CN112816711A - Molecular marker for prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate and application thereof - Google Patents
Molecular marker for prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a molecular marker for prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate and application thereof. The molecular marker for prenatal noninvasive diagnosis of neural tube deformity, congenital heart disease and cleft lip and palate is one or more of CORO1A, DNM2 and ACTR2 proteins. The prenatal noninvasive diagnosis molecular marker is applied to the preparation of prenatal screening, early warning, clinical diagnosis and biochemical inspection products for neural tube malformation, congenital heart disease and cleft lip and palate. The invention discovers and verifies that the expression abnormality of proteins (including CORO1A, DNM2 and ACTR2) in the blood of pregnant women has close correlation with the occurrence of neural tube malformation, congenital heart disease and cleft lip and palate fetuses for the first time, has large amount of verified samples and accurate results, and provides a new way for prenatal screening, early warning and diagnosis of the neural tube malformation, congenital heart disease and cleft lip and palate fetuses.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a molecular marker (CORO1A, DNM2 and ACTR2 protein) for prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate fetus and application thereof.
Background
Neural tube deformity, congenital heart disease, cleft lip and palate and the like are common major fetal developmental deformities in China and seriously harm the improvement of the life quality of the population at birth. Therefore, the research on the method for the early noninvasive diagnosis of the congenital malformation embryos can make the diagnosis before serious structural abnormality or irreversible damage, and develop corresponding new strategies for early embryo treatment and prevention, and has extremely important significance for reducing the disability rate of the malformation and improving population quality.
Establishing a non-invasive early screening method for congenital malformation is always an ideal target continuously pursued by people. Although the development of imaging techniques (ultrasound and MRI) is very rapid, and the diagnosis time of some congenital malformations is advanced, the requirements of early screening and diagnosis cannot be met. Maternal serology examination is a noninvasive prenatal diagnosis method, is easily accepted by pregnant women, and is suitable for large-scale prenatal screening. Therefore, many scholars at home and abroad are dedicated to research on finding new specific diagnostic markers, but no diagnostic molecular marker for clinical application of birth defects is available so far except that neural tube malformation and Down syndrome can be screened by using serum alpha-fetoprotein.
With the rapid development of various omics technologies in recent years, a series of new technologies are integrated into high-throughput omics research, so that a new breakthrough is brought to the molecular marker screening work of disease diagnosis, and an extremely important means is provided for the transformed medical research which is very important at present. The high-flux omics technology can fully consider two factors of a mother and a fetus, a group of key molecules with obvious change is screened from a plurality of complex proteins, the change rule of the key molecules is comprehensively analyzed, the molecular markers which are beneficial to determining early diagnosis and prognosis judgment of diseases are determined, and the trend of converting the complex molecular markers into clinical application in the future is also provided.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide molecular markers for prenatal noninvasive diagnosis of neural tube deformity, congenital heart disease and cleft lip and palate and application thereof, wherein the molecular markers are CORO1A, DNM2 and ACTR2 proteins, can be used for prenatal screening of congenital deformity alone or in combination, and provide new targets for treatment of neural tube deformity, congenital heart disease and cleft lip and palate.
In order to achieve the purpose, the invention adopts the following technical scheme.
The molecular marker for prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate is composed of one or more of CORO1A, DNM2 and ACTR2 proteins.
The molecular marker is applied to the preparation of prenatal screening, early warning, clinical diagnosis and biochemical test products for neural tube malformation, congenital heart disease and cleft lip and palate.
Furthermore, the product comprises a reagent, a kit, a chip, test paper and a high-throughput sequencing platform, and protein molecular markers related to prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate fetus are detected by using a mass spectrometry technology, a PCR (polymerase chain reaction), in-situ hybridization, fluorescence in-situ hybridization, an immunotransmission turbidimetry, a radioimmunoassay and other related methods.
Furthermore, the prenatal screening, early warning, clinical diagnosis and biochemical examination of the neural tube malformation, congenital heart disease and cleft lip and palate fetus comprise pregnant woman blood (and exosomes thereof), urine (and exosomes thereof), amniotic fluid (and exosomes thereof), a fetus specimen and the like.
The application of a reagent for detecting molecular markers for prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate in the preparation of prenatal noninvasive fetal diagnosis tools.
Further, the reagent for detecting prenatal noninvasive diagnosis molecular markers of neural tube malformation, congenital heart disease and cleft lip and palate comprises a reagent capable of quantifying the protein.
Further, the reagent capable of quantifying the protein may be a primer specific to a gene or a transcript, a specific recognition probe, or both a primer and a probe.
A tool for prenatal screening, early warning and diagnosis of neural tube malformations, congenital heart diseases and cleft lip and palate fetus, wherein the tool can detect the expression amount of the molecules.
Further, the tools include prenatal noninvasive diagnostic protein molecular markers including CORO1A, DNM2 and ACTR2 that can be quantified and correlated with neural tube malformations, congenital heart disease and cleft lip and palate.
Further, the tool for prenatal screening, early warning and diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate comprises a chip, a kit, test paper and a high-throughput sequencing platform.
A method for prenatal screening, early warning and diagnosis of neural tube abnormalities, congenital heart disease and cleft lip and palate, said method comprising the steps of.
(1) A sample of the subject is obtained.
(2) Detecting the expression level of the above-mentioned molecule in a sample of the subject.
(3) Correlating the trend of the measured expression level of the above-mentioned molecules with the disease-related condition of the subject.
(4) Abnormal expression of the above molecules, as compared to normal controls, indicates that the subject is at high risk of harboring a fetus with neural tube malformations, congenital heart disease, and cleft lip and palate.
The reagent for quantifying a protein of the present invention can exert its function based on a known method other than an antibody: such as mass spectrometry techniques, including MRM and PRM, among others.
The reagent for quantifying a protein of the present invention can exert its function based on a known method using an antibody: for example, chips (protein chips, microfluidic chips, etc.), digital single molecule microarrays, ELISAs, radioimmunoassays, immunotransmission turbidimetry, immunohistochemistry, Western blots, etc. may be included.
The reagent for quantifying a protein of the present invention includes an antibody or a fragment thereof that specifically binds to the protein. An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc., may be used so long as it isBinding to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')2Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The reagent for quantifying a protein of the present invention may include an isolated nucleic acid encoding an amino acid sequence of an antibody or encoding a fragment of an antibody, a vector comprising the nucleic acid, and a cell carrying the vector.
Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against the protein of the present invention can be obtained by subjecting the obtained antibody to antigen-specific purification using the protein or a portion thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.
Binding of the label to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, proteins or peptides may be fluorescently labeled as follows: after washing proteins or peptides with phosphate buffer, DMSO, buffer, etc. were added, the solution was mixed, and the mixture was left at room temperature for 10 minutes. In addition, labeling can be carried out using a commercially available labeling kit, such as a biotin labeling kit, e.g., biotin labeling kit-NH2Biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kit such as alkaline phosphatase labeling kit-NH2Alkali, alkaliSex phosphatase labeling kit-SH (Dojindo laboratories); peroxidase labeling kit such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH2(Dojindo Laboratories); phycobiliprotein labeling kit such as phycobiliprotein labeling kit-NH2Phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH2R-phycoerythrin labeling kit SH (Dojindo laboratories); fluorescent labeling kit such as fluorescein labeling kit-NH2HiLyte Fluor (TM)555 labeling kit-NH2HiLyte Fluor (TM)647 marker kit-NH2(Dojindo Laboratories); and DyLight 547 and DyLight647(Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation), and EZ-marker protein labeling kit (Funakoshi Corporation). For proper labeling, a suitable instrument can be used to detect the labeled antibody or fragment thereof.
The obtaining of the sample for detecting the expression level of the above-mentioned molecule according to the present invention is a routine technique in the art, and preferably can be achieved by a method selected to be non-invasive or minimally invasive.
The sample of the invention may be (but is not limited to): pregnant woman blood, urine, amniotic fluid and abnormal fetus or infant specimen. In a specific embodiment of the invention, the sample is from a tissue of a subject.
The high-throughput proteomics detection platform is a special tool, and by comparing the protein expression difference of disease patients and normal people, which protein expression abnormality is related to diseases can be easily separated out. Therefore, the knowledge of the abnormal expression of the above molecules in high-throughput proteomic detection and the correlation between neural tube malformation, congenital heart disease and cleft lip and palate also belong to the novel application using the invention, and are also within the protection scope of the invention.
The kit of the present invention may contain a plurality of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed so far, and is included in the scope of the present invention as long as the reagents are used for determining neural tube malformation, congenital heart disease, and cleft lip and palate based on the above-mentioned molecular detection.
In the context of the present invention, "prenatal screening, early warning and diagnosis of neural tube malformations, congenital heart disease and cleft lip and palate" includes determining whether a subject fetus has developed neural tube malformations, congenital heart disease and cleft lip and palate, and determining whether the subject fetus is at risk of developing neural tube malformations, congenital heart disease and cleft lip and palate.
Compared with the prior art, the invention has the following beneficial effects.
The invention discovers and verifies that the expression abnormality of proteins (including CORO1A, DNM2 and ACTR2) in the blood of pregnant women has close correlation with the occurrence of neural tube malformation, congenital heart disease and cleft lip and palate fetuses for the first time, and has a large number of verified samples and accurate results.
The protein marker related to prenatal noninvasive diagnosis of neural tube deformity, congenital heart disease and cleft lip and palate fetus provided by the invention provides a service for prenatal diagnosis or risk monitoring of neural tube deformity, congenital heart disease and cleft lip and palate fetus, and consultative services for diagnosis and prognosis are cooperatively or independently sold to hospitals and clinics.
The protein marker related to prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate provided by the invention provides a new way for prenatal screening, early warning and diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate fetus.
Drawings
FIGS. 1 to 3 show that 33 differentially expressed proteins were selected from peripheral blood of neural tube malformation using proteomics, and bioinformatics analysis was performed to find three interacting differentially expressed proteins (CORO1A, DNM2, and ACTR 2).
FIGS. 4-1 to 4-4 show that the Western-blot method is used to carry out quantitative detection and verification on three differentially expressed proteins CORO1A, DNM2 and ACTR2 in the peripheral blood of a pregnant mouse with a neural tube defect of embryos at days E12, E14 and E18, and the significant low expression of CORO1A, DNM2 and ACTR2 in the peripheral blood of the pregnant mouse with the neural tube defect is confirmed.
FIGS. 5-1 to 5-4 show that the Western-blot method is used for carrying out quantitative detection and verification on three differentially expressed proteins CORO1A, DNM2 and ACTR2 in spinal cord tissues with neural tube deformities at embryonic days E12 and E18 and exosomes derived from embryonic neural tissues of peripheral blood of pregnant mice at day E18, and meanwhile, the immunohistochemical method is used for carrying out detection and analysis on the expression conditions of CORO1A, DNM2 and ACTR2 in spinal cord tissues with neural tube deformities, so that the CORO1A, DNM2 and ACTR2 are obviously and lowly expressed in the neural tube deformities.
FIGS. 6-1 to 6-3 show that the quantitative detection of CORO1A and DNM2 in the peripheral blood of pregnant women with neural tube defects by ELISA method proves that CORO1A and DNM2 are obviously low expressed in the peripheral blood of pregnant women with neural tube defects and are changed more obviously in the early pregnancy.
Fig. 7-1 to 7-3 are results of ROC curve analysis showing that cor 1A and DNM2 are analyzed for the diagnosis accuracy, sensitivity and specificity of pregnant women with a neural tube malformed fetus, showing that cor 1A and DNM2 are more effective in diagnosing neural tube malformations in early pregnancy.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The animal model used in the examples was derived from the animal center of the Shengjing hospital, and the peripheral blood specimens of the pregnant women were derived from the sample bank of the Shengjing birth queue and approved by the ethical committee of the Shengjing hospital affiliated to the university of medical science in China (approval No.: 2017PS 264K).
Example 1 screening of differentially expressed proteins in peripheral blood of pregnant mice with neural tube defects using proteomics techniques and bioinformatic analysis.
Separating plasma exosomes of pregnant mice with neural tube malformation by adopting an ultra-high speed centrifugation method, and completing the work of related identification of the exosomes. Proteomics screening was performed using a non-labeled quantitative technique (label-free) and combined with bioinformatics analysis to initially screen for potential protein markers, as shown in FIGS. 1-3.
1. Plasma was separated.
Collecting a whole blood sample, gently inverting and uniformly mixing in an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, centrifuging for 10min at 1600 Xg by using a 4-DEG C low-temperature centrifuge, collecting supernatant (blood plasma) to a new EP (ethylene propylene glycol) tube after centrifugation, centrifuging for 10min at 16000 Xg to remove cell debris, and subpackaging the blood plasma into a plurality of centrifuge tubes.
2. And (4) separating and identifying exosomes.
Separating plasma exosome by ultra-high speed centrifugation. The separation method comprises the following steps: centrifuging at 10000 Xg for 1h at 4 deg.C, transferring the supernatant to a new ultra-high speed centrifuge tube, and centrifuging at 100000 Xg for 4h at 4 deg.C. The supernatant was discarded and the exosomes were resuspended in 100. mu.l of cold PBS. The isolated exosome particles were identified using three standard methods (transmission electron microscopy, particle size analysis and exosome surface marker protein).
3. Proteomics detection and bioinformatics analysis.
Proteomics detection of non-labeled quantitative technology (label-free) is carried out on 3 pregnant mice with neural tube malformation and 3 normal pregnant mice, omics data are input into TBtools software to make a heat map, bioinformatics analysis is carried out on a webgetatal website to carry out enrichment analysis on the biological process of gene GO, and then protein interaction network is carried out on differential proteins on the String website.
4. Exosome identification and proteome detection results.
(1) And (3) exosome identification: transmission electron microscope and particle diameter detection prove that the exosome finds a peak at the diameter of 100nm, which indicates that the exosome extracted by the ultracentrifugation method has good purity. Thereafter, Western blot assays were performed on Alix, CD63 and CD9 using exosomes at different concentrations to identify exosome marker expression, and the results showed that Alix, CD63 and CD9 bands exhibited correct trends in changes with increasing concentration gradient.
(2) Proteomics results: a total of 3 serum samples from E18 normal mice and 3 serum exosomes samples from neural tube malformations were used for proteomic testing, a total of 63 enriched proteins were found, and 33 proteins differed between the normal and malformed groups. There were 7 proteins specifically expressed in NTDs, 12 proteins specifically expressed in the normal group, and 2 unknown proteins. In addition, 14 proteins were expressed in both the NTDs group and the normal group, of which 1 protein was highly expressed in the neural tube malformation and 13 proteins were lowly expressed. GO analysis found the enrichment of 4 biological processes, respectively: regulation of actin polymerization (depolymerization), regulation of anatomical morphological structure development, response to oxygen-containing substances and cellular structure assembly. Wherein ACTR2, CORO1A, DNM2, AGT, GLUL, LIMS1, PDCD6 and PKM are involved in 2 or more biological processes, wherein ACTR2, CORO1A, GLUL and DNM2 are associated with neural development and have not been reported in neural tube malformations. The 4 proteins were analyzed for protein interaction on the String website, and the results showed that ACTR2, cor o1A, and DNM2 could be clustered into the same interactive network cluster.
Example 2 the expression of ACTR2, cor 1A, and DNM2 was verified in neural tube malformation serum exosomes.
1. The expression of ACTR2, cor 1A and DNM2 was verified in embryonic E18 day serum exosomes.
And selecting 19 samples except the omics detection to carry out Western-blot amplification sample size verification on the embryo E18 day samples. This validation detects ACTR2 at 45kDa, CORO1A at 57kDa and DNM2 at 98kDa using Alix as an internal control. A total of 19 samples derived from pregnant mouse serum exosomes (19 normal, 19 neural tube malformations) were tested and found to be consistent with the trend of omics results, all with low expression in the malformed group. Statistical results also showed that there was a statistical difference in the low expression of ACTR2, cor 1A, and DNM2 in the neural tube malformation group. The detection of CORO1A by ELISA also confirmed the consistency of the Western-blot results.
2. Expression of ACTR2, cor 1A and DNM2 in embryonic day E12, E14 and E16 serum exosomes.
To verify whether ACTR2, cor 1A, and DNM2 were also altered in the early pregnancy, serum exosomes from days E12, E14, and E16 were tested using the Western-blot method. The result shows that the expression condition of the band and the statistic result of the unpaired T test show that the band is obviously reduced in the neural tube malformation group. This part of the results clearly shows that the expression trend of these proteins in the early pregnancy is consistent with that of E18 days. In addition, the results of the CORO1A assay on E12 by ELISA showed a significant down-regulation trend, confirming the consistency of the Western-blot results. This section clearly demonstrates that ACTR2, cor 1A and DNM2 also have a significant down-regulation tendency during the period from E12 to E16, suggesting that they may be diagnostic indicators of early pregnancy neural tube malformations.
The results of the proteomics, namely ACTR2, CORO1A and DNM2, were verified in neural tube malformation serum exosomes by Western-blot method, as shown in FIGS. 4-1 to 4-4.
Example 3 expression of ACTR2, cor 1A and DNM2 in spinal cord tissue and embryonic neurogenic exosomes.
1. ACTR2, cor 1A, and DNM2 are expressed in spinal cord tissue.
In order to investigate the relationship between ACTR2, CORO1A and DNM2 and neural tube malformation and to clarify the expression in spinal cord tissue, expression in spinal cord tissue was examined by Western-blot at embryonic days E18 and E12, and beta-actin was used as an internal reference for tissue detection. The results show that ACTR2, cor o1A, and DNM2 all show statistically significant down-regulation trends in spinal cord tissue with neural tube abnormalities consistent with serum exosome trends. Immunohistochemical staining showed expression of ACTR2, cor o1A and DNM2 in spinal cord tissue with neural tube malformations at day E18, confirming whether these proteins were specifically expressed in neural tube tissue. ACTR2 is expressed in nerve cells, neuroepithelium, and on the neural crest side and is significantly down-regulated in neural tube malformed tissues; CORO1A is expressed on nerve fibers and nerve cells obviously, and has down-regulation expression in neural tube malformed tissues; DNM2 was expressed on nerve fibers, neuroepithelium, nerve cells, and neural crest, and showed a down-regulation tendency in neural tube malformed tissues. Suggesting that changes in these 3 markers in serum exosomes are likely to result from changes in spinal cord tissue.
2. ACTR2, cor 1A, and DNM2 were expressed in fetal neurogenic exosomes.
In order to verify whether the low expression of the ACTR2, the CORO1A and the DNM2 in the serum exosomes of the pregnant mouse with the neural tube deformity is caused by the low expression of the fetal neural exosomes, the serum exosomes of the pregnant mouse are further separated, the fetal neural exosomes are separated by using a separation kit combined with specific antibodies, and the quantitative detection of the ACTR2, the CORO1A and the DNM2 is carried out by using a Western-blot method, so that the result shows that the expression of the CORO1A and the DNM2 is obviously reduced in the neural tube deformity group, and the expression level of the ACTR2 is not statistically different between the neural tube deformity group and a normal control group. The results suggest that the reduced expression of CORO1A and DNM2 in pregnant mouse serum exosomes is due to reduced expression in exosomes produced by embryonic spinal cord tissue.
The above ACTR2, cor o1A and DNM2, which were tested in pregnant mouse serum exosomes, were tested in neural tube malformed spinal cord tissue and in embryonic neurogenic exosomes using Western-blot and immunohistochemical methods, as shown in fig. 5-1 to 5-4.
Example 4 the diagnostic efficacy of cor 1A and DNM2 as molecular markers was demonstrated in mother peripheral blood exosomes of neural tube malformations, congenital heart disease and cleft lip and palate.
1. Study samples were included.
19 cases of peripheral blood of fetal mothers with neural tube malformation, 100 cases of peripheral blood of fetal mothers with congenital heart diseases and 40 cases of peripheral blood of fetal mothers with cleft lip and palate were collected from Shengjing birth queue, and 159 cases of normal pregnant women with age similar to gestational age were taken as normal controls.
2. And (4) separating exosomes.
Separating plasma exosome by ultra-high speed centrifugation. The separation method comprises the following steps: centrifuging at 10000 Xg for 1h at 4 deg.C, transferring the supernatant to a new ultra-high speed centrifuge tube, and centrifuging at 100000 Xg for 4h at 4 deg.C. The supernatant was discarded and the exosomes were resuspended in 100. mu.l of cold PBS.
3. ELISA was used to detect the expression level of CORO1A and DNM 2.
Diluting 50 microliters of serum exosomes into 100 microliters in PBS (phosphate buffer solution), then operating by using a human CORO1A ELISA kit and a human DNM2 ELISA kit according to the steps of the instruction, adding a reaction termination solution, detecting at 450 nanometers by using a multifunctional enzyme-linked immunosorbent assay, and calculating the expression quantity of CORO1A and DNM2 according to a standard curve.
4. Diagnostic efficacy of CORO1A and DNM2 as molecular markers in mother peripheral blood exosomes of neural tube malformations, congenital heart disease and cleft lip and palate.
The DNM2 test result shows that the expression amount of the exosome in the peripheral blood of the mother of the fetus with the neural tube malformation is obviously reduced, and the statistical difference exists in comparison with the normal pregnant woman group. ROC curve analysis DNM2 diagnosed neural tube malformations with a sensitivity of 73.68% and a specificity of 78.95%. Further analysis of the gestational period of the mother with the neural tube malformation shows that the DNM2 has better diagnosis efficiency on the early pregnancy (12-18 weeks of pregnancy) and the sensitivity and the specificity reach 100%. The diagnosis efficiency in the late pregnancy is poor, the sensitivity is 68.42 percent, and the specificity is 89.47 percent. The expression quantity of peripheral blood exosomes of the congenital heart disease fetal mother is obviously reduced, and compared with a normal pregnant woman group, the fetal heart disease fetal maternal exosomes have statistical difference. ROC curve analysis DNM2 diagnosed congenital heart disease with 78.68% sensitivity and 80.65% specificity. The expression level of the exosome in the peripheral blood of the mother of the fetus with cleft lip and palate is also obviously reduced, and the exosome is statistically different from the exosome in the group of normal pregnant women. ROC curve analysis DNM2 diagnosed neural tube malformations with a sensitivity of 70.38% and a specificity of 75.55%.
The CORO1A test result shows that the expression level of exosome in the peripheral blood of a mother of a fetus with a neural tube malformation is also obviously reduced, and the statistical difference exists compared with that in a normal pregnant woman group. ROC curve analysis cor 1A diagnosed neural tube malformations with a sensitivity of 73.68% and a specificity of 78.95%. Further analysis of the gestational week of the mother with the neural tube malformation revealed that CORO1A was also better in diagnosis efficiency in the early stage of pregnancy (12-18 weeks of pregnancy), with sensitivity of 85.71% and specificity of 85.71%. The diagnosis efficiency is poor in the late pregnancy, the sensitivity is 75.00%, and the specificity is 83.33%. The expression quantity of peripheral blood exosomes of the congenital heart disease fetal mother is obviously reduced, and compared with a normal pregnant woman group, the fetal heart disease fetal maternal exosomes have statistical difference. ROC curve analysis cor 1A diagnosed congenital heart disease with a sensitivity of 80.11% and specificity of 79.28%. The expression level of the exosome in the peripheral blood of the mother of the fetus with cleft lip and palate is also obviously reduced, and the exosome is statistically different from the exosome in the group of normal pregnant women. ROC curve analysis cor 1A diagnosed neural tube malformations with a sensitivity of 71.22% and a specificity of 77.78%.
The molecular markers selected in the above animal models (CORO1A and DNM2) were verified in peripheral blood exosomes of mother of neural tube malformations, congenital heart disease and cleft lip and palate fetuses by ELISA method, as shown in FIGS. 6-1 to 6-3 and FIGS. 7-1 to 7-3.
Claims (9)
1. The molecular marker for prenatal noninvasive diagnosis of neural tube malformation, congenital heart disease and cleft lip and palate fetus is characterized by consisting of one or more of CORO1A, DNM2 and ACTR2 proteins.
2. Use of the prenatal noninvasive diagnosis molecular marker of claim 1 for the preparation of prenatal screening, early warning, clinical diagnosis and biochemical test products for neural tube malformations, congenital heart disease and cleft lip and palate.
3. The use of claim 2, wherein said product comprises reagents, kits, chips, test strips, high throughput sequencing platforms, and is used for the detection of molecular markers of proteins associated with prenatal non-invasive diagnosis of neural tube abnormalities, congenital heart disease and cleft lip and palate via mass spectrometry, PCR, in situ hybridization, fluorescence in situ hybridization, immunoturbidimetry, radioimmunoassay, and the like.
4. The use of claim 2, wherein the prenatal screening, prewarning, clinical diagnosis and biochemical testing of neural tube malformations, congenital heart disease and cleft lip and palate of a fetus comprises pregnant woman blood and its exosomes, urine exosomes, amniotic fluid and its exosomes and fetal specimens.
5. Use of an agent for detecting the molecular markers of claim 1 for prenatal noninvasive diagnosis of neural tube malformations, congenital heart disease, and cleft lip and palate for the preparation of a prenatal noninvasive diagnosis tool for infants.
6. The use of claim 5, wherein said means for detecting molecular markers for prenatal non-invasive diagnosis of neural tube abnormalities, congenital heart disease, and cleft lip and palate comprises means for quantifying the molecular markers for prenatal non-invasive diagnosis of neural tube abnormalities, congenital heart disease, and cleft lip and palate according to claim 1.
7. The use of claim 6, wherein the agent capable of quantifying the molecular markers of claim 1 for prenatal noninvasive diagnosis of neural tube malformations, congenital heart disease, and cleft lip and palate is a specific primer for a gene or transcript, or a specific recognition probe, or both a primer and a probe.
8. A tool for prenatal screening, pre-warning and diagnosis of neural tube abnormalities, congenital heart diseases and cleft lip and palate, said tool being capable of detecting the molecular expression levels of the molecular markers of claim 1 for prenatal non-invasive diagnosis of neural tube abnormalities, congenital heart diseases and cleft lip and palate.
9. The tool of claim 8, comprising molecular markers capable of quantifying the prenatal noninvasive diagnosis of neural tube malformations, congenital heart disease, and cleft lip and palate of claim 1, said tool comprising a chip, a kit, a test strip, and a high throughput sequencing platform.
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