KR101073653B1 - Specific marker genes for diagnosing pregnancy-induced hypertension and methods for analyzing gene expression levels related to pregnancy-induced hypertension using the specific marker genes - Google Patents

Abstract

The present invention relates to a diagnostic marker gene specific for preeclampsia and a method for analyzing preeclampsia-related gene expression using the same, and analyzes a preeclampsia-related gene expression using a marker gene for increasing the accuracy of predicting preeclampsia risk in pregnant women. The preeclampsia related gene expression analysis method of the present invention comprises the steps of preparing the RNA of the pregnant woman, the expression level of at least one gene selected from the group consisting of OXGR1, GALK2 and HBG2 genes specific for preeclampsia from the prepared RNA. And measuring the difference between expression of at least one gene selected from the group consisting of the OXGR1, GALK2, and HBG2 genes, expression level, and expression level with the normal group. Therefore, the present invention can predict the risk of preeclampsia in pregnant women early.

Preeclampsia, marker genes, risk prediction, gene expression patterns, early prediction

Description

Specific marker genes for diagnosing pregnancy-induced hypertension and methods for analyzing gene expression levels related to pregnancy-induced hypertension using the specific marker genes}

The present invention relates to a diagnostic marker gene specific to preeclampsia and a method for analyzing preeclampsia-related gene expression using the same, selecting marker genes to increase the accuracy when prematurely predicting the risk of preeclampsia in pregnant women using blood of the pregnant woman. In addition, the present invention relates to providing a method for analyzing gene expression patterns related to preeclampsia.

Preeclampsia is a serious complication of pregnancy that is defined as high blood pressure, edema, and proteinuria after 20 weeks of pregnancy. If the pregnant woman shows preeclampsia, the blood supply to the placenta and fetus is impaired, causing fetal growth failure and, in severe cases, causing fetal death.

Preeclampsia usually occurs in unborn babies and increases in frequency at the age of teenagers or over 35 years of age, and usually varies according to race, socioeconomic status, and age, but the frequency is approximately 5-8%. Risk factors for preeclampsia include first pregnancies, chronic hypertension, family history of preeclampsia, and preeclampsia in previous pregnancies; multiple pregnancies, diabetes mellitus, autoimmune diseases, kidney disease, and thrombophilic disorders. Black women and Asian women are two times more likely to have preeclampsia than white women.

The cause and etiology of preeclampsia have not yet been clearly identified as a "hypothesis" despite numerous studies. The pathogenesis is known as shallow endovascular cytotrophoblast invasion of spiral arteries and endothelial cell dysfunction.

Four hypotheses have been suggested for the cause of preeclampsia: placental ischemia, very low-density lipoproteins (VLDL), and VLDL versus toxicity preventing activity and immunological potential. Immune maladaptation, and finally genetic imprinting, has been discussed.

On the other hand, severe preeclampsia has been reported to have genetic predisposition. The incidence of preeclampsia in pregnant women is 8%, while the frequency of preeclampsia in daughters is 26% and that of sisters with preeclampsia is 37% (Chesley LC, Annitto JE, Cosgrove). RA Obstet Gynecol. 1968; 32: 303-11).

In addition, evidence has been reported that the genetic predisposition of preeclampsia may be associated with implantation and placental formation (Arngrimsson R, Connor JM, Geirsson RT, Brennecke S, Cooper DW. Lancet. 1994; 343: 1643-4), HLA- DR4 has been reported to be closely associated with the development of preeclampsia (Kilpatrick DC, Gibson F, Livingston J, Liston WA. Tissue Antigens. 1990; 35: 178-8).

In a 1993 study of preeclamptic pregnancies in Utah, U.S.A, a comparison of the normal pregnancies and the pregnancies with an M235T point mutation of angiotensinogen exon 2 was performed. It has been reported that gene point mutations are significantly frequent in preeclamptic pregnant women (Ward K, Hata A, Jeunemaitre X. Nature Genet 1993; 4: 59-61). However, in Korean preeclamptic pregnant women, preeclampsia has not been associated with an M235T gene point mutation of angiotensinogen exon 2 (Cho Yong-gyun, 1995. Ph.D. Thesis).

As can be seen, if a specific gene associated with the occurrence of preeclampsia is found, it is considered to be useful for the early prediction of preeclampsia. However, the marker gene related to preeclampsia has not been clearly identified. . This is because the cause of preeclampsia may vary among races and patients, and accordingly, enormous research results need to be collected to database the expression patterns of variously varying genes.

Under these backgrounds, the inventors of the present invention first discovered genes showing different expression patterns in preeclamptic pregnant women than in normal pregnant women using DNA chips to develop biological markers for the diagnosis of preeclampsia using blood of pregnant women. PCR confirmed that they were highly significant genetic markers for identifying preeclampsia.

As gene markers for identifying preeclampsia of the present invention, genes OXGR1 (GenBank Accession No .: NM_080818), GALK2 (GenBank Accession No .: NM_002044), and HBG2 (GenBank Accession No .: NM_000184) were identified and identified. The risk may be used for early prediction.

An object of the present invention is to provide marker genes to increase the accuracy in predicting the risk of preeclampsia of pregnant women by collecting blood of pregnant women.

In addition, an object of the present invention is to provide a method for analyzing preeclampsia-related gene expression patterns using diagnostic marker genes specific for preeclampsia and early diagnosis of preeclampsia risk in pregnant women.

Diagnostic marker genes specific for preeclampsia in pregnant women of the present invention is at least selected from the group consisting of OXGR1 (GenBank Accession No .: NM_080818), GALK2 (GenBank Accession No .: NM_002044) and HBG2 (GenBank Accession No .: NM_000184) genes As a gene, they can increase the accuracy of predicting the risk of preeclampsia.

Gene expression pattern analysis method related to preeclampsia of the present invention,

Preparing RNA for pregnant women,

From the prepared RNA, at least one gene selected from the group consisting of OXGR1 (GenBank Accession No .: NM_080818), GALK2 (GenBank Accession No .: NM_002044), and HBG2 (GenBank Accession No .: NM_000184) genes specific for preeclampsia Measuring the expression level,

Analyzing whether the expression of at least one gene selected from the group consisting of the OXGR1, GALK2 and HBG2 genes, the degree of expression and the difference between the expression level and the normal group.

Preeclampsia related gene expression analysis method of an embodiment of the present invention,

Preparing RNA for pregnant women,

Synthesizing a fluorescently labeled complementary nucleotide sequence using the prepared RNA;

The fluorescently labeled complementary sequences and selected from the group consisting of OXGR1 (GenBank Accession No .: NM_080818), GALK2 (GenBank Accession No .: NM_002044) and HBG2 (GenBank Accession No .: NM_000184) genes specific for preeclampsia Hybridizing oligonucleotides of at least one gene,

After the hybridization, confirming fluorescence intensity by binding of the fluorescently labeled complementary nucleotide sequence to oligonucleotide,

And analyzing the difference between expression of genes specific for preeclampsia, expression level, and expression level with the normal group by the identified fluorescence intensity.

In the preeclampsia related gene expression analysis method of an embodiment of the present invention, the fluorescently labeled complementary base sequence may be cDNA or aRNA. In addition, the oligonucleotide of at least one gene selected from the group consisting of OXGR1, GALK2 and HBG2 genes specific for preeclampsia may be a probe that is part of at least one gene selected from the group consisting of OXGR1, GALK2 and HBG2 genes. have.

In another embodiment of the present invention, the preeclampsia related gene expression analysis method,

Preparing RNA for pregnant women,

At least one selected from the group consisting of OXGR1 (GenBank Accession No .: NM_080818), GALK2 (GenBank Accession No .: NM_002044), and HBG2 (GenBank Accession No .: NM_000184) genes using the prepared RNA as a template and specific for preeclampsia Performing RT PCR (Reverse Transcriptase Polymerase Chain Reaction) using a primer for the gene of

Performing electrophoresis on the RT PCR product to analyze whether the expression of at least one gene selected from the group consisting of OXGR1, GALK2, and HBG2 genes specific for preeclampsia, the difference in expression level, and the expression level with the normal group It includes.

In a method for analyzing preeclampsia-related gene expression according to another embodiment of the present invention, the primer may be at least one oligonucleotide primer selected from the oligonucleotide group including the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 6.

Through such preeclampsia related gene expression analysis methods, the present invention can predict the risk of preeclampsia in pregnant women early.

The present invention enables the early prediction of the risk of preeclampsia by first discovering marker genes for improving the prediction accuracy of preeclampsia risk in pregnant women.

In addition, the present invention is to analyze the expression patterns of the preeclampsia-related genes (difference in expression, degree of expression and expression with normal group) using marker genes for improving the accuracy of preeclampsia risk prediction in pregnant women from the blood samples of pregnant women Early prediction of preeclampsia risk.

Hereinafter, it demonstrates in detail based on the Example of this invention. The following examples of the present invention are not intended to limit or limit the scope of the present invention only to embody the present invention. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. References cited in the present invention are incorporated herein by reference.

Example 1 RNA Isolation and Quantitation from Specimen

Example 1-1 RNA isolation and quantitation in patient blood

Blood samples from 9 normal pregnant women and 11 preeclamptic patients were provided from CHA. RNA from preeclampsia patient samples immediately before delivery (33 weeks or more) was extracted by the manufacturer's suggested method using the QiaAmp RNA blood mini kit (Qiagen, Cat # 52304). The patient's sample (whole blood) was collected at about 5cc. The extracted RNA was quantitatively analyzed using a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies).

Example 1-2 RNA Separation and Quantitation in Patients Placenta

Samples of 16 normal pregnant women and 17 preeclamptic patients were provided by CHA. The placenta of delivery patients with preeclampsia is immediately extracted and stored in liquid nitrogen, and the tissue is ground by a homogenizer (Omnigen) using the Trizol reagent (Invitrogen) by the method suggested by the manufacturer. After separating only the RNA portion of chloroform (Chloroform, Sigma) of vol. 10 ml of Trizol reagent per patient sample cut to 1 cm ³ was used. The extracted RNA was quantitatively analyzed using a NanoDrop spectrophotometer (ND-1000, NanoDrop Technologies).

Example 2: Microarray Experiment and Results Analysis

Example 2-1 Chip Testing and Analysis of Blood Samples

Codelink human whole genome microarray chips (Applied Microarrays) were used to examine the gene expression patterns of the samples. To compare the gene expression patterns of the samples, the results were divided into normal and preeclamptic patients. The experiment was carried out using a single fluorescent dye (Single-dye).

Samples were prepared by amplifying aRNA with an amino Allyl MessageAmp II aRNA Amplification kit (Ambion Inc.), starting with 2 μg of total RNA, and combining fluorescent dyes by chemical methods.

In the hybridization process, the hybridization solution component A and component B provided by Applied Microarrays were used, and the final solution was mixed with the sample prepared above to make 260 μl. Samples mixed with the hybridization solution were placed in an array chamber where airtightness was maintained, and the hybridization reaction was performed at 37 ° C. for 18 hours in a shaking incubator rotating at 300 rpm. Then, the chips were washed by soaking at 46 ° C. for 1 hour using a 0.75 × TNT washing solution.

Both normal and preeclampsia patients were labeled with Cy5 fluorescent labeling reagents (Applied Microarrays, Cat # 28900224). The chip was placed in 3.5 ml of Cy5-Streptavidin working solution and allowed to stand at room temperature for 30-40 minutes to bind with streptavidin-dye. In order to remove the remaining unbound fluorescent labeling reagent, it was washed four times for 5 minutes with 1X TNT solution (0.10M Tris-HCl, pH7.6, 0.15M NaCl, 0.05% Tween20), and finally 0.1 × SSC / 0.05% Tween20. The solution was washed for 30 seconds.

The washed chip was scanned at a wavelength of 635 nm using a laser scanner (GenePix 4000B, Molecular Devices) to obtain the data of the fluorescence present in each spot and stored as an image file in the form of TIFF.

1A and 1B illustrate image results obtained by scanning after performing a hybridization reaction through a chip experiment using a blood sample of a normal pregnant woman, and FIGS. 2A and 2B illustrate a chip experiment using a blood sample of a preeclampsia patient The image results obtained by scanning after performing the hybridization reaction are shown.

The TIFF image file thus obtained was quantified by GenePix 6.0 software (Molecular Devices) to quantify the fluorescence value of each spot.

Meanwhile, in the present embodiment, the CodeLink human hole genomic microarray chip is used, but the present invention is not limited thereto. It can be used in the present invention if a microarray chip can contain thousands of human genes known to be associated with or not related to preeclampsia, and can comprise a number of different human genes known to be associated with preeclampsia. Those skilled in the art will readily understand.

Example 2-2 Chip Experiments and Analysis of Placental Samples

Chip experiments were also performed on the aRNA amplified from the RNA of the placental sample prepared in Example 1-2 in the same manner as in Example 2-1, and the fluorescence value thereof was analyzed. In this example, the gene expression pattern of each spot was determined according to the fluorescence intensity of the a5-labeled Cy5 fluorescent label at each spot on the TIFF image obtained by scanning the hybridized DNA chip as in Example 2-1. Quantification using 6.0 software.

Example 2-3 Analysis of Quantified Chip Fluorescence Data and Change Analysis of Gene Expression Patterns

Typically, thousands of tens of thousands of genes are planted in DNA chip microarrays, which can cause errors when analyzing fluorescence data due to the large amount of data and various forms. Therefore, analytical tools and statistical techniques that correct for the differences between these fluorescence data should be used. That is, it is necessary to remove the background fluorescence value from the actual fluorescence data value and to remove spots that are found to be errors. In addition, it is necessary to use a global normalization technique that adjusts the difference between two different fluorescence values in order to easily understand the difference in fluorescence data values between contrasted genes and use them in gene analysis.

Specifically, in Example 2-1 and Example 2-2, "Codelink Gene Expression Analysis Software (Codelink Gene Expression Analysis Software)" is used for quantitative results obtained through GenePix 6.0 software (Molecular Devices). Applied Microarrays ”were used to remove the spots that were found to be error and global normalization was performed. Afterwards, GeneSpring GX software (Agilent Technologies) was used to analyze the hierarchical clustering and to determine the difference in the overall expression patterns between normal and patient groups.

3 and 4 show the results of applying the hierarchical clustering assay after quantifying the expression level of each gene, ie, the fluorescence intensity of the fluorescent label, from the DNA chip hybridization results for the blood sample and the placenta sample, respectively. . It can be seen from FIG. 3 and FIG. 4 that the normal and preeclamptic pregnant women are distinguished.

And, Figure 5 is a chart of the change in the overall gene expression pattern between the normal pregnant group and preeclampsia patient group. 5A corresponds to the results of the blood sample, and FIG. 5B corresponds to the results of the placental sample.

Example 3: Screening for Genes with Significant Differences in Expression Between Normal and Preeclamptic Patients

Various t-tests (Student t-test, Welch t-test, Nonparametric t-test) among statistical methods provided by GeneSpring GX software for screening genes with significant differences in expression between normal pregnant women and preeclampsia patients. Was used.

All of the statistical methods used were selected for genes whose FDR (false discovery rate) <0.05 or less, with more than twofold differences in expression between normal and patient groups.

Among them, three genes of which the difference in expression levels in blood samples were remarkable and related to preeclampsia were selected (see Table 1), and the expression patterns were examined through RT-PCR.

On the other hand, Figure 6 is a graph of the change in the expression pattern for the gene group, the difference in gene expression between the normal pregnant women group and preeclampsia patients group more than two times and its statistical significance is FDR <0.05 level. Figure 6a corresponds to the results of blood samples, Figure 6b corresponds to the results of placental samples.

Table 1. Genes not yet known to be associated with preeclampsia, with significant changes in expression between normal and preeclamptic pregnancies.

Serial Number Expression difference
(Preeclampsia / normal) Gene name GenBank
Accession
Number UniGene
Cluster ID Genetic symbol
(Gene Symbol) One 3.595 oxoglutarate
(alpha-ketoglutarate)
receptor 1 NM_080818 Hs.352218 OXGR1 2 3.871 galactokinase 2 NM_002044 Hs.122006 GALK2 3 8.428 hemoglobin, gamma G NM_000184 Hs.302145 HBG2

That is, as shown in Table 1, OXGR1 (GenBank Accession No .: NM_080818), GALK2 (GenBank Accession No .: NM_002044) and HBG2 (GenBank Accession No .: NM_000184) genes genes in preeclamptic pregnant women compared to normal pregnant women Expression was more than two times higher. Therefore, these three genes were found to be suitable as marker genes for increasing the early prediction accuracy of preeclampsia risk in pregnant women.

Example 4 Verification of Gene Expression Patterns Using RT-PCR

Gene expression levels were confirmed by RT-PCR method to verify the efficacy of preeclampsia as a biomarker for the genes of Table 1. RT-PCR was performed by pooling samples of total RNA from normal and preeclamptic patients, which were used in microarray experiments, and also separately from each of the RNAs extracted from normal pregnant and preeclamptic patients. ) Was performed (see FIG. 7).

The RT-PCR reaction was performed using Maxim RT-PCR PreMix kit (Intron biotechnology), which proceeds from cDNA synthesis to PCR process in one step. The amount was 10ng, 200ng. As primers for RT-PCR for each gene identified in Table 1, primer base sequences as shown in Table 2 were used.

Table 2. Primer Sequences for RT-PCR

Then, 7 μl of the final PCR reaction product was taken and electrophoresed on a 2% agarose gel in the presence of 0.5 μg / ml of ethidium bromide to observe the band (see FIG. 7).

As a result, it was confirmed that the RT-PCR results for all three genes in Table 1 are exactly the same as the microarray chip results, and the marker genes OXGR1 (GenBank Accession No .: NM_080818) and GALK2 (GenBank Accession No .: NM_002044) and HBG2 (GenBank Accession No .: NM_000184) were able to confirm that there is a significant difference in gene expression between normal and patient groups (see FIG. 7). Therefore, three genes of OXGR1 (GenBank Accession No .: NM_080818), GALK2 (GenBank Accession No .: NM_002044), and HBG2 (GenBank Accession No .: NM_000184) were found to be suitable as biomarker genes for preeclampsia.

The present invention predicts lethal preeclampsia prematurely to pregnant women using marker genes of OXGR1 (GenBank Accession No .: NM_080818), GALK2 (GenBank Accession No .: NM_002044), and HBG2 (GenBank Accession No .: NM_000184) as described above. Diagnosis can be made. That is, the present invention analyzes the expression patterns (differences of expression, degree of expression and degree of expression with a normal group) of preeclampsia-related genes by using the marker genes to increase the accuracy of predicting preeclampsia risk in pregnant women. It is possible to predict early, and to easily diagnose the expected group of patients at risk of preeclampsia among pregnant women.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. It will be understood by those skilled in the art that modifications and variations may be made without departing from the spirit and scope of the invention, and that such modifications and variations are also contemplated by the present invention.

1 is a photograph of amplified from RNA isolated from a normal pregnant woman blood sample and hybridized with a fluorescently labeled aRNA and a Codelink human hole genomic microarray chip, followed by scanning fluorescence from fluorescent labels. FIG. 1A is a whole photograph, and FIG. 1B is an enlarged photograph of a portion.

FIG. 2 is a photograph of amplified from RNA isolated from blood samples of preeclampsia patients and hybridized with a fluorescence-labeled aRNA and a Codelink human hole genomic microarray chip, followed by scanning fluorescence from a fluorescent label. FIG. 2A is a whole photograph and FIG. 2B is an enlarged photograph of a portion.

FIG. 3 shows the results of dividing normal and preeclamptic pregnant women using hierarchical clustering assay after quantifying the expression level of each gene, ie, the fluorescence intensity of fluorescent labels, from DNA chip hybridization results of blood samples. have.

Figure 4 shows the results of dividing the normal and preeclamptic pregnant women group using a hierarchical clustering assay after quantifying the expression level of each gene, that is, the fluorescence intensity of the fluorescent label from the DNA chip hybridization results of the placenta sample have.

FIG. 5 is a chart of the changes in overall gene expression patterns between normal pregnant and preeclamptic patients. 5A corresponds to the results of the blood sample, and FIG. 5B corresponds to the results of the placental sample.

FIG. 6 is a diagram of the change in expression patterns for gene groups in which the difference in gene expression is more than doubled between normal pregnant group and preeclampsia patients group and its statistical significance is FDR <0.05. Figure 6a corresponds to the results of blood samples, Figure 6b corresponds to the results of placental samples.

Figure 7 shows the results of electrophoresis after performing Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) for the OXGR1, GALK2 and HBG2 genes selected as marker genes.

<110> DIGITAL GENOMICS INC. <120> Specific marker genes for diagnosing pregnancy-induced          hypertension and methods for analyzing gene expression levels          related to pregnancy-induced hypertension using the specific          marker genes <130> P09-0147KR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> OXGR1 sense primer <400> 1 aaggctaacc attctgctac tc 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> OXGR1 anti-sense primer <400> 2 gtcgctgacc accacatata g 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GALK2 sense primer <400> 3 taatccaaga ggcacttcca ag 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GALK2 anti-sense primer <400> 4 tagtccatcc acaagagaaa gc 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HBG2 sense primer <400> 5 aggacaaggc tactatcaca ag 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HBG2 anti-sense primer <400> 6 ccactccagt caccatcttc 20  

Claims (6)

In the diagnostic composition specific to preeclampsia comprising a diagnostic marker gene specific for preeclampsia of a pregnant woman as an active ingredient, A diagnostic composition specific to preeclampsia comprising a GALK2 (GenBank Accession No .: NM_002044) gene as an active ingredient. delete delete Preparing RNA for pregnant women, Using the prepared RNA as a template and performing RT PCR (Reverse Transcriptase Polymerase Chain Reaction) using primers for GALK2 (GenBank Accession No .: NM_002044) gene specific for preeclampsia, An electrophoresis of the RT PCR product to analyze whether the expression of GALK2 gene specific to preeclampsia, the difference between the expression level and the expression level with the normal group genotype-specific marker genotype analysis method. 5. The method of claim 4, Said primer is a marker genotype analysis method specific to preeclampsia, characterized in that the primer having a nucleotide sequence of SEQ ID NO: 3 and a primer having a nucleotide sequence of SEQ ID NO: 4. delete

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