Single-gene hereditary cardiovascular disease related gene detection probe group, kit and application of single-gene hereditary cardiovascular disease related gene detection probe group

Abstract

The invention discloses a single-gene hereditary cardiovascular disease related gene detection probe group, a kit and an application of the single-gene hereditary cardiovascular disease related gene detection probe group. The probe group is a specific probe which is selected from exon regions of genes such as ABCC9, ABCG8, ACADM, ACADS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1 or ALMS1 and the like, and 20-30 bp intron regions on the upstream and downstream of the exon regions of the genes such as ABCC9, ABCG8, ACADM, ACADDS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1 or ALMS1. The detection probe and the kit provided by the invention can accurately detect exon regions of 268 pathogenic gene variations related to the single-gene hereditary cardiovascular disease, intron regions of 20-30 bp upstream and downstream of exon and copy number variations of pathogenic genes, under the average 1G data volume, the nuclear genome coverage depth reaches 200 * on average, the 30 * coverage degree reaches 99%, and the detection sensitivity is high. The detection sensitivity of low-frequency mutation in a target area is improved, the detection cost is effectively controlled, an experimental system is stable, and the discrete coefficient of library results among samples is less than 10%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a single-gene hereditary cardiovascular disease-related gene detection probe set, kit, and their applications. Background Technology

[0002] Precision medicine for cardiovascular diseases is an inevitable trend for the future, both from the perspective of advancements in molecular biology technologies and the current severe situation of clinical cardiovascular disease prevention and treatment. Epidemiological data shows that hypertrophic cardiomyopathy and dilated cardiomyopathy are the most common cardiomyopathy, accounting for more than 90% of all outpatient and inpatient cardiomyopathy patients. The outpatient and inpatient rates for arrhythmogenic cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction are all between 2.1% and 3.6%. These patients can potentially receive early intervention and treatment in the following four stages (preconception-pregnancy-early childhood-adulthood), improving current cardiac health management and disease outcomes. Genetic testing is the main method for achieving this goal. Genetic testing not only helps in the early diagnosis and differential diagnosis of patients with monogenic hereditary cardiovascular diseases and their families, but also plays an important guiding role in prognostic risk stratification, treatment strategy formulation, genetic screening, and selective reproduction.

[0003] Currently, clinical testing for single-gene genetic diseases mainly employs liquid-phase hybridization capture sequencing, multiplex PCR sequencing, and whole-genome sequencing. For some specific diseases, additional technologies, such as methylation MLPA, may be necessary. Liquid-phase hybridization capture sequencing can achieve larger target regions with good uniformity, such as WES (Whole-exome sequencing). Multiplex PCR sequencing offers advantages such as simplicity, flexibility, speed, and low starting amount, making it suitable for detecting smaller gene regions. Whole-genome sequencing can achieve complete coverage of the entire genome.

[0004] While whole-genome sequencing (WGS) and whole-exome sequencing (WES) offer high accuracy and comprehensiveness, their relatively limited sequencing depth can lead to a decrease in the detection rate of low-frequency mutations. Furthermore, whole-genome sequencing is expensive, imposing a significant financial burden on patients. In contrast, panel assays target a specific range of genes, achieving greater coverage depth at the same sequencing throughput. This improves the sensitivity of detecting low-frequency mutations within the target region and effectively controls testing costs.

[0005] While there are currently marketed single-gene hereditary cardiovascular disease testing products based on next-generation sequencing (NGS) technology, some products cover an overly broad range of genes, with insufficient or even controversial evidence linking certain genes to hereditary cardiovascular diseases. These over-amplified gene panels not only increase the complexity of interpreting clinical reports and pose challenges to genetic counseling, but may also cause unnecessary psychological distress and burden on patients. Other products may only cover a single type of cardiovascular disease, forcing patients to undergo multiple tests and incurring financial burdens.

[0006] Therefore, there is an urgent need to develop a highly efficient, accurate, and rapid gene detection kit for monogenic hereditary cardiovascular diseases, which is of great significance for gene screening and molecular genetic diagnosis of monogenic hereditary cardiovascular diseases. Summary of the Invention

[0007] In response to the shortcomings of existing technologies and practical needs, this invention provides a probe set, kit, and application for detecting genes related to single-gene hereditary cardiovascular diseases, which solves the problem and achieves the desired effect.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a detection probe for genes related to single-gene hereditary cardiovascular diseases. The probe is a specific probe for the exon region and intron regions 20-30 bp upstream and downstream of the exon region of the following genes: ABCC9, ABCG8, ACADM, ACADS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1, ALMS1, ALPK3, ANKRD1, APOB, ARSB, ATP13A3, ATP5MK, BAG3, BMPR2, BTD, BVES, CACNA1C, CACNA1H, CALM1, CALM2, CALM3, CASQ2, CAV1, CAV3, CLCN2, COL3A1, COL5A1, COL5... A2, COX10, COX15, CPT1A, CPT2, CRYAB, CSRP3, CTNNA3, CUL3, CYP11B1, CYP11B2, CYP17A1, DES, DLAT, DLD, DSC2, DSG2, DSP, EARS2, ECHS1, EFEM P2, EIF2AK4, ELN, EMD, ENG, EPAS1, ETFA, ETFB, ETFDH, ETHE1, EYA4, F2, F5, F9, FAH, ARS2, FBN1, FBXL4, FH, FHL1, FLNA, FLNC, FOXE3, FOXF1, FO XRED1, FXN, GAA, GALNS, GBA, GBE1, GDF2, GFM1, GFM2, GLA, GLB1, GNB2, GNPTAB, GNPTG, GNS, GRHPR, GTPBP3, GUSB, GYS1, HADH, HADHA, HAMP, HCN 4. HEXB, HFE, HJV, HGSNAT, HIBCH, HOGA1, HPD, HPDL, HRG, HSD11B2, IDS, IDUA, JPH2, JUP, KCNE1, KCNH2, KCNJ2, KCNJ5, KCNK3, KCNQ1, KDR, KLF1 0. KLHL3, LAMP2, LDB3, LDLR, LDLRAP1, LIPT1, LMBRD1, LMNA, LOX, LPL, LRPPRC, LZTR1, MAX, MECR, MFAP5, MFF, MIB1, MLYCD, MMACHC, MMUT, MRPS 34. MTFMT, MTRFR, MYBPC3, MYH11, MYH6, MYH7, MYL2, MYL3, MYLK, MYO6, NAGLU, NDUFA1, NDUFA13, NDUFA2, NDUFA9, NDUFAF2, NDUFAF5, NDUFAF6,NDUFAF8, NDUFB8, NDUFC2, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NEXN, NF1, NKX2-5, NR0B1, NR3C1, NUBPL, OPA1, PCCA, PCCB, PCSK9, PDHA1, PDHB, PDHX, PDSS2, PET100, PEX7, P HKA1, PHKA2, PHKB, PHKG2, PHYH, PIEZO2, PKP2, PLN, PNPLA2, PNPT1, PRDM16, PRKAG2, PRKG1, PROC , PROS1, PSEN2, PTPN11, RAF1, RBM20, RET, RIT1, RYR2, SCN1B, SCN5A, SCNN1A, SCNN1B, SCNN1G, SCO 2. Any one or a combination of at least two of the following: SDHA, SDHAF2, SDHB, SDHC, SDHD, SERAC1, SERPINC1, SGCD, SGSH, SKI, SLC19A3, SLC22A5, SLC25A20, SLC25A4, SLC40A1, SLC4A3, SMAD2, SMAD3, SMAD9, SUCLA2, SUCLG1, SURF1, TACO1, TAFAZZIN, TAT, TBX4, TCAP, TECRL, TFR2, TGFB2, TGFBR1, TGFBR2, TMEM127, TMEM43, TNNC1, TNNI3, TNNT2, TPK1, TPM1, TRDN, TRIM63, TRMU, TRPM4, TSFM, TTC19, TTN, TTR, VCL, VHL, WNK1, or WNK4.

[0010] The detection probes and kits of this invention can accurately detect exon regions, intron regions 20-30 bp upstream and downstream of exons, and copy number variations of pathogenic genes associated with 268 pathogenic gene variants in monogenic hereditary cardiovascular diseases. With an average data volume of 1G, the nuclear genome coverage depth reaches an average of 200×, and the 30× coverage reaches 99%, which improves the detection sensitivity of low-frequency mutations in the target region and effectively controls the detection cost; the experimental system is stable, and the coefficient of variation of library results between samples is <10%.

[0011] Preferably, the monogenic hereditary cardiovascular diseases include cardiomyopathy, ion channelopathies, metabolic cardiomyopathy, genetically related hypertension, familial hypercholesterolemia, pulmonary hypertension, hereditary thrombophilia, and aortic disease.

[0012] Preferably, the screening criteria for the single-gene hereditary cardiovascular disease-related genes are any one of the following:

[0013] (1) After rigorous evaluation of the recommended test genes using the ClinGen database, select genes that are recommended for testing by guidelines or expert consensus and whose evidence strength for gene-disease relationship in the ClinGen database is limited or greater;

[0014] (2) The test is recommended by guidelines or expert consensus. The ClinGen database has no evidence of gene-disease relationship, but it is included in the OMIM database. The MalaCards database score is >1000, indicating a clear inheritance pattern and a known genetic molecular basis.

[0015] (3) The guidelines or expert consensus do not recommend testing, but it is related to the clinical phenotype of cardiovascular disease, and it is included in the OMIM database, with a clear inheritance pattern, known genetic molecular basis, and strong evidence of gene-disease relationship in the ClinGen database. Select genes with moderate or higher evidence of gene-disease relationship.

[0016] Preferably, the screening criteria for single-gene hereditary cardiovascular disease-related genes, under the conditions of (1), (2) or (3), are combined with the ClinGen database to select genes with a level of limited or higher clinical operability evidence in children or adults.

[0017] In a second aspect, the present invention provides the application of the detection probe for monogenic hereditary cardiovascular disease-related genes described in the first aspect in the preparation of products for detecting monogenic hereditary cardiovascular diseases.

[0018] Preferably, the product includes any one of a reagent, a kit, or a chip.

[0019] Thirdly, the present invention provides a kit for detecting genes related to monogenic hereditary cardiovascular diseases, the kit comprising the detection probes for monogenic hereditary cardiovascular disease-related genes described in the first aspect.

[0020] Preferably, the kit further includes any one or a combination of at least two of the following: terminal repair enzyme, terminal repair buffer, deoxyadenosine triphosphate, ligation premix, ligation enhancer, adapter, hybridization buffer, hybridization enhancer, blocking agent, streptavidin capture beads, or elution buffer.

[0021] Fourthly, the present invention provides a method for constructing a high-throughput sequencing library of genes related to single-gene hereditary cardiovascular diseases, the method comprising the following steps:

[0022] (1) Fragment the genomic DNA to obtain DNA fragments;

[0023] (2) The DNA fragment is repaired at the end, a dA tail is added, and the adapter is ligated to obtain the ligation product;

[0024] (3) Using the detection probe of the single-gene hereditary cardiovascular disease-related gene described in the first aspect, the ligation product is hybridized and captured to obtain the target fragment;

[0025] (4) Perform PCR amplification on the target fragment to obtain the amplification product;

[0026] (5) Separate and purify the amplification products to form a high-throughput sequencing library.

[0027] Preferably, the connector in step (2) contains a label.

[0028] Fifthly, the present invention provides a method for detecting genes related to single-gene hereditary cardiovascular diseases for purposes other than disease treatment and diagnosis, the method comprising the following steps:

[0029] (1) Construct a high-throughput sequencing library of genes related to single-gene hereditary cardiovascular diseases according to the method described in the fourth aspect;

[0030] (2) Sequencing the high-throughput sequencing library to obtain sequencing results;

[0031] (3) Perform data analysis on the sequencing results to determine the gene information related to single-gene hereditary cardiovascular diseases.

[0032] In a sixth aspect, the present invention provides an apparatus for detecting genes related to single-gene hereditary cardiovascular diseases, the apparatus comprising: a high-throughput sequencing library construction unit, a sequencing unit, a gene mutation analysis unit, and a pathogenicity classification unit;

[0033] The high-throughput sequencing library construction unit is used to perform the following: tagging sample DNA and capturing the target DNA fragment of a selected exon region of a single-gene hereditary cardiovascular disease-related gene using the detection probe of the single-gene hereditary cardiovascular disease-related gene described in the first aspect;

[0034] The sequencing unit is connected to the high-throughput sequencing library construction module and is used to sequence the DNA target fragment to obtain sequence information of the exon region and the intron region 20-30 bp upstream and downstream of the exon of the gene related to single-gene hereditary cardiovascular disease.

[0035] The gene mutation analysis unit is connected to the sequencing unit and performs data analysis on the sequence information obtained by the sequencing unit to obtain information on the exon region and the intron region 20-30 bp upstream and downstream of the exon of genes related to single-gene hereditary cardiovascular diseases.

[0036] The pathogenicity classification unit is connected to the gene mutation analysis unit. The variant annotation information obtained by the gene mutation analysis unit is combined with the ClinVar and HGMD databases, and the pathogenicity of the variants is initially classified according to the "ACMG Genetic Variation Classification Standards and Guidelines". The pathogenicity (P), probable pathogenicity (LP), undetermined significance (VUS), probable benignity (LB), and benignity (B) classification results of variants in the exon regions and intron regions 20-30 bp upstream and downstream of the exons of genes related to hereditary cardiovascular diseases are obtained.

[0037] In a seventh aspect, the present invention provides a gene panel for detecting monogenic hereditary cardiovascular diseases, the gene panel comprising the following genes: ABCC9, ABCG8, ACADM, ACADS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1, ALMS1, ALPK3, ANKRD1, APOB, ARSB, ATP13A3, ATP5MK, BAG3, BMPR2, BTD, BVES, CACNA1C, CACNA1H, CALM1, CALM2, CALM3, CASQ2, CAV1, CAV3, CLCN2, COL3A 1. COL5A1, COL5A2, COX10, COX15, CPT1A, CPT2, CRYAB, CSRP3, CTNNA3, CUL3, CYP11B1, CYP11B2, CYP17A1, DES, DLAT, DLD, DSC2, DSG2, DSP, EARS2, ECHS1 , EFEMP2, EIF2AK4, ELN, EMD, ENG, EPAS1, ETFA, ETFB, ETFDH, ETHE1, EYA4, F2, F5, F9, FAH, ARS2, FBN1, FBXL4, FH, FHL1, FLNA, FLNC, FOXE3, FOXF1, FOXRE D1, FXN, GAA, GALNS, GBA, GBE1, GDF2, GFM1, GFM2, GLA, GLB1, GNB2, GNPTAB, GNPTG, GNS, GRHPR, GTPBP3, GUSB, GYS1, HADH, HADHA, HAMP, HCN4, HEXB, HFE, HJV, HGSNAT, HIBCH, HOGA1, HPD, HPDL, HRG, HSD11B2, IDS, IDUA, JPH2, JUP, KCNE1, KCNH2, KCNJ2, KCNJ5, KCNK3, KCNQ1, KDR, KLF10, KLHL3, LAMP2, LDB3, LDLR, LDLRAP1, LIPT1, LMBRD1, LMNA, LOX, LPL, LRPPRC, LZTR1, MAX, MECR, MFAP5, MFF, MIB1, MLYCD, MMACHC, MMUT, MRPS34, MTFMT, MTRFR, MYBPC3, MYH11 , MYH6, MYH7, MYL2, MYL3, MYLK, MYO6, NAGLU, NDUFA1, NDUFA13, NDUFA2, NDUFA9, NDUFAF2, NDUFAF5, NDUFAF6, NDUFAF8, NDUFB8, NDUFC2, NDUFS1, NDUFS2,NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NEXN, NF1, NKX2-5, NR0B1, NR3C1, NUBPL, OPA1, PCCA, PCCB, PCSK9, PDHA1, PDHB, PDHX, PDSS2, PET100, PEX7, PHKA1, PHKA2, PHKB, PHKG2, P HYH, PIEZO2, PKP2, PLN, PNPLA2, PNPT1, PRDM16, PRKAG2, PRKG1, PROC, PROS1, PSEN2, PTPN11, RAF1, RBM20, RET, RIT1, RYR2, SCN1B, SCN5A, SCNN1A, SCNN1B, SCNN1G, SCO2, SDHA, SDHA F2, SDHB, SDHC, SDHD, SERAC1, SERPINC1, SGCD, SGSH, SKI, SLC19A3, SLC22A5, SLC25A20, SLC25A4, SLC40A1, SLC4A3, SMAD2, SMAD3, SMAD9, SUCLA2, SUCLG1, SURF1, TACO1, TAFAZZIN , TAT, TBX4, TCAP, TECRL, TFR2, TGFB2, TGFBR1, TGFBR2, TMEM127, TMEM43, TNNC1, TNNI3, TNNT2, TPK1, TPM1, TRDN, TRIM63, TRMU, TRPM4, TSFM, TTC19, TTN, TTR, VCL, VHL, WNK1 and WNK4. ,

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) With an average data volume of 1G, the present invention achieves an average nuclear genome coverage depth of 200× and a 30× coverage of 99%, which improves the detection sensitivity of low-frequency mutations in the target region and effectively controls the detection cost; the experimental system is stable and the coefficient of variation of library results between samples is <10%;

[0040] (2) This invention enables simultaneous detection of seven types of cardiovascular diseases (cardiomyopathy, ion channelopathies, metabolic cardiomyopathy, genetically related hypertension, familial hypercholesterolemia, pulmonary hypertension, hereditary thrombophilia and aortic disease), clarifies the patient’s carrier status, reduces the cost of multiple tests for different single diseases or direct detection using WES, and is easy to promote and apply.

[0041] (3) Based on expert consensus or guidelines, this invention combines the ClinGen database, MalaCards database and OMIM database to exclude genes for which the clinical association evidence between hereditary cardiovascular diseases is insufficient, thereby reducing the complexity of interpreting clinical reports and genetic counseling, and thus reducing unnecessary distress and anxiety for patients.

[0042] (4) The present invention, combined with the clinical operability of the ClinGen database for children and adults, enables early detection, early intervention, and early treatment;

[0043] (5) The present invention can realize the simultaneous detection of pathogenic gene SNVs, Indels and gene copy number variations (CNVs). Attached Figure Description

[0044] Figure 1 This is a graph showing the Sanger sequencing validation results. Detailed Implementation

[0045] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0046] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0047] To facilitate a clearer understanding of this invention, certain terms are first defined. As used herein, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0048] The term "PCR" refers to polymerase chain reaction.

[0049] The term "MLPA" refers to multiple ligation-dependent probe amplification.

[0050] The term "SNV" refers to a single nucleotide variant.

[0051] The term "Indels" refers to insertion / deletion variants.

[0052] The term "CNV" refers to copy number variation.

[0053] Example 1

[0054] This embodiment provides a gene panel and a set of probes for detecting genes related to single-gene hereditary cardiovascular diseases.

[0055] Contact Us A2、ACTC1、ACTN2、ACVRL1、AGK、AGL、AGXT、AIFM1、ALMS1、ALPK3、ANKRD1、APO B、ARSB、ATP13A3、ATP5MK、BAG3、BMPR2、BTD、BVES、CACNA1C、CACNA1H、CALM 1、CALM2、CALM3、CASQ2、CAV1、CAV3、CLCN2、COL3A1、COL5A1、COL5A2、COX10、 COX15, CPT1A, CPT2, CRYAB, CSRP3, CTNNA3, CUL3, CYP11B1, CYP11B2, CYP17A1, DES, DLAT, DLD, DSC2, DSG2, DSP, EARS2, ECHS1, EFEMP2, EIF2AK4, ELN, EM D, ENG, EPAS1, ETFA, ETFB, ETFDH, ETHE1, EYA4, F2, F5, F9, FAH, ARS2, FBN1, FBXL4, FH, FHL1, FLNA, FLNC, FOXE3, FOXF1, FOXRED1, FXN, GAA, GALNS, GBA, G BE1、GDF2、GFM1、GFM2、GLA、GLB1、GNB2、GNPTAB、GNPTG、GNS、GRHPR、GTPBP3 、GUSB、GYS1、HADH、HADHA、HAMP、HCN4、HEXB、HFE、HJV、HGSNAT、HIBCH、HOGA1 、HPD、HPDL、HRG、HSD11B2、IDS、IDUA、JPH2、JUP、KCNE1、KCNH2、KCNJ2、KCNJ 5、KCNK3、KCNQ1、KDR、KLF10、KLHL3、LAMP2、LDB3、LDLR、LDLRAP1、LIPT1、LMB RD1、LMNA、LOX、LPL、LRPPRC、LZTR1、MAX、MECR、MFAP5、MFF、MIB1、MLYCD、MM ACHC、MMUT、MRPS34、MTFMT、MTRFR、MYBPC3、MYH11、MYH6、MYH7、MYL2、MYL3、M YLK、MYO6、NAGLU、NDUFA1、NDUFA13、NDUFA2、NDUFA9、NDUFAF2、NDUFAF5、ND UFAF6、NDUFAF8、NDUFB8、NDUFC2、NDUFS1、NDUFS2、NDUFS3、NDUFS4、NDUFS7、NDUFS8, NDUFV1, NEXN, NF1, NKX2-5, NR0B1, NR3C1, NUBPL, OPA1, PCCA, PCCB, PCSK9, PDHA1, PDHB, PDHX, PDSS2, PET100, PEX7, PHKA1, PHKA2, PHKB, PHKG2, PHYH, PIEZO2, PKP2, P LN, PNPLA2, PNPT1, PRDM16, PRKAG2, PRKG1, PROC, PROS1, PSEN2, PTPN11, RAF1, RBM20, RET, RIT1, RYR2, SCN1B, SCN5A, SCNN1A, SCNN1B, SCNN1G, SCO2, SDHA, SDHAF2, SDHB, SDHC , SDHD, SERAC1, SERPINC1, SGCD, SGSH, SKI, SLC19A3, SLC22A5, SLC25A20, SLC25A4, SLC40A1, SLC4A3, SMAD2, SMAD3, SMAD9, SUCLA2, SUCLG1, SURF1, TACO1, TAFAZZIN, TAT, TBX 4. Combination of TCAP, TECRL, TFR2, TGFB2, TGFBR1, TGFBR2, TMEM127, TMEM43, TNNC1, TNNI3, TNNT2, TPK1, TPM1, TRDN, TRIM63, TRMU, TRPM4, TSFM, TTC19, TTN, TTR, VCL, VHL, WNK1 and WNK4. ,

[0056] This invention provides a probe set for detecting single-gene hereditary cardiovascular disease-related genes based on the aforementioned preferred genes. The exon sequences of all the aforementioned genes are obtained from data published in the Human Genome Database (GRCh38 / hg38). Probes are designed using these exon sequences as target sequences, with the probe sequences complementary to the target regions. The probes designed in this invention cover all exon regions and 25 bp intron regions upstream and downstream of the exons of all the aforementioned genes. The probe design and preparation method described in this invention belongs to conventional techniques in the field of multi-gene capture sequencing probe preparation. In a specific embodiment, the probes are designed in a shingled manner, with the overlap between probes being less than 30 bases. According to the method described, the probe volume obtained by this invention is relatively large, and is not specifically shown in the specification. Based on the 268 single-gene hereditary cardiovascular disease-related genes disclosed in this invention, those skilled in the art can obtain the probes through conventional techniques.

[0057] Example 2

[0058] This embodiment uses the detection probe described in Example 1 to detect and verify 30 peripheral blood samples with known gene variation information.

[0059] Thirty peripheral blood DNA samples from our hospital in recent years, whose genetic variation information has been independently confirmed by whole-exome sequencing (WES) and Sanger assay, were selected. Among them, 24 were positive samples: patients with hereditary cardiovascular diseases carrying pathogenic (P) or probable pathogenic (LP) loci identified by WES and Sanger assay; 6 were negative samples: non-patients (including patients with other diseases or healthy controls) for whom no relevant pathogenic loci were found by WES and Sanger assay. Peripheral blood gDNA was extracted using a blood genomic DNA extraction kit (centrifuge column type) (YDP348) (Beijing Medical Device Registration No. 20200001). Using a blinded method, the targeted panel of this invention was used to test the 30 samples to evaluate its consistency, sensitivity, and specificity with the reference method. The specific detection method is as follows:

[0060] 1. Pre-capture library construction

[0061] (1) Fragmentation, terminal repair and A tail

[0062] (a) After briefly centrifuging the DNA fragmentation and end-repair enzyme, place the mixture on an ice box and prepare the system shown in Table 1 in a PCR tube:

[0063] Table 1

[0064]

[0065] (b) Gently pipette 10 times, centrifuge briefly, and immediately place in a PCR instrument pre-cooled to 4°C. Run the PCR program shown in Table 2 below (heated lid temperature 75°C):

[0066] Table 2

[0067]

[0068] (2) Connector connection and purification

[0069] (a) Prepare the following systems in PCR tubes according to Table 3 on an ice box:

[0070] Table 3

[0071]

[0072] (b) Vortex mixing, instantaneous centrifugation.

[0073] (c) Place the PCR tube on the PCR instrument and run the PCR program shown in Table 4 below (the hot cap temperature is set to 30°C).

[0074] Table 4

[0075]

[0076] (d) Centrifuge instantly and proceed to the next step immediately.

[0077] DNA purification magnetic beads (Ampure XP beads, A63882) need to be taken out in advance, vortexed for 30 seconds to mix, and equilibrated at room temperature for 30 minutes. When using, slowly aspirate. Prepare a fresh 80% ethanol solution for later use.

[0078] (e) Add 55 μL of DNA purification magnetic beads to each adapter ligation product PCR tube, mix well, let stand at room temperature for 5 min, then place on a magnetic rack for 5 min until the liquid is clear, and discard the supernatant (be careful not to pick up the magnetic beads).

[0079] (f) Add 200 μL of 80% ethanol, let stand for 30 s, and discard the supernatant (be careful not to pick up the magnetic beads).

[0080] (g) Repeat the previous step.

[0081] (h) Centrifuge briefly, then place on a magnetic rack for 2 min until the liquid is clear. Use a 10 μL pipette to remove any residual ethanol at the bottom (be careful not to pick up the magnetic beads).

[0082] (i) Keep the PCR tube on the magnetic rack and place it at room temperature for 3 minutes to allow the residual ethanol to evaporate completely until there are no obvious water stains on the surface (be careful not to dry it too much).

[0083] (j) Remove the PCR tube from the magnetic rack, add 22 μL of DNA elution buffer, resuspend the magnetic beads, and let stand at room temperature for 5 min. Place the PCR centrifuge tube back on the magnetic rack for 5 min until the liquid is clear, and take 20 μL of the elution product into a new PCR tube (be careful not to pick up the magnetic beads).

[0084] (3) Amplification, enrichment and purification

[0085] (a) Prepare the following systems in PCR tubes according to Table 5 on an ice box:

[0086] Table 5

[0087]

[0088] (b) Vortex mixing, instantaneous centrifugation.

[0089] (c) Place the PCR tubes on the PCR instrument and run the PCR program shown in Table 6 below (with the hot cap temperature set to 105°C):

[0090] Table 6

[0091]

[0092] DNA purification magnetic beads (Ampure XP beads, A63882) need to be taken out in advance, vortexed for 30 seconds to mix, and equilibrated at room temperature for 30 minutes. When using, slowly aspirate. Prepare a fresh 80% ethanol solution for later use.

[0093] (d) Centrifuge briefly, add 50 μL of DNA purification magnetic beads to each PCR tube containing amplified enriched product, vortex thoroughly to mix, let stand at room temperature for 5 min, then place on a magnetic rack for 5 min until the liquid is clear, and discard the supernatant (be careful not to pick up the magnetic beads).

[0094] (e) Add 200 μL of 80% ethanol, let stand for 30 s, and discard the supernatant (be careful not to pick up the magnetic beads).

[0095] (f) Repeat the previous step.

[0096] (g) Centrifuge briefly, then place on a magnetic rack for 2 min until the liquid is clear. Use a 10 μL pipette to remove any remaining ethanol at the bottom (be careful not to pick up the magnetic beads).

[0097] (h) Keep the PCR tube on the magnetic rack and place it at room temperature for 3 minutes to allow the residual ethanol to evaporate completely until there are no obvious water stains on the surface (be careful not to dry it too much).

[0098] (i) Remove the PCR tube from the magnetic rack, add 30 μL of DNA elution buffer, resuspend the magnetic beads, and let stand at room temperature for 5 min.

[0099] (j) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 min, wait for the solution to become clear, and then transfer 28 μL of the supernatant into a new EP tube (be careful not to pick up the magnetic beads).

[0100] (4) Document quality inspection

[0101] Take 1 μL of the library and quantify it using the Qubit dsDNA HS Assay Kit. The sample concentration should be ≥20 ng / μL and the library yield should be ≥500 ng. Only if both conditions are met is the result acceptable.

[0102] 2. Hybrid capture

[0103] (1) Probe hybridization

[0104] Preheat blocking solutions 1, 2, and 3 to an ice box to thaw, vortex thoroughly to mix, and briefly centrifuge. Take the probe immediately before use, thaw it to an ice box, gently tap to mix, and briefly centrifuge.

[0105] (a) Prepare the following systems in PCR tubes on an ice box according to Table 7 below:

[0106] Table 7

[0107]

[0108] (b) Take 500 ng of each of the eight libraries with different paired-end tag primers and mix them into the same EP tube.

[0109] (c) Add 1.8 times the volume of DNA purification magnetic beads (Ampure XP beads, A63882) to each EP tube, vortex thoroughly to mix, let stand at room temperature for 5 min, then place on a magnetic rack for 5 min until the liquid is clear, and discard the supernatant (be careful not to pick up the magnetic beads).

[0110] (d) Add 200 µL of 80% ethanol, let stand at room temperature for 30 s, and discard the supernatant.

[0111] (e) Repeat the previous step.

[0112] (f) Centrifuge briefly, then place the PCR tube back on the magnetic rack for 2 min. Use a 10 μL pipette to remove any residual ethanol from the bottom (be careful not to pick up the magnetic beads).

[0113] (g) Keep the EP tube on the magnetic rack and place it at room temperature for about 3 minutes to allow the residual ethanol to evaporate fully until there are no obvious water stains on the surface (be careful not to dry it too much).

[0114] (h) Remove the EP tube from the magnetic rack, add 26 µL of hybridization reaction system, gently blow to mix, and let stand at room temperature for 5 min.

[0115] (i) Instant centrifugation: Place the sample tube on a magnetic rack for 3 min and wait for the solution to become clear.

[0116] (j) Pipette 26 µL of supernatant into a new PCR tube, add 2 µL of blocking buffer 2 and 2 µL of hybridization capture probe, gently pipette to mix, and centrifuge briefly.

[0117] (k) Place the PCR tube on the PCR instrument and run the program shown in Table 8 below to incubate overnight (heat cap temperature is 85°C).

[0118] Table 8

[0119]

[0120] (2) Cleaning of captured magnetic beads

[0121] Remove the capturing magnetic beads in advance, vortex them to mix, and let them equilibrate at room temperature for 30 minutes before use; prepare a fresh 80% ethanol solution for later use.

[0122] (a) Add 50 µL of capture magnetic beads to the PCR tube, place it on a magnetic rack for 1 min, wait for the solution to become clear, and discard the supernatant (be careful not to pick up the magnetic beads).

[0123] (b) Remove the PCR tube from the magnetic rack, add 180 µL of washing buffer 1, gently pipette several times to resuspend the magnetic beads, centrifuge briefly, place the PCR tube on the magnetic rack, and carefully discard the supernatant after the solution becomes clear (be careful not to pick up the magnetic beads).

[0124] (c) Repeat step (b) twice, for a total of three cleanings of the magnetic beads.

[0125] (d) Remove the PCR tube from the magnetic rack, add 180 µL of washing buffer 1, and gently pipette to resuspend the magnetic beads for later use.

[0126] (3) Enrichment of DNA library in target region

[0127] (a) Keep the hybridization product on the PCR instrument, add 180 µL of resuspended capture magnetic beads to the hybridization product, and gently mix with a pipette (be careful to avoid generating a lot of air bubbles).

[0128] (b) Remove the PCR tube and place it on a rotary mixer (10 rpm / min) for 30 min at room temperature.

[0129] (c) Remove the PCR tube, centrifuge briefly, place the PCR tube on a magnetic rack for 2 min, wait for the solution to become clear, and discard the supernatant (be careful not to pick up the magnetic beads).

[0130] (d) Add 150 µL of washing buffer 2, gently pipette to mix, and then place on a rotary mixer (speed: 10 rpm / min) to wash at room temperature for 15 min.

[0131] (e) Remove the PCR tube, centrifuge briefly, place on a magnetic rack for 2 min, wait for the solution to become clear, and discard the supernatant (be careful not to pick up the magnetic beads).

[0132] (f) Add 150 µL of preheated 50°C washing buffer 3, gently pipette several times to mix (avoid generating a large number of bubbles), centrifuge briefly, and incubate at 50°C for 10 min.

[0133] (g) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 min, wait for the solution to become clear, and discard the supernatant.

[0134] (h) Repeat steps (f) and (g) twice, for a total of three cleanings of the magnetic beads.

[0135] (i) Add 150 µL of 80% ethanol solution, let stand at room temperature for 30 s, completely remove the ethanol solution, and air dry at room temperature (be careful not to over-dry).

[0136] (j) Add 24 µL of nuclease-free water, and gently resuspend and mix the magnetic beads with a pipette.

[0137] (4) Post-capture PCR amplification and purification

[0138] (a) Prepare the capture-after PCR amplification system on an ice box according to Table 9 below.

[0139] Table 9

[0140]

[0141] (b) Gently blow and mix.

[0142] (c) Place the PCR tube on the gene amplification instrument and run the program in Table 10 (heat cap temperature 105°C).

[0143] Table 10

[0144]

[0145] (d) Centrifuge briefly, place the PCR tube on a magnetic rack, let it stand at room temperature for 3 min, aspirate 50 µL of supernatant and transfer it to a new PCR tube, and discard the magnetic beads.

[0146] (e) Add 55 µL of DNA purification magnetic beads to a new PCR tube, vortex thoroughly to mix, and let stand at room temperature for 5 min.

[0147] (f) Centrifuge briefly, place the PCR tube on a magnetic rack for 3 min, and carefully remove the supernatant after the solution has cleared (be careful not to pick up the magnetic beads).

[0148] (g) Add 200 µL of 80% ethanol solution, let stand at room temperature for 30 s, and discard the supernatant.

[0149] (h) Repeat the previous step.

[0150] (i) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 min, use a 10 μL pipette to remove the residual ethanol at the bottom (be careful not to pick up the magnetic beads), place at room temperature for about 3 min to allow the residual ethanol to evaporate fully until there are no obvious water stains on the surface (be careful not to dry it too much).

[0151] (j) Add 25 µL of nuclease-free water, vortex thoroughly to mix, and let stand at room temperature for 2 min.

[0152] (k) Centrifuge briefly, place the PCR tube on a magnetic rack for 2 min, wait for the solution to become clear, and then transfer 23 μL of the supernatant into a new EP tube (be careful not to pick up the magnetic beads).

[0153] (5) Quality inspection of the captured document

[0154] Library concentration determination: Take 1 μL of the library and quantify it using the Qubit dsDNA HS Assay Kit. The library concentration is approximately ≥6 ng / μL.

[0155] 3. Sequencing

[0156] Library circularization was performed according to the instructions of the MGIEAsy double barcode circularization kit (BGI Genomics, catalog number: 1000020570), and sequencing was performed using the DA500 / DA5000 platform (Suzhou Beikang Medical Devices Co., Ltd.).

[0157] The test results are shown in Table 11 below:

[0158] Table 11

[0159]

[0160]

[0161]

[0162] As shown in Table 1, the detection results of the probe set of the present invention are consistent with the known results, with a compliance rate of 100%.

[0163] Example 3

[0164] This embodiment uses a patient sample. MRI revealed left atrial and left ventricular enlargement, decreased left ventricular systolic function, and slight delayed enhancement in the interventricular septum, possibly indicating dilated cardiomyopathy or mild mitral regurgitation. Echocardiography revealed left atrial and left ventricular enlargement, mild mitral regurgitation, severe left ventricular systolic dysfunction, and a small amount of pericardial effusion. Electrocardiogram suggested possible right ventricular hypertrophy; therefore, gene detection was performed using the gene panel described in Embodiment 1 of this invention. The detection results revealed a TTN gene mutation. Data quality control results are shown in Table 12, and specific detection results are shown in Table 13. This mutation was verified using Sanger sequencing (F primer: AACTGGATGCTGACCTGCG, R primer: ATGTCTTAGTGACCTCTTTTACCTTC), and the verification results are as follows. Figure 1 As shown, the gene panel of the present invention can effectively, accurately, and conveniently detect genes related to single-gene hereditary cardiovascular diseases.

[0165] Table 12

[0166]

[0167] Table 13

[0168]

[0169] Comparative Example 1

[0170] The difference between this comparative example and Example 2 is that the SCN5A gene is replaced with the GPD1L gene, while other genes remain unchanged as in Example 1. Sequencing probes for single-gene hereditary cardiovascular disease-related genes were obtained according to the general method described in Example 1. Peripheral blood genomic DNA samples from 24 patients with hereditary cardiovascular diseases in Example 2, identified by WES and Sanger validation as carrying pathogenic (P) or probable pathogenic (LP) loci, were tested for single-gene hereditary cardiovascular disease-related genes according to the method described in Example 2. The results showed that positive samples numbered DSUH-17 and DSUH-18 were not detected, causing the positive detection rate to decrease from 100% to 91.7%, i.e., a concordance rate of 93.3%. Although GPD1L has been reported in Brugada syndrome, the strength of evidence for its gene-disease relationship is controversial according to the ClinGen database. Furthermore, the ACMG, based on the ClinGen evaluation system, incorporates gene-disease validity into variant classification, providing a clear technical reference standard. The strength of evidence for GPD1L gene-Brugada syndrome is controversial, and GPD1L gene variants are generally not included in Brugada syndrome patient test reports to avoid clinical misrepresentation.

[0171] Comparative Example 2

[0172] The difference between this comparative example and Example 2 is that the LDLR gene is replaced with the EPHX2 gene, while other genes remain unchanged as in Example 1. Sequencing probes for single-gene hereditary cardiovascular disease-related genes were obtained according to the general method described in Example 1. Peripheral blood genomic DNA samples from 24 patients with hereditary cardiovascular diseases carrying pathogenic (P) or probable pathogenic (LP) loci, as verified by WES and Sanger assays in Example 2, were tested for single-gene hereditary cardiovascular disease-related genes. The results showed that positive samples numbered DSUH-22 and DSUH-23 were not detected, causing the positive detection rate to decrease from 100% to 91.7%, i.e., a concordance rate of 93.3%. The EPHX2 gene is included in the OMIM database, and its inheritance pattern and molecular basis are known. There is no strong evidence of a gene-disease relationship in the ClinGen database, and its score in the MalaCards database is 145.47. Although related variants can cause dyslipidemia, it is only a risk factor, and there are few related reports in databases such as ClinVar and HGMD.

[0173] Comparative Example 3

[0174] The difference between this comparative example and Example 2 is that the TNNI3 gene is replaced with the DMD gene, while other genes remain unchanged as in Example 1. Sequencing probes for single-gene hereditary cardiovascular disease-related genes were obtained according to the general method described in Example 1. Peripheral blood genomic DNA samples from 24 patients with hereditary cardiovascular disease carrying pathogenic (P) or probable pathogenic (LP) loci, as verified by WES and Sanger assays in Example 2, were tested for single-gene hereditary cardiovascular disease-related genes. The result was that the positive sample numbered DSUH-1 was not detected, causing the positive detection rate to decrease from 100% to 95.8%, i.e., the concordance rate was 96.7%. Although pathogenic mutations in the DMD gene can lead to cardiac involvement in some patients, with a significant decline in cardiac function and cardiomyopathy typically occurring after age 10, the core and early symptoms of this disease are progressive muscle weakness, delayed motor development, and cognitive impairment, and patients are mostly diagnosed in childhood. Patients usually develop the disease before the age of 5, with most starting to show symptoms and signs of limb weakness from the age of 3-4. They usually lose the ability to walk independently by the age of 9-10, and a few patients lose the ability to walk independently before the age of 8.

[0175] In summary, the detection probes and kits of this invention can accurately detect exon regions, intron regions 20-30 bp upstream and downstream of exons, and copy number variations of pathogenic genes associated with 268 pathogenic gene variants in monogenic hereditary cardiovascular diseases. With an average data volume of 1G, the nuclear genome coverage depth reaches an average of 200×, and the 30× coverage reaches 99%. This improves the detection sensitivity of low-frequency mutations in the target region, effectively controls the detection cost, and the experimental system is stable with a coefficient of variation of <10% for library results among samples.

[0176] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A detection probe for a gene associated with a monogenic inherited cardiovascular disease, characterized in that, The probes are specific probes for the exon region and the intron region 20-30 bp upstream and downstream of the exon region of a gene selected from the group consisting of ABCC9, ABCG8, ACADM, ACADS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1, ALMS1, ALPK3, ANKRD1, APOB, ARSB, ATP13A3, ATP5MK, BAG3, BMPR2, BTD, BVES, CACNA1C, CACNA1H, CALM1, CALM2, CALM3, CASQ2, CAV1, CAV3, CLCN2, COL3A1, COL5A1, COL5A2, COX10, COX15, CPT1A, CPT2, CRYAB, CSRP3, CTNNA3, CUL3, CYP11B1, CYP11B2, CYP17A1, DES, DLAT, DLD, DSC2, DSG2, DSP, EARS2, ECHS1, EFEMP2, EIF2AK4, ELN, EMD, ENG, EPAS1, ETFA, ETFB, ETFDH, ETHE1, EYA4, F2, F5, F9, FAH, ARS2, FBN1, FBXL4, FH, FHL1, FLNA, FLNC, FOXE3, FOXF1, FOXRED1, FXN, GAA, GALNS, GBA, GBE1, GDF2, GFM1, GFM2, GLA, GLB1, GNB2, GNPTAB, GNPTG, GNS, GRHPR, GTPBP3, GUSB, GYS1, HADH, HADHA, HAMP, HCN4, HEXB, HFE, HJV, HGSNAT, HIBCH, HOGA1, HPD, HPDL, HRG, HSD11B2, IDS, IDUA, JPH2, JUP, KCNE1, KCNH2, KCNJ2, KCNJ5, KCNK3, KCNQ1, KDR, KLF10, KLHL3, LAMP2, LDB3, LDLR, LDLRAP1, LIPT1, LMBRD1, LMNA, LOX, LPL, LRPPRC, LZTR1, MAX, MECR, MFAP5, MFF, MIB1, MLYCD, MMACHC, MMUT, MRPS34, MTFMT, MTRFR, MYBPC3, MYH11, MYH6, MYH7, MYL2, MYL3, MYLK, MYO6, NAGLU, NDUFA1, NDUFA13, NDUFA2, NDUFA9, NDUFAF2, NDUFAF5, NDUFAF6, NDUFAF8, NDUFB8, NDUFC2, NDUFS1, NDUFS2,Any one of, or a combination of at least two of, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NEXN, NF1, NKX2-5, NR0B1, NR3C1, NUBPL, OPA1, PCCA, PCCB, PCSK9, PDHA1, PDHB, PDHX, PDSS2, PET100, PEX7, PHKA1, PHKA2, PHKB, PHKG2, PHYH, PIEZO2, PKP2, PLN, PNPLA2, PNPT1, PRDM16, PRKAG2, PRKG1, PROC, PROS1, PSEN2, PTPN11, RAF1, RBM20, RET, RIT1, RYR2, SCN1B, SCN5A, SCNN1A, SCNN1B, SCNN1G, SCO2, SDHA, SDHAF2, SDHB, SDHC, SDHD, SERAC1, SERPINC1, SGCD, SGSH, SKI, SLC19A3, SLC22A5, SLC25A20, SLC25A4, SLC40A1, SLC4A3, SMAD2, SMAD3, SMAD9, SUCLA2, SUCLG1, SURF1, TACO1, TAFAZZIN, TAT, TBX4, TCAP, TECRL, TFR2, TGFB2, TGFBR1, TGFBR2, TMEM127, TMEM43, TNNC1, TNNI3, TNNT2, TPK1, TPM1, TRDN, TRIM63, TRMU, TRPM4, TSFM, TTC19, TTN, TTR, VCL, VHL, WNK1, or WNK4.

2. The detection probe for a gene related to a monogenic inherited cardiovascular disease according to claim 1, characterized in that, The single gene hereditary cardiovascular disease includes cardiomyopathy, ion channel disease, metabolic cardiomyopathy, hereditary related hypertension, familial hypercholesterolemia, pulmonary arterial hypertension, hereditary thrombophilia and aortic disease.

3. The detection probe for a gene associated with a monogenic inherited cardiovascular disease according to claim 1 or 2, characterized in that, The screening criteria of the single gene hereditary cardiovascular disease related gene meet any of the following: (1) after strict evaluation of the recommended detection gene using the ClinGen database, the gene recommended for detection by the guidelines or expert consensus and the evidence intensity of the gene-disease relationship in the ClinGen database is limited and above; (2) the guidelines or expert consensus recommend detection, the ClinGen database has no evidence intensity of gene-disease relationship, but the OMIM database includes, the MalaCards database score is >1000, the genetic mode is clear, and the genetic molecular basis is known; (3) the guidelines or expert consensus are not recommended for detection, but are related to the clinical phenotype of cardiovascular disease, and the OMIM database includes, the genetic mode is clear, the genetic molecular basis is known, the ClinGen database has evidence intensity of gene-disease relationship, and the evidence intensity of gene-disease relationship is selected to be moderate and above; Preferably, the screening criteria of the single gene hereditary cardiovascular disease related gene meet the conditions of (1), (2) or (3), and the ClinGen database is selected to have a child or adult clinical operability evidence level of limited and above.

4. The use of the detection probe of the single gene hereditary cardiovascular disease related gene in any one of claims 1-3 in the preparation of a product for detecting single gene hereditary cardiovascular disease.

5. A kit for detecting a gene associated with a monogenic inherited cardiovascular disease, characterized in that, The kit comprises the detection probe of the single gene hereditary cardiovascular disease related gene in any one of claims 1-3.

6. The kit of claim 5, wherein The kit further comprises any one or a combination of at least two of the following: terminal repair enzyme, terminal repair buffer, deoxyadenosine triphosphate, ligation premix, ligation enhancer, linker, hybridization buffer, hybridization enhancer, blocking agent, streptavidin capture magnetic bead or elution buffer.

7. A method of constructing a high-throughput sequencing library of a gene associated with a monogenic inherited cardiovascular disease, characterized in that, The method comprises the following steps: (1) fragmenting genomic DNA to obtain DNA fragments; (2) performing terminal repair on the DNA fragments, adding a dA tail, and connecting the linker to obtain a ligation product; (3) hybridizing and capturing the ligation product using the detection probe of the single gene hereditary cardiovascular disease related gene in any one of claims 1-3 to obtain the target fragment; (4) performing PCR amplification on the target fragment to obtain an amplification product; (5) separating and purifying the amplification product to form a high-throughput sequencing library; Preferably, the linker in step (2) contains a label.

8. A method for detecting a monogenic inherited cardiovascular disease-related gene for non-therapeutic and diagnostic purposes, characterized in that, The method comprises the following steps: (1) constructing a single gene hereditary cardiovascular disease related gene high-throughput sequencing library according to the method of claim 7; (2) sequencing the high-throughput sequencing library to obtain sequencing results; (3) analyzing the sequencing results to determine single gene hereditary cardiovascular disease related gene information.

9. An apparatus for detecting a gene associated with a monogenic inherited cardiovascular disease, characterized by, The device comprises a high-throughput sequencing library construction unit, a sequencing unit, a gene mutation analysis unit and a pathogenicity classification unit; The high-throughput sequencing library construction unit is configured to perform the following functions: adding a label to the sample DNA and capturing the DNA target fragments of the selected exon regions of the monogenic genetic cardiovascular disease related genes using the detection probes of the monogenic genetic cardiovascular disease related genes according to any one of claims 1-3; The sequencing unit is connected to the high-throughput sequencing library construction module and is configured to sequence the DNA target fragments to obtain sequence information of the exon regions of the monogenic genetic cardiovascular disease related genes and the intron regions 20-30 bp upstream and downstream of the exons; The gene mutation analysis unit is connected to the sequencing unit and is configured to analyze the sequence information obtained by the sequencing unit to obtain variation information of the exon regions of the monogenic genetic cardiovascular disease related genes and the intron regions 20-30 bp upstream and downstream of the exons; The pathogenicity classification unit is connected to the gene mutation analysis unit and is configured to combine the variation annotation information obtained by the gene mutation analysis unit with the ClinVar and HGMD databases, and preliminarily classify the pathogenicity of the variations according to the “ACMG Genetic Variation Classification Standards and Guidelines” to obtain classification results of the pathogenicity (P), possible pathogenicity (LP), unknown significance (VUS), possible benignity (LB), and benignity (B) of the variations in the exon regions of the genetic cardiovascular disease related genes and the intron regions 20-30 bp upstream and downstream of the exons.

10. A gene panel for detecting monogenic inherited cardiovascular diseases, characterized in that, The gene panel includes the following genes: ABCC9, ABCG8, ACADM, ACADS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1, ALMS1, ALPK3, ANKRD1, APOB, ARSB, ATP13A3, ATP5MK, BAG3, BMPR2, BTD, BVES, CACNA1C, CACNA1H, CALM1, CALM2, CALM3, CASQ2, CAV1, CAV3, CLCN2, COL3A1, COL5A1, COL5A2, COX10, COX15, CPT1A, CPT2, CRYAB, CSRP3, CTNNA3, CUL3, CYP11B1, CYP11B2, CYP17A1, DES, DLAT, DLD, DSC2, DSG2, DSP, EARS2, ECHS1, EFEMP2, EIF2AK4, ELN, EMD, ENG, EPAS1, ETFA, ETFB, ETFDH, ETHE1, EYA4, F2, F5, F9, FAH, ARS2, FBN1, FBXL4, FH, FHL1, FLNA, FLNC, FOXE3, FOXF1, FOXRED1, FXN, GAA, GALNS, GBA, GBE1, GDF2, GFM1, GFM2, GLA, GLB1, GNB2, GNPTAB, GNPTG, GNS, GRHPR, GTPBP3, GUSB, GYS1, HADH, HADHA, HAMP, HCN4, HEXB, HFE, HJV, HGSNAT, HIBCH, HOGA1, HPD, HPDL, HRG, HSD11B2, IDS, IDUA, JPH2, JUP, KCNE1, KCNH2, KCNJ2, KCNJ5, KCNK3, KCNQ1, KDR, KLF10, KLHL3, LAMP2, LDB3, LDLR, LDLRAP1, LIPT1, LMBRD1, LMNA, LOX, LPL, LRPPRC, LZTR1, MAX, MECR, MFAP5, MFF, MIB1, MLYCD, MMACHC, MMUT, MRPS34, MTFMT, MTRFR, MYBPC3, MYH11, MYH6, MYH7, MYL2, MYL3, MYLK, MYO6, NAGLU, NDUFA1, NDUFA13, NDUFA2, NDUFA9, NDUFAF2, NDUFAF5, NDUFAF6, NDUFAF8, NDUFB8, NDUFC2, NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NEXN, NF1,NKX2-5, NR0B1, NR3C1, NUBPL, OPA1, PCCA, PCCB, PCSK9, PDHA1, PDHB, PDHX, PDSS2, PET100, PEX7, PHKA1, PHKA2, PHKB, PHKG2, PHYH, PIEZO2, PKP2, PLN, PNPLA2, PNPT1, PRDM16, PRKAG2, PRKG1, PROC, PROS1, PSEN2, PTPN11, RAF1, RBM20, RET, RIT1, RYR2, SCN1B, SCN5A, SCNN1A, SCNN1B, SCNN1G, SCO2, SDHA, SDHAF2, SDHB, SDHC, SDHD, SERAC1, SERPINC1, SGCD, SGSH, SKI, SLC19A3, SLC22A5, SLC25A20, SLC25A4, SLC40A1, SLC4A3, SMAD2, SMAD3, SMAD9, SUCLA2, SUCLG1, SURF1, TACO1, TAFAZZIN, TAT, TBX4, TCAP, TECRL, TFR2, TGFB2, TGFBR1, TGFBR2, TMEM127, TMEM43, TNNC1, TNNI3, TNNT2, TPK1, TPM1, TRDN, TRIM63, TRMU, TRPM4, TSFM, TTC19, TTN, TTR, VCL, VHL, WNK1, and WNK4.

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