Notch3 mutants and uses thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to NOTCH3 mutants and their applications. This invention discloses for the first time a protein with the following mutation compared to the wild-type NOTCH3 protein: p.Val1186Met (p.V1186M), and the nucleic acid encoding this protein. This protein and / or the nucleic acid encoding it are pathogenic factors of lymphocystic hygroma. Detection of this protein and / or the nucleic acid encoding it can be used for the diagnosis or prognostic assessment of lymphocystic hygroma. Specific alterations to this protein and / or the substance encoding it can achieve the effects of preventing and / or treating lymphocystic hygroma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to NOTCH3 mutants and their applications. Background Technology

[0002] Cystic hygroma (CH) is a common congenital developmental abnormality. Its main clinical feature is the formation of cystic lymphatic vessels filled with abnormally accumulated lymph fluid, resulting from lymphatic vessel obstruction and dilation due to abnormal lymphatic vessel development. It most commonly occurs in early pregnancy, with over 90% occurring in the neck, especially the posterior neck. The incidence of fetal neck cystic hygroma in spontaneous abortions is 1 in 750. In high-risk pregnancies undergoing prenatal diagnosis, the incidence reaches 6.8%. In clinical practice, prenatal ultrasound scans are primarily used to diagnose fetal neck cystic hygroma. Based on ultrasound characteristics, it can be divided into septated and non-septated subtypes. The non-septated type is difficult to distinguish from typical nuchal translucency (NT). Therefore, in clinical practice, the majority of fetal neck cystic hygromas are of the septated type, accounting for over 85%. Unseptated cystic hygromas primarily present as unilocular cystic masses, mostly located on both sides of the neck, appearing as a small cystic mass surrounding the neck, with good internal sound transmission, making them easily missed. Unseptated cystic hygromas without other abnormalities and with normal chromosomal karyotypes have a better prognosis and can be treated intrauterinely or surgically removed during the neonatal period. Septated cystic hygromas have an extremely poor prognosis and are extremely dangerous. The most common comorbid malformations are hydrops fetus, cardiovascular malformations, and skeletal malformations. Pregnancy outcomes are poor, with most pregnant women choosing to terminate the pregnancy. In the few pregnancies that continue, there is either spontaneous abortion or intrauterine fetal death. Some surviving newborns die in the perioperative period or are left with sequelae of psychomotor developmental delay. Completely healthy cases are extremely rare, accounting for less than 4%. If various causes lead to delayed lymphatic and jugular sinus return or increased fetal venous pressure, the lymphatic fluid in the cystic hygroma cannot drain into the lymphatic system and internal jugular vein, resulting in excessive accumulation of cervical lymphatic fluid in the neck, and a significant thickening of the nuchal translucency (NT). Severe lymphatic circulation disorders or congenital malformations of the reticular lymphatic vessels that transport tissue fluid to the venous system can develop into chronic hemorrhage (CH). Fetal nuchal translucency (NT) thickening has a similar pathological basis to CH, only differing in degree.

[0003] Currently, chromosomal aberrations are known to be the main cause of fetal neck cystic ovary syndrome, accounting for approximately 40%–45% of cases. The most common cause is Turner syndrome (congenital ovarian hypoplasia), followed by trisomy 21, trisomy 18, and trisomy 13. Another 5% of cases are caused by chromosomal microdeletions or microduplications, and some are related to genetic syndromes, such as Noonan syndrome, multiple pterygoid syndrome, and musculoskeletal abnormalities-related syndromes. In clinical practice, the etiology and molecular alterations of 60% of fetal cases remain unknown. The discovery and detection of genes causing fetal neck cystic ovary syndrome can enhance our understanding of this disease and provide guidance for prenatal diagnosis. The recently developed next-generation sequencing (NGS) technology can directly and accurately detect gene variants within the 3 billion base pairs of the whole genome, providing technical convenience and feasibility for exploring and discovering pathogenic genes of genetic diseases. Among them, whole exome sequencing (WES) has been widely used in the research of genetic diseases and complex diseases due to its economic and efficient advantages. As WES develops, more genes associated with fetal neck cystic lymphoma will be discovered, leading to a deeper understanding of the genetic causes of this disease. Summary of the Invention

[0004] The first aspect of the present invention is to provide a protein.

[0005] A second aspect of the present invention is to provide a nucleic acid encoding the first aspect of the present invention.

[0006] A third aspect of the present invention aims to provide the use of substances for detecting proteins of the first aspect of the present invention and / or substances for detecting substances of the second aspect of the present invention in the preparation of products for the diagnosis or prognostic assessment of lymphocystoma.

[0007] A fourth aspect of the present invention is to provide a method for constructing a biological model.

[0008] A fifth aspect of the present invention aims to provide the application of biological models in drug screening.

[0009] A sixth aspect of the present invention is to provide the use of substances that specifically alter the proteins of the first aspect of the present invention and / or the nucleic acids of the second aspect of the present invention in the preparation of medicaments for the prevention and / or treatment of lymphocystic hygroma.

[0010] The seventh aspect of the present invention is to provide a medicine.

[0011] An eighth aspect of the present invention is to provide a product.

[0012] The object of the ninth aspect of the present invention is to provide nucleic acid-related biomaterials related to the second aspect of the present invention.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides a protein having the following mutation compared to the wild-type NOTCH3 protein: p.Val1186Met (p.V1186M).

[0015] Preferably, the amino acid sequence of the wild-type NOTCH3 protein is shown in SEQ ID NO.2.

[0016] A second aspect of the invention provides a nucleic acid encoding a protein of the first aspect of the invention.

[0017] Preferably, the nucleic acid has the following mutation compared to the wild-type NOTCH3 gene: c.3556G>A.

[0018] Preferably, the nucleotide sequence of the wild-type NOTCH3 gene is shown in SEQ ID NO.1.

[0019] A third aspect of the invention provides the use of at least one of (e1) to (e3) in the preparation of products for the diagnosis or prognostic assessment of lymphocystoma;

[0020] (e1) A substance for detecting the protein of the first aspect of the present invention;

[0021] (e2) A substance for detecting nucleic acids according to the second aspect of the present invention;

[0022] (e3) Detection of substances in wild-type NOTCH3.

[0023] Preferably, the substance for detecting the protein of the first aspect of the present invention is a substance for quantitatively detecting the protein of the first aspect of the present invention.

[0024] Preferably, the substance used to detect the protein of the first aspect of the present invention is selected from substances of one or more detection methods from the group consisting of: immunohistochemistry, Western blotting, and biochip methods.

[0025] Preferably, the immunohistochemical method is selected from: immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), and immunogold assay.

[0026] Preferably, the substance for detecting the protein of the first aspect of the present invention comprises a substance that is specific to the protein of the first aspect of the present invention.

[0027] Preferably, the substance specific to the protein of the first aspect of the present invention is any one of (a1) to (a3):

[0028] (a1) An antibody that specifically binds to the protein of the first aspect of the present invention;

[0029] (a2) A ligand protein or polypeptide that specifically binds to the protein of the first aspect of the present invention;

[0030] (a3) Non-protein compounds that specifically recognize the proteins of the first aspect of the present invention.

[0031] Preferably, the antibody includes at least one of polyclonal antibody, monoclonal antibody, single-chain antibody, functional antibody fragment, antibody Fab region, nanobody, chimeric antibody, and multispecific antibody.

[0032] Preferably, the substance for detecting the nucleic acid of the second aspect of the present invention is a substance for quantitatively detecting the nucleic acid of the second aspect of the present invention.

[0033] Preferably, the substance used to detect the nucleic acid of the second aspect of the present invention is selected from one or more detection techniques or methods from the group consisting of: Northern blotting, PCR, and biochip methods.

[0034] Preferably, the substance for detecting the nucleic acid of the second aspect of the present invention includes probes, gene chips, PCR primers, etc. of the nucleic acid of the second aspect of the present invention.

[0035] Preferably, the substance used to detect the nucleic acid of the second aspect of the present invention comprises NOTCH3 primers.

[0036] Preferably, the sequences of the NOTCH3 primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0037] Preferably, the substance for detecting wild-type NOTCH3 includes a substance for quantitatively detecting wild-type NOTCH3.

[0038] Preferably, the substance for detecting wild-type NOTCH3 includes a substance that detects wild-type NOTCH3 at the gene level and / or protein level.

[0039] Preferably, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: immunohistochemistry, Western blotting, Northern blotting, PCR, and microarrays.

[0040] Preferably, the immunohistochemical method is selected from at least one of the following: immunofluorescence analysis, reverse enzyme-linked immunosorbent assay (ELISA), and immunogold assay.

[0041] Preferably, the substance for detecting wild-type NOTCH3 is selected from at least one of the following: substances specific to wild-type NOTCH3, such as antibodies (preferably monoclonal antibodies); probes, gene chips, PCR primers, etc., specific to wild-type NOTCH3.

[0042] Preferably, the product comprises at least one of reagents, reagent kits, test strips, chips, and systems.

[0043] Preferably, the test sample for the product is selected from at least one of blood, amniotic fluid, chorionic villus, umbilical cord blood, tissue, and cell samples.

[0044] Preferably, the lymphocystic hygroma includes at least one of septated lymphocystic hygroma and non-septated lymphocystic hygroma; more preferably, the lymphocystic hygroma includes septated lymphocystic hygroma.

[0045] The inventors discovered that patients with lymphocystic hygroma have the aforementioned gene mutations in their exons, specifically heterozygous and / or homozygous mutations; and / or decreased expression of wild-type NOTCH3; and / or decreased activity of wild-type NOTCH3 protein. Products prepared from substances for detecting the aforementioned nucleic acids and / or the aforementioned proteins and / or for detecting wild-type NOTCH3 can be used for the diagnosis or prognostic assessment of lymphocystic hygroma.

[0046] A fourth aspect of the present invention provides a method for constructing a biological model, comprising the following steps: causing the biological model to carry at least one of (b1) to (b4):

[0047] (b1) The protein of the first aspect of the present invention;

[0048] (b2) Nucleic acid of the second aspect of the present invention;

[0049] (b3) The expression level of wild-type NOTCH3 was reduced;

[0050] (b4) The activity of wild-type NOTCH3 protein is reduced.

[0051] Preferably, the wild-type NOTCH3 described in (b3) comprises protein and nucleic acid.

[0052] Preferably, the expression level of wild-type NOTCH3 is reduced by substances that degrade wild-type NOTCH3 and / or substances that reduce the expression level of wild-type NOTCH3.

[0053] Preferably, the activity of wild-type NOTCH3 protein is reduced by substances that degrade wild-type NOTCH3 protein and / or substances that inhibit the activity of wild-type NOTCH3 protein.

[0054] Preferably, the biological model includes at least one of an animal model and a cell model; more preferably, the biological model includes an animal model.

[0055] Preferably, the animal model includes at least one of a mouse model and a rat model.

[0056] Preferably, the biological model is a lymphocystic hygroma model.

[0057] Preferably, the lymphocystic hygroma includes at least one of septated lymphocystic hygroma and non-septated lymphocystic hygroma; more preferably, the lymphocystic hygroma includes septated lymphocystic hygroma.

[0058] A fifth aspect of the invention provides the application of a biological model in drug screening, said biological model carrying at least one of (b1) to (b4):

[0059] (b1) The protein of the first aspect of the present invention;

[0060] (b2) Nucleic acid of the second aspect of the present invention;

[0061] (b3) The expression level of wild-type NOTCH3 was reduced;

[0062] (b4) The activity of wild-type NOTCH3 protein is reduced.

[0063] Preferably, the wild-type NOTCH3 described in (b3) comprises protein and nucleic acid.

[0064] Preferably, the expression level of wild-type NOTCH3 is reduced by substances that degrade wild-type NOTCH3 and / or substances that reduce the expression level of wild-type NOTCH3.

[0065] Preferably, the activity of wild-type NOTCH3 protein is reduced by substances that degrade wild-type NOTCH3 protein and / or substances that inhibit the activity of wild-type NOTCH3 protein.

[0066] Preferably, the biological model includes at least one of an animal model and a cell model; more preferably, the biological model includes a cell model.

[0067] Preferably, the animal model includes at least one of a mouse model and a rat model.

[0068] Models carrying the aforementioned nucleic acids, proteins, reduced expression levels of wild-type NOTCH3, and reduced activity of wild-type NOTCH3 protein can serve as c.3556G>A-related disease models. These disease models can then be used for scientific research, such as screening drugs to treat c.3556G>A-related diseases.

[0069] Preferably, the biological model is a lymphocystic hygroma model.

[0070] Preferably, the drug is used for the prevention and / or treatment of lymphocystoma.

[0071] Preferably, the lymphocystic hygroma includes at least one of septated lymphocystic hygroma and non-septated lymphocystic hygroma; more preferably, the lymphocystic hygroma includes septated lymphocystic hygroma.

[0072] A sixth aspect of the present invention is to provide the use of at least one of (f1) to (f3) in the preparation of a medicament for the prevention and / or treatment of lymphocystic hygroma;

[0073] (f1) Substances that increase the expression level of wild-type NOTCH3;

[0074] (f2) Substances that enhance the activity of wild-type NOTCH3 protein;

[0075] (f3) Substances that specifically alter the protein of the first aspect of the present invention and / or the nucleic acid of the second aspect of the present invention.

[0076] As mentioned above, patients with lymphocystic hygroma carry the aforementioned gene mutations, which are heterozygous and / or homozygous mutations; and / or have reduced expression levels of wild-type NOTCH3; and / or reduced activity of wild-type NOTCH3 protein. If a drug can restore the aforementioned nucleic acid or protein to wild-type or non-pathogenic, and / or increase the expression level of wild-type NOTCH3, and / or increase the activity of wild-type NOTCH3 protein, it has the potential to treat and / or prevent lymphocystic hygroma.

[0077] Preferably, the specificity change is to restore the protein of the first aspect of the present invention and / or the nucleic acid of the second aspect of the present invention to the wild type.

[0078] Preferably, the substance that specifically alters the protein of the first aspect of the present invention and / or the nucleic acid of the second aspect of the present invention is a substance based on at least one gene editing method, including single-base gene editing, zinc finger nuclease (ZFN), transcription activation-like effector nuclease (TALEN), CRISPR / Cas9 (which can be combined with iPSC and AAV vector technology simultaneously).

[0079] Preferably, the lymphocystic hygroma includes at least one of septated lymphocystic hygroma and non-septated lymphocystic hygroma; more preferably, the lymphocystic hygroma includes septated lymphocystic hygroma.

[0080] A seventh aspect of the present invention is to provide a medicament comprising at least one of (f1) to (f3):

[0081] (f1) Substances that increase the expression level of wild-type NOTCH3;

[0082] (f2) Substances that enhance the activity of wild-type NOTCH3 protein;

[0083] (f3) Substances that specifically alter the protein of the first aspect of the present invention and / or the nucleic acid of the second aspect of the present invention.

[0084] Preferably, the specificity change is to restore the protein of the first aspect of the present invention and / or the nucleic acid of the second aspect of the present invention to the wild type.

[0085] Preferably, the substance that specifically alters the protein of the first aspect of the present invention and / or the nucleic acid of the second aspect of the present invention is a substance based on at least one gene editing method, including single-base gene editing, zinc finger nuclease (ZFN), transcription activation-like effector nuclease (TALEN), CRISPR / Cas9 (which can be combined with iPSC and AAV vector technology simultaneously).

[0086] Preferably, the drug is used for the prevention and / or treatment of lymphocystoma.

[0087] Preferably, the lymphocystic hygroma includes at least one of septated lymphocystic hygroma and non-septated lymphocystic hygroma; more preferably, the lymphocystic hygroma includes septated lymphocystic hygroma.

[0088] An eighth aspect of the present invention is to provide a product comprising at least one of (g1) to (g3):

[0089] (g1) Substances for detecting the protein of the first aspect of the present invention;

[0090] (g2) A substance for detecting nucleic acids according to the second aspect of the present invention;

[0091] (g3) Detection of substances in wild-type NOTCH3.

[0092] Preferably, the product comprises (g1) or (g2).

[0093] Preferably, the product comprises at least two of (g1) to (g3).

[0094] Preferably, the product comprises at least one of a substance for detecting proteins of the first aspect of the present invention, a substance for detecting nucleic acids of the second aspect of the present invention, and a substance for detecting wild-type NOTCH3.

[0095] Preferably, the substance for detecting wild-type NOTCH3 includes a substance for quantitatively detecting wild-type NOTCH3.

[0096] Preferably, the substance for detecting wild-type NOTCH3 includes a substance that detects wild-type NOTCH3 at the gene level and / or protein level.

[0097] Preferably, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: immunohistochemistry, Western blotting, Northern blotting, PCR, and microarrays.

[0098] Preferably, the immunohistochemical method is selected from at least one of the following: immunofluorescence analysis, reverse enzyme-linked immunosorbent assay (ELISA), and immunogold assay.

[0099] Preferably, the substance for detecting wild-type NOTCH3 is selected from at least one of the following: substances specific to wild-type NOTCH3, such as antibodies (preferably monoclonal antibodies); probes, gene chips, PCR primers, etc., specific to wild-type NOTCH3.

[0100] Preferably, the substance for detecting the protein of the first aspect of the present invention is a substance for quantitatively detecting the protein of the first aspect of the present invention.

[0101] Preferably, the substance used to detect the protein of the first aspect of the present invention is selected from substances of one or more detection methods from the group consisting of: immunohistochemistry, Western blotting, and biochip methods.

[0102] Preferably, the immunohistochemical method is selected from: immunofluorescence assay, enzyme-linked immunosorbent assay (ELISA), and immunogold assay.

[0103] Preferably, the substance for detecting the protein of the first aspect of the present invention comprises a substance that is specific to the protein of the first aspect of the present invention.

[0104] Preferably, the substance specific to the protein of the first aspect of the present invention is any one of (a1) to (a3):

[0105] (a1) An antibody that specifically binds to the protein of the first aspect of the present invention;

[0106] (a2) A ligand protein or polypeptide that specifically binds to the protein of the first aspect of the present invention;

[0107] (a3) Non-protein compounds that specifically recognize the proteins of the first aspect of the present invention.

[0108] Preferably, the antibody includes at least one of polyclonal antibody, monoclonal antibody, single-chain antibody, functional antibody fragment, antibody Fab region, nanobody, chimeric antibody, and multispecific antibody.

[0109] Preferably, the substance for detecting the nucleic acid of the second aspect of the present invention is a substance for quantitatively detecting the nucleic acid of the second aspect of the present invention.

[0110] Preferably, the substance used to detect the nucleic acid of the second aspect of the present invention is selected from one or more detection techniques or methods from the group consisting of: Northern blotting, PCR, and biochip methods.

[0111] Preferably, the substance for detecting the nucleic acid of the second aspect of the present invention includes probes, gene chips, PCR primers, etc. of the nucleic acid of the second aspect of the present invention.

[0112] Preferably, the substance used to detect the nucleic acid of the second aspect of the present invention comprises NOTCH3 primers.

[0113] Preferably, the sequences of the NOTCH3 primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0114] Preferably, the product is used for the diagnosis or prognostic assessment of lymphocystic hygroma.

[0115] Preferably, the product comprises at least one of reagents, reagent kits, test strips, chips, and systems.

[0116] Preferably, the test sample for the product is selected from at least one of blood, amniotic fluid, chorionic villus, umbilical cord blood, tissue, and cell samples.

[0117] Preferably, the product also contains other substances for diagnosing lymphocystic fibrosis.

[0118] Preferably, the lymphocystic hygroma includes at least one of septated lymphocystic hygroma and non-septated lymphocystic hygroma; more preferably, the lymphocystic hygroma includes septated lymphocystic hygroma.

[0119] A ninth aspect of the present invention provides a nucleic acid-related biomaterial related to the second aspect of the present invention, said biomaterial comprising at least one of (c1) to (c7):

[0120] (c1) An expression cassette containing the nucleic acid of the second aspect of the present invention;

[0121] (c2) A vector comprising the nucleic acid of the second aspect of the present invention;

[0122] (c3) A carrier containing the expression box described in (c1);

[0123] (c4) A transgenic cell line comprising the nucleic acid of the second aspect of the present invention;

[0124] (c5) A transgenic cell line containing the expression cassette described in (c1);

[0125] (c6) A transgenic cell line containing the vector described in (c2);

[0126] (c7) A transgenic cell line containing the vector described in (c3).

[0127] Preferably, the transgenic cell line does not contain propagation material.

[0128] The beneficial effects of this invention are:

[0129] This invention discloses for the first time a protein with the following mutation compared to the wild-type NOTCH3 protein: p.Val1186Met (p.V1186M), and the nucleic acid encoding the protein. This protein and / or the nucleic acid encoding the protein are pathogenic factors of lymphocystic hygroma. By detecting this protein and / or the nucleic acid encoding the protein, lymphocystic hygroma can be diagnosed or its prognosis can be assessed. By specifically altering this protein and / or the substance encoding the protein, the effects of preventing and / or treating lymphocystic hygroma can be achieved. Attached Figure Description

[0130] Figure 1 The images show fetal pathology images of non-syndromic segmented lymphocystic cystic cysts: A is a fetal microscopic image of the overall appearance of the fetus with lymphocystic ...

[0131] Figure 2 This is a volcano plot of protein expression results: In the figure, proteins with no significant changes in expression are shown in black, upregulated expression is shown in green, and downregulated expression is shown in red. The NOTCH3 protein is located in the red area on the right, indicating significant downregulated expression.

[0132] Figure 3 This is an image of the F2 generation of the NOTCH3 gene mutant C57BL / 6 mouse model.

[0133] Figure 4 This is a partial anatomical diagram of the neck of a F2 generation mouse model of the NOTCH3 gene mutation C57BL / 6 mouse. Detailed Implementation

[0134] The present invention will be further described in detail below through specific embodiments.

[0135] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0136] Terminology Explanation:

[0137] 1. gnomAD: https: / / gnomad.broadinstitute.org / , a population database that has currently collected data on 15,708 whole genomes and 125,748 exomes;

[0138] 2.1000 Genomes: https: / / www.internationalgenome.org / , the 1000 Genomes database, containing 2,504 sequencing samples;

[0139] 3. OMIM: https: / / www.omim.org / , an online database of human Mendelian genetics;

[0140] 4. BWA: http: / / bio-bwa.sourceforge.net / , software that aligns raw sequencing data to a reference genome;

[0141] 5. GATK: https: / / gatk.broadinstitute.org / hc / en-us, short for Genome Analysis Tool Kit, is a software for analyzing variant information from high-throughput sequencing data;

[0142] 6. VEP: Variant Effect Predictor, which annotates different types of mutations generated by second-generation testing, https: / / asia.ensembl.org / info / docs / tools / vep / index.html;

[0143] 7. SIFT, Polyphen2_HDIV, REVEL, MutationTaster, MetaSVM: Various hazard prediction software used to predict the hazards caused by mutations;

[0144] 8. ClinVar: https: / / www.ncbi.nlm.nih.gov / clinvar / , stores the relationship between human variant sites and phenotypes and provides supporting evidence;

[0145] 9. HGMD: http: / / www.hgmd.cf.ac.uk / ac / gene.php?, The Human Gene Mutation Database, collects mutations related to human genetic diseases;

[0146] 10. Orphanet: https: / / www.orpha.net / consor / cgi-bin / index.php, a database of rare human diseases;

[0147] 11. DDG2P: https: / / decipher.sanger.ac.uk / ddd / ddgenes, a database of genotypes and phenotypes related to developmental disorders;

[0148] 12. PCR: Polymerase chain reaction is a molecular biology technique used to amplify specific DNA fragments;

[0149] 13. CRISPR-Cas9: A genome editing technology developed from the acquired immune system unique to microorganisms such as bacteria and archaea;

[0150] 14. C57BL / 6: This is a common inbred strain of laboratory mice, widely used in genetic experiments as transgenic mice to simulate human genetic defects.

[0151] Example 1: Identification of the pathogenic gene for fetal cervical cystic lymphoma

[0152] 1. Sample collection

[0153] The inventors collected 52 cases of fetal cystic hygroma of unknown cause and their families for whole-exome sequencing (WES). WES results identified and screened the NOTCH3 gene and its mutation site in 7 fetal cases. Simultaneously, Sanger sequencing was used to extend the screening of the identified NOTCH3 gene and its mutation site in 100 normal controls and 103 fetal cases of cystic hygroma. Therefore, a total of 52+103 fetal cases of complete non-syndromic septate cystic hygroma were collected. Peripheral blood samples were drawn from all individuals (patients and normal individuals), and chorionic villus sampling, amniotic fluid sampling, and umbilical cord blood sampling were collected from fetal cases. Neck tissue was also collected from some cases of induced labor. Sample collection was approved by the Ethics Committee of Guangzhou Women and Children's Medical Center, adhering to the standards established in the 1964 Declaration of Helsinki and its subsequent revisions. Informed consent was obtained from all participating individuals or their guardians. Clinical information, including age, sex, and clinical symptoms, was collected. Immunofluorescence staining was used to conduct pathological studies on fetal tissues from non-syndromic chronic hemangiomas (CH) resulting from termination of pregnancy. Specific lymphatic and vascular staining antibodies (LYVE1, PROX1, CD31, etc.) confirmed that the pathological features of cystic hygroma of the neck in these fetal cases are lymphatic dysplasia and abnormal development of the basal smooth muscle of capillaries. Furthermore, these abnormalities were not correlated with gestational age. Figure 1 Whole-exome sequencing (WES) was used to perform whole-exome sequencing analysis on 52 fetuses with unexplained cervical lymphocystic hygroma and their core family members, and in-depth analysis was conducted from different levels, including biological significance, genetic significance, and clinical characteristics (related to lymphatic vessel development and capillary development).

[0154] 2. Whole exome sequencing

[0155] All DNA samples were hybridized using the SureSelect XT Human All Exon 50Mb V5 Kit (Agilent) to obtain target region sequences, which were then sequenced at 150bp paired ends on an Illumina HiSeq 2500 sequencer. The raw data obtained after sequencing were subjected to raw data quality control using FASTP (V0.20) to remove reads containing adapters or low quality. The clean data after quality control was aligned to the human reference genome GRCH37 (hg19) using BWA (V0.7.17). Samtools (V1.9) and Picard software were used to sort the aligned data and label repetitive sequences. Finally, the HaplotypeCaller module of GATK (V3.8) was used to detect variant information in the samples. The initial variant results were filtered to retain high-quality information, removing variants that met one of the following conditions: total mutation depth less than 10, genotype quality value less than 50, and alignment quality value less than 30. Variants were annotated using VEP (Variant Effect Predictor), along with information from disease databases, population frequency databases, hazard or conserved prediction software, and variant site databases. Population databases included 1000genomics, gnomAD, and local laboratory frequency databases. Hazard or conserved prediction software included SIFT, Polyphen2_HDIV, REVEL, MutationTaster, and MetaSVM. Variant site databases included ClinVar and HGMD, while disease databases included OMIM, Orphanet, MedGen, and DDG2P.

[0156] Analysis of WES data from the core families of the aforementioned 52 patients ( Figure 2

[0157] Example 2: Sanger sequencing verification

[0158] 1. DNA extraction: DNA was extracted from all samples using the QIAamp DNA Blood Mini Kit (method as per the instruction manual).

[0159] 2. Primer design and PCR reaction

[0160] a) Primer design: Based on the human genome reference sequence Hg19 / GRCH37, mutation site-specific primers were designed, as shown in Table 1.

[0161] Table 1. Specific primers for NOTCH3_3556G>A

[0162]

[0163] b) Reaction system: 3 μL MgCl2 (25 mM), 15 μL GC Buffer I, 3 μL dNTP (2.5 mM), 2 μL each of upstream and downstream primers (10 μmol / L), 0.1 μL Promega Taq DNA polymerase, 1 μL template DNA, and 7 μL ddH2O.

[0164] c) Reaction program: as follows: 94℃ for 3 min; 94℃ for 30 s, 59℃ for 30 s, 72℃ for 100 s, 30 cycles; 72℃ for 5 min; 10℃ for ∞.

[0165] 3. Sanger sequencing

[0166] The PCR amplification products from the 52 patient families, 103 sporadic patients, and 100 healthy individuals obtained in step 2 were directly sequenced using an ABI 3730 DNA Analyzer to obtain sequencing results. Based on the sequencing results, the NOTCH3 gene sequence of the above samples was aligned. The results are shown in Table 2: 7 patients (from the 52 patients) and 4 patients (from the 103 sporadic patients) had a missense mutation c.3556G>A in the NOTCH3 gene, while none of the 100 healthy individuals had the c.3556G>A missense mutation in their NOTCH3 gene. Therefore, the c.3556G>A missense mutation in the NOTCH3 gene is a pathogenic mutation in septate lymphocystic hygroma and can be used as a diagnostic marker for septate lymphocystic hygroma.

[0167] Table 2. NOTCH3 gene sample information from 11 patients and 100 healthy individuals.

[0168] Sample number Illness Mutant genes Mutant exons cDNA sequence Mutation type P1 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P2 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P3 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P4 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P5 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P6 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P7 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P8 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P9 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P10 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding P11 Neck lymphocystic fibroma NOTCH3 Exon 22 c.3556G>A Hybrid Misunderstanding 100 normal controls normal NOTCH3 Exon 22 No mutation No mutation

[0169] Example 3: A method for constructing a mouse model of lymphocystic hygroma.

[0170] A method for constructing a lymphocystic hygroma mouse model was proposed. This method involved directly inserting a missense mutation c.3556G>A (p.Val leucine 1186Met methionine) into the NOTCH3 gene of C57BL / 6 mice using CRISPR / Cas9 gene knockout technology, resulting in a lymphocystic hygroma (NOTCH3 heterozygous mutant) mouse model (F1 generation). The appearance and local neck anatomy of the F2 generation mice were then observed, and the results are as follows: Figure 3 , 4As shown: F2 generation NOTCH3 mutant mice exhibited significant cervical lymphadenopathy, abnormal cardiac development, and curled tail. By constructing a mouse model, this study explored the molecular mechanisms by which NOTCH3 mutations affect lymphatic vessel and capillary development and the Notch signaling pathway. Clinical fetal samples and mouse tissues from fetal lymphocystic tumors were used, and neck tissues from F2 generation NOTCH3-mutant mice at different embryonic developmental stages were collected, with wild-type C57BL / 6 embryonic mouse neck tissues serving as controls. The expression patterns of NOTCH3 at each embryonic developmental stage were observed. Simultaneously, transcriptome RNA sequencing analysis was performed, and GO and KEGG enrichment analyses were conducted on differentially expressed genes at the transcriptome level. RT-PCR, immunohistochemistry, and Western blotting were used to observe the expression levels of ligands JAG1 and D114 proteins and other key molecules (LYVE-1, prox-1, VEGF-3, VEGFA, etc.) in the Notch signaling pathway, changes in intracellular effector molecules (CSL-DNA binding proteins), and downstream target genes (Hes, Hey, Herp) to analyze the mechanism of action of the Notch signaling pathway induced by NOTCH3 mutations in fetal lymphocystic tumors. Finally, using human umbilical vein endothelial cells, a lentiviral vector interfering with the NOTCH3 gene was constructed to silence and overexpress the NOTCH3 gene in umbilical vein endothelial cells (experimental groups: normal control group, negative plasmid group, NOTCH3 gene interference group, NOTCH3 gene overexpression group). Western blotting, immunohistochemistry and other techniques were used to detect the expression of cell growth and proliferation-related proteins (VEGF, etc.), apoptosis activating factors and anti-apoptotic factors (Apaf-1, caspase-9, caspase-3, Bcl-2 and Bcl-xL), endothelial angiogenesis factors (LYVE-1, prox-1, VEGF-3, VEGFA, etc.), and key Notch signaling pathway factors JAG1, D114, CSL-DNA binding protein, Hes, Hey, Herp in each group to evaluate the regulatory mechanism of NOTCH3 gene deficiency on lymphatic vessel and capillary development and differentiation. Finally, normal concentrations of wild-type NOTCH3-mRNA (normal NOTCH3 concentration is 20 pmol / L) were added to each group of cells, and the expression changes of the above indicators were observed. The aim was to explore the use of normal wild-type NOTCH3-mRNA to repair the abnormal activation of the NOTCH3-Notch signaling pathway caused by NOTCH3 gene defects, and to evaluate the regulatory mechanism of NOTCH3 gene defects on lymphatic vessel and capillary development and differentiation.This indicates that NOTCH3 protein is associated with lymphocysticercosis (LCB), and mutations or deletions of NOTCH3 protein can cause LCB. LCB can be diagnosed or its prognosis assessed by detecting the expression levels of NOTCH3 protein and / or the nucleic acid encoding NOTCH3 protein, and / or the NOTCH3 protein with the aforementioned point mutation (p.V1186M) and / or the NOTCH3 gene (c.3556G>A). Correspondingly, increasing the expression levels of NOTCH3 protein and / or the nucleic acid encoding NOTCH3 protein, and / or restoring the NOTCH3 protein with the aforementioned point mutation (p.V1186M) and / or the NOTCH3 gene (c.3556G>A) to wild-type can achieve the effects of preventing and / or treating LCB.

[0171] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A protein, which, compared with the wild-type NOTCH3 protein, has only the following mutation: p.V1186M, wherein the amino acid sequence of the wild-type NOTCH3 protein is shown in SEQ ID NO:

2.

2. The nucleic acid encoding the protein of claim 1.

3. The use of at least one of the reagents for detecting the protein of claim 1 or the nucleic acid of claim 2 in the preparation of products for the diagnosis or prognostic assessment of lymphocystoma.

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