Application of KCNJ5 gene p.R242Q mutation site

Abstract

The invention discloses application of a KCNJ5 gene p.R242Q mutation site, relates to the technical field of biology, and is technically characterized in that the invention provides application of the KCNJ5 gene p.R242Q mutation site as a molecular marker in preparation of hypertension combined thoracic aortic aneurysm and dissection diagnosis products, the application can be used for screening high-risk individuals with hypertension combined thoracic aortic aneurysm dissection, and the application is applicable to screening of high-risk individuals with hypertension combined thoracic aortic aneurysm dissection diagnosis products and diagnosis of hypertension combined thoracic aortic aneurysm dissection diagnosis products. And early drug intervention can be conveniently carried out on patients and improvement of living habits of the patients can be guided. The invention also provides an application of the p.R242Q mutation site of the KCNJ5 gene in preparation / screening of drugs for treating hypertension combined with thoracic aortic aneurysm and dissection, and can guide clinical selection of corresponding hypertension drugs or design of novel hypertension drugs, thereby realizing accurate treatment of hypertension and prevention of dissection.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the application of a KCNJ5 gene p.R242Q mutation site. Background Art

[0002] Thoracic aortic aneurysm (TAA), characterized by dilation of the aorta above the diaphragm, poses a significant clinical challenge as it enlarges over time, ultimately leading to catastrophic events such as aortic dissection. Aortic dissection requires urgent medical intervention, often surgical repair, and is associated with both acute and long-term mortality risk. Hypertension is the most prominent population-attributable risk factor for the development of TAA. It is prevalent in patients with TAA, predicts the development of acute aortic syndrome, and adversely affects overall survival.

[0003] Approximately 20% of thoracic aortic aneurysm and dissection (TAAD) cases show familial aggregation, with autosomal dominant inheritance observed in 76.9% of cases. The current understanding of the pathophysiology of TAAD involves genetic variants that disrupt key functions in vascular smooth muscle cell contraction, extracellular matrix stabilization, or transforming growth factor (TGF)-β signaling pathways. Several defined genetic syndromes have been identified—such as Marfan syndrome, Loeys-Dietz syndrome, Ehlers-Danlos syndrome type IV, and TGFβ-associated vasculopathy—as well as TAAD with a family history (FTAAD), in which causative genes play an important role in guiding personalized medical care. However, these known causative genes account for only approximately 20% of FTAAD cases.

[0004] Recently, genome-wide association studies (GWAS) on TAAD have suggested that the genetic architecture of TAAD is similar to other complex traits, in which hypertension plays a minor pathogenic role. Therefore, identifying pathogenic variants in TAAD families, especially mutations in genes associated with inherited hypertension, is crucial to elucidate the potential Mendelian genetic link between TAAD and hypertension.

[0005] KCNJ5 encodes the G-activated inwardly rectifier potassium channel subunit 4 (GIRK4), which is essential for aldosterone secretion from the adrenal cortex. Pathogenic variants in KCNJ5, specifically in the extracellular pore-forming loop domain, are prevalent in a cohort of patients with primary aldosteronism (PA), who typically present with adrenal aldosterone-producing adenomas and hereditary hypertension characterized by familial hyperaldosteronism type III. These variants impair the channel's K + Selectivity, leading to Ca 2+ Increased influx and subsequent upregulation of aldosterone synthase (CYP11B) transcription.

[0006] To this end, the present invention sought to elucidate the genetic landscape of an autosomal dominant TAAD family with asymptomatic hypertension using whole exome sequencing (WES) and genotype-phenotype co-segregation approaches, as well as to identify a novel causative gene responsible for FTAAD through functional experiments. Summary of the invention

[0007] The purpose of the present invention is to provide an application of a KCNJ5 gene p.R242Q mutation site, and a specific purpose is to provide an application of a KCNJ5 gene p.R242Q mutation site in the preparation / screening of drugs for treating hypertension combined with thoracic aortic aneurysm and dissection and / or as a molecular marker in the preparation of a detection reagent for hypertension combined with thoracic aortic aneurysm and dissection, which can be used to screen high-risk individuals with hypertension combined with thoracic aortic aneurysm and dissection, so as to facilitate early drug intervention and improvement of lifestyle habits; and to guide clinical selection of hypertension drugs to achieve precise treatment of hypertension and prevent the occurrence of dissection.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] A KCNJ5 gene p.R242Q mutation site is used in the preparation / screening of drugs for treating hypertension combined with thoracic aortic aneurysm and dissection and / or as a molecular marker in the preparation of a detection reagent for hypertension combined with thoracic aortic aneurysm and dissection.

[0010] Furthermore, the p.R242Q mutation site is located at the 725th base in the non-coding sequence of the KCNJ5 gene, and a G>A mutation occurs.

[0011] The use of a reagent for detecting the above-mentioned KCNJ5 gene p.R242Q mutation site in the preparation of a reagent for detecting hypertension combined with thoracic aortic aneurysm and dissection.

[0012] Furthermore, the detection reagent includes detecting the p.R242Q mutation site of the KCNJ5 gene.

[0013] Furthermore, the detection reagent includes at least one pair of primers for specifically amplifying the amino acid mutation site p.R242Q encoded by the KCNJ5 gene and / or a capture probe for capturing the p.R242Q mutation site of the KCNJ5 gene.

[0014] Application of reagents for detecting hypertension combined with thoracic aortic aneurysm and dissection in the preparation of drugs for treating hypertension combined with thoracic aortic aneurysm and dissection.

[0015] The invention discloses an application of the inhibitor of expression of the KCNJ5 gene p.R242Q mutation site in the preparation of a drug for treating hypertension combined with thoracic aortic aneurysm and dissection.

[0016] A preventive and therapeutic drug for hypertension combined with thoracic aortic aneurysm and dissection, the preventive and therapeutic drug comprising a drug that inhibits the expression of the KCNJ5 gene p.R242Q mutation site.

[0017] Furthermore, the preventive and therapeutic drugs include drugs that relieve / inhibit the occurrence of isolated systolic hypertension.

[0018] Furthermore, the preventive and therapeutic drugs include drugs that inhibit the release of adrenaline from the adrenal medulla.

[0019] Compared with the prior art, the solution of the present invention has the following beneficial effects:

[0020] 1. The present invention utilizes the KCNJ5 gene p.R242Q mutation site as a molecular marker to prepare a diagnostic product for hypertension combined with thoracic aortic aneurysm and dissection, which can be used to screen high-risk individuals with hypertension combined with thoracic aortic aneurysm and dissection, facilitate early drug intervention for patients and guide the improvement of patients' living habits, and reduce the incidence of hypertension combined with thoracic aortic aneurysm and dissection.

[0021] 2. The pathogenic mechanism of KCNJ5 p.R242Q determined in the present invention is related to the binding of arginine in the inner helix of IRK_C region of KCNJ5 protein to GPCR. By using the KCNJ5 gene p.R242Q mutation site as a target, drugs for treating hypertension combined with thoracic aortic aneurysm and dissection can be prepared / screened, guiding the clinical selection of corresponding hypertension drugs or the design of new hypertension drugs, so as to achieve precise treatment of hypertension and prevent the occurrence of dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the correlation diagram of FTAAD family and KCNJ5 p.R242Q (A is CT angiography in the past three years showing the stents and vascular grafts of the proband (II:4) and his two brothers (II:1, II:3) with acute thoracic aortic dissection (preoperative images are no longer available); B is the proband family showing that KCNJ5p.R242Q co-segregates with TAAD in an autosomal dominant inheritance pattern, red symbols indicate individuals affected by TAAD, blue symbols indicate people with hypertension, green boxes indicate the absence of mutations, brown boxes indicate the presence of mutations, and black boxes indicate individuals not involved in genetic testing; C is Sanger sequencing to verify the genotype of full exome sequencing; D is interspecies Blat alignment showing that R242 is conserved, indicated by a red box);

[0023] Figure 2 Figure 1 is the result of TADD caused by KCNJ5 p.R242Q in mice (A is a representative ultrasound image of the thoracic aorta of 48-week-old mice; B is the statistical result of the diameter of the ascending, arch and descending arteries of 48-week-old mice (Kcnj5 + / +n=10,Kcnj5 + / R242Q C is the experimental procedure for the incidence of BAPN-induced TADD and the survival curves of the three genotypes during the 28-day observation period (Kcnj5 + / + 、Kcnj5 + / R242Q and Kcnj5 R242Q / R242Q ); D is the diameter of the thoracic aorta at three sites and the corresponding statistical results in mice fed with BAPN that survived for 28 days (Kcnj5 + / + n=8,Kcnj5 + / R242Q n=25 and Kcnj5 R242Q / R242Q n = 15); error bars indicate SEM, P values ​​are shown using two-tailed unpaired t test for A and C, and chi-square distribution test for comparison between groups for B);

[0024] Figure 3 It is Kcnj5 + / R242Q The mice showed increased systolic blood pressure (A) systolic blood pressure, diastolic blood pressure and heart rate of 8-week-old male mice (Kcnj5 + / + n = 18, Kcnj5 + / R242Q B is the systolic and diastolic blood pressure of 8-week-old male mice (Kcnj5 + / + n=26, Kcnj5 + / R242Q B is the systolic, diastolic and pulse pressures of male mice aged 8, 24 and 48 weeks (Kcnj5 + / + n = 9, Kcnj5 + / R242Q n = 19), for AC, P values ​​are shown using unpaired two-tailed Student t test);

[0025] Figure 4 is a 48-week-old Kcnj 5+ / R242Q Characteristic images of the thoracic aorta wall of mice (A is EVG staining showing the elastic lamella of the thoracic aorta, the red arrow indicates the anatomical area, the black arrow indicates the break point of the elastic lamella, and the bar graph shows the number of break points (Kcnj5 + / + n=8,Kcnj5 + / R242Q B is Masson staining showing collagen deposition in the thoracic aorta, represented by blue, and the bar graph shows the collagen content (Kcnj5 + / + n=8,Kcnj5 + / R242Qn = 11); C is a bubble plot showing the significant Reactome pathway enrichment of upregulated genes in the thoracic aorta transcriptome analysis, and red fonts indicate pathways related to extracellular matrix remodeling; D is a heat map of 20 genes in the extracellular matrix organization pathway in Reactome, and red fonts highlight the 7 enriched genes whose signals are mediated by TGFB family members; E is a schematic diagram of the TGFβ signaling pathway and a heat map of the top 17 genes in the KEGG pathway, including classical and non-classical pathways. In the classical pathway, TGFβ receptor signaling activates SMADs* (marked in red), which is associated with four genes. The non-classical pathway involves TGFβ receptor signaling activating MAPK cascades (marked in red), which is associated with 12 genes. The non-classical pathway is marked in red; F is a Western Blot and quantitative analysis of Tgfb levels, with Kcnj5 as the + / + Controls were used for normalization, and Gapdh was used as a loading control; for A, B, and F, data are expressed as mean ± standard error, and P values ​​are shown using an unpaired two-tailed Student t test; scale bars, 100 μm);

[0026] Figure 5 The systolic and diastolic blood pressure and heart rate of the surgical group and the sham surgery group 28 weeks after bilateral adrenalectomy (Kcnj5 + / + and Kcnj5 + / R242Q 6 mice each, data are presented as mean ± SEM, unpaired two-tailed Student t test shows P value);

[0027] Figure 6 It is Kcnj5 + / R242Q Autonomy of the mouse adrenal medulla regulated by the sympathetic nervous system (A is the systolic and diastolic blood pressure and heart rate of 28-week-old mice before and after restraint; B is the differential effects of blood pressure and heart rate on wild-type and heterozygous mice before and after restraint; C is a schematic diagram of the physiological pathways that regulate blood pressure increase in the restraint response. Mental stress is transmitted to the brain through the sympathetic-adrenal medulla axis, and the hypothalamus-pituitary-adrenal cortex axis plays a promoting role. The cross indicates that the sympathetic-adrenal medulla axis is inhibited in heterozygous mice. Normally, activation of the sympathetic nerves prompts the adrenal medulla to release catecholamines, including adrenaline, which increases blood pressure. Hypothalamic corticotropin-releasing hormone (CRH) stimulates the release of pituitary adrenocorticotropic hormone (ACTH), initiating the production of mineralized corticosteroids in the adrenal cortex, such as aldosterone. For A and B, data are expressed as mean ± SEM, where A shows P values ​​using unpaired two-tailed Student t test and B shows P values ​​using paired two-tailed Student t test);

[0028] Figure 7 Figure 3: Adrenal lesions in individuals with the KCNJ5 p.R242Q mutation evaluated using CT imaging. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0030] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0031] Example:

[0032] The following is the specific implementation and test of the implementation scheme of the present invention:

[0033] 1. Experimental methods

[0034] 1.1 The experiments in this invention adhere to the ethical principles of the Declaration of Helsinki. All participants obtained informed consent for the protocol approved by the Ethics Review Committee of the First Affiliated Hospital of Gannan Medical College (approval number: LLSC-2021120902). The design and execution of animal studies complied with the "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T 35892-2018, National Standard of the People's Republic of China) and obtained ethical approval from the Ethics Committee of Gannan Medical College (approval number: 2022038).

[0035] 1.2 The research data of the present invention are expressed as mean ± standard error (SEM). The Shapiro-Wilk test was used for normal distribution test. When comparing two groups, an unpaired two-tailed Student t test was performed. If the comparison was between the same subject before and after treatment, a paired Student t test was used. For non-normally distributed data, the non-parametric Mann-Whitney U test was used to compare the two groups. Survival curves were constructed using the Kaplan-Meier method, and statistical analysis was performed using the log-rank test. The incidence of aneurysms was analyzed using Fisher's exact test. All graphics were generated using Graphpad Prism9 software. All analyses were performed using SPSS20 software. P values ​​less than 0.05 were considered statistically significant.

[0036] 1.3 The present invention uses whole exome sequencing (WES) and genotype-phenotype co-segregation methods to elucidate the genetic landscape of an autosomal dominant TAAD family with asymptomatic hypertension. Specifically, a missense mutation (p.R242Q) of the KCNJ5 gene was selected to co-segregate with the occurrence of TAAD in the family. Using the CRISPR / Cas9 system, a mouse model carrying this specific human mutation was engineered to explore its effects on the thoracic aorta and hypertension in living animals, and further analyze the mechanism of hypertension-induced TAAD.

[0037] 1.4 Blood pressure measurement

[0038] Arterial blood pressure (BP) of mice was measured using the automatic tail cuff BP-2010A blood pressure analysis system of Softron and evaluated using the BP-2010 series system (Softron). The measurements were performed in a quiet and temperature-controlled environment (±22°C), and the animals were preheated (39°C, 15 minutes) before the measurements and maintained at this temperature. The results were expressed as the mean of all measurements. In the statistical analysis, when comparing two different groups, the unpaired tail Student's t-test was used, while the paired Student's t-test was used to evaluate the changes before and after treatment in the same individual.

[0039] 2 Experimental results

[0040] 2.1 Features of the FTAAD family

[0041] The present invention discovered a TAAD family through data screening, in which three individuals underwent thoracic aortic repair surgery and no Marfan-like symptoms were observed. Figure 1 A, individual II:4) is a 51-year-old Chinese male who presented to the Cardiac Medical Center for evaluation of TAAD due to family history. His medical history included an acute thoracic aortic dissection repaired at age 42. The dissection was classified as De Bakey type I with a ruptured aneurysm located above the sinotubular junction and approximately 5 cm in length in the ascending aorta. The proband's two brothers ( Figure 1 A, Individuals II:1 and II:3) also experienced acute thoracic aortic dissection at ages 42 and 45, respectively. Brother II:1 had a type De Bakey dissection with rupture in the ascending aorta, while brother II:3 had a type De Bakey III dissection with rupture near the left subclavian artery. All three brothers had a history of hypertension that began in their 30s to early 40s. Their father died suddenly in his 50s with symptoms consistent with thoracic aortic dissection.

[0042] 2.1 Identification of the KCNJ5 p.R242Q missense mutation as a candidate variant for FTAAD

[0043] Targeted exome sequencing of known TAAD-related genes was performed in the proband, and no deleterious mutations in known pathogenic genes were found. Then, whole exome sequencing was performed in the proband (II:4), two brothers with TAAD (II:1 and II:3), and one unaffected brother (II:5) to identify the pathogenic mutations in TAAD in the family ( Figure 1 B). A total of nine variants were found by genotype-phenotype co-segregation analysis with 100% penetrance in a dominant inheritance pattern (Table S2). Further screening based on rarity and functional prediction narrowed the gap to two potential variants: KCNJ5c.725G>A (p.R242Q) and MEF2Bc.29G>C (p.R10P). Neither variant has been found to be involved in arterial wall biology. Given the presence of hypertension in the affected individuals, KCNJ5 p.R242Q was selected as a candidate variant due to the known association of KCNJ5 with hypertension. The variant was detected in all three cases but not in the unaffected brother and was validated by Sanger sequencing ( Figure 1 C) The arginine residue at position 242 is highly conserved ( Figure 1 D), indicating that this missense mutation may disrupt the normal function of KCNJ5. However, due to the lack of functional evidence supporting the occurrence of TAAD in variant carriers, the pathogenic link between KCNJ5 p.R242Q and TAAD has not been conclusively established.

[0044] 2.3 TAAD in mice carrying the KCNJ5 p.R242Q mutation

[0045] To investigate the functional relevance of the KCNJ5 p.R242Q variant in TAAD, the present invention used CRISPR / Cas9 to introduce the human variant into the corresponding amino acid position in the mouse Kcnj5 gene. Male mice were selected for aorta experiments due to the potential sex-related vascular protection mechanism in females. Among them, heterozygous mice carrying this mutation (Kcnj5 + / R242Q ) were the experimental group, and wild-type mice that did not carry this mutation were the control group (Kcnj5 + / + ).

[0046] The results showed that the heterozygous mice were phenotypically normal at 6 months of age, with no increased mortality. By 48 weeks of age, the diameter of the arch and descending thoracic aorta of heterozygous mice had increased by approximately 16% ( Figure 2 A and Figure 2 B), one of the mice developed thoracic aortic dissection. In addition, the BAPN-induced aortic dissection model showed that the incidence of thoracic aortic dissection in heterozygous mice was significantly higher than that in wild-type mice ( Figure 2 C). The diameters of the arch and descending thoracic aorta of surviving heterozygous mice were significantly larger than those of wild-type mice ( Figure 2 D). The thoracic aorta phenotype of heterozygous mice is consistent with the clinical phenotype of TAAD in this family, indicating that KCNJ5 p.R242Q is the causative mutation of TAAD in this family.

[0047] 2.4Kcnj5 + / R242Q Mice exhibit isolated systolic hypertension

[0048] Since TAAs are absent in Kcnj5 knockout mice, hypertension is a known risk factor for TAAs, and KCNJ5 gene variation is associated with hypertension, the blood pressure phenotype of heterozygous mice was evaluated. Blood pressure measurements showed that Kcnj5 was downregulated compared with wild-type controls. + / R242Q Male mice had a significant increase in systolic blood pressure at 8 weeks ( Figure 3 A), while diastolic blood pressure did not differ significantly. The same blood pressure pattern was observed in 8-week-old female mice ( Figure 3 B) This elevated systolic pressure is Kcnj 5+ / R242Q Isolated systolic hypertension persisted from 8 to 48 weeks of age in animals throughout their lifespan ( Figure 3 These results suggest that the KCNJ5 p.R242Q variant specifically causes isolated systolic hypertension and that the increase in systolic blood pressure occurs even in Kcnj5 + / R242Q Before mice reach sexual maturity.

[0049] 2.5Kcnj5 + / R242Q Features of the mouse thoracic aorta wall resemble those of hypertensive aorta

[0050] Histological analysis of the thoracic aorta of 48-week-old mice showed that Kcnj5 + / R242Q The aorta wall of mice was thickened with appropriate elastic layers (5 to 6) and vascular smooth muscle cell layers. + / R242Q Two of the samples had more elastic layer breakpoints (Z scores > 2), but there was no significant difference in breakpoints between the two genotypes (Figure 4A). + / R242Q The collagen deposition in the aorta of mice was increased (Figure 4B). + / R242Q Mice and Kcnj5 + / +Transcriptome analysis of the thoracic aorta of littermate control mice identified 283 upregulated genes and 205 downregulated genes. The upregulated genes were enriched in 14 significant pathways in the Reactome enrichment analysis (Padjust < 0.05), including five pathways directly involved in extracellular matrix remodeling - three related to extracellular matrix organization and two related to TGFβ signaling (Figure 4C). The most enriched pathway among the upregulated genes was extracellular matrix organization, which was enriched with 17 upregulated genes, which also contributed to the other four pathways - elastic fiber formation (8 genes), molecules related to elastic fibers (6 genes), signaling of TGFβ family members (7 genes), and TGFβ receptor signaling activation of SMADs (4 genes) (Figure 4D). Among them, the TGFβ receptor signaling activation of SMADs pathway represents the classical TGFβ signaling pathway. At the same time, the upregulated non-classical TGFβ pathway, p38 MAPK, was the top KEGG pathway. Increased RNA expression of genes in the upregulated canonical and non-canonical TGFβ pathways indicated overactivation of the TGFβ pathway, a known mechanism for promoting TAA ( Figure 4E , Figure 4E ). The results of overactivation of the TGFβ pathway were confirmed by using Western blotting to detect TGFβ overload, which was consistent with the morphological features of hypertension-induced aortas ( Figure 4F ).

[0051] 2.6Kcnj5 + / R242Q Systolic hypertension in mice is associated with adrenal medullary dysfunction

[0052] Kcnj5 is highly expressed in the adrenal gland and pancreas, and the adrenal gland plays a crucial role in blood pressure regulation. To investigate the role of adrenal dysfunction in Kcnj5 + / R242Q To investigate the role of Kcnj5 in the elevated blood pressure in mice, we performed bilateral adrenalectomy. Blood pressure monitoring showed that adrenalectomy resulted in + / R242Q The systolic and diastolic blood pressures of the mice decreased significantly, while there were no such changes in wild-type mice ( Figure 5 This suggests that adrenal dysfunction may contribute to Kcnj5 + / R242Q Hypertension observed in mice.

[0053] 2.7 Adrenal medulla in Kcnj5 + / R242Q Autonomous secretion of adrenaline in mice

[0054] To determine whether adrenal medullary pathology was due to sympathetic overactivity, the authors placed mice in 30-minute restraints to induce a stress response and monitored their blood pressure before and after the procedure. Restraint resulted in a significant increase in blood pressure in both genotypes. However, in contrast to Kcnj5 + / + Compared with mice, Kcnj5 + / R242Q The mice showed less change in blood pressure, refuting the hypothesis that Kcnj5+ / R242Q Sympathetic nerve hyperactivity in mice Figure 6 To this end, the present invention treated mice with the non-selective adrenergic blocker propranolol and the mineralocorticoid receptor antagonist spironolactone, which is a standard treatment for PA associated with KCNJ5 mutations. Propranolol effectively alleviated the Kcnj5 mutation. + / R242Q The systolic blood pressure of mice increased, unlike spironolactone, which did not change the systolic blood pressure level. + / + Compared with mice, Kcnj5 + / R242Q Pnmt expression is upregulated in the adrenal gland, and Pnmt is an enzyme directly involved in the synthesis of epinephrine in the adrenal medulla. This pattern is consistent with Kcnj5 + / R242Q These results suggest that the KCNJ5 p.R242Q variant is directly responsible for the observed increase in systolic blood pressure by mediating SNS-independent adrenal medullary epinephrine release.

[0055] In summary, the animal model experiments of the present invention verified that KCNJ5 p.R242Q is the pathogenic mutation of TAAD in this family, and Kcnj5 p.R242Q heterozygous mice showed greater thoracic aortic dilatation than wild-type controls under normal conditions. In addition, under BAPN-induced conditions, the mutant allele was positively correlated with the incidence of TAAD. The significant increase in systolic blood pressure in heterozygous mice is the cause of this vulnerability, which is mainly reflected in: the gradual increase in systolic blood pressure throughout the life cycle, hypertension-induced aortic dilatation, accompanied by increased collagen deposition, and autonomous release of adrenaline from the adrenal medulla, bypassing SNS regulation. The adrenal medulla of heterozygous mice expressed higher levels of Kcnj5 than wild-type, and adrenergic blockade alleviated the increase in systolic blood pressure in these mice, supporting the hypothesis that the R242Q replacement upregulates Kcnj5 in the adrenal medulla, leading to uncontrolled adrenaline release and the occurrence of isolated systolic hypertension-a key pathway for the development of TAAD.

[0056] It was further clarified that KCNJ5 was identified as a novel pathogenic gene for isolated TAAD. The results of the present invention showed that a sustained increase in systolic blood pressure can activate TGFβ signaling, indicating that certain risk factors can affect TGFβ signaling in a manner similar to mutations in the cascade.

[0057] The present study highlights the importance of considering genes with major effects. + / R242QThe blood pressure phenotype observed in mice is defined by elevated systolic and pulse pressures, which result from enhanced adrenaline secretion from the adrenal medulla. Epinephrine activates beta-adrenergic receptors in the heart, leading to increased cardiac output, which is the primary factor in elevated central systolic and pulse pressures. This suggests that higher central systolic and pulse pressures are associated with larger thoracic aortic aneurysms and more rapid aneurysm growth.

[0058] The present invention depicts a unique blood pressure phenotype in patients with the KCNJ5 p.R242Q mutation, which is different from the KCNJ5-related hypertension subtypes described in the prior art, including familial and sporadic primary aldosteronism (PA), both in clinical manifestations and pathogenesis. Phenotypically, individuals with this mutation exhibit isolated systolic hypertension, which is different from the combined systolic and diastolic hypertension observed in PA. Mechanistically, the mutation leads to increased adrenaline levels, which is different from the PA mutation triggering excessive aldosterone production. TAAD in KCNJ5 p.R242Q mutation carriers is associated with vascular remodeling caused by high-velocity blood flow, while PA mutations cause vascular remodeling by increasing blood volume. This explains why there is a specific susceptibility to TAAs, rather than the risk of abdominal aortic aneurysm associated with PA. Clinically, the diagnostic criteria for adrenal-related hypertension, including pheochromocytoma and aldosteronoma, are not consistent with the characteristics of patients with the KCNJ5 p.R242Q mutation, who present with only slightly elevated plasma methylnorephinephrine levels and lack overt adrenal hyperplasia on CT imaging ( Table 1 and Figure 7 ). Patients carrying the KCNJ5p.R242Q mutation face an increased risk of adverse long-term outcomes, as demonstrated in the present TAAD family. In this TAAD family, three mutation carriers under the age of 50 experienced acute thoracic aortic dissection after a diagnosis of hypertension. After surgical repair, these patients (II:1, II:3, and II:4) were repeatedly hospitalized for complications such as cerebral infarction, valvular calcification, and pseudoaneurysm at the anastomotic site - a consequence of poor blood pressure control.

[0059] Table 1 Norepinephrine levels outside the reference range in KCNJ5 p.R242Q carrier III:2

[0060]

[0061] Remarks: Four individuals carrying the KCNJ5 p.R242Q mutation were included in the follow-up study. Testing was not performed because three patients (II:1, II:3, II:4) underwent thoracic aortic repair surgery and were receiving long-term hypertension and coagulation treatment involving drugs that may affect adrenal cortical and medullary function, such as metoprolol and rivaroxaban. Although individual III:2 had leukemia and was receiving doxorubicin, this drug has limited effects on the adrenal glands and was therefore suitable for testing. Ultimately only III:2 was included in the study. Norepinephrine is an intermediate metabolite of epinephrine that is more stable than epinephrine and is commonly used as a biomarker for abnormalities in pheochromocytoma.

[0062] In the clinical setting, this specific hypertension phenotype is easily misdiagnosed as common underlying hypertension. The reasons are: (1) isolated systolic hypertension is a common form of underlying hypertension, and (2) the modest elevation of adrenaline levels and the lack of obvious adrenal hyperplasia on CT imaging. In addition, the presence of traditional risk factors such as smoking and high-salt diet in mutation carriers and the absence of known TAAD gene mutations also lead clinicians to overlook this patient group.

[0063] Therefore, the present invention uses the KCNJ5 gene p.R242Q mutation site as a molecular marker to prepare a diagnostic product for hypertension combined with thoracic aortic aneurysm and dissection, which is used to screen high-risk individuals with hypertension combined with thoracic aortic aneurysm and dissection, and uses the KCNJ5 gene p.R242Q mutation site as a target to prepare / screen drugs for treating hypertension combined with thoracic aortic aneurysm and dissection, which is of great significance to high-risk individuals suffering from dissection among blood pressure patients.

[0064] The above-described embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. Use of a KCNJ5 gene p.R242Q mutation site in the preparation / screening of drugs for the treatment of hypertension combined with thoracic aortic aneurysm and dissection and / or as a molecular marker in the preparation of a detection reagent for hypertension combined with thoracic aortic aneurysm and dissection.

2. The use according to claim 1, characterized in that: The p.R242Q mutation site is located at the 725th base of the non-coding sequence of the KCNJ5 gene, and a G>A mutation occurs.

3. Use of a reagent for detecting the KCNJ5 gene p.R242Q mutation site described in claim 1 or claim 2 in the preparation of a reagent for detecting hypertension combined with thoracic aortic aneurysm and dissection.

4. The use according to claim 3, characterized in that: The detection reagent comprises detecting the KCNJ5 gene p.R242Q mutation site described in claim 1 or claim 2.

5. The use according to claim 4, characterized in that: The detection reagent comprises at least one pair of primers for specifically amplifying the amino acid mutation site p.R242Q encoded by the KCNJ5 gene and / or a capture probe for capturing the p.R242Q mutation site of the KCNJ5 gene.

6. Use of the detection reagent described in claim 4 or claim 5 in the preparation of a drug for treating hypertension combined with thoracic aortic aneurysm and dissection.

7. Use of the inhibitor of expression of the KCNJ5 gene p.R242Q mutation site as claimed in claim 1 in the preparation of a drug for treating hypertension combined with thoracic aortic aneurysm and dissection.

8. A drug for the prevention and treatment of hypertension combined with thoracic aortic aneurysm and dissection, characterized in that: The preventive and therapeutic drugs include drugs that inhibit the expression of the KCNJ5 gene p.R242Q mutation site of claim 1.

9. The drug for preventing and treating hypertension combined with thoracic aortic aneurysm and dissection according to claim 8, characterized in that: The preventive and therapeutic drugs include drugs that relieve / inhibit the occurrence of isolated systolic hypertension.

10. The drug for preventing and treating hypertension combined with thoracic aortic aneurysm and dissection according to claim 8, characterized in that: The preventive and therapeutic drugs include drugs that inhibit the release of adrenaline from the adrenal medulla.

Images