BMP10 gene mutation related to congenital heart disease complicated with pulmonary arterial hypertension and application of BMP10 gene mutation

Abstract

The invention discloses BMP10 gene mutation related to congenital heart disease combined with pulmonary hypertension and application thereof, the invention discovers pathogenic BMP10 variants related to congenital heart disease combined with pulmonary hypertension for the first time, including missense mutations p.Q74R, p.I335S and p.R96Q and nonsense mutations p.Q116Ter and p.R313Ter, and functional experiments confirm that the mutations are pathogenic mutations, and the BMP10 variants can be used for treating pulmonary hypertension associated with the congenital heart disease combined with the pulmonary hypertension associated with the congenital heart disease combined with the pulmonary hypertension associated with the congenital heart disease combined with the pulmonary hypertension associated with the congenital heart disease combined with the pulmonary hypertension associated with the congenital heart disease combined with the pulmonary hypertension. A new target spot is provided for effective diagnosis or prediction of the congenital heart disease combined with the pulmonary hypertension, and an important foundation is laid for pathogenesis research of the congenital heart disease combined with the pulmonary hypertension.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically, relates to a BMP10 gene mutation associated with congenital heart disease combined with pulmonary hypertension, and more specifically, relates to a BMP10 gene mutation associated with congenital heart disease combined with pulmonary hypertension and a use thereof. Background Art

[0002] Congenital heart disease associated pulmonary arterial hypertension (CHD-PAH) is a type of pulmonary hypertension, accounting for 40%-50% of the first category of pulmonary arterial hypertension. The pathogenesis of CHD-PAH patients is unclear, there is a lack of effective treatment, and the prognosis of patients is poor. Compared with patients with simple CHD, the risk of death in CHD-PAH patients is increased by 4 times. In the late stage of CHD-PAH, bidirectional cardiac shunt will occur, and patients will be in a state of hypoxia for a long time, with a low quality of life and a 10-year survival rate of only 57%. Therefore, CHD-PAH urgently needs to study the disease mechanism in depth, find genes and targets with important functions, explore the causes and laws of the occurrence and development of the disease, and then provide new and effective treatment methods for clinical practice.

[0003] CHD-PAH patients have strong clinical heterogeneity: some patients with similar shunt positions and calibers develop severe PAH or even Eisenmenger syndrome in adolescence, while others do not develop PAH until middle age; some CHD-PAH patients of similar age and similar congenital heart disease defects have good outcomes after surgery, with pulmonary artery pressure almost returning to normal, while others have continued to increase pressure after surgery and have a very poor prognosis. Therefore, in addition to structural defects in congenital heart disease, genetic factors also play an important role in the onset of CHD-PAH. Studies have shown that 6%-7.5% of CHD-PAH patients carry BMPR2 gene mutations, and 3.3% of CHD-PAH patients carry SOX17 gene mutations, suggesting that genetic factors are indeed involved in the course of CHD-PAH. However, overall, there are few studies on the genetics of CHD-PAH, and the existing pathogenic genes can only explain the etiology of a small number of patients, and the genetic etiology of a large number of patients is completely unknown.

[0004] BMP10 is the main ligand of BMPR2. After the Pre-Pro-BMP10 full-length protein is specifically expressed in the right atrium, it is secreted out of the cells under the guidance of the signal peptide and cut by the Fusion enzyme at the 313-316 amino acid (RXXR) position to form the pro-domain and mature-domain. The two domains are covalently bound to form a dimer, which is transported throughout the body through the blood circulation. After binding to receptors such as BMPR2 and ALK1, it plays a role in stabilizing myocardial cells and endothelial cells and resisting pathological damage.

[0005] BMP10 is an important regulatory factor in cardiac development. BMP10 knockout mice have arrested heart development at embryonic E9.0-9.5, accompanied by severe ventricular wall dysplasia and ventricular trabeculae deficiency, and embryonic death at E10.5. After birth, BMP10 protects cardiomyocytes by activating the classical BMPR2 pathway and the non-classical STAT3 pathway, resisting cardiomyocyte apoptosis and damage caused by pathological stimulation. Supplementation of BMP10 can treat heart diseases such as heart failure with preserved ejection fraction (HFpEF), left ventricular hypertrophy, and left ventricular noncompaction. The receptor BMPR2 of BMP10 is highly expressed in pulmonary vascular endothelial cells. BMP10 also plays an important role in maintaining the homeostasis of pulmonary vascular endothelial cells and regulating pulmonary vascular remodeling function by activating the BMPR2 signaling pathway.

[0006] Given that BMP10 plays an important role in cardiac development and pulmonary vascular function, we speculate that this gene mutation is likely to be involved in the disease process of congenital heart disease combined with pulmonary hypertension. We searched the literature and found that BMP10 mutations are extremely rare. So far, only 6 BMP10 mutations have been reported, including 5 missense mutations (p.T53I, p.M416I, p.R353C, p.E83K, p.V407I) and one truncated mutation (p.R124Ter). These mutation carriers show hereditary pulmonary hypertension, idiopathic pulmonary hypertension, and left ventricular noncompaction disease phenotypes, and no CHD-PAH related reports have been found.

[0007] In this project, the inventors first established a CHD-PAH clinical cohort and biological sample library in the Fuwai Hospital of the Chinese Academy of Medical Sciences, and detected rare and harmful variants of the BMP10 gene in CHD-PAH patients through whole genome sequencing; then, we detected rare and harmful variants of BMP10 in internal healthy controls and external general population controls, and clarified the diagnostic value of BMP10 mutation for CHD-PAH by comparing the mutation frequency of BMP10 in CHD-PAH patients and the two groups of controls; finally, we detected the effect of BMP10 mutation on protein expression and secretion at the molecular level to clarify the harmfulness of the mutation. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a BMP10 gene mutation associated with congenital heart disease combined with pulmonary hypertension and uses thereof.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect of the present invention, the present invention provides a biomarker associated with congenital heart disease combined with pulmonary hypertension.

[0011] Further, the biomarkers include mutant BMP10 protein and mutant BMP10 gene;

[0012] The mutant BMP10 protein has any one or more of the following mutation sites relative to the wild-type BMP10 protein: p.Q74R, p.I335S, p.Q116Ter, p.R313Ter, p.R96Q;

[0013] The mutant BMP10 gene has any one or more of the following mutation sites relative to the wild-type BMP10 gene: c.221A>G, c.1004T>G, c.346C>T, c.937C>T, c.287G>A.

[0014] In a specific embodiment of the present invention, the wild-type BMP10 gene has a Gene ID of 27302 in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and its full name is bone morphogenetic protein 10 [Homo sapiens (human)], and the wild-type BMP10 protein is the protein corresponding to the wild-type BMP10 gene with Gene ID 27302 as described above. The detailed information of the wild-type BMP10 gene and the wild-type BMP10 protein are recorded in the NCBI database, and those skilled in the art can obtain the required information from the database.

[0015] In some embodiments, the mutation site or sequence is referenced to the content included in the NCBI database. Those skilled in the art should understand that due to updates to the database or differences in databases, the mutation sites and sequences shown may be slightly different or changed. These differences or changes can be found based on the content of the database given as a standard, and these differences or changes are also included in the scope of protection of the present invention.

[0016] In some embodiments, those skilled in the art should understand that the wild-type BMP10 gene sequence position described herein is based on the sequence of the wild-type BMP10 gene in the human genome, but when the wild-type BMP10 gene exists in other species, the sequence may be different, and the wild-type BMP10 gene of the species can be compared with the wild-type BMP10 gene in the human genome to obtain the corresponding position in the wild-type BMP10 gene of the species.

[0017] In a specific embodiment of the present invention, the inventors of the present invention discovered for the first time that the following mutations of BMP10 protein: p.Q74R, p.I335S, p.Q116Ter, p.R313Ter, p.R96Q, and the following mutations of BMP10 gene: c.221A>G, c.1004T>G, c.346C>T, c.937C>T, c.287G>A, are closely related to the onset of congenital heart disease combined with pulmonary hypertension. Therefore, by detecting whether the above-mentioned protein mutations or gene mutations exist in biological samples derived from the subject, it is possible to effectively detect whether the subject suffers from congenital heart disease combined with pulmonary hypertension.

[0018] In addition, the present invention also provides a construct, which comprises the gene mutation as described above.

[0019] Further, the construct containing the gene mutation as described above means that compared with the wild-type BMP10 gene, the nucleic acid contained therein has any one or more of the following mutations: c.221A>G, c.1004T>G, c.346C>T, c.937C>T, c.287G>A, and thus, the recombinant cells obtained by transforming the receptor cells with the construct provided by the present invention can be effectively used as a model for related research on congenital heart disease combined with pulmonary hypertension. The type of the receptor cell is not particularly limited, for example, it can be an Escherichia coli cell or a mammalian cell, and preferably, the receptor cell is derived from a mammal.

[0020] In some embodiments, the form of the construct is not particularly limited, and it can be at least one of a plasmid, a bacteriophage, an artificial chromosome, a cosmid, and a virus, preferably a plasmid. Plasmid, as a genetic vector, has simple operation and can carry the property of a larger fragment, which is convenient for operation and processing. The form of the plasmid is also not particularly limited, and can be either a circular plasmid or a linear plasmid, and can be either single-stranded or double-stranded. Those skilled in the art can select as needed.

[0021] In the present invention, the nucleic acid can be any polymer containing deoxyribonucleotides or ribonucleotides, including but not limited to: modified or unmodified DNA, RNA, and its length is not subject to any special restrictions. For constructs used to construct recombinant cells, it is preferred that the nucleic acid is DNA, because DNA is more stable and easy to handle than RNA.

[0022] In addition, the present invention also provides a recombinant cell, which is obtained by transforming the construct as described above into a receptor cell. The recombinant cell provided by the present invention can be effectively used as a model for related research on congenital heart disease combined with pulmonary hypertension.

[0023] In a second aspect of the present invention, the present invention provides the use of a reagent for detecting biomarkers associated with congenital heart disease combined with pulmonary hypertension in the preparation of a product for diagnosing congenital heart disease combined with pulmonary hypertension;

[0024] The biomarkers include mutant BMP10 protein and mutant BMP10 gene;

[0025] The mutant BMP10 protein has any one or more of the following mutation sites relative to the wild-type BMP10 protein: p.Q74R, p.I335S, p.Q116Ter, p.R313Ter, p.R96Q;

[0026] The mutant BMP10 gene has any one or more of the following mutation sites relative to the wild-type BMP10 gene: c.221A>G, c.1004T>G, c.346C>T, c.937C>T, c.287G>A;

[0027] Preferably, the diagnosis of congenital heart disease combined with pulmonary hypertension includes any one or more of the following aspects:

[0028] (1) Diagnose whether the subject has congenital heart disease combined with pulmonary hypertension;

[0029] (2) assess the risk of subjects suffering from congenital heart disease combined with pulmonary hypertension;

[0030] (3) Indicates the risk of offspring of carriers of the mutant BMP10 gene developing congenital heart disease combined with pulmonary hypertension.

[0031] In the present invention, the diagnosis of whether a subject suffers from congenital heart disease combined with pulmonary hypertension includes diagnosing whether a subject with congenital heart disease has pulmonary hypertension and diagnosing whether a subject has congenital heart disease combined with pulmonary hypertension; the assessment of the risk of a subject suffering from congenital heart disease combined with pulmonary hypertension includes assessing the risk of a subject with congenital heart disease combined with pulmonary hypertension and assessing the risk of a subject suffering from congenital heart disease combined with pulmonary hypertension.

[0032] Furthermore, the reagent includes a detection reagent that specifically recognizes the mutant BMP10 protein and / or a detection reagent for the mutant BMP10 gene.

[0033] Furthermore, the detection reagent that specifically recognizes the mutant BMP10 protein includes an antibody, an antibody fragment and / or an affinity protein that specifically binds to the mutant BMP10 protein.

[0034] Furthermore, the detection reagent for the mutant BMP10 gene includes primers for specifically amplifying the mutant BMP10 gene and / or probes for specifically identifying the mutant BMP10 gene.

[0035] Furthermore, the products include detection kits, detection chips, and detection test strips.

[0036] In some embodiments, the reagents include (but are not limited to) at least one of antibodies, probes, primers, and mass spectrometry detection reagents specific for the gene mutation and / or protein mutation.

[0037] In certain specific embodiments, a person skilled in the art can detect whether the above-mentioned protein mutation exists in a test sample derived from a subject by specifically binding an antibody that specifically recognizes the protein mutation to the protein mutation site, that is, detecting whether the above-mentioned protein mutation exists in a test sample derived from a subject by the interaction between the specific antibody and the antigen.

[0038] In certain specific embodiments, those skilled in the art may also pre-design a probe that specifically recognizes the gene mutation, and identify whether the gene mutation is present in the test sample from the subject by complementary pairing of the probe with the nucleic acid fragment where the gene mutation site is located.

[0039] In certain specific embodiments, a person skilled in the art may also design specific primers for amplifying the exons where the above-mentioned gene mutations are located, and then determine whether the above-mentioned gene mutations exist in the test sample derived from the subject through gene amplification and sequencing.

[0040] In certain specific embodiments, a person skilled in the art may also detect the m / z of a protein (polypeptide) by mass spectrometry to determine whether the polypeptides with the p.Q74R, p.I335S, p.Q116Ter, p.R313Ter, and p.R96Q mutations are present in the test sample derived from the subject.

[0041] In some embodiments, the detection kit may also include a variety of different reagents suitable for practical use (such as for different detection methods), and is not limited to the reagents currently listed in the present invention. As long as it is a reagent for diagnosing congenital heart disease combined with pulmonary hypertension based on the detection of gene mutations and / or protein mutations described in the first aspect of the present invention, it is included in the scope of the present invention.

[0042] In some embodiments, the preparation of the detection chip can adopt the conventional preparation method of biochip known to those skilled in the art, including but not limited to: using a solid phase carrier of a modified glass slide or silicon wafer, the 5' end of the probe contains an amino-modified poly dT string, the oligonucleotide probe is prepared into a solution, and then it is spotted on the modified glass slide or silicon wafer using a spotter, arranged into a predetermined sequence or array, and then fixed by leaving it overnight to obtain the chip of the present invention; if the nucleic acid does not contain amino modification, its preparation method can also refer to: "Gene Diagnosis Technology-Non-Radioactive Operation Manual" edited by Wang Shenwu; JLerisi, VRIyer, POBROWN. Exploring the metabolic and genetic control of gene expression on agenomic scale. Science, 1997; 278: 680 and Ma Liren, Jiang Zhonghua, eds. Biochip. Beijing: Chemical Industry Press, 2000, 1-130.

[0043] In some embodiments, at least one of the antibodies, probes, primers and mass spectrometry detection reagents described above can specifically and highly sensitively screen out the gene mutations or protein mutations described above, and then specifically and highly sensitively screen out patients with congenital heart disease and pulmonary hypertension among subjects, and can then be effectively used to prepare a kit or device for screening patients with congenital heart disease and pulmonary hypertension.

[0044] In a third aspect of the present invention, the present invention provides a method for screening candidate drugs for treating and / or preventing congenital heart disease combined with pulmonary hypertension.

[0045] Further, the method comprises the following steps:

[0046] (1) placing a first biological sample capable of expressing the mutant BMP10 protein and / or mutant BMP10 gene described in the first aspect of the present invention in the presence of a drug to be tested for culturing;

[0047] (2) culturing a second biological sample capable of expressing the mutant BMP10 protein and / or mutant BMP10 gene described in the first aspect of the present invention in the absence of the drug to be tested;

[0048] (3) Detecting the expression level of BMP10 in the first biological sample and the second biological sample. If the expression level of BMP10 in the first biological sample is higher than that in the second biological sample, it indicates that the drug to be tested can be used as a candidate drug for treating and / or preventing congenital heart disease combined with pulmonary hypertension.

[0049] In the fourth aspect of the present invention, the present invention provides the use of the mutant BMP10 protein and / or mutant BMP10 gene described in the first aspect of the present invention in screening candidate drugs for treating and / or preventing congenital heart disease combined with pulmonary hypertension.

[0050] Furthermore, the method for screening candidate drugs is as described in the third aspect of the present invention.

[0051] In a fifth aspect of the present invention, the present invention provides a diagnostic reagent for diagnosing congenital heart disease combined with pulmonary hypertension.

[0052] Further, the diagnostic reagent includes a reagent that can be used to detect the mutant BMP10 protein and / or mutant BMP10 gene described in the first aspect of the present invention;

[0053] Preferably, the reagent includes a detection reagent that specifically recognizes the mutant BMP10 protein and / or a detection reagent for the mutant BMP10 gene;

[0054] More preferably, the detection reagent that specifically recognizes the mutant BMP10 protein includes an antibody, an antibody fragment and / or an affinity protein that specifically binds to the mutant BMP10 protein;

[0055] More preferably, the detection reagent for the mutant BMP10 gene includes primers for specifically amplifying the mutant BMP10 gene and / or probes for specifically recognizing the mutant BMP10 gene;

[0056] Preferably, the diagnosis of congenital heart disease combined with pulmonary hypertension includes any one or more of the following aspects:

[0057] (1) Diagnose whether the subject has congenital heart disease combined with pulmonary hypertension;

[0058] (2) assess the risk of subjects suffering from congenital heart disease combined with pulmonary hypertension;

[0059] (3) Indicates the risk of offspring of carriers of the mutant BMP10 gene developing congenital heart disease combined with pulmonary hypertension.

[0060] In a sixth aspect of the present invention, the present invention provides an in vitro diagnostic kit for diagnosing congenital heart disease combined with pulmonary hypertension.

[0061] Further, the in vitro diagnostic kit comprises the diagnostic reagent described in the fifth aspect of the present invention;

[0062] Preferably, the in vitro diagnostic kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant or a solvent;

[0063] Preferably, the in vitro diagnostic kit further comprises a buffer, dNTPs, and a polymerase;

[0064] Preferably, the diagnosis of congenital heart disease combined with pulmonary hypertension includes any one or more of the following aspects:

[0065] (1) Diagnose whether the subject has congenital heart disease combined with pulmonary hypertension;

[0066] (2) assess the risk of subjects suffering from congenital heart disease combined with pulmonary hypertension;

[0067] (3) Indicates the risk of offspring of carriers of the mutant BMP10 gene developing congenital heart disease combined with pulmonary hypertension.

[0068] In addition, the present invention also provides a medicine for treating and / or preventing congenital heart disease combined with pulmonary hypertension.

[0069] Furthermore, the drug comprises an agent capable of specifically changing the gene mutation or protein mutation described in the first aspect of the present invention.

[0070] Furthermore, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease.

[0071] In a specific embodiment of the present invention, the specific change refers to the ability to restore the site of gene mutation and / or protein mutation to the original wild state or other non-pathogenic state, without having a substantial impact on other sequences of the individual genome. As mentioned above, the gene mutation and / or protein mutation described in the first aspect of the present invention are closely related to the onset of congenital heart disease combined with pulmonary hypertension. Therefore, the drugs prepared by these reagents that can specifically change the aforementioned gene mutation and / or protein mutation can be effectively used to treat and / or prevent congenital heart disease combined with pulmonary hypertension.

[0072] In some embodiments, the reagent is a reagent based on at least one of shRNA, antisense nucleic acid, ribozyme, dominant negative mutation, CRISPR-Cas9, CRISPR-Cpf1 and zinc finger nuclease. For example, CRISPRs technology is a technology that guides Cas protein to modify targeted genes by RNA. CRISPR-Cas9 mainly achieves genome modification through three approaches: gene knockout, introduction of special mutations and site-directed transgenics. Based on the CRISPR-Cas9 method, technicians in this field can design sgRNA and synthesize gRNA of the sequence, and then co-express gRNA with d Cas9 in cells, and mediate the binding of d Cas9 protein to the target DNA region through gRNA, thereby achieving repair or change of specific sites.

[0073] In addition, the present invention also provides the use of the reagent as described above that can specifically change the gene mutation or protein mutation described in the first aspect of the present invention in the preparation of a drug for treating and / or preventing congenital heart disease combined with pulmonary hypertension.

[0074] In addition, the present invention also provides an application of a biological model in screening drugs for treating and / or preventing congenital heart disease combined with pulmonary hypertension.

[0075] Further, the biological model carries the gene mutation and / or protein mutation described in the first aspect of the present invention;

[0076] Preferably, the biological model is a cell model or an animal model.

[0077] In a specific embodiment, the biological model carrying the gene mutation described in the first aspect of the present invention refers to a biological model in which the BMP10 gene carried by the biological model has any one or more of the following mutation sites compared with the wild-type BMP10 gene: c.221A>G, c.1004T>G, c.346C>T, c.937C>T, c.287G>A.

[0078] In a specific embodiment, the biological model carrying the protein mutation described in the first aspect of the present invention refers to the BMP10 protein carried by the biological model having any one or more of the following mutation sites compared with the wild-type BMP10 protein: p.Q74R, p.I335S, p.Q116Ter, p.R313Ter, p.R96Q.

[0079] In addition, the present invention also provides a biological model for screening drugs for treating and / or preventing congenital heart disease combined with pulmonary hypertension.

[0080] Furthermore, the biological model is as described above, and carries the gene mutation and / or protein mutation described in the first aspect of the present invention.

[0081] In addition, the present invention also provides a method for diagnosing whether a subject in need suffers from congenital heart disease combined with pulmonary hypertension, the method comprising the following steps:

[0082] (1) extracting nucleic acid samples from biological samples obtained from subjects;

[0083] (2) judging whether the subject suffers from congenital heart disease combined with pulmonary hypertension based on the nucleic acid sequence or its complementary sequence of the nucleic acid sample and whether it has c.221A>G, c.1004T>G, c.346C>T, c.937C>T and / or c.287G>A mutations compared with the wild-type BMP10 gene;

[0084] Among them, the nucleic acid sequence or its complementary sequence of the nucleic acid sample has c.221A>G, c.1004T>G, c.346C>T, c.937C>T and / or c.287G>A mutations compared with the wild-type BMP10 gene, which is an indication that the subject suffers from congenital heart disease combined with pulmonary hypertension.

[0085] In some embodiments, the biological sample described in the present invention is the same as the biological specimen, and is derived from a subject in need. The type of biological sample is not particularly limited, as long as the biological sample can be extracted to obtain a gene reflecting whether the BMP10 gene in the biological sample has the mutation described above. In a specific embodiment, the biological sample includes but is not limited to: at least one of blood, tissue, and serum from a subject. In a preferred embodiment, the biological sample is blood.

[0086] In some embodiments, the subject of the present invention is not particularly limited, including but not limited to: mammals, such as primates (e.g., humans, chimpanzees or monkeys), rodents (e.g., rats, mice, guinea pigs, gerbils or hamsters), lagomorphs (e.g., rabbits), porcine animals (e.g., pigs), equines (e.g., horses), canines (e.g., dogs) or felines (e.g., cats). In a specific embodiment of the present invention, the subject is preferably a human.

[0087] In some embodiments, the nucleic acid sample should be understood in a broad sense, and it can be any sample that can reflect whether the BMP10 gene in the biological sample has the mutation, for example, it can be the whole genome DNA directly extracted from the biological sample, or it can be a part of the whole genome containing the BMP10 gene coding sequence, it can be the total RNA extracted from the biological sample, or it can be the mRNA extracted from the biological sample. In a specific embodiment, the nucleic acid sample is the whole genome DNA. Thus, the source range of the biological sample can be expanded, and multiple information of the biological sample can be determined at the same time, so as to improve the efficiency of screening biological samples with congenital heart disease and pulmonary hypertension. In addition, for the use of RNA as a nucleic acid sample, extracting a nucleic acid sample from a biological sample can further include: extracting an RNA sample from the biological sample, preferably the RNA sample is mRNA; and based on the obtained RNA sample, obtaining a cDNA sample through a reverse transcription reaction, and the obtained cDNA sample constitutes a nucleic acid sample. Thus, the efficiency of using RNA as a nucleic acid sample to screen biological samples with congenital heart disease and pulmonary hypertension can be further improved.

[0088] In some embodiments, after obtaining the nucleic acid sample, the nucleic acid sample can be analyzed so as to determine the nucleic acid sequence of the obtained nucleic acid sample. The method and device for determining the nucleic acid sequence of the obtained nucleic acid sample are not particularly limited. In a specific embodiment, the nucleic acid sequence of the nucleic acid sample can be determined by a sequencing method. It should be noted that the method and device for sequencing are not particularly limited. The second generation sequencing technology can be used, and the third generation and fourth generation or more advanced sequencing technology can be used. The nucleic acid sequence can be sequenced using at least one selected from BGISEQ-500, BGISEQ-500RS, HISEQ2000, SOLiD, 454 and single molecule sequencing devices. Thus, combined with the latest sequencing technology, a higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved, so that the high throughput and deep sequencing characteristics of these sequencing devices can be used to further improve the efficiency of detecting and analyzing nucleic acid samples. Thus, the precision and accuracy of subsequent analysis of sequencing data can be improved.

[0089] In some embodiments, regarding the method and process of constructing a sequencing library for a nucleic acid sample, a person skilled in the art can make an appropriate selection according to different sequencing technologies. For details of the process, reference can be made to the procedures provided by the manufacturer of the sequencing instrument, such as Illumina, for example, see Illumina's Multiplexing Sample Preparation Guide (Part#1005361; Feb 2010) or Paired-End Sample Prep Guide (Part#1005063; Feb 2010), which are incorporated herein by reference. In the present invention, the method and apparatus for extracting nucleic acid samples from biological samples are not particularly limited, and can be performed using a commercial nucleic acid extraction kit.

[0090] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0091] The inventors of the present invention have discovered pathogenic BMP10 variants associated with congenital heart disease combined with pulmonary hypertension for the first time through extensive and in-depth research, including missense mutations p.Q74R, p.I335S, p.R96Q and nonsense mutations p.Q116Ter, p.R313Ter, and confirmed through functional experiments that the mutations can lead to reduced BMP10 protein expression, thereby leading to the occurrence of congenital heart disease combined with pulmonary hypertension. The diagnostic reagent or kit prepared with the newly discovered mutations for the first time as the target can be used for the effective diagnosis or prediction of congenital heart disease combined with pulmonary hypertension, and has good application prospects. In addition, the present invention lays an important foundation for the study of the pathogenesis of congenital heart disease combined with pulmonary hypertension, and provides a new theoretical basis for the diagnosis and treatment of congenital heart disease combined with pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 : Topological analysis of human BMP10 indicates the locations of the five rare variants in the BMP10 gene found in this study. The locations of BMP10 mutations are represented by lollipops of equal size. The key protein domains are: signal peptide, prodomain, and mature domain. Variants are grouped by color i.e. red circles indicate missense and blue circles indicate nonsense. Abbreviation: LoF, loss of function.

[0093] Figure 2 : Distribution of five rare BMP10 variants found in the CHD-PAH cohort on the 3D structure of the BMP10 dimer. Pro-domain: purple and blue, Mature-domain: yellow and green.

[0094] Figure 3: The full-length BMP10 between different species was aligned using the pairwise alignment tool of the Protein Information Resource. Five rare BMP10 mutations were highly conserved between different species, shown in red boxes (http: / / pir.georgetown.edu / pirwww / search / ).

[0095] Figure 4 : Genetic profile of a core family of CHD-PAH patients carrying BMP10 mutations.

[0096] Figure 5 :BMP10 mutant plasmids and WT plasmids were transfected into HEK293E cells and cell lysate proteins were collected and immunoblotted with mature BMP10 or BMP10 precursor specific antibodies. The different forms of WT BMP10 protein were three bands of BMP10 precursor (120 kDa), partially processed BMP10 precursor (60 kDa) and pro domain monomer (43 kDa). GAPDH was used as an internal reference. WT: wild type; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

[0097] Figure 6 :BMP10 mutant plasmids and WT plasmids were transfected into HEK293E cells and the supernatants were collected and immunoblotted with mature BMP10 or BMP10 precursor specific antibodies. WT BMP10 protein has four bands of BMP10 precursor (120kDa), partially processed BMP10 precursor (60kDa), pro domain monomer (43kDa) and mature BMP10 dimer (17kDa). HSP70 was used as an internal reference. WT: wild type; HSP: heat shock protein.

[0098] Figure 7 : After hPAECs were stimulated with or without 20 ng / mL BMP10 for 6 h, the expressions of BMPR2, Smad1 / 5 / 8, and phosphorylated Smad1 / 5 / 8 were detected by Western blot.

[0099] Figure 8 : Schematic diagram of combined surgery; 2 weeks after the combined surgery, echocardiography showed that the anterior wall of the right ventricle of the rats was significantly thickened and the right heart cavity diameter was significantly enlarged. DETAILED DESCRIPTION

[0100] The present invention is further described below in conjunction with specific examples, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0101] Example Discovery of BMP10 gene mutation associated with congenital heart disease and pulmonary hypertension and its functional verification I. Experimental methods

[0102] 1. Study population

[0103] (1) Patient cohort with congenital heart disease and pulmonary arterial hypertension (CHD-PAH): From May 2020 to December 2021, we enrolled 378 patients with CHD-PAH at the Fuwai Hospital of the Chinese Academy of Medical Sciences. All patients underwent detailed clinical examinations, including echocardiography, right cardiac catheterization, blood routine, blood biochemistry, heart failure marker NT-proBNP, etc. All patients signed informed consent and voluntarily provided peripheral blood for genetic testing. The research plan of this subject has been reviewed by the Ethics Committee of the Fuwai Hospital of the Chinese Academy of Medical Sciences.

[0104] Inclusion criteria for CHD-PAH patients: echocardiography was used to detect the patient's cardiac structure and function, to identify the type of congenital heart disease and the location and size of the heart defect. Patients with elevated pulmonary artery pressure indicated by echocardiography underwent right cardiac catheterization. According to the 2018 Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension in China, pulmonary hypertension was diagnosed if the patient's mean pulmonary artery pressure at rest was ≥25 mmHg and pulmonary vascular resistance was ≥3 Wood Units. There were no restrictions on gender, age, or cardiac function classification for the selection of patients.

[0105] Exclusion criteria for CHD-PAH patients: Exclude other types of pulmonary hypertension, such as pulmonary hypertension caused by immune diseases, left heart-related pulmonary hypertension, and pulmonary hypertension related to respiratory diseases.

[0106] (2) Control population: This project includes two groups of control populations. The first group is the internal control, which consists of 1,586 healthy people selected from the community in Beijing. These controls all underwent echocardiography, electrocardiography, and physical examination to exclude congenital heart disease and pulmonary hypertension. The second group is the external control, which downloads the whole exome sequencing data of East Asians from the world's largest genetic database (GnomAD) (https: / / gnomad.broadinstitute.org / ), including a total of 18,403 research subjects without specific cardiovascular diseases.

[0107] (3) Collection of biological samples: 4 mL of overnight fasting venous blood was collected from all cases and controls and anticoagulated with sodium citrate. Within 30 minutes after the blood was removed from the body, the plasma and lymphocytes were separated by centrifugation. The plasma was divided into 2 mL cryovials, quickly frozen in liquid nitrogen, and stored at -80°C.

[0108] (4) Genetic testing: The second-generation sequencing panel established by our research group was used to detect rare harmful variants in all exon regions and exon-intron splicing regions of 22 PAH-related genes. The genetic rare variant analysis software established by the research group was used to annotate the genetic variants of BMP10. Variants that met the following five criteria were defined as rare, harmful variants:

[0109] a. The minimum allele frequency (MAF) in the 1000 Genomes Database and GnomAD Database is < 0.005;

[0110] b. The variant should be a missense variant, nonsense variant, small insertion / deletion variant, loss-of-function variant, or variant affecting RNA splicing, excluding synonymous variants;

[0111] c. The mutation should be located in a highly conserved region;

[0112] d. SIFT, Polyphen-2, Mutation Taster, PROVEAN and other four bioinformatics software were used to evaluate the harmfulness of the variant site, and at least two of them were considered harmful;

[0113] e. Those annotated as harmful in ClinVar and HGMD databases are given priority.

[0114] We performed whole exome sequencing (WES) on 378 CHD-PAH patients and 1586 internal healthy controls. Sequencing experiments were performed using Agilent SureSelect Human All Exon V6 libraries and sequenced using the Illumina Nova-seq platform. Sequencing data were mapped to the human reference genome (GRCh38 / hg38) using the Burrows–Wheeler Aligner (BWA, v0.7.17), and variants were discovered using GATK (v4.3.0.0). Variant and sample quality control was performed using PLINK (v1.90b) and vcftools (v0.1.16). Variants were annotated using the Variant Effect Predictor (VEP, v105) and LOFTEE. This project focused on analyzing genomic intervals related to the BMP10 gene (Chromosome 2, NC_000002.12, 68860909..68871397). We defined variants with a minor allele frequency (MAF) ≤ 0.001 in public population databases as rare variants, and retained frameshift variants, truncating variants, significantly deleterious variants such as changes in stop codons and start codons, and missense mutations that affect amino acid coding among these rare variants. Subsequently, we evaluated rare variants using the 2015 guidelines of the American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP). According to the ACMG recommendations, variants were classified as pathogenic (P, ACMG category 5), likely pathogenic (LP, ACMG category 4), uncertain significance (VUS, ACMG category 3), likely benign (LB, ACMG category 2), or benign (B, ACMG category 1). We only retained variants rated as UVS, LP, and P by ACMG.

[0115] (5) For CHD-PAH patients found to carry rare deleterious variants in BMP10, we will further screen whether the patient carries mutations in 27 other known pathogenic genes of pulmonary arterial hypertension, including BMPR2, EIF2AK4, TBX4, ATP13A3, GDF2, SOX17, AQP1, ACVRL1, SMAD9, ENG, KCNK3, CAV1, SMAD4, SMAD1, KLF2, BMPR1B, KCNA5, KDR, FBLN2, PDGFD, PTGIS, ABCA3, ABCC8, GGCX, KLK1, TOPBP1 and SMAD5. For the rare deleterious variants of BMP10 found in patients by next-generation sequencing, we use Sanger sequencing for verification.

[0116] (6) External controls included 18,403 East Asians. We downloaded exome sequencing data of these controls from the GnomAD database and used a strategy that was exactly the same as the genetic variation analysis of the CHD-PAH patient cohort described above to discover rare, deleterious variants associated with BMP10.

[0117] 2. Cell culture

[0118] HEK-EBNA cell culture

[0119] HEK-EBNA cells were cultured in 10 cm culture dishes using DMEM high-glucose complete medium containing 10% FBS and 1% double antibody. The complete medium was replaced one day after cell recovery. Cells were subcultured after the cell density reached 90%, with an average of one subculture every 2-3 days. When the cell number and state grew well, the cells were subcultured and inoculated into the corresponding culture plates for experiments.

[0120] 3. Construction and transfection of wild-type and mutant plasmids of BMP10

[0121] To construct five mutant plasmids, human wild-type (WT) BMP10 cDNA (1275 bp, NM_014482.3, Tianyi Huiyuan Company) was cloned into the EcoRI / BamHI site of the pcDNA3.1 plasmid. Five mutant plasmids (Q74R, R96Q, I335S, Q116Ter, R313Ter) were constructed based on the WT plasmid using the Quick Change site-directed mutagenesis kit (Stratagene, LaJolla, CA, USA), and all constructs were sequenced to confirm that the plasmid construction was successful and the mutation site was correct. HEK-EBNA cells were plated in 6 cm culture dishes and cultured to 70%-80% confluence. All constructed expression plasmids were transfected into HEK-EBNA cells using Lipofectamine 3000 (Invitrogen, L3000150). After 6 h, complete culture medium without dual antibodies was replaced and cultured for a further 24 h. Then, CDCHO medium (Gbico, A11483-01) was replaced and cultured for 48 h. Cell supernatants and cell lysates were collected and stored at -80°C.

[0122] 4. Non-reducing denaturing Western Blot

[0123] The supernatant and lysate of HEK-EBNA cells transfected with wild-type or mutant BMP10 plasmids were collected, electrophoresed at a constant voltage of 80V for 2h using non-reducing NuPAGE precast gel (Invitrogen, NP0336BOX), and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes under constant current conditions of 250mA. The PVDF membrane was then blocked with 5% skim milk powder for 1h, and immunoblotted using anti-human BMP10 propeptide antibody (R&D Systems, BAF3956) and anti-human mature BMP10 antibody (R&D Systems, MAB2926), and the target protein was detected by chemiluminescence. The internal reference for all cell lysate proteins was GAPDH (proteintech) and the internal reference for all cell supernatant proteins was Hsp70 (BioSS, bs-0244R).

[0124] 5. Statistical analysis

[0125] Graphpad prism software was used for statistical processing and analysis. Mean ± standard deviation (x ± s) was used to express continuous variables. One-way ANOVA was used to compare the mean differences among multiple groups. Independent sample t-test was used to analyze the mean differences between two groups. P < 0.05 indicated that the difference reached a significant level.

[0126] 2. Experimental Results

[0127] 1. A novel potentially pathogenic BMP10 mutation was found in CHD-PAH patients

[0128] Among 378 CHD-PAH patients, we found a total of 5 patients carrying 5 BMP10 mutations, with a mutation rate of (1.32%). Specifically, 3 of these 5 mutations were missense mutations (p.Ile335Ser, p.Arg96Gln, p.Gln74Arg), and two were truncating mutations (p.Arg313Ter, p.Gln116Ter). All of these mutations are extremely rare (MAF < 0.0001), affect protein function or integrity, and are potentially harmful (ACMG scores are VUS, likelypathogenic or pathogenic) (Table 1). The above mutations are distributed in the two main domains of BMP10 (see Figure 1 and Figure 2). All five CHD-PAH patients carrying mutations were diagnosed with severe pulmonary hypertension by right cardiac catheterization. Their pulmonary artery mean pressure ranged from a minimum of 52 mmHg to a maximum of 82 mmHg, and pulmonary vascular resistance ranged from a minimum of 7.6 Wood units to a maximum of 23.8 Wood units (Table 1). Regarding the three BMP10 missense mutations, prediction software analysis showed that these mutations were deleterious and located in a highly conserved region of BMP10 in various species (see Figure 3 ). This suggests that BMP10 gene mutations are likely to be involved in the onset of CHD-PAH. We reviewed 27 known pulmonary hypertension pathogenic gene mutations in 5 patients with BMP10 mutations (see the Methodology section for a list of 27 genes) and found that they only carried BMP10 mutations and no mutations in other known genes, suggesting that BMP10 mutations are the main genetic cause of these patients.

[0129] Table 1 Clinical phenotype and genotype of 5 CHD-PAH patients carrying BMP10 gene mutation

[0130]

[0131] Notes: F: female, M: male. VSD: ventricular septal defect; ASD: atrial septal defect; PAH: pulmonary arterial hypertension; MAF: minor allele frequency. VUS: undetermined clinical significance; LP: possibly harmful; LB: probably benign, where, in the amino acid change, "Ter" is used to indicate protein mutation when a stop codon appears, and there is no corresponding amino acid. NA: The mutation has not been reported in the database.

[0132] 2. BMP10 mutation has significant molecular diagnostic value for CHD-PAH

[0133] (1) Comparison between CHD-PAH patients and healthy controls

[0134] To clarify the molecular diagnostic value of BMP10 mutations in CHD-PAH patients, we enrolled 1586 healthy controls and performed whole exome sequencing. Using genetic variation screening conditions consistent with those of CHD-PAH patients, we found only one rare, deleterious variation (BMP10, c.A1106G, p.H369R) in 1586 healthy controls.

[0135] Furthermore, we analyzed the diagnostic value of BMP10 mutations for CHD-PAH patients. Taking all BMP10 mutations (including truncating mutations and missense mutations) as the analysis object, the mutation rate of CHD-PAH patients was 1.32%, while that of healthy controls was only 0.06%. The overall mutation rate of BMP10 in CHD-PAH was much higher than that in controls (P = 6.6 × 10 -5)(Table 2). Using BMP10 mutation as a diagnostic indicator for CHD-PAH, the specificity was 0.83, the sensitivity was 0.81, the positive predictive value was 0.19, and the negative predictive value was 0.81, which has good molecular diagnostic value.

[0136] Table 2BMP10 mutation frequency in CHD-PAH patients and healthy controls

[0137] CHD-PAH patients Healthy controls Number of people carrying BMP10 mutations 5 1 Number of people without BMP10 mutation 373 1586 Mutation rate 1.32％ 0.06％

[0138] (2) Comparison between CHD-PAH patients and the general population

[0139] We downloaded whole exome sequencing data of 18,403 East Asians from the public database GnomAD. Using genetic variation screening conditions consistent with those of CHD-PAH patients, we found only 9 subjects carrying rare harmful mutations in BMP10, with a mutation rate of 0.05% (9 / 18,403) (Table 3). The BMP10 mutation rate in the GnomAD database (0.05%) is basically consistent with that in our internal database (0.06%), suggesting that BMP10 mutations are extremely rare and relatively stable in healthy people.

[0140] The mutation rate of BMP10 gene in CHD-PAH patients was much higher than that in the general population (1.32% vs 0.05%, P < 1×10 -8 ). Using BMP10 mutation as a diagnostic indicator for CHD-PAH, compared with the general population of GnomAD, the specificity was 0.35, the sensitivity was 0.98, the positive predictive value was 0.02, and the negative predictive value was 0.98, which has a good molecular diagnostic value.

[0141] Table 3 Mutation frequency of BMP10 in CHD-PAH patients and the general population

[0142] CHD-PAH patients GnomAD Database Number of people carrying BMP10 mutations 5 9 Number of people without BMP10 mutation 373 18394 Mutation rate 1.32％ 0.05％

[0143] 3. BMP10 mutation can directly cause PAH, and BMP10 mutation plus CHD will further aggravate the patient's PAH

[0144] In order to clarify the inheritance pattern of the mutation, the present invention tested the direct relatives (father, mother or grandparents) of the patients carrying the mutation. Among the 5 patients carrying the mutation, a total of 4 patients provided core family samples (see Figure 4). All patients inherited the mutation from their father or mother. In another family (No. 1), the father's sample was not detected, but the grandmother also suffered from CHD. Families 1-3 all showed a consistent inheritance pattern: family members carrying the mutation will only have pulmonary hypertension if they have CHD, and there will be no pulmonary hypertension if they do not have CHD, suggesting that BMP10 mutation plus CHD is the real cause of PAH in patients. In family No. 4, the patient and his mother both carried the same rare BMP10 variant (c.1004T>Gp.Ile335Ser), and the patient and his mother both suffered from CHD-PAH.

[0145] 4. BMP10 mutation affects protein expression, maturation and secretion

[0146] The five BMP10 mutations we found in CHD-PAH patients were distributed in two major domains: pro-BMP10 and mature-BMP10 ( Figure 1 and Figure 2 ). The amino acids affected by the five mutations are all highly conserved in biological evolution, suggesting that they may play an important role in protein function.

[0147] To clarify the effect of BMP10 mutation on protein, we constructed wild-type BMP10 and five mutant BMP10 plasmids, transfected them into human embryonic kidney HEK293E cells, and detected the expression of Pro-BMP10 and BMP10 in cell lysate and cell supernatant, respectively. Western blotting results showed that wild-type (WT) and R96Q missense plasmids expressed similar levels of BMP10 proprotein in cell supernatant and cell lysate (see Figure 5 Top right, bottom right and Figure 6 The Q74R and I335S missense plasmids did not express or expressed very low levels of any BMP10 protein in the supernatant (see Figure 6 ), the BMP10 proprotein encoded by the I335S plasmid in the cell lysate was abnormal in form and had an increased molecular weight (see Figure 5 and Figure 6 The possible reason is that the expression level of BMP10 preprotein is reduced and the amino acid is mutated from isoleucine to methionine, which is easy to form disulfide bonds with other short peptides. The two truncated plasmids Q116Ter and R313Ter do not express any form of BMP10 protein (see Figure 5 and Figure 6), the above results suggest that the four mutants p.Q74, p.I335S, p.Q116Ter, and p.R313Ter significantly affect the expression of BMP10 in cells. Although the mutant plasmid p.R96Q can be expressed in cells, the amount of BMP10 in the cell supernatant is significantly reduced. This suggests that the mutation is likely to affect the exocytosis of BMP10, thereby weakening the function of BMP10. In addition, the present invention also proves through animal experiments that the loss of BMP10 will significantly increase vascular remodeling and right ventricular hypertrophy in pulmonary hypertension.

[0148] The above results indicate that the BMP10 mutations (BMP10 mutations p.Q74R, p.I335S, p.Q116Ter, p.R313Ter or p.R96Q) carried by the above five types of CHD-PAH patients can lead to decreased BMP10 protein expression, thereby leading to the occurrence of congenital heart disease combined with pulmonary hypertension. Therefore, the BMP10 mutations p.Q74R, p.I335S, p.Q116Ter, p.R313Ter or p.R96Q are pathogenic mutations.

[0149] 5. Wild-type BMP10 has the function of activating and upregulating the BMPR2 pathway

[0150] The present invention uses synthetic wild-type human BMP10 to stimulate human pulmonary artery endothelial cells (PAEC). Compared with the control group, the expression of BMPR2 and downstream target gene ID-1 in the BMP10 stimulation group was significantly upregulated (see Figure 7 ), suggesting that wild-type BMP10 has the function of activating the BMPR2 pathway.

[0151] 6. Cardiac remodeling and increased mean pulmonary artery pressure occurred in rat models of congenital heart disease and pulmonary hypertension

[0152] The present invention constructs a rat model of congenital heart disease combined with pulmonary hypertension. The model is divided into two stages of surgery. In the first stage of surgery, the right pulmonary artery of the rat is ligated to increase the blood volume of the left lung. One week later, the second stage of surgery is performed to anastomose the left carotid artery and jugular vein of the rat to create a left carotid artery-venous shunt. After the two operations, the pulmonary artery pressure of the rat gradually increased. Two weeks after the operation, echocardiography showed that the right heart free wall of the rats in the surgery group was thickened and the cavity diameter was enlarged (see Figure 8 ).

[0153] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A biomarker related to congenital heart disease combined with pulmonary hypertension, characterized in that: The biomarkers include mutant BMP10 protein and mutant BMP10 gene; The mutant BMP10 protein has any one or more of the following mutation sites relative to the wild-type BMP10 protein: p.Q74R, p.I335S, p.Q116Ter, p.R313Ter, p.R96Q; The mutant BMP10 gene has any one or more of the following mutation sites relative to the wild-type BMP10 gene: c.221A>G, c.1004T>G, c.346C>T, c.937C>T, c.287G>A.

2. Use of reagents for detecting biomarkers related to congenital heart disease combined with pulmonary hypertension in the preparation of products for diagnosing congenital heart disease combined with pulmonary hypertension; The biomarkers include mutant BMP10 protein and mutant BMP10 gene; The mutant BMP10 protein has any one or more of the following mutation sites relative to the wild-type BMP10 protein: p.Q74R, p.I335S, p.Q116Ter, p.R313Ter, p.R96Q; The mutant BMP10 gene has any one or more of the following mutation sites relative to the wild-type BMP10 gene: c.221A>G, c.1004T>G, c.346C>T, c.937C>T, c.287G>A.

3. The use according to claim 2, characterized in that: The diagnosis of congenital heart disease combined with pulmonary hypertension includes any one or more of the following aspects: (1) Diagnose whether the subject has congenital heart disease combined with pulmonary hypertension; (2) assess the risk of subjects suffering from congenital heart disease combined with pulmonary hypertension; (3) Indicates the risk of offspring of carriers of the mutant BMP10 gene developing congenital heart disease combined with pulmonary hypertension.

4. The use according to claim 2, characterized in that: The reagents include a detection reagent that specifically recognizes the mutant BMP10 protein and / or a detection reagent targeting the mutant BMP10 gene.

5. The use according to claim 4, characterized in that: The detection reagent that specifically recognizes the mutant BMP10 protein includes an antibody, an antibody fragment and / or an affinity protein that specifically binds to the mutant BMP10 protein.

6. The use according to claim 4, characterized in that: The detection reagent for the mutant BMP10 gene includes primers for specifically amplifying the mutant BMP10 gene and / or probes for specifically identifying the mutant BMP10 gene.

7. The use according to claim 2, characterized in that: The products include detection kits, detection chips or detection test strips.

8. A diagnostic reagent for diagnosing congenital heart disease combined with pulmonary hypertension, characterized in that: The diagnostic reagent includes a reagent that can be used to detect the mutant BMP10 protein and / or mutant BMP10 gene described in claim 1.

9. The diagnostic reagent according to claim 8, characterized in that The reagents include a detection reagent that specifically recognizes the mutant BMP10 protein and / or a detection reagent for the mutant BMP10 gene; Optionally, the detection reagent that specifically recognizes the mutant BMP10 protein includes an antibody, an antibody fragment and / or an affinity protein that specifically binds to the mutant BMP10 protein; Optionally, the detection reagent for the mutant BMP10 gene includes primers for specifically amplifying the mutant BMP10 gene and / or probes for specifically recognizing the mutant BMP10 gene; Optionally, the diagnosis of congenital heart disease combined with pulmonary hypertension includes any one or more of the following aspects: (1) Diagnose whether the subject has congenital heart disease combined with pulmonary hypertension; (2) assess the risk of subjects suffering from congenital heart disease combined with pulmonary hypertension; (3) Indicates the risk of offspring of carriers of the mutant BMP10 gene developing congenital heart disease combined with pulmonary hypertension.

10. An in vitro diagnostic kit for diagnosing congenital heart disease combined with pulmonary hypertension, characterized in that: The in vitro diagnostic kit comprises the diagnostic reagent according to claim 8 or 9; Optionally, the in vitro diagnostic kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant or a solvent; Optionally, the in vitro diagnostic kit further comprises a buffer, dNTPs, and a polymerase; Optionally, the diagnosis of congenital heart disease combined with pulmonary hypertension includes any one or more of the following aspects: (1) Diagnose whether the subject has congenital heart disease combined with pulmonary hypertension; (2) assess the risk of subjects suffering from congenital heart disease combined with pulmonary hypertension; (3) Indicates the risk of offspring of carriers of the mutant BMP10 gene developing congenital heart disease combined with pulmonary hypertension.

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