WO2020091173A1 - Biomarker for prediction of gestational diabetes and hypertension in pregnancy - Google Patents

Abstract

The present invention relates to a composition and a kit for diagnosis of gestational diabetes or hypertension in pregnancy and a method for diagnosis of gestational diabetes or hypertension in pregnancy, using same. According to an embodiment, a composition and a kit comprising a selenoprotein P-measuring agent for diagnosis of gestational diabetes or hypertension in pregnancy and a method for diagnosis of gestational diabetes or hypertension in pregnancy, using same allows the easy diagnosis of gestational diabetes or hypertension in pregnancy with high accuracy and specificity.

Description

Biomarkers for predicting gestational diabetes and hypertension during pregnancy

It relates to a biomarker for predicting gestational diabetes and hypertension during pregnancy, a composition, kit for diagnosing gestational diabetes and hypertension using the same, and a method for diagnosing gestational diabetes and hypertension during pregnancy.

Pregnancy associated hypertension during pregnancy includes hypertension combined with pregnancy, such as preeclampsia, preeclampsia, gestational hypertension, and the like. It is a major cause of perinatal death of the mother and fetus, and accounts for a large proportion of 5-8% of all pregnant women. Representative major symptoms include hypertension, edema and increased toxic protein levels during pregnancy, severe headaches and sudden And weight gain. Gestational diabetes mellitus (GDM) also refers to a decrease in glucose tolerance that occurred during pregnancy or was first identified.

Hypertensive disease during pregnancy usually occurs after 20 weeks and is a major cause of preterm birth. However, the exact cause and treatment method of the disease have not been clearly identified to date, and there is no clear treatment method other than delivery. However, if hypertension or gestational diabetes occurs during pregnancy in the early stages of pregnancy, early delivery may increase the mortality rate of the fetus, so it may be the best way to diagnose patients early and manage the patient while maintaining pregnancy as much as possible. Therefore, early diagnosis is required to reduce the premature birth of the fetus caused by the disease and the mortality rate of the fetus.

Research has been conducted to find biomarkers for predicting and diagnosing hypertensive disease or gestational diabetes during pregnancy, and several markers have been proposed until recently, but the usefulness of clinical prediction or diagnosis has not been proven. Continuous research needs to be done.

One aspect is to provide a composition for diagnosing gestational diabetes or hypertensive disease during pregnancy, comprising an agent that measures the expression level of selenoprotein P, or a fragment thereof.

Another aspect is to provide a kit for diagnosing gestational diabetes or hypertensive disease during pregnancy, comprising an agent that measures the expression level of selenoprotein P, or a fragment thereof.

Another aspect includes measuring the expression level of selenoprotein P, or a fragment thereof, in a biological sample isolated from a subject suspected of gestational diabetes or hypertensive disease during pregnancy; And it provides a method for diagnosing gestational diabetes or hypertensive disease during pregnancy comprising comparing the measured expression level with the protein expression level of the normal control.

One aspect provides a composition for diagnosing gestational diabetes or hypertensive disease during pregnancy, comprising an agent that measures the expression level of selenoprotein P (Senoprotein P) or a fragment thereof.

As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, diagnosis may mean confirming the onset or possibility of developing gestational diabetes or hypertensive disease during pregnancy.

As used herein, the term "pregnancy diabetes" is a disease that occurs in about 3 to 14% of pregnant women, regardless of the degree of glucose in the blood is higher than the normal range, refers to the case that is first discovered or started during pregnancy .

As used herein, the term "pregnancy associated hypertension" refers to pre-eclampsia, eclampsia, gestational hypertension, chronic hypertension, HELLP syndrome. ) May be selected from the group consisting of. In addition, it may include a pregnancy with a small fetal (SGA) compared to the gestational period that occurs in connection with hypertensive disease during pregnancy.

The pre-eclampsia shows symptoms of hypertension, edema, and proteinuria, affecting 5-10% of pregnancy, with significant maternal and fetal morbidity and mortality. Can cause. Pre-eclampsia causes the death of more than 200,000 mothers worldwide each year. Symptoms of preeclampsia typically appear after 20 weeks of pregnancy and are usually detected by regular measurements of blood pressure and urine in women. However, these monitoring methods are ineffective for diagnosis in the early stages of symptoms and therefore require early diagnosis markers. Preeclampsia is also referred to as addiction to pregnancy and preeclampsia.

The eclampsia is a case in which convulsion occurs in addition to hypertension and proteinuria. Around 6-7 months of pregnancy or the delivery is imminent, suddenly dizziness and fainting, causing impotence, convulsions of the limbs, biting of the mouth, and vomiting of bubbles in the mouth.

The gestational hypertension refers to a case of newly occurring pregnancy (after 20 weeks of pregnancy) and becoming normal within 12 weeks of childbirth, and refers to hypertension without proteinuria.

The chronic hypertension (chronic hypertension) refers to a case where hypertension occurred before or 20 weeks before pregnancy.

In order to diagnose the gestational diabetes or hypertensive disease during pregnancy, the mother visits the gynecologist to measure blood pressure at every regular checkup and to perform a urine test. However, a biomarker that can be quickly and accurately diagnosed using a sample that is easy to collect has not been developed.

As used herein, the term "marker" or biomarker is a substance that can be diagnosed by distinguishing between a normal group individual and an individual having pregnancy-related hypertensive disease, and increases in the gestational diabetes of the present invention or an individual having hypertensive disease during pregnancy. And organic biomolecules such as polypeptides, proteins or nucleic acids, genes, lipids, glycolipids, glycoproteins or sugars. In particular, in the present specification, the present invention may be a protein or gene that is changed in an individual having gestational diabetes or hypertension during pregnancy, but is not limited thereto.

The selenoprotein P (selenoprotein P) is a glycoprotein found in plasma, and its exact function is unknown, but it is considered to be related to antioxidant. Prior studies on the relationship between selenoprotein P and hypertensive disease during pregnancy are lacking. The selenoprotein P is SeP; SELP; SEPP; It can also be indicated as SEPP1. The selenoprotein P is a GenBank Accession No. NM_001085486.2, NM_001093726.2, NM_005410.3 It may be a protein encoded from a nucleic acid comprising the nucleotide sequence of. The selenoprotein P is a GenBank Accession No. The amino acid sequence of NP_001078955.1, NP_001087195.1, NP_005401.3 or UniProt No. It may be a protein comprising the amino acid sequence of P49908.3.

The selenoprotein P may be a protein comprising any one or more amino acid sequences of SEQ ID NOs: 1-3. The selenoprotein P protein may be a homozygous protein of selenoprotein P. The term "isoform" refers to different forms of the same protein.

The fragment of selenoprotein P may be an immunogenic fragment. It may be a fragment having one or more epitopes recognizable by an antibody against the selenoprotein P.

The expression level may be a protein expression level. The expression level can be measured by measuring the amount or concentration of protein in the sample. The expression level can be expressed as the absolute (eg, μg / mL) or relative (eg, relative intensity of the signal) difference of the protein described above.

The amount of the selenoprotein P or a fragment thereof can be measured through electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, mass spectrometry, or a combination thereof.

The agent measuring the expression level refers to a substance that can be used for the detection or quantification of a protein or fragment thereof. The agent may be an antibody or antigen-binding fragment thereof that specifically binds selenoprotein P or a fragment thereof. The term "antibody" can be used interchangeably with the term "immunoglobulin". The antibody can be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide comprising an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab ', or scFv. The antibody or antigen-binding fragment may be a commercially available antibody or antigen-binding fragment for selenoprotein P.

In addition, the agent measuring the expression level refers to a substance that can be used for the detection or quantification of polynucleotides. The agent may be a primer, probe, or antisense nucleotide that specifically binds to a polynucleotide encoding selenoprotein P, or a fragment thereof.

The term “primer” refers to a short nucleic acid sequence with a short free 3 ′ terminal hydroxyl group that can form a complementary template and base pair and functions as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures and four different nucleoside triphosphates.

The term "probe" refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically identifying the presence of a target substance in a sample through the binding. The type of probe molecule is a material commonly used in the art, and is not limited, but may be preferably peptide nucleic acid (PNA), locked nucleic acid (LNA), peptide, polypeptide, protein, RNA or DNA. More specifically, the probe is a biomaterial, which is derived from an organism or similar, or is prepared in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and RNA. It may be, DNA includes cDNA, genomic DNA, oligonucleotide, RNA includes genomic RNA, mRNA, oligonucleotide, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.

The term "antisense oligonucleotide" is a DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a sequence of a specific mRNA, and functions to bind to a complementary sequence in the mRNA and inhibit the translation of the mRNA into a protein. An antisense oligonucleotide sequence refers to a DNA or RNA sequence complementary to mRNA and capable of binding to the mRNA. This can inhibit the essential activity for the translation of the gene mRNA, translocation into the cytoplasm, maturation or any other overall biological function.

The composition may further include a sample necessary for diagnosing gestational diabetes or hypertensive disease during pregnancy.

Another aspect provides a kit for diagnosing gestational diabetes or hypertensive disease during pregnancy, comprising an agent that measures the expression level of selenoprotein P or a fragment thereof.

Selenoprotein P, fragments of selenoprotein P, expression levels, and measurement of expression levels are as described above.

The kit may further include a sample necessary for diagnosing gestational diabetes or hypertensive disease during pregnancy. The kit may include an antibody, a substrate for immunological detection of the antibody, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, or a chromogenic substrate.

In another embodiment, the kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit or multiple reaction monitoring ) Can be a kit.

Another aspect includes measuring the expression level of selenoprotein P or a fragment thereof in a biological sample isolated from a subject suspected of gestational diabetes or hypertensive disease during pregnancy; And it provides a method for providing information necessary for the diagnosis of gestational diabetes or hypertensive disease during pregnancy, comprising comparing the measured expression level with the protein expression level of a normal control.

Selenoprotein P, fragments of selenoprotein P, expression levels, and measurement of expression levels are as described above.

The method includes measuring the expression level of selenoprotein P or a fragment thereof in a biological sample isolated from a subject suspected of gestational diabetes or hypertensive disease during pregnancy.

The subject can be a mammal, including a human.

The term "biological sample" refers to a sample obtained from an organism. The biological sample may be blood, plasma, platelets, serum, ascites fluid, bone marrow fluid, lymph fluid, saliva, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof. When the biological sample is blood or plasma, since blood and plasma that are easy to collect are used as samples, it is easy to analyze without burdening the patient, unlike conventional methods of diagnosing diabetes during pregnancy or hypertension during pregnancy. In addition, it can be useful for early diagnosis of gestational diabetes or hypertensive disease during pregnancy due to its high accuracy and sensitivity.

The biological sample may include an intact protein. The intact protein may be a protein isolated from a biological sample without modification of a separate protein. The intact protein can be a protein isolated from a biological sample, without proteolysis, for example, by proteolytic enzymes.

The measuring may include incubating a biological sample and an antibody that specifically binds to selenoprotein P, or a fragment thereof. Incubation can be performed in vitro. The measurement method may be, for example, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, or a combination thereof.

The measuring may include mass analyzing the biological sample. The mass spectrometry of the biological sample may be mass spectrometry of the intact protein.

The method includes comparing the measured expression level with that of the normal control selenoprotein P.

The normal control group may be a biological sample isolated from an individual who is not a patient with gestational diabetes or hypertension during pregnancy, or an individual who is not at risk of gestational diabetes or hypertension during pregnancy.

The method may further include a step of determining gestational diabetes or hypertensive disease during pregnancy when the measured expression level of selenoprotein P is higher than that of a normal control sample. The expression level of selenoprotein P, or a fragment thereof, measured in a biological sample isolated from a subject suspected of gestational diabetes or hypertensive disease during pregnancy, or a fragment thereof, measured in a sample separated from a normal control sample When the expression level is increased, the subject may be determined to have a high probability of having gestational diabetes or hypertensive disease during pregnancy or gestational diabetes or hypertensive disease during pregnancy. For example, if the selenoprotein P is higher than 90 percentile, the risk of developing about 3.4 times gestational diabetes or hypertensive disease during pregnancy increases.

According to one aspect, the composition, kit for diagnosing gestational diabetes or hypertension during pregnancy, and a method for diagnosing gestational diabetes or hypertension during pregnancy using the same, can be easily diagnosed with the disease and can be used to diagnose early with high accuracy and specificity have.

1 is a graph showing the results of measuring the expression level of selenoprotein P in a patient group with gestational diabetes and a normal group without gestational diabetes.

Figure 2 is a graph showing the results of measuring the expression level of selenoprotein P in a patient group with hypertension during pregnancy and a normal group without hypertension during pregnancy at the beginning of 10-14 weeks of pregnancy.

3 is a graph showing the results of measuring the expression level of selenoprotein P in a patient group with hypertension during pregnancy and a normal group without hypertension during pregnancy in the middle of 22-30 weeks of pregnancy.

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Confirmation of the diagnosis effect of selenoprotein P on gestational diabetes

1.1. Patient preparation

We analyzed 572 normal patients and 36 patient groups in pregnant women with single mothers with similar age, obstetric history, past history of pre-diabetic diabetes, family history of diabetes, waist circumference, number of pregnancies drawn, and body mass index. In the experiment, the mothers who were in the patient group were diagnosed with gestational diabetes by using a two-step method (50g-100g Glucose Tolerance Test (OGTT)) between 24-28 weeks and had a history of diabetes before diabetic diagnosis or diabetes in oral glucose tolerance test after delivery. Cases were excluded. Therefore, these patient groups can be said to be gestational diabetics who have temporarily suffered from glucose dysfunction due to pregnancy.

1.2. Sample preparation and storage

Peripheral blood of pregnant women visited at 10-14 weeks of pregnancy and 22-30 weeks of pregnancy was collected and centrifuged to obtain plasma by taking supernatant, which was stored in a polypropylene tube at -70 ° C until the experiment. In the pregnant women group using the stored samples, the gestational diabetes group used insulin, fasting serum glucose, and an enzymatic method (Glucose HK, Roche diagnostics, IN) using a Roche / Hitachi 911 (Roche diagnostics) chemistry analyzer. It was measured through. Thereafter, plasma insulin concentration was analyzed using an immunometric assay (IRMA) in duplicate using the same batch of a kit (INS-Irma, DIAsource ImmouAssays S.A., Belgium).

1.3. Diagnosis of gestational diabetes by measuring the expression level of selenoprotein P

It was confirmed by ELISA whether gestational diabetes could be diagnosed by measuring the expression level of selenoprotein P.

Specifically, an ELISA kit (cat No. SEB809Hu, cloud-clone corp. # SEB809Hu) was used. The plasma sample prepared in the above 1.2 is diluted 1000 times and used. A composition of 600 ml of wash buffer was prepared by mixing 580 ml of distilled water and 20 ml of concentrated wash buffer.

In order to make 500 ng / ml of standard diluent among human selenoprotein P1 standard, selenoprotein P standard was made with 1 ml of standard diluent and gently shaken for 10 minutes. After the standard dilution was made, serial dilution was performed. Standard, blank, and samples were added at 100 μl per well. The cover was covered and incubated at 37 ° C. for 1 hour. After incubation, discard the solution in 4 wells and do not wash. After adding 100 μl of detection reagent A per well, the cover was covered and incubated at 37 ° C. for 1 hour. Washing was performed 3 times, and the wells were washed with 400 μl of washing buffer at the last washing. After adding 100 μl of detection reagent B per well, the cover was covered and incubated at 37 ° C for 30 minutes. Washing was repeated 5 times in the same manner as above. Substrate solution was added at 90 μl per well, and then covered and incubated at 37 ° C. for 10 minutes. At this time, the shading was maintained. After the reaction, 50 μl per well of a stop solution was added. Thereafter, absorbance was measured at 450 nm.

Table 1 below shows the results of measuring the expression level of selenoprotein P in the patient group with gestational diabetes and the normal group without gestational diabetes. In addition, the results of Table 1 are shown graphically in FIG. 1.

Characteristic	Normal group (n = 572)	Patient group (n = 36)	P- value
Selenoprotein P (mg / mL)	5.83 (2.9014.51)	10.01 (6.7234.86)	<.005

As shown in Table 1 and FIG. 1, the selenoprotein P level was measured to be about twice as high in the gestational diabetes patient group as compared to the normal group. In addition, the possibility of gestational diabetes expression according to the level of selenoprotein P is shown in Table 2.

Seleno Protein P (mg / ml)	Odds ratio (95% CI)	P-value
= 6.08	1.00 (reference)	-
> 6.08	3.40 (1.08-10.69)	<0.05

As shown in Table 2, when the selenoprotein P is 90 percentile or more, it was confirmed that the likelihood of gestational diabetes is about 3.4 times higher than that of the normal group (OR (Odds ratio), 3.40; 95% CI). , 1.08-10.69; P <.05). Thus, gestational diabetes can be diagnosed by measuring the level of selenoprotein P.

Example 2. Confirmation of the diagnosis effect of selenoprotein P during hypertension during pregnancy

2.1. Experimental group

We analyzed 857 normal patients and 20 patient groups in single-pregnant pregnant women with similar age, obstetric history, history of hypertension before pregnancy, waist circumference, number of pregnancies collected, and body mass index. Among the target groups defined as the patient group in the experiment, gestational hypertension was normal blood pressure before 20 weeks of pregnancy, and newly confirmed systolic blood pressure of 140 mmHg or more or diastolic blood pressure of 90 mmHg or more and not accompanied by proteinuria after 20 weeks of pregnancy. It was defined as the case of normal blood pressure within 12 weeks after the preeclampsia, and the preconfirmed systolic blood pressure of 140 mmHg or more or diastolic blood pressure of 90 mmHg or more was confirmed after 20 weeks of pregnancy, accompanied by proteinuria, decreased platelets, elevated blood creatinine, and elevated liver value. Eclampsia was defined as a case of convulsion in pre-eclampsia.

2.2. Sample preparation and storage

Peripheral blood of pregnant women visited at 10-14 weeks of pregnancy and 22-30 weeks of pregnancy was collected and centrifuged to obtain plasma by taking supernatant, which was stored in a polypropylene tube at -70 ° C until the experiment.

2.3. Diagnosis of hypertension during pregnancy by measuring the expression level of selenoprotein P

The expression level of selenoprotein P was measured, and it was confirmed by ELISA whether it was possible to diagnose hypertensive disease during pregnancy. The method was performed in the same manner as in 1.3 above, and Table 2 below shows the results of measuring the expression level of selenoprotein P in the patient group with hypertension during pregnancy and the normal group without hypertension during pregnancy at the beginning of 10-14 weeks of pregnancy. . In addition, the results of Table 3 are shown graphically in FIG. 2.

Characteristic	Normal group (n = 633)	Patient group (n = 17)	P- value
Selenoprotein P (ng / mL)	5820	16764	<0.01

Table 4 below shows the results of measuring the expression level of selenoprotein P in the patient group with hypertension during pregnancy and the normal group without hypertension during pregnancy in the middle of 22-30 weeks of pregnancy. In addition, the results of Table 3 are shown graphically in FIG. 3.

Characteristic	Normal group (n = 633)	Patient group (n = 17)	P -value
Selenoprotein P (ng / mL)	12616	75023	<0.01

As shown in Table 3-4 and Figure 2-3, it was confirmed that the selenoprotein P measured in the early and middle of pregnancy is significantly higher in the hypertensive patient group during pregnancy. Therefore, by measuring the level of selenoprotein P, it is possible to diagnose hypertensive disease during pregnancy in both early and midterm pregnant women.

Claims (12)

1. A composition for diagnosing gestational diabetes or hypertensive disease during pregnancy, comprising an agent that measures the expression level of selenoprotein P or a fragment thereof.
2. The method according to claim 1, wherein the selenoprotein P is a composition for diagnosing gestational diabetes or hypertension during pregnancy, which comprises at least one amino acid sequence of SEQ ID NOs: 1-3.
3. The composition for diagnosing gestational diabetes or hypertensive disease during pregnancy according to claim 1, wherein the agent is an antibody or an antigen-binding fragment thereof that specifically binds to selenoprotein P or a fragment thereof.
4. The method according to claim 3, wherein the antibody is a polyclonal antibody or monoclonal antibody composition for diagnosing gestational diabetes or hypertension during pregnancy.
5. The composition for diagnosing gestational diabetes or hypertensive disease during pregnancy according to claim 1, wherein the agent is a primer, probe, or antisense nucleotide that specifically binds to a polynucleotide encoding selenoprotein P.
6. A kit for diagnosing gestational diabetes or hypertension during pregnancy, comprising an agent that measures the expression level of selenoprotein P or a fragment thereof.
7. Measuring the expression level of selenoprotein P or a fragment thereof in a biological sample isolated from a subject suspected of gestational diabetes or hypertensive disease during pregnancy; And

   A method of providing information necessary for diagnosing gestational diabetes or hypertensive disease during pregnancy, comprising comparing the measured expression level with the protein expression level of a normal control.
8. The method of claim 7, wherein the biological sample is blood, plasma, platelets, serum, ascites fluid, bone marrow fluid, lymph fluid, saliva, tear fluid, mucous fluid, amniotic fluid, or a combination thereof.
9. The method according to claim 7, wherein the step of measuring comprises incubating the biological sample with an antibody that specifically binds to selenoprotein P or a fragment thereof.
10. The method of claim 7, wherein the measuring comprises mass spectrometry of the biological sample.
11. The method of claim 10, wherein the mass spectrometry of the biological sample is mass spectrometry of the intact protein.
12. The method according to claim 7, If the expression level of the measured selenoprotein P protein is higher than the protein expression level of the normal control sample, a method comprising determining gestational diabetes or hypertensive disease during pregnancy.

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