of protein into the urine (proteinuria). Further, preeclampsia is one of the pregnancy complications that bring hypertension, proteinuria and traumatism to the mother. It is known that preeclampsia occurs to only about 3-5% of pregnant women, but it can seriously affect both the mother and her unborn (or newborn) baby, and thus, acts as a major cause of increasing perinatal mortality and morbidity rates.
 Globally, at least 200,000 pregnant women die from preeclampsia every year. Its symptoms typically become evident after the 20th week of pregnancy. Preeclampsia is usually diagnosed by detecting high blood pressure of a pregnant woman or by checking her urine for protein. Early diagnosis and timely treatment of preeclampsia can remarkably reduce risks to the mother and her unborn baby, but such a monitoring method by using those symptoms as criteria is not effective for an early diagnosis of preeclampsia. Further, no treatments are currently available to cure preeclampsia. Preeclampsia can be mild, but potentially life-threatening depending on the severity of the disease. Despite such clinical risks, however, it is difficult to find the cause or the pathogenesis of preeclampsia at an early stage, or to make an early diagnosis and prognosis.
 Therefore, if it becomes possible to suggest the pathogenesis of preeclampsia and make an early diagnosis and prognosis based on the same, the mother having preeclampsia and her unborn baby can be protected, and the death rate would be reduced. Even if many researches have been conducted to monitor and predict the occurrence of preeclampsia, they are limited to using a specific protein or substance, which is not sufficient to explain the whole phenomenon about the occurrence of preeclampsia and the pathogenesis thereof.
 While the inventors of the present invention are trying to discover the pathogenesis of preeclampsia through the comparison between normal pregnant women and patients with preeclampsia (whether mild or severe), they have learned that the patients with preeclampsia express lower amounts of diverse glycosaminoglycans (GAGs) in the placenta and placenta enzymes responsible for sulfonation of GAGs. Based on this, the inventors developed a new way for the early diagnosis and prognosis of preeclampsia by preparing a biomarker and a composition.
SUMMARY OF THE INVENTION
 It is, therefore, a primary object of the present invention to provide a novel biomarker to detect preeclampsia developed in the placenta of a pregnant woman.
 It is another object of the present invention to provide a composition for the diagnosis of preeclampsia based on the amounts of diverse GAGs in the placenta and placenta enzymes responsible for sulfonation of GAGs.
 It is still another object of the present invention to provide a predictive biomarker kit capable of predicting a risk of preeclampsia.
 In accordance with one aspect of the present invention, there is provided a biomarker for diagnosis of preeclampsia using an enzyme selected from the group consisting of placental chondroitin 4-O-sulfotransferase 1 (C4ST), chondroitin 6-sulfotransferase (C6S), heparan sulfate 6-O-sulfotransferase 1 (HS6S), and dermatan/chondroitin sulfate 2-sulfotransferase (CS-2OST), or uronic acid-2-sulfate (UA2S).
 In accordance with another aspect of the present invention, there is provided a composition for diagnosis of preeclampsia, comprising one or more primer pairs selected from the group consisting of primer pairs with the sequences of: (Primer Pair No. 1) Forward: 5'GTGGGGAGAGGGAGAGAATC3' (Sequence No. 1), Reverse: 5'ACAGACAAGAACGACCCATC3' (Sequence No. 2); (Primer Pair No. 2) Forward: 5'CCCAAAGTCAGAAAGCGAAG3' (Sequence No. 3), Reverse: 5'ACAAGCAAACCCACCAACTC3' (Sequence No. 4); (Primer Pair No. 3) Forward: 5'TCTGAGCCTGACCACAGATG3' (Sequence No. 5), Reverse: 5'CACCTGCACAGAACTCAGGA3' (Sequence No. 6); (Primer Pair No. 4) Forward: 5'CCCAGTGGCCCTAAAGTACA3' (Sequence No. 7), Reverse: 5'GTCCATCACTTTGGCAGGTT3' (Sequence No. 8); and (Primer Pair No. 5) Forward: 5'GTACAACCTGGCCAACAACC3' (Sequence No. 9), Reverse: 5'CGCGTGCTATTGTACTGCAT3' (Sequence No. 10).
BRIEF DESCRIPTION OF THE DRAWINGS
 The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
 The above and other objects and features of the present invention will become apparent from the following description of the preferred examples given in conjunction with the accompanying drawings, in which:
 FIG. 1 shows a polyacrylamide gel electrophoresis (PAGE) assay picture of a GAG isolated from other tissue (where lane std denotes a heparin oligosaccharide standard, lanes 1 to 12 correspond to GAGs from sample #1 to #12 (refer to Table 2), and lane HS denotes a heparan sulfate control group);
 FIG. 2 represents a chromatographic result of CS (chondroitin sulfates) disaccharide standard; and
 FIG. 3 presents a chromatographic result of Hep/HS disaccharide standard.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 In the following detailed description, reference is made to the accompanying drawings and sequence listing that show, by way of illustration, specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. It is to be understood that the various embodiments of the invention, although different, are not necessarily mutually exclusive. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims that should be appropriately interpreted along with the full range of equivalents to which the claims are entitled.
 Then, experiments performed for better understanding the present invention will be described in detail as follows, which are set forth to illustrate, but are not to be construed to limit the present invention.
 The inventors confirmed by using RT-PCR that patients who developed preeclampsia expressed a lower amount of placenta enzymes, which are involved in sulfonation of GAGs. GAGs cover plasma membranes of all cells and fill extracellular matrix composed of heparin sulfate (HS), CS, and dermatan sulfate (DS). The inventors confirmed that mRNA levels of C4ST, C6S, HS6S and CS-2OST were reduced in patients with preeclampsia. Particularly, the inventors turned out that patients with preeclampsia did not have other isoforms of the last enzyme (i.e., CS-2OST), which cause C2-sulfonation on uronic acid or iduronic acid in CS with disaccharide repeat units. This decrease in the amount of the enzyme was confirmed through the lack of UA2S in a placental sample tested for preeclampsia.
 Further, the inventors learned from RT-PCR assay results that the amount of CS-2OST was noticeably decreased in the placenta of a woman affected by preeclampsia (this was also the same for other GAG sulfotransferases). The inventors also confirmed through the disaccharide assay that UA2S (the final product of sulfate 2-sulfotransferase), which is the disaccharide repeat unit of placental GAGs, was completely disappeared from a disease-infected sample.
 The inventors assumed that preeclampsia might be associated with the progress of glycomics of the placental abnormalities. This assumption was confirmed by monitoring decrease in the placental mRNA expression of diverse GAG sulfotransferases in preeclamptic pregnancy.
 One of the important findings through the analysis of CS disaccharide composition for this invention is that preeclamptic pregnancies lack the expression of UA2S, and this supports parallel decrease observed in the expression of CS-2OST. This is very clear in that the enzyme has only a single heteroplasm in most entities with known genome sequences, which is contradictory to its various heteroplasms in almost all other chondroitin sulfotransferases (see Xu, D. et al., J. Biol. Chem., Mar. 16, 2007; 282(11): 8356-67). Along with complicated functions that are verified in diverse manners (see Bao, X. et al., (2005), J. Biol. Chem., 280, 23184-23193), CS-2OST carries a sulfo group to the 2-OH position of hexauronic acid adjacent to a N-acetylated galactosamine residue carrying 6-0 or 4-0 sulfo group (see Kobayashi, M. et al., (1999), J. Biol. Chem., 274, 10474-10480; Ohtake, S. et al., (2005), J. Biol. Chem., 280, 39115-39123).
 Interestingly enough, the clear superimposing image of a decreased CS-2OST mRNA expression, as the last step of CS variation in addition to the absence of UA2S subcluster in CS under preeclampsia, reflects a poor, final tuning of CS that occurs commonly in the placenta of a pregnant woman.
 No one has yet discovered like the inventors that there is decrease in the expression of sulfotransferases under preeclampsia. What is new and interesting is that the decrease in the mRNA expression of CS-2OST enzyme in the placenta of the pregnant woman with preeclampsia is in parallel with complete absence of its byproduct, UA2S.
 The use of such data makes it possible to develop a predictive biomarker kit through which the risk of preeclampsia in a pregnant woman can be predicted.
 To this end, the inventors obtained placental samples from normal pregnant women and from patients who are epidemiologically diagnosed with preeclampsia. Those samples were ground, fats were removed therefrom, proteins were hydrolyzed, and various GAGs were isolated by using a column chromatography. These isolated GAGs were quantified by carbazole assay (Blyscan assay) (refer to Table 1). In result, the average amount of GAGs (mg/g; dry sample) was lower in preeclampsia samples than that of the control group.
 In addition, for the analysis of a molecular weight and polydispersity of a sample, the inventors conducted PAGE, and computed an average molecular weight of GAGs based on the heparin oligosaccharide standard (refer to FIG. 1). In result, the average molecular weight of GAGs was slightly lower in preeclampsia samples than that of the control group (refer to Table 2).
 Then, the inventors depolymerized the GAG complex enzymatically, and conducted a disaccharide analysis using LC-MS (refer to FIG. 2). In doing so, 8 kinds of disaccharides of Hep/HS were isolated (refer to FIG. 3).
 A total of 12 samples were subjected to a compositional analysis of CS disaccharides (refer to FIG. 2), wherein the compositional analysis was measured in terms of Mean/SD (refer to Table 3). The comparison of CS disaccharides between the preeclampsia sample group and the control group shows that samples in the control group have more UA2S and TriS than samples in the preeclampsia group (refer to Table 4). On the other hand, Hp/HS disaccharides have N-sulfo disaccharides that are more abundant in the control group than in the preeclampsia sample group. This implies that translocation of the N-sulfo group under preeclampsia occurs most frequently at the sixth carbon position. The preeclampsia samples are very rich in tri-sulfo disaccharides (refer to Table 5).
 The inventors also examined the variation in the amount of mRNA of diverse GAG synthesis regulatory enzymes by Quantitative Real-Time PCR (qRT-PCR). All primers were designed using gene-specific sequences fostered by GenBank (refer to Table 6). The RT-PCR result confirms that the preeclampsia samples, compared with the control group placenta, showed noticeable decrease in the expression of diverse chondroitin sulfotransferases enzymes (refer to Table 7).
 Hereinafter, the present invention will be explained in more detail through examples. However, it will be apparent to those skilled in the art that these examples are only for the purpose of explaining the present invention in detail, but not intended to limit the scope of the invention.
Acquisition--Purchase of Samples
 Placental samples used for the present invention (e.g., placental samples from normal pregnant women and from patients who are epidemiologically diagnosed with preeclampsia) were provided by the department of obstetrics and gynecology in Inje University Hospital.
 Actinase E was purchased from Kaken Biochemicals (Tokyo, Japan), CS, chondroitin lyases and heparin lyases were purchased from Seikagaku (Tokyo, Japan), and polyacrylamide, urea, CHAPS, Alcian blue dye, 2-cyanoacetamide and tetra-n-butylamonium hydrogen sulfate were purchased from Sigma Chemical Company (St. Louis, Mo.). All other samples were of reagent grade. Vivapure MAXI QH columns were purchased from Viva science (Edgewood, N.J.).
 Unsaturated disaccharide standards from CS (Di-0S ΔUA-Gal, Di-4S ΔUA-Gal4S, Di-6S ΔUA-Gal6S, Di-UA2S ΔUA2S-Gal, Di-diSB ΔUA2S-Gal4S, Di-diSD ΔUA 2S-Gal6S, Di-diSE ΔUA-Gal4S6S, Di-triS ΔUA2S-Gal4S6S) were purchased from Seikagaku Corporation (Japan), and chondroitinase ABC and ACII were also purchased from Seikagaku Corporation (Japan).
 Unsaturated disaccharide standards from Hep/HS (Di-0S ΔUA-GlcNAc, Di-NS ΔUA-GlcNS, Di-6S ΔUA-GlcNAc6S, Di-UA2S ΔUA2S-GlcNAc, Di-UA2SNS ΔUA2S-GlcNS, Di-NS6S ΔUA-GlcNS6S, Di-UA2S6S ΔUA2S-GlcNAc6S, Di-triS ΔUA2S-GlcNS6S) were purchased from Seikagaku Corporation (Japan).
 A mixture (150 ng/μl) of disaccharide standards containing Di-0S, Di-4S, Di-6S, Di-diSD, Di-diSE, and Di-triS was indicated as std1. A mixture of disaccharide standards containing Di-0S, Di-4S, Di-UA2S, Di-diSD, Di-diSE, and Di-triS was indicated as std2. A mixture of disaccharide standards containing Di-0S, Di-4S, Di-6S, Di-diSB, Di-diSD, and Di-triS was indicated as std3. A mixture of disaccharide standards containing Di-0S, Di-4S, Di-UA2S, Di-diSB, Di-diSE, and Di-triS was indicated as std4. A mixture of disaccharide standards containing Di-4S, Di-diSE, and double the amount of Di-6S and Di-diSD was indicated as std5. A mixture of disaccharide standards containing Di-6S, Di-diSD, and double the amount of Di-4S and Di-diSE was indicated as std6. A mixture of disaccharide standards containing Di-UA2S and double the amount of Di-6S was indicated as std7.
Isolation and Purification of GAG
 With the use of mortars and pestles, the samples were pulverized together with dry ice to very fine particles of uniform size. Tissues were rinsed with a mix solution of chloroform/methanol (2:1, 1:1, and 1:2) (v/v), respectively, overnight, and thus defatted.
 In 5 ml water, the fat-free samples were treated with 1% Actinase E (20 mg/ml) at 55° C. for 18 hours to hydrolyze protein. Following the hydrolysis of protein, dry urea and dry'CHAPS (2 wt % CHAPS and 8 M urea) were added to each of the samples. The obtained blurring solutions passed through a syringe filter having a 0.2 μm film, and thus became transparent. Vivapure MAXI QH spin columns reached the equilibrium state with the addition of 3 ml of 8 M urea (pH 8.3) containing 2% CHAPS. The clear, filtered samples were loaded on the Vivapure MAXI QH spin columns in a centrifugal separator (500×g) and then perfused. First, the columns were rinsed with 3 ml of 8 M urea (pH 8.3) containing 2% CHAPS at pH 8.3. Then, they were rinsed five times with 5 ml of 200 mM NaCl. Further, the samples were rinsed three times with 1 ml of 16% NaCl to emit GAG from the spin column. To prepare an 80 vol % solution, 12 ml of methanol was added to each sample, and the mix solutions were allowed to reach the equilibrium state over the period of 18 hours at 4° C. The obtained precipitates were centrifuged at 2500×g for 15 min and collected. The precipitates were dissolved in 0.5 ml water and collected. The collected precipitates were put in a freezer for additional analysis on the collected GAGs.
Quantification of GAGS by Carbazole Assay (Blyscan Assay)
 With the isolated GAGs as a marker dye, the inventors conducted Blyscan assay using 1,9-dimethylmethylene blue (DMB) and quantified the amount of GAG in each of the samples by using standard CS.
 As shown in Table 1 below, the inventors turned out that the average amount of GAGs (mg/g; dry samples) in the control group was 2.78±0.48 mg/g, and the average amount of GAGs in the preeclampsia samples was 2.26±0.21 mg/g.
TABLE-US-00001 TABLE 1 Description on Original Dry GAG GAG MW No. sample mass (g) mass (g) (mg) (mg)/g (KD) 1 Normal control 0.9729 0.1094 0.205 1.87 8.4 group A, Sample 1 2 Normal control 1.0842 0.1136 0.305 2.68 8.7 group A, Sample 2 3 Normal control 0.78 0.0817 0.226 2.77 1.3 group B, Sample 1 4 Normal control 0.73 0.081 0.283 3.49 10.0 group B, Sample 2 5 Normal control 0.769 0.0694 0.197 2.84 10.0 group C, Sample 1 6 Normal control 0.545 0.063 0.190 3.0 10.0 group C, Sample 2 7 Preeclampsia 1.26 0.109 0.211 1.94 9.3 patient A, Sample 1 8 Preeclampsia 1.1 0.099 0.233 2.35 9.7 patient A, Sample 2 9 Preeclampsia 0.795 0.094 0.200 2.13 9.6 patient B, Sample 1 10 Preeclampsia 0.93 0.109 0.229 2.1 9.7 patient B, Sample 2 11 Preeclampsia 1.18 0.116 0.287 2.47 9.3 patient C, Sample 1 12 Preeclampsia 1.05 0.116 0.295 2.54 9.3 patient C, Sample 2
 For the analysis of a molecular weight and polydispersity of each sample, PAGE assay was conducted. Electrophoresis of about 5 mg of GAG isolated to each lane was performed on the enzymatically prepared heparin oligosaccharide standard from bovine lung heparin. Then, gel visualized by Alcian blue was digitized with UN-Scan-it software (Silk Scientific, Utah, US), and the average molecular weight of GAGs was computed based on the heparin oligosaccharide standard (refer to FIG. 1).
 In result, as described in Table 2, an average molecular weight of GAGs from the control group and an average molecular weight of GAGs from the preeclampsia samples were 9.57±0.73 kD and 9.48±0.19 kD, respectively.
Disaccharide Assay Using LC-MS
5-1 Enzymatic De-Polymerization of Complex GAG
 The purified GAGs (20 μg/μl) were decomposed additionally by chondroitinase ABC 10U and ACII (SIGMA) and dissolved in 500 μl of 0.1% BSA, respectively. 5 μl of chondroitinase ABC and 5 μl of ACII were added, as enzyme solutions, to 20 μl of a substrate and allowed for the reaction overnight at 37° C. The products were then filtered by a centrifugal filter device (3000 Da cutoff, Millipore Corporation) to thus yield CS disaccharides. An accurately measured 100 μl of dd H2O was added, and thereafter, the disaccharides were freeze-dried. Then, heparinase I, II, and III (Sigma) were added to the residual and allowed for the reaction overnight at 37° C. Also, the products were filtered by the centrifugal filter device (3000 Da cutoff, Millipore Corporation) to thereby yield Hep/HS disaccharides.
5-2 LC-MS Assay
 The LC-MS assay was conducted through LC-MS system (Agilent LC/MSD trap MS). Solutions A and B for HPLC were 15% and 70%, respectively, each containing 37.5 mM NH4CO3 and 11.25 mM Tributylamine at the same concentration. The pH values of the solutions were adjusted to 6.5 with acetic acid. A flow rate was 10 μl/min. Separation was conducted using a C-18 column (Agilent) for 20 min for the solution A, followed by 20-45 min for the solution B with the slope of a straight line from 0% to 50%. Column effluent returned to the source of EMI-MS to continue monitoring.
 In result, in standard chromatography (refer to the top panel in FIG. 2), Di-0S appeared at 5.3-5.4 min. Disaccharides having a single sulfate, such as Di-UA2S, Di-4S and Di-6S, appeared at 10.3-12.5 min. Disulfated disaccharides like Di-diSB, Di-diSD and Di-diSE appeared at 10.3-12.5 min. Trisulfated disaccharide Di-triS appeared lastly at about 43.7 min. According to standard chromatograph of another mix solution, Di-4S appeared at 11 min, Di-6S appeared at 10.3 min, and the peak at the center was Di-UA2S. Di-diSE appeared at about 38.6 min, and Di-diSE and Di-diSD appeared together at 39.5 min.
 FIG. 3 shows 8 kinds of disaccharides well isolated from Hep/HS. All fractions were confirmed by MS (where data is not shown).
 It is evident from the comparison on CS disaccharides between the sample group and the control group that the control group samples are more abundant in UA2S and TriS than the preeclampsia samples (refer to Table 4). In addition, it was verified that Hp/HS disaccharides have N-sulfo disaccharides that are more abundant in the control group compared with the preeclampsia sample group. This result supports that translocation of the N-sulfo group under preeclampsia occurs most frequently at the sixth carbon position. Interestingly enough, as shown in Table 5, one of the preeclampsia samples had a very large amount of tri-sulfo disaccharide.
 Tables 2 to 5 below show compositional analysis of CS disaccharides on all 12 samples, compositional analysis of CS disaccharides in terms of Mean/SD, compositional analysis of Hp/HS disaccharides on all 12 samples, and compositional analysis of Hp/HS disaccharides in terms of Mean/SD, respectively.
TABLE-US-00002 TABLE 2 diSD or 0S 6S UA2S 4S diSE diSD triS TPA Control group 1 n.d 42.6% n.d 57.4% n.d n.d n.d 1715 2 n.d 16% 39.3% 42.7% n.d n.d 2% 24637 Mean n.d 29.3% 19.7% 50.1% n.d n.d 1% 13176 3 1.3% 4.1% 8.8% 83.7% n.d n.d 2.1% 16627 4 n.d 37.2% n.d 62.1% n.d n.d 0.7% 32478 Mean 0.7% 20.7% 4.4% 72.9% n.d n.d 1.4% 24552 5 2.9% 24.4% n.d 66.5% n.d n.d 6.1% 5926 6 n.d 46.2% n.d 6.8% n.d n.d 7% 5443 Mean 1.5% 35.3% n.d 56.7% n.d n.d 6.5% 5684 Pre-eclampsia 7 n.d 44.7% n.d 54.7% n.d n.d 0.6% 68532 8 n.d 49.2% n.d 50.8% n.d n.d 0.2% 169810 Mean n.d 47% n.d 52.7% n.d n.d 0.4% 119171 9 n.d 32.2% n.d 66.5% n.d n.d 1.2% 30556 10 0.3% 37% n.d 62.4% n.d n.d 0.2% 101902 Mean 0.15% 34.6% n.d 64.5% n.d n.d 0.7% 66229 11 n.d 43.3% n.d 56.7% n.d n.d n.d 236590 12 n.d 42.4% n.d 57.6% n.d n.d n.d 187563 Mean n.d 42.9% n.d 57.1% n.d n.d n.d 212076
TABLE-US-00003 TABLE 3 diSD or 0S 6S UA2S 4S diSE diSD triS TPA Control Mean 0.7% 28.4% 8% 59.9% n.d n.d 2.98% 14471 group ±SD 0.7% 0.7% 10.3% 11.8% 0 0 3.1% Preeclampsia Mean n.d 41.5% n.d 58.1% n.d n.d 0.3% 132492 ±SD 0.1% 6.3% 0 5.9% 0 0 0.3%
TABLE-US-00004 TABLE 4 0S NS 6S UA2S UA2SNS NS6S UA2S6S triS TPA Control group 1 n.d 63.6% 36.4% n.d n.d n.d n.d n.d 11579 2 n.d 74.3% 25.7% n.d n.d n.d n.d n.d 5114 Mean n.d 69.0% 31.0% n.d n.d n.d n.d n.d 8346 3 n.d 48.2% 51.8% n.d n.d n.d n.d n.d 25163 4 n.d 34.7% 60.7% n.d 0.4% n.d n.d 4.3% 54561 Mean n.d 41.5% 56.2% n.d 0.2% n.d n.d 2.1% 39862 5 n.d 69.0% 26.0% 5.0% n.d n.d n.d n.d 17876 6 n.d 75.5% 24.5% n.d n.d n.d n.d n.d 9447 Mean n.d 72.3% 25.2% 2.5% n.d n.d n.d n.d 13661 Pre- eclampsia 7 n.d 49.0% 51.0% n.d n.d n.d n.d n.d 45024 8 n.d 28.8% 50.9% 20.3% n.d n.d n.d n.d 104083 Mean n.d 38.9% 51.0% 10.1% n.d n.d n.d n.d 74553 9 n.d 12.4% 84.6% n.d 3.0% n.d n.d n.d 3586 10 n.d 16.9% 78.2% n.d 1.9% n.d n.d 3.0% 11064 Mean n.d 14.7% 81.4% n.d 2.5% n.d n.d 0.1% 7325 11 n.d 10.8% 69.9% n.d 1.2% n.d n.d 18.1% 43001 12 n.d 12.6% 16.7% 9.1% 8.4% n.d n.d 53.1% 3740 Mean n.d 11.7% 43.3% 4.6% 4.8% n.d n.d 35.6% 23374
TABLE-US-00005 TABLE 5 0S NS 6S UA2S UA2SNS NS6S UA2S6S triS TPA Control Mean n.d 60.8% 37.5% 0.8% n.d n.d n.d n.d 20623 group ±SD 0 16.9% 16.5% 1.4% 0.1% 0 0 0 Pre- Mean n.d 21.8% 58.6% 4.9% 2.4% n.d n.d 12.4% 35083 eclampsia ±SD 0 10.5% 14.2% 3.6% 1.7% 0 0 14.2%
 Variation in the amount of mRNA of diverse GAG synthesis regulatory enzymes was examined by qRT-PCR. Total RNA amount was extracted, using an RNA Ambion RiboPure total RNA isolation kit, from preeclampsia-infected placentas and normal placentas. Following the treatment with deoxyribonuclease I (Takara Holdings Inc., Japan), total RNA was reversely transcribed by SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif.). Next, qRT-PCR was conducted using iCycler iQ system (Bio-Rad, Hercules, Calif.). Primers were designed using software Primer 3 (developed by Steve Rozen and Helen J. Skaletsky). All primers were designed using gene-specific sequences fostered by GenBank. The gene expression data was normalized to GAPDH as much as house keeping gene used as an internal standard for research. Primer sequences are presented in Table 6 (forward primers and reverse primers used for qRT-PCR) as follows.
TABLE-US-00006 TABLE 6 GenBank Primer Sequence Accession Forward Reverse Predicted # direction direction size(bp) (GAPDH) NM_002046.3 5' 5' 452 ACCACAGTCCATGCCA TCCACCACCCTGTTGCTGT TCAC3' A3' Sequence No. 1 Sequence No. 2 Chondroitin NM_018413 5' 5' 200 4-0- GTGGGGAGAGGGAGAG ACAGACAAGAACGACCCAT sulfo- AATC3' C3' transferase Sequence No. 3 Sequence No. 4 (C4ST) Chondroitin NM_004273 5' 5' 189 6- CCCAAAGTCAGAAAGC ACAAGCAAACCCACCAACT sulfo- GAAG3' C3' transferase Sequence No. 5 Sequence No. 6 (C6S) Dermatan/ NM_005715 5' 5' 153 chondroitin TCTGAGCCTGACCACA CACCTGCACAGAACTCAGG sulfate GATG3' A3' 2 sulfo- Sequence No. 7 Sequence No. 8 transferase (CS-20ST) Heparan NM_001543 5' 5' 205 N- CCCAGTGGCCCTAAAG GTCCATCACTTTGGCAGGT deacetylase/ TACA3' T3' N-sulfo- Sequence No. 9 Sequence No. 10 transferase-1 (NDST-1) Heparan NM_004807 5' 5' 243 sulfate GTACAACCTGGCCAAC CGCGTGCTATTGTACTGCA 6-0- AACC3' T3' sulfo- Sequence No. 11 Sequence No. 12 transferase 1 (HS6S)
 It was verified from the RT-PCR result that the preeclampsia samples, compared with the control group placenta, showed noticeable decrease in the expression of diverse chondroitin sulfotransferases enzymes (refer to Table 7). Although the expression of heparan sulfate 6-O-sulfotransferase in preeclampsia was markedly decreased, NDST-1 mRNA expression showed no significant change even in preeclampsia.
TABLE-US-00007 TABLE 7 Quantitative analysis of relative change in mRNA expression of diverse GAG synthesis enzymes based on qRT-PCR of placenta of women with preeclampsia Relative expres- siond to control Gene CTa ΔCTb ΔΔCTc group chondroitin 4-O-sulfotransferase 1 (C4ST) Pre- GAPDH 16.54 ± 0.38 5.25 ± 0.61 1.35 0.39 eclampsia C4ST 21.79 ± 0.24 Control GAPDH 17.43 ± 0.17 3.9 ± 0.25 group C4ST 21.33 ± 0.18 chondroitin 6-sulfotransferase (C6S) Pre- GAPDH 16.54 ± 0.38 7.913 ± 0.45 2.9 0.134 eclampsia C6S 24.45 ± 0.61 Control GAPDH 17.43 ± 0.17 .sup. 5 ± 0.6 group C6S 22.43 ± 0.43 dermatan/chondroitin sulfate 2-sulfotransferase (CS-2OST) Pre- GAPDH 16.54 ± 0.38 8.45 ± 0.64 2.3 0.2 eclampsia CS2S 24.99 ± 0.49 Control GAPDH 17.43 ± 0.17 6.15 ± 0.70 group CS2S 23.58 ± 0.53 heparan N-deacetylase/N-sulfotransferase-1 (NDST-1) Pre- GAPDH 16.54 ± 0.38 3.85 ± 0.29 -0.167 1.123 eclampsia HDS-1 20.53 ± 0.48 Control GAPDH 17.43 ± 0.17 4.02 ± 1.03 group HDS-1 21.45 ± 0.86 heparan sulfate 6-O-sulfotransferase 1 (HS6ST) Pre- GAPDH 16.54 ± 0.38 7.21 ± 1.29 1.78 0.29 eclampsia HS6S 23.75 ± 1.12 Control GAPDH 17.43 ± 0.17 5.43 ±±.02 group HS6S 22.87 ± 0.85 where aCT data average for each sample, bΔCT value is calculated by subtracting GAPDH C1 from each sample CT, cΔΔCT value is calculated by subtracting control group ΔCT from each preeclampsia sample ΔCT, and drelative expression to the control group is calculated using equation 2.sup.-ΔΔCT.
 As discussed earlier, the biomarker and composition for diagnosis of preeclampsia according to the present invention are not only effective in the diagnosis of preeclampsia, but also useful for developing a predictive biomarker kit through which the risk of preeclampsia in a pregnant woman can be predicted.
 While the invention has been shown and described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and the scope of the invention as defined in the following claims.
12120DNAArtificial SequenceGAPDH forward 1accacagtcc atgccatcac 20220DNAArtificial SequenceGAPDH backward 2tccaccaccc tgttgctgta 20320DNAArtificial SequenceC4ST forward 3gtggggagag ggagagaatc 20420DNAArtificial SequenceC4ST backward 4acagacaaga acgacccatc 20520DNAArtificial SequenceC6S forward 5cccaaagtca gaaagcgaag 20620DNAArtificial SequenceC6S backward 6acaagcaaac ccaccaactc 20720DNAArtificial SequenceCS-2OST forward 7tctgagcctg accacagatg 20820DNAArtificial SequenceCS-2OST backward 8cacctgcaca gaactcagga 20920DNAArtificial SequenceNDST-1 forward 9cccagtggcc ctaaagtaca 201020DNAArtificial SequenceNDST-1 backward 10gtccatcact ttggcaggtt 201120DNAArtificial SequenceHS6S forward 11gtacaacctg gccaacaacc 201220DNAArtificial SequenceHS6S backward 12cgcgtgctat tgtactgcat 20
Patent applications by Jin Han, Busan KR
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