Application of TMPRSS9 gene in the diagnosis and treatment of venous thromboembolism

Abstract

The present invention discloses the application of TMPRSS9 gene in the diagnosis and treatment of venous thromboembolism, and belongs to the field of medical diagnosis and treatment technology. The present invention verifies the association between missense mutations of TMPRSS9 gene and thrombophilia in human population, in vivo and in vitro, and draws the following conclusions: nonsense mutations and missense mutations at the sites of c.334C>T, c.692C>T, c.917G>A, and c.1709C>T of TMPRSS9 gene are representative in the effect of TMPRSS9 gene mutation on the occurrence of venous thrombosis. The discovery of the above-mentioned pathogenic gene mutations can prevent and treat thrombophilia at an early stage, which is of great significance for the diagnosis, treatment and management of thrombophilia. At the same time, the inclusion of the new TMPRSS9 gene enriches the genetic variation spectrum of venous thromboembolic diseases, which is of guiding significance for the screening of diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis and treatment technology, and in particular relates to the application of TMPRSS9 gene in the diagnosis and treatment of venous thromboembolism. Background Art

[0002] Thrombophilia refers to a pathological state in which thrombosis and thromboembolism are prone to occur due to various genetic or acquired factors. The main clinical manifestations of thrombophilia are venous thromboembolism (VTE): such as deep vein thrombosis, pulmonary embolism, intracranial venous thrombosis, portal vein formation, etc. Some types of thrombophilia can manifest as early-onset acute coronary syndrome, ischemic stroke and other arterial thrombotic events in young people. The repeated onset of thrombotic events caused by thrombosis significantly increases the disability and mortality rates of patients, seriously endangering human health. Therefore, early screening for thrombophilia is of great significance for improving clinical diagnosis and treatment. Genetic molecular diagnostic technology can screen high-risk thrombophilia patients more comprehensively and accurately. At present, the molecular diagnostic technologies for thrombophilia include PCR, gene chips and second-generation sequencing (NGS). The application of these technologies is very important. The basis for the completeness of the genetic variation spectrum of thrombophilia diseases. At present, the number of candidate genes for molecular diagnostic technology of thrombophilia is still at a low level (currently there are only dozens of candidate genes), and the analysis of new genes and new mutation sites and functional validation are needed to accelerate the advancement of molecular diagnostic technology of thrombophilia. In the past 50 years, researchers have continuously explored the genetic determinants of thrombophilia risk. It was not until the last decade that these studies expanded our biological knowledge base of venous thrombosis beyond the well-characterized coagulation / anticoagulation and fibrinolysis / antifibriolysis pathways. Serine proteases participate in many reactions in the human body, especially the coagulation and complement systems. Several activated coagulation factors are serine proteases (including coagulation factor X, coagulation factor XI, thrombin, plasmin, etc.). In the past few decades, many cell surface-anchored serine proteases have been identified. Transmembrane serine proteases (TMPRSSs), also known as type II transmembrane serine proteases, are a family of proteins with conserved serine protease domains located on the cell membrane. Since the discovery of the first transmembrane serine protease TMPRSS1 in 1988, more than 20 members have been found in humans, mice and rats, including more than a dozen in humans. The TMPRSS9 gene is located on chromosome 19p13 and is a sub-member of the TMPRSSs family. It has three unique tandem serine protease domains (serase-1, serase-2, serase-3) and an LDLR domain. The addition and detection of new pathogenic genes can increase the detection rate of thrombophilia, which has important clinical significance for the early diagnosis and prevention of thrombophilia. Summary of the invention

[0003] The present invention provides an application of a detection reagent for the TMPRSS9 gene in the preparation of a venous thromboembolism diagnosis and treatment product, wherein the detection reagent can detect at least one of the following mutation sites of the TMPRSS9 gene: c.334C>T, c.692C>T, c.917G>A, and c.1709C>T.

[0004] Preferably, the product is a reagent.

[0005] Preferably, the product is a kit.

[0006] Preferably, the detection reagent for the TMPRSS9 gene contains a primer set for detecting at least one of the following mutation sites of the TMPRSS9 gene: c.334C>T, c.692C>T, c.917G>A, c.1709C>T.

[0007] More preferably, the primer set sequence for detecting the mutation site c.334C>T is as shown in SEQ ID NO.7, 8.

[0008] More preferably, the primer set sequence for detecting the mutation site c.692C>T is shown in SEQ ID NOs.13 and 14.

[0009] More preferably, the primer set sequence for detecting the mutation site c.917G>A is as shown in SEQ ID NO.15, 16.

[0010] More preferably, the primer set sequence for detecting the mutation site c.1709C>T is shown in SEQ ID NOs.23 and 24.

[0011] The present invention further provides a kit, which contains the detection reagent for the TMPRSS9 gene.

[0012] Preferably, the kit contains a primer set for detecting at least one of the following mutation sites c.334C>T, c.692C>T, c.917G>A, and c.1709C>T of the TMPRSS9 gene; the primer set sequence for detecting the mutation site c.334C>T is shown in SEQ ID NOs.7 and 8, the primer set sequence for detecting the mutation site c.692C>T is shown in SEQ ID NOs.13 and 14, the primer set sequence for detecting the mutation site c.917G>A is shown in SEQ ID NOs.15 and 16, and the primer set sequence for detecting the mutation site c.1709C>T is shown in SEQ ID NOs.23 and 24.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention verifies the association between missense mutations of the TMPRSS9 gene and thrombophilia in humans, in vivo, and in vitro.

[0015] (2) Nonsense and missense mutations at the c.334C>T, c.692C>T, c.917G>A, and c.1709C>T sites of the TMPRSS9 gene are representative of the effects of TMPRSS9 gene mutations on the occurrence of venous thrombosis.

[0016] (3) Discovery of pathogenic gene mutations and early prevention and treatment are of great significance for the diagnosis, treatment and management of thrombophilia.

[0017] (4) The inclusion of the new TMPRSS9 gene enriches the genetic variation spectrum of venous thromboembolism and has guiding significance for disease screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a disease type distribution diagram of the venous thrombosis group in Example 1.

[0019] Figure 2 The statistical results of missense mutations in the VTE group and the control group in Example 1, wherein A: missense mutation ratio, B: mutation type ratio in missense mutations.

[0020] Figure 3 This is the peak diagram for the alignment of the c.334C>T, c.692C>T, c.917G>A, and c.1709C>T mutation sites in Example 1.

[0021] Figure 4 This is a molecular simulation diagram of the c.334C>T, c.692C>T, c.917G>A, and c.1709C>T mutant proteins in Example 1.

[0022] Figure 5 This is a map of mutation sites in Example 1.

[0023] Figure 6 This is the in vivo phenotype verification result of KO mice in Example 1.

[0024] Figure 7 The expression of EGFP green fluorescent protein after the wild-type and mutant plasmids of TMPRSS9 gene in Example 1 were transfected into HEK-293T cells respectively.

[0025] Figure 8 The changes in the mRNA expression levels of the TMPRSS9 gene mutation sites in Example 1, wherein MOCK represents an empty plasmid; * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

[0026] Fig. 9The changes in protein expression levels at the TMPRSS9 gene mutation site in Example 1, wherein MOCK represents an empty plasmid; * represents P<0.05; ** represents P<0.01; *** represents P<0.001. DETAILED DESCRIPTION

[0027] Example 1

[0028] 1. Population-based case-control

[0029] 1.1 Collection of clinical samples

[0030] The subjects were divided into two groups (venous thrombosis group (VTE group) vs normal health group (control group)). The venous thrombosis group included patients diagnosed with venous thrombosis (VTE) in Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from March 2022 to October 2023. The normal health group included healthy physical examination patients in the same region and time period with no history of thrombotic diseases and family history. This study was reviewed by the Ethics Committee of Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology (Ethics Approval No.:

[2022] Lun Shen Zi (0157)), in accordance with the Declaration of Helsinki, and the epidemiological data collection and clinical blood sample collection processes of this study were carried out after signing the informed consent.

[0031] 1.2 Inclusion and Exclusion Criteria

[0032] Venous thrombosis group (VTE group):

[0033] Inclusion criteria: (1) local clinical manifestations of venous thrombosis, refer to the clinical diagnosis of VTE in the "Chinese Guidelines for the Diagnosis and Prevention of Thrombophilia (2021 Edition)"; (2) plasma D-dimer>0.5mg / L; (3) imaging examinations, such as compression ultrasound or venography to confirm deep vein thrombosis.

[0034] Exclusion criteria: (1) patients with hemorrhagic diseases; (2) patients with acute or chronic bacterial or viral infections; (3) patients suspected of having gas, amniotic fluid or fat embolism; (4) patients with incomplete epidemiological data; and (5) patients who refused to participate in this study.

[0035] Normal healthy group (control group):

[0036] Inclusion criteria: (1) no history of thrombotic disease or family history; (2) no history of medication that affects coagulation within 1 month; (3) gender and age stratification matched the venous thrombosis group;

[0037] Exclusion criteria: (1) patients with a history of thrombosis or family history; (2) patients with incomplete epidemiological data; (3) patients who refused to participate in this study.

[0038] 1.3 Blood sample collection and preparation

[0039] 3 ml of cubital venous blood was collected from all subjects and injected into EDTA anticoagulant tubes. The collected venous blood samples were centrifuged at 2000 g for 10 min at 4 °C to separate two portions of plasma and blood cells, which were stored in 2 ml cryopreservation tubes at -80 °C. The samples were numbered in code sequence and stored in 100-well centrifuge tube boxes. DNA samples were extracted for subsequent sequencing preparation.

[0040] 1.4 Sequencing primer design

[0041] Primer5 software was used to design PCR primers. Primer design principles: (1) The length of the primer is about 15-30 bp; (2) The GC content of the primer is between 40% and 60%, and the Tm value is preferably close to 58°C-60°C; (3) Avoid repeated bases, especially G; (4) The length of the PCR amplification product should be within 300 bp as much as possible; (5) For the specificity of the primers, we analyzed the designed primers in the NCBI online BLAST analysis and found that there were no other non-specific products and the primers had good specificity. However, in actual design, if non-specific fragments that are significantly different from the target fragment and can be distinguished are found, the primers are also considered to have good specificity. The sequencing primers are as follows:

[0042] Table 1

[0043]

[0044]

[0045] 1.5 Sample sequencing results

[0046] A total of 618 participants were included in this case-control study, including 325 in the venous thrombosis group and 293 in the normal control group (see Table 2). The average age was 44.96±15.97, and the age was divided into four levels according to different age groups: <30 (115 cases), 30-40 (156 cases), 40-60 (224 cases), >60 (123 cases). There was no significant difference in the number of people in the VTE group and the control group in each age group (P=0.319). Among all the participants, there were 332 males and 286 females. The proportion of males in the VTE group and the control group was similar, 53.23% and 54.27%, respectively. The proportion of females in the two groups was 46.77% and 45.73%, respectively. There was no significant difference in gender between the two groups (P>0.05). We also counted whether the participants in the two groups were pregnant and had a history of cancer. The results showed that there was no significant difference between the two groups in terms of pregnancy and history of cancer (P>0.05). In addition, we classified and counted the disease types of the participants in the VTE group (see Figure 1In our venous thrombosis cohort, patients with lower extremity deep vein thrombosis accounted for the largest proportion (72.6%), followed by patients with pulmonary embolism (27.4%), and the smallest proportions were femoral vein thrombosis, visceral thrombosis, and pregnancy-related deep vein thrombosis, all at 0.3%.

[0047] Table 2 Basic information and statistical data of venous thrombosis group and normal control group

[0048]

[0049] By performing PCR amplification and Sanger sequencing on DNA samples extracted from the blood of 618 participants, we found 33 single nucleotide mutations in the TMPRSS9 gene through sequence comparison, including 18 missense or nonsense mutations and 15 synonymous mutations. We focused on missense mutations and nonsense mutations and compared the VTE group with the control group (see Figure 2 A). We found that in the VTE group, missense mutations accounted for 36%, of which the proportion of heterozygous missense mutations was 27.3% and the proportion of homozygous missense mutations was 8.6%. In the control group, the proportion of missense mutations was 30.6%, of which the proportion of heterozygous missense mutations was 24% and the proportion of homozygous missense mutations was 6.5%. Overall, the proportion of missense mutations in the TMPRSS9 gene in the VTE group was higher than that in the control group. We counted the types of missense mutations (see Figure 2 B), we found that cytosine (C) to thymine (T) mutation was the most frequent missense mutation type in both the VTE group and the control group, followed by guanine (G) to adenine (A), and the lowest mutation frequency was cytosine (C) to guanine (G).

[0050] To further analyze the frequency of missense mutations, we performed genotype and allele analysis (see Table 3). Among the 18 missense mutations (rs891174, rs17685098, rs372254805, rs773453462, rs758572916, rs757239911, rs117767265, rs200206860, rs62120714, rs1281796844, Among the mutation sites (rs762535649, rs146529608, rs735911, rs369734929, rs199503794, rs765439744, rs7247162 and rs752580832), except rs7247162 (c.2812G>A) which did not conform to the Hardy-Weinberg genetic equilibrium (P<0.05), the other mutation sites (SNPs) reached genetic equilibrium (P>0.05). In the TMPRSS9 gene, the frequency of T allele in the rs200206860 (c.692C>T) VTE group participants was significantly higher than that in the control group (χ2=0.057; OR:1.907; 95%CI=1.808-2.011), the frequency of A allele in the rs62120714 (c.917G>A) VTE group participants was significantly higher than that in the control group (χ2=0.004; OR:1.460; 95%CI=1.128-1.889), and the frequency of T allele in the rs146529608 (c.1709C>T) VTE group was higher than that in the control group (χ2=0.1; OR:1.906; 95%CI=1.807-2.010). We noticed that the arginine mutation of rs372254805 (c.334C>T) is a stop codon and is a nonsense mutation in a protein truncation mutation. The mutations at the above four sites are statistically significant, and we focus on these four mutations in the TMPRSS9 gene. Sequence alignment was performed using SnapGene 6.0 software (see Figure 3 ), and visualized the four mutations. Among them, c.334C>T, c.692C>T, and c.1709C>T are all heterozygous mutations, and c.917G>A has heterozygous mutations and homozygous mutations.

[0051] Table 3 Genotype and allele frequencies of TMPRSS9 missense mutations

[0052]

[0053]

[0054] We used MutationTaster, SIFT, PolyPhen-2, CADD (PHRED), and GERP++score online software to predict the pathogenicity of all missense mutation sites in TMPRSS9 (see Table 4). MutationTaster, SIFT, and PolyPhen-2 can automatically evaluate whether a mutation is harmful or benign, and believe that missense mutation sites may be harmful (Damaging) when CADD (PHRED)>15 and GERP++score>3. We combined the prediction results of various prediction tools and considered sites with two or more predicted results as harmful mutations to be pathogenic mutations. The results showed that a total of 10 sites may be pathogenic mutations, namely rs17685098 (c.217C>T), rs372254805 (c.334C>T), rs758572916 (c.562C>T), rs200206860 (c.692C>T), rs62120714 (c.917G>A), rs146529608 (c.1709C>T), rs369734929 (c.2399C>T), rs199503794 (c.2545C>T), rs7247162 (c.2812G>A), and rs752580832 (c.3087C>G).

[0055] Table 4 Pathogenicity prediction of missense mutation sites

[0056]

[0057] We visualized the mutant proteins at the mutation sites we focused on (see Figure 4 ).

[0058] In order to better demonstrate the location of the possible pathogenic mutations we found in the TMPRSS9 gene and protein molecular structure, we visualized the overall structure (see Figure 5). The TMPRSS9 gene (NM_182973.3 transcript) is located on chromosome 19 and has 18 exons, encoding polyserase-1. Polyserase-1 has three serine protein domains (serase-1, serase-2, serase-3), so it is named polyserase-1. In addition, the stem region of polyserase-1 has a low-density lipoprotein receptor type A (LDLA) domain. Because the active site of the third protease domain contains an alanine residue instead of a serine residue, serase-3 is considered to be catalytically inactive. The mutations we focused on were c.692C>T (p.Ala231Val), c.917G>A (p.Arg306Gln), c.1709C>T (p.Pro570Leu) and the nonsense mutation c.334C>T (p.Arg112Ter). We found that the three sites c.692C>T (p.Ala231Val), c.917G>A (p.Arg306Gln), and c.1709C>T (p.Pro570Leu) are all in the catalytically active region of the TMPRSS9 encoded protein. The truncated mutation c.334C>T (p.Arg112Ter) occurs before the LDLA region, causing the protein translation to terminate here, also leading to inactivation of the catalytically active region. Mutations at these sites are likely to affect the structure and function of the TMPRSS9 encoded protein and are pathogenic.

[0059] 2. Phenotypic Verification of KO Mice in Vivo

[0060] We constructed C57 mice with Tmprss9- / - and Tmprss9+ / - genetic backgrounds and verified the effect of Tmprss9 knockout on venous thromboembolism in mice. We constructed an inferior vena cava thrombosis model and compared the weight and length of thrombi in Tmprss9- / - (10 mice), Tmprss9+ / - (10 mice) and wild-type WT (10 mice) to determine whether there were phenotypic differences. We also performed HE staining on the cross-sections of embolic thrombi to observe whether there were differences in the pathological characteristics of thrombi. Results (e.g. Figure 6 ) As shown in Figure 2, the thrombus length and weight of Tmprss9- / - mice and Tmprss9+ / - mice were higher than those of wild-type WT mice. The HE staining results of the inferior vena cava thrombosis model showed that the thrombus density and lumen filling degree of Tmprss9- / - mice and Tmprss9+ / - mice were greater than those of wild-type WT mice.

[0061] 3. In vitro verification of pathogenic mutation sites

[0062] 3.1 Construction of plasmids

[0063] PGMLV-6395 was used as a plasmid vector to construct wild-type and mutant plasmids of TMPRSS9 gene. HEK-293T cells were transfected and the expression of EGFP green fluorescent protein was observed (see Figure 7 ), the results showed that the green fluorescence after transfection indicated that the quantity reached the standard and the fluorescence intensity was strong, indicating that the TMPRSS9 gene plasmid was successfully transiently transfected into HEK-293T cells and successfully expressed.

[0064] 3.2 Effect of TMPRSS9 gene mutation sites on mRNA expression

[0065] Plasmids carrying empty vector, wild type and 10 missense mutation sites (c.217C>T, c.334C>T, c.562C>T, c.692C>T, c.917G>A, c.1709C>T, c.2399C>T, c.2545C>T, c.2812G>A, c.3087C>G) were transfected into HEK-293T cells, and the changes in TMPRSS9 gene mRNA transcription levels were detected by RT-PCR 48 h after transfection (see Figure 8 ). The mRNA of TMPRSS9 gene in the empty plasmid was basically not expressed. Compared with the wild-type plasmid, the mRNA expression of TMPRSS9 gene in the missense mutation plasmids of c.334C>T, c.692C>T, and c.1709C>T was significantly decreased, with statistical differences. Except for the c.217C>T mutation plasmid, the mRNA expression of the other mutant plasmids decreased, but there was no significant statistical difference. The RT-PCR results were repeated 3 times independently.

[0066] 3.3 Effect of TMPRSS9 gene mutation sites on the expression of encoded proteins

[0067] After HEK-293T cells were transfected with plasmids carrying empty vector, wild type, and 10 missense mutation sites (c.217C>T, c.334C>T, c.562C>T, c.692C>T, c.917G>A, c.1709C>T, c.2399C>T, c.2545C>T, c.2812G>A, c.3087C>G), the mean fluorescence intensity of TMPRSS9 protein expression was detected by flow cytometry 48 h after transfection. The results showed (see Fig. 9 ) The protein expression levels of c.217C>T, c.334C>T, c.692C>T, c.917G>A, and c.1709C>T mutant plasmids were significantly lower than those of the unmutated WT group. Results The experiment was repeated three times independently.

[0068] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a TMPRSS9 gene detection reagent in the preparation of a venous thromboembolism diagnosis and treatment product, characterized in that: The detection reagent can detect at least one of the following mutation sites of the TMPRSS9 gene: c.334C>T, c.692C>T, c.917G>A, and c.1709C>T.

2. The use according to claim 1, characterized in that: The product is a reagent.

3. The use according to claim 1, characterized in that: The product is a test kit.

4. The use according to claim 1, characterized in that: The detection reagent of the TMPRSS9 gene contains a primer set for detecting at least one of the following mutation sites of the TMPRSS9 gene: c.334C>T, c.692C>T, c.917G>A, and c.1709C>T.

5. The use according to claim 4, characterized in that: The primer set sequences for detecting the mutation site c.334C>T are shown in SEQ ID NOs. 7 and 8.

6. The use according to claim 5, characterized in that The primer set sequences for detecting the mutation site c.692C>T are shown in SEQ ID NOs. 13 and 14.

7. The use according to claim 6, characterized in that The primer set sequences for detecting the mutation site c.917G>A are shown in SEQ ID NOs. 15 and 16.

8. The use according to claim 7, characterized in that The primer set sequences for detecting the mutation site c.1709C>T are shown in SEQ ID NOs. 23 and 24.

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