Application of Dimethylglycine as a Serum Marker for Fetal Congenital Heart Disease

Abstract

The present application discloses the use of dimethylglycine as a serum biomarker for fetal congenital heart disease. For the first time, the present application screens, identifies, and finally confirms through metabolomics that dimethylglycine in serum can be used as a biomarker for the early diagnosis of fetal congenital heart disease, and develops a diagnostic product for the early diagnosis of fetal congenital heart disease based on this biomarker. This diagnostic product has high sensitivity and high specificity, and can solve the problem of missed diagnosis in the early stage of fetal congenital heart disease. At the same time, it also has the characteristics of low detection cost and good repeatability, and verifies the stability and reliability of using dimethylglycine in serum for the diagnosis of fetal congenital heart disease through discovery and verification methods, and has important clinical development and application value.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biological detection, and particularly to the use of dimethylglycine as a biomarker in the preparation of products for diagnosing or treating fetal congenital heart disease. Background Art

[0002] Congenital heart disease (CHD) is the most common congenital malformation, which is caused by genetic factors, environmental factors, or the combined action of both, resulting in abnormal development of the patient's cardiovascular system. According to statistics, 8-10 out of every 1000 live births (0.8%-1%) have CHD, and its incidence rate is even higher up to 8.3% in premature infants. Among these newborns (50%-60%), they need to take medicine for life and are accompanied by repeated surgical treatments, bringing a heavy burden to families and society.

[0003] Currently, fetal ultrasound examination plays a crucial role in the prenatal diagnosis of CHD. The operator can not only clearly observe the morphological structure of the fetal heart, but also it is safe and non-invasive for pregnant women and fetuses. However, due to various factors such as the operator's experience, fetal position, and instrument probe frequency, missed diagnoses often occur clinically. Magnetic resonance imaging (MRI) can overcome the disadvantages of small diagnostic field of view and poor soft tissue contrast of ultrasound diagnosis, and the image quality is not affected by factors such as amniotic fluid, bones, and maternal obesity. However, there is a time difference between MRI image acquisition and real-time imaging, and it cannot be examined in real time, and it is also easily affected by fetal movement.

[0004] The malformations of CHD patients are sometimes not limited to the heart. Many are accompanied by malformations outside the heart, and some are accompanied by changes in genetic material, including chromosomal aberrations, single-gene genetic defects, polygenic genetic defects, etc. Therefore, genetic examinations are also particularly important. Chromosome karyotype analysis is a classic genetic examination technique for diagnosing chromosomal number abnormalities and microscopic structural abnormalities, but this technique has a long detection period, low resolution, and high technical requirements for operators. Copy number variation detection (CNV-seq) technology has the advantages of rapidity, accuracy, and low operating cost. However, this technology cannot detect uniparental disomy and triploidy. Chromosomal microarray analysis (CMA) has advantages such as high throughput and high sensitivity, but it can only detect and analyze known loci, and the price is relatively expensive.

[0005] Therefore, early prenatal diagnosis is crucial for improving the outcomes of both the mother and the fetus. It helps to provide optimal perinatal and perioperative management and reduce postpartum morbidity and mortality. Therefore, in order to improve the sensitivity and specificity of early clinical diagnosis of fetal congenital heart disease, it is imperative to develop new diagnostic products. Summary of the Invention

[0006] The present application discovers a biomarker for the early diagnosis of fetal congenital heart disease - serum N,N-dimethylglycine, and develops a diagnostic product for the early diagnosis of fetal congenital heart disease based on this biomarker, which can solve the problem of easy misdiagnosis in the early stage of fetal congenital heart disease.

[0007] Based on the discovery that dimethylglycine can be used as a biomarker for the early diagnosis of fetal congenital heart disease:

[0008] The present application provides the use of dimethylglycine as a diagnostic marker for fetal congenital heart disease in the preparation of a diagnostic product for fetal congenital heart disease.

[0009] Optionally, the test sample of the diagnostic product for fetal congenital heart disease is the serum of the subject.

[0010] Optionally, the diagnostic product for fetal congenital heart disease is a diagnostic kit, and the diagnostic kit contains a detection reagent for specifically detecting dimethylglycine in a biological sample.

[0011] The present application also provides the use of a reagent for in vitro detection of dimethylglycine in serum in the preparation of a diagnostic product for fetal congenital heart disease.

[0012] Optionally, the diagnostic product is a diagnostic kit.

[0013] Optionally, the reagent can be a reagent for detecting the content level of dimethylglycine in serum based on the principles of chromatography and mass spectrometry separation.

[0014] The present application also provides the use of dimethylglycine as a drug target for fetal congenital heart disease in the preparation of a drug for treating fetal congenital heart disease. The present application also provides a diagnostic kit for fetal congenital heart disease based on the detection of dimethylglycine in human serum, and the dimethylglycine diagnostic kit contains a detection reagent for specifically detecting dimethylglycine in the serum of the subject.

[0015] Optionally, the diagnostic kit for fetal congenital heart disease includes:

[0016] (1) Dimethylglycine standard;

[0017] (2) Diluent;

[0018] (3) Internal standard solution;

[0019] (4) Eluent;

[0020] Optionally, the diluent is an acetonitrile solution; the internal standard solution is an isotopically labeled dimethylglycine solution diluted with ammonium formate or an isotopically labeled choline (choline-d9) solution diluted with ammonium formate; the mobile phase A of the eluent is an ammonium formate solution containing formic acid, and the mobile phase B is acetonitrile.

[0021] Furthermore, the acetonitrile solution of the diluent is an 80% by mass acetonitrile solution and a 100% by mass acetonitrile solution. In the internal standard solution, the concentration of isotopically labeled dimethylglycine or isotopically labeled choline is 200 - 500 ng / ml, and the concentration of ammonium formate is 10 mmol / L; in the mobile phase A, the mass percentage of formic acid is 0.005% - 0.02%.

[0022] Most preferably, the diluent is an 80% by mass acetonitrile solution and a 100% by mass acetonitrile solution; the internal standard solution is an isotopically labeled dimethylglycine solution diluted with ammonium formate, where the concentration of the isotopically labeled dimethylglycine diluted with ammonium formate is 500 ng / ml, and the concentration of ammonium formate is 10 mmol / L; the mobile phase A of the eluent is an ammonium formate solution containing 0.01% formic acid, and the mobile phase B is acetonitrile.

[0023] This application also provides a dimethylglycine detection system in serum, including:

[0024] A standard curve plotting module, which plots a quantitative standard curve with the ratio of the peak areas of dimethylglycine and the internal standard as the vertical axis and the concentration of dimethylglycine as the horizontal axis, based on using dimethylglycine as the standard product and isotopically labeled dimethylglycine or isotopically labeled choline as the internal standard.

[0025] A separation module, which is used to extract and elute dimethylglycine from a serum sample of a subject and record its peak area.

[0026] A calculation module, which calculates the concentration of dimethylglycine in the serum sample of the subject based on the standard curve plotted by the standard curve plotting module and the peak area recorded by the separation module.

[0027] Optionally, the separation module includes a HILIC chromatographic column and an ultra-high performance liquid chromatograph tandem mass spectrometer.

[0028] Compared with the prior art, this application has the following effects:

[0029] For the first time, this application screens, identifies, and finally confirms through metabolomics that dimethylglycine in serum can be used as a biomarker for the early diagnosis of fetal congenital heart disease, for the diagnosis of fetal congenital heart disease, with high sensitivity and high specificity, and at the same time has the characteristics of low detection cost and good repeatability. Moreover, the stability and reliability of using dimethylglycine in serum for the diagnosis of fetal congenital heart disease are verified through the discovery and verification methods, which has important clinical development and application value. Description of the Drawings

[0030] Figure 1 It is the chromatogram-mass spectrometry diagrams of 6 choline metabolism-related substances in serum.

[0031] Figure 2 In it, A is the graph of the content change of dimethylglycine in the serum samples of pregnant women with fetuses diagnosed as CHD by ultrasound and confirmed by follow-up and pregnant women with healthy fetuses (represented by mean ± standard error) (in the figure, CHDP represents pregnant women with fetuses diagnosed as CHD by ultrasound and confirmed by follow-up, and ZCP represents pregnant women with healthy fetuses).

[0032] Figure 2 In it, B is the ROC curve graph of using dimethylglycine for pregnant women with fetuses diagnosed as CHD by ultrasound and confirmed by follow-up and pregnant women with healthy fetuses. Detailed Embodiments

[0033] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0035] Metabolomics is a new discipline following genomics, proteomics, and transcriptomics, and is a scientific field aimed at studying small molecule metabolites. Since the presence of small molecule metabolites in body fluids is relatively stable, the analysis of some endogenous small molecule metabolites by metabolomics has been widely used in the early diagnosis of diseases. In this application, metabolomics methods using ultra-high performance liquid chromatography-mass spectrometry were used to detect metabolites in serum. Through bioinformatics analysis, biomarkers suitable for the early diagnosis of fetal congenital heart disease were screened. This method has the advantages of fast detection, good repeatability, high sensitivity, and low cost. A strategy from discovery to verification was adopted to screen potential diagnostic markers, and an early diagnostic marker for fetal congenital heart disease based on dimethylglycine in human serum was successfully screened. The diagnostic sensitivity and specificity of this diagnostic marker are good, and no kit for this diagnostic marker has been reported yet.

[0036] The process by which this application discovered that dimethylglycine can be used as an early diagnostic biomarker for fetal congenital heart disease is as follows:

[0037] (1) Using metabolomics techniques based on nuclear magnetic resonance and tandem mass spectrometry with ultra-high performance liquid chromatography, metabolomic fingerprint analysis was performed on the sera of 55 pregnant women with fetuses diagnosed by ultrasound and followed up as having CHD (CHDP group) and 49 pregnant women with healthy fetuses (ZCP group). It was found that the choline metabolic pathway in the sera of patients in the CHDP group changed, and quantitative analysis was performed on 6 choline-related metabolites in the sera of patients in the CHDP group and patients in the ZCP group (see Figure 1 ), and it was found that 5 choline metabolites all had significant differences.

[0038] (2) Using the data statistical software SPSS, ROC analysis (Receiver Operating Characteristic Curve) was performed on the above 5 metabolites to screen out the metabolite with the best diagnostic performance for fetal congenital heart disease, and its sensitivity and specificity were evaluated. The results are as Figure 2 shown, showing the ROC curve of dimethylglycine in the experimental group for differentiating patients in the ZCP group and patients in the CHDP group. Finally, it was determined that dimethylglycine has the best diagnostic performance for diagnosing fetal congenital heart disease, with an AUROC of 0.883. When the cut-off value is 1.36 ug / mL, its sensitivity and specificity are 85.2% and 82.0% respectively, that is, when the content of dimethylglycine in the patient's serum is higher than 1.36 ug / mL, it can be diagnosed as fetal congenital heart disease.

[0039] Based on this discovery:

[0040] The present application provides: the use of dimethylglycine as a diagnostic marker for fetal congenital heart disease in the preparation of a diagnostic product for fetal congenital heart disease; the use of a reagent for in vitro detection of dimethylglycine in serum in the preparation of a diagnostic product for fetal congenital heart disease; the use of dimethylglycine as a drug target for fetal congenital heart disease in the preparation of a drug for treating fetal congenital heart disease; a diagnostic kit for fetal congenital heart disease; and a detection system for dimethylglycine in serum.

[0041] The diagnostic product can be a reagent, test strip, diagnostic kit, etc. The diagnostic product is a diagnostic kit, and the diagnostic kit contains a detection reagent for specifically detecting dimethylglycine in a biological sample. The detection sample of the diagnostic product is the serum of the subject.

[0042] As an implementation manner of the diagnostic kit for fetal congenital heart disease, the diagnostic kit for fetal congenital heart disease includes:

[0043] (1) Dimethylglycine standard;

[0044] (2) Diluent;

[0045] (3) Internal standard solution;

[0046] (4) Eluent

[0047] The dimethylglycine standard is used for the qualitative analysis of dimethylglycine in serum and the drawing of a standard curve. The mass numbers of the parent ion and its main fragment ions of dimethylglycine in the MRM mode of mass spectrometry are 104.10 and 58.07 respectively.

[0048] The diluent is used for pretreatment of the extract and standard of the serum sample from the subject, and pure acetonitrile solution and 80% acetonitrile solution can be selected.

[0049] The internal standard solution can be an isotopically labeled dimethylglycine solution diluted with ammonium formate or an isotopically labeled choline solution diluted with ammonium formate. In the internal standard solution, the concentration of ammonium formate is 10 mmol / L, and the final concentration of the isotopically labeled dimethylglycine or isotopically labeled choline is 300 - 500 ng / ml. Quantitative analysis is carried out in the SRM or MRM mode of mass spectrometry. The mass numbers of the parent ion and its main fragment ions of dimethylglycine in the MRM mode of mass spectrometry are 113.16 and 69.13 respectively.

[0050] The eluent is used to elute the chromatographic column. Mobile phase A can be an ammonium formate solution containing 0.01% formic acid, and mobile phase B can be acetonitrile. The chromatographic column can be a C18 or HILIC chromatographic column.

[0051] The kit of the present application can be applied to an ultra-high performance liquid chromatography tandem mass spectrometer, and can also simultaneously detect other choline-related metabolites such as choline, carnitine, acetylcholine, dimethylglycine, etc., and can more comprehensively analyze the choline metabolism disorder of pregnant women with fetal congenital heart disease. Serum samples are used to test the application effect of the present invention. Using the serum samples of pregnant women with fetal CHD and pregnant women with healthy fetuses, and using dimethylglycine in the serum as the diagnostic criterion, the two groups of patients can be well distinguished.

[0052] Specifically, a method for detecting dimethylglycine in a serum sample of a subject using the kit of the present application includes the following steps:

[0053] (1) Using dimethylglycine as a standard, with the internal standard being isotopically labeled dimethylglycine diluted with ammonium formate (the concentration of isotopically labeled dimethylglycine is 500 ng / ml), then adding an acetonitrile solution and directly injecting for detection to draw the corresponding quantitative standard curve;

[0054] (2) Pretreatment of the serum sample from the subject: Thaw the serum sample at room temperature, take 50 μL of serum and add 30 μL of internal standard solution (isotopically labeled dimethylglycine diluted with ammonium formate), 30 μL of 80% acetonitrile, and 340 μL of acetonitrile, vortex mix vigorously, after ultrasonic treatment, centrifuge at 12000 g for 10.0 min, take 200.0 μL of the supernatant and place it in an auto-sampler vial for the detection of dimethylglycine in the serum, and record the ratio of the elution peak area of dimethylglycine in the serum sample to the internal standard peak area;

[0055] (3) According to the quantitative standard curve in step (1) and the ratio of the peak areas in step (2), calculate the content of dimethylglycine in the serum sample of the subject.

[0056] The diagnostic kit detects the content of dimethylglycine in the serum of pregnant women (pregnant women with fetal CHD and pregnant women with healthy fetuses), and uses a cut-off value of 1.36 μg / mL. When the content of dimethylglycine in the patient's serum is higher than 1.36 μg / mL, it is diagnosed that the patient has fetal congenital heart disease.

[0057] As an implementation manner of a detection system for dimethylglycine in serum, it includes:

[0058] A standard curve drawing module, based on using dimethylglycine as a standard and isotopically labeled dimethylglycine or isotopically labeled choline (choline-d9) as an internal standard, drawing a quantitative standard curve with the ratio of the peak areas of dimethylglycine and the isotopically labeled internal standard as the vertical axis and the concentration of dimethylglycine as the horizontal axis;

[0059] A separation module, used to extract and elute dimethylglycine from the serum sample of the subject and record its peak area;

[0060] A calculation module calculates the concentration of dimethylglycine in the serum sample of the subject based on the standard curve drawn by the standard curve drawing module and the peak area recorded by the separation module.

[0061] The devices included in the diagnostic system: Use a UPLC chromatograph of Waters Corporation in the United States combined with an Xevo G2-XS QTof mass spectrometer to detect metabolites in the multiple reaction monitoring mode (MRM). The chromatographic column is a SeQuant ZIC-HILIC hydrophilic chromatographic column (3.5 μm, 2.1×150 mm) of Merck KGaA in Germany, and the electrospray ionization source uses positive ions.

[0062] The following takes a specific example of detection using this kit for illustration. The concentrations defined by percentages in the following examples refer to mass percentage concentrations unless otherwise specified.

[0063] Example 1:

[0064] 1. Serum sample collection

[0065] Before collection, informed consent forms signed by all subjects included in this study were obtained.

[0066] The 104 serum samples in this study were from Jiaxing Maternal and Child Health Hospital and Jiangxi Maternal and Child Health Hospital, including 55 pregnant women with fetuses diagnosed with CHD by ultrasound and confirmed by follow-up (CHDP group) and 49 pregnant women with healthy fetuses (ZCP group).

[0067] Inclusion criteria: ① Singleton pregnancy; ② Complete B-ultrasound examination data, and the fetus is confirmed to have CHD by follow-up after birth or induction of labor; ③ The pregnant woman or her family member signs an informed consent form; ④ The pregnant woman is 20-40 years old and in the second trimester of pregnancy (20-24 weeks).

[0068] Exclusion criteria: ① Unable to have a physical examination at the specified time or not cooperate with follow-up; ② The fetus has other congenital malformations; ③ The pregnant woman has severe diseases of organs such as the heart, brain, liver, and kidneys; ④ The pregnant woman has massive vaginal bleeding, acute fetal distress in utero, fetal death, twin and multiple pregnancies, etc.; ⑤ Pregnant women with pregnancy complications or complications such as gestational diabetes and gestational hypertension.

[0069] To eliminate dietary interference, all pregnant women collected blood samples on an empty stomach in the morning and left them standing in a 4°C refrigerator for 2 hours. The blood samples were centrifuged at 4000 r for 15 minutes, and the serum was collected and stored in a -80°C refrigerator. It was thawed at room temperature when used. This study has obtained the approval of the Ethics Committee of Jiaxing Maternal and Child Health Hospital (No. 2020-5) and obtained the informed consent of the patients.

[0070] 2. Analysis method

[0071] 2.1 Plotting of the standard curve

[0072] Weigh a certain amount of dimethylglycine and dissolve it in distilled water to a certain concentration as the stock solution, which is stored at 4 °C for later use. The stock solution is diluted successively by 2-fold with distilled water and vortexed thoroughly to obtain standard solutions of dimethylglycine with different gradients. Add 50 μL of standard solutions of dimethylglycine with different concentrations, 30 μL of internal standard solution (isotope-labeled dimethylglycine solution diluted with ammonium formate, where the concentration of isotope-labeled dimethylglycine is 500 ng / ml and the concentration of ammonium formate is 10 mmol / L), 30 μL of 80% acetonitrile, and 340 μL of acetonitrile into an EP tube in sequence, vortex vigorously, ultrasonicate, and then centrifuge at 12000 g for 10.0 min. Take 200.0 μL of the supernatant and transfer it to an auto-sampler vial for plotting the standard curve.

[0073] 2.2 Pretreatment of serum samples

[0074] Thaw the serum samples at room temperature. Add 50 μL of serum, 30 μL of internal standard solution (the same as in 2.1), 30 μL of 80% acetonitrile, and 340 μL of acetonitrile into an EP tube in sequence, vortex vigorously to precipitate proteins, ultrasonicate, and then centrifuge at 12000 g for 10.0 min. Take 200.0 μL of the supernatant and transfer it to an auto-sampler vial for the detection of dimethylglycine in serum.

[0075] 2.3 Analysis by ultra-high performance liquid chromatography-tandem mass spectrometry

[0076] (1) Liquid phase conditions: The chromatograph is Waters ultra-high performance liquid chromatography; SeQuant ZIC-HILIC hydrophilic chromatographic column (3.5 μm, 2.1×150 mm) from Merck, USA; The mobile phase composition is: 10.0 mmol / L ammonium formate aqueous solution containing 0.01% formic acid (A) - acetonitrile (B); The flow rate is 0.3 mL / min, the injection volume is 4.0 μL; The separation time is 8.0 min; The following gradient elution is adopted: 0 - 2.0 min, 10% A; 2.0 - 4.0 min, 10% - 70% A; 4.0 min - 5.0 min, 70% A; 5.0 min - 5.5 min, 70% - 10% A; 5.5 min - 8 min, 10% A; The column temperature is set at 45 °C, and the auto-sampler temperature is set at 8 °C.

[0077] (2) Mass spectrometry conditions: The mass spectrometer is Xevo QTof (Waters, US), and metabolites are detected in the multiple reaction monitoring mode (MRM), and positive ion detection is used for the electrospray ionization source; The electrospray capillary voltage is set at 3.0 kV, nitrogen is used as the drying gas for solvent evaporation, and the flow rate is 50 L / h; The ion source temperature is 100 °C.

[0078] 2.4 Serum test results

[0079] Taking the standard product concentration as the abscissa and the ratio of the standard product peak area to the internal standard peak area as the ordinate, a standard curve was plotted; the detection results of dimethylglycine in the serum samples were substituted into the standard curve, and finally the contents of dimethylglycine in each sample were obtained. The serum test results are as Figure 1 shown

[0080] It was found that the content of dimethylglycine in the serum of patients in the CHDP group increased significantly compared with that of patients in the ZCP group. When the cut-off value was 1.36 ug / mL, the sensitivity and specificity were 85.2% and 82.0% respectively. That is, when the content of dimethylglycine in the patient's serum is higher than 1.36 ug / mL, fetal congenital heart disease can be diagnosed.

[0081] The above-described embodiments merely represent several implementation manners of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. Use of dimethylglycine as a diagnostic marker for fetal congenital heart disease in the preparation of a diagnostic product for fetal congenital heart disease, wherein the test sample of the diagnostic product for fetal congenital heart disease is the serum of a subject, and the subject is a pregnant woman in the second trimester of pregnancy; the diagnostic product for fetal congenital heart disease is a diagnostic kit, and the diagnostic kit contains a detection reagent for specifically detecting dimethylglycine in a biological sample.

2. Use of a reagent for in vitro detection of dimethylglycine in serum in the preparation of a diagnostic product for fetal congenital heart disease, wherein the test sample of the diagnostic product is the serum of a subject, and the subject is a pregnant woman in the second trimester of pregnancy; the diagnostic product for fetal congenital heart disease is a diagnostic kit.

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