Biomarker combination for early screening and diagnosis of transthyretin cardiac amyloidosis and use thereof

Abstract

This invention provides the application of a combination of biomarkers for early screening and diagnosis of transthyretin-mediated cardiac amyloidosis (ATTR-CA) in the preparation of products for early screening and diagnosis of ATTR-CA in high-risk populations. Generally, one or more biomarkers are used, such as total prealbumin (total PA), transthyretin (TTR) tetramer, misfolded proteins, protein misfolding rate, and soluble growth-stimulating gene 2 protein (sST2). Significant differences in these biomarkers are observed between high-risk and non-high-risk ATTR-CA populations, suggesting an association between the biomarkers and ATTR-CA. The combined use of multiple biomarkers demonstrates high sensitivity and specificity in early screening and diagnosis of ATTR-CA in high-risk populations, exhibiting high diagnostic efficacy and providing new ideas and strategies for the early diagnosis and treatment of ATTR-CA.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to the application of a combination of biomarkers for early screening and diagnosis of transthyretin cardiac amyloidosis (ATTR-CA) in the preparation of products for early screening and diagnosis of ATTR-CA high-risk populations. Background Technology

[0002] Transthyretin cardiac amyloidosis (ATTR-CA) is an infiltrative cardiomyopathy, also known as transthyretin amyloid cardiomyopathy (ATTR-CM). It is the most common type of cardiac amyloidosis (CA) besides immunoglobulin light chain cardiac amyloidosis (AL-CA). It is caused by the dissociation and misfolding of transthyretin (TTR, also known as prealbumin (PA)) tetramers, forming insoluble amyloid fibers that deposit in the myocardial interstitium. Based on the presence or absence of TTR gene mutations, the disease is classified into hereditary / mutant (ATTRm) and wild-type (ATTRwt).

[0003] ATTR-CA is a progressive, debilitating, and fatal disease characterized by poor quality of life, low survival rates, and poor prognosis. Previously considered a rare disease, it is now recognized as a more common cause of heart failure and death worldwide. Due to a lack of awareness and the heterogeneity of symptoms, ATTR-CA remains underdiagnosed, leading to an underestimated incidence and frequent missed or misdiagnosis. Furthermore, the lack of specific clinical manifestations in the early stages of ATTR-CA poses a significant challenge to early screening and diagnosis. Therefore, focusing on screening high-risk populations and clinically identifying and diagnosing ATTR-CA in its early stages is crucial to providing patients with broader treatment options, improving survival rates, and preventing potentially irreversible functional loss and a decline in quality of life.

[0004] With a deeper understanding of ATTR-CA disease and advancements in endocardial myocardial biopsy and radionuclide bone scintillation scanning (…), 99 Tc mThe development of diagnostic technologies such as PYP (Prognostics and Biopsy) and gene testing has made early diagnosis of ATTR-CA possible. However, existing examination methods still have limitations. Endocardial biopsy is the "gold standard" for ATTR-CA diagnosis, but myocardial biopsy is an invasive procedure that requires a specialized medical team and equipment, and the cost of surgery and pathological analysis is high. 99 Tc m - PYP can be used for non-invasive diagnosis of ATTR-CA. Although the positive rate is high, it involves radiation exposure, high cost, poor accessibility, and strong dependence on technology and interpretation. Genetic testing is the gold standard for distinguishing between ATTRm and ATTRwt, but it is costly and technically challenging. Conventional imaging examinations such as echocardiography and cardiac magnetic resonance (CMR) can show characteristic changes of myocardial amyloidosis, but they may be insensitive in the early stages. Imaging features, such as myocardial thickness and signal abnormalities, may be helpful in the diagnosis of ATTR-CA, but they may also be seen in other cardiomyopathy. Biomarkers, such as B-type natriuretic peptide (BNP), N-terminal pro-B-type natriuretic peptide (NT-proBNP), and troponin, may be helpful in the diagnosis of ATTR-CA, but their sensitivity is limited and may be affected by other heart diseases. Furthermore, there is currently a lack of clinical methods to directly detect TTR in the blood.

[0005] Therefore, there is an urgent need for new, highly sensitive, and specific biomarkers for the identification, early screening, and diagnosis of high-risk populations for ATTR-CA, without the need for imaging examinations and invasive tissue biopsies. Summary of the Invention

[0006] The purpose of this invention is to provide a combination of blood biomarkers for early screening and diagnosis of ATTR-CA and its application, in order to solve the problems existing in the prior art. This combination of biomarkers can realize the identification, early screening and diagnosis of high-risk groups for ATTR-CA.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of a combination of biomarkers for early screening and diagnosis of transthyretin-cardiac amyloidosis (ATTR-CA) in the preparation of products for early screening and diagnosis of ATTR-CA high-risk populations; generally, one or more biomarkers, such as total prealbumin (total PA), transthyretin (TTR) tetramer, misfolded protein, protein misfolding rate, and soluble growth-stimulating gene 2 protein (sST2); the ATTR-CA high-risk population is a group of people who have ATTR-CA warning signs or whose biomarker values ​​are not within the reference range when compared with the reference range.

[0009] A further preferred embodiment: the biomarker combination consists of TTR tetramer, protein misfolding rate, and sST2.

[0010] A further preferred option: the product includes the ATTR-CA high-risk population diagnostic model.

[0011] A further preferred embodiment: The model uses the detected or calculated values ​​of biomarkers as input variables to construct a diagnostic model, and the model uses the equation: Logit(p)=2.067-1.843×[TTR tetramer content]+0.178×[protein misfolding rate]+0.035×[sST2 content].

[0012] A further preferred option: the cutoff value for evaluating the model's diagnostic results for high-risk ATTR-CA populations is 0.62. When p>0.62, the individual is diagnosed as a high-risk ATTR-CA population; otherwise, the individual is diagnosed as a non-high-risk ATTR-CA population.

[0013] A further preferred embodiment: the product is a kit containing reagents for detecting TTR tetramer.

[0014] A further preferred embodiment: the kit is an ultra-high performance liquid chromatography kit.

[0015] A further preferred embodiment: the sample of the subject to be tested in the kit is serum or plasma.

[0016] A further preferred embodiment: the control samples for the kit are derived from non-high-risk individuals with ATTR-CA, and the samples are serum or plasma.

[0017] Furthermore, the diagnosis of high-risk individuals for ATTR-CA includes the following steps:

[0018] Step 1: Collect samples from the subjects to be tested and control samples;

[0019] Step 2: Detect the total PA content in the test subject samples and control samples;

[0020] Step 3: Detect the TTR tetramer content in the test subject samples and control samples;

[0021] Step 4: Detect the sST2 content in the test subject samples and control samples;

[0022] Step 5: Calculate the misfolded protein concentration and protein misfolding rate based on the total PA and TTR tetramer content, i.e., misfolded protein content = total PA content - TTR tetramer content; protein misfolding rate (%) = misfolded protein content / total PA content;

[0023] Step Six: Substitute the detected or calculated values ​​of the biomarkers into the model equations to obtain the calculation results;

[0024] Step 7: Compare with the diagnostic cutoff value to diagnose whether the subject is at high risk for ATTR-CA.

[0025] As used in this article, the subjects are human.

[0026] As used herein, the sample of the subject to be tested is a clinical biological sample of the subject, namely serum or plasma.

[0027] As used herein, the detection of total PA content is performed using a PA assay kit and an immunoturbidimetric assay.

[0028] Furthermore, the TTR tetramer detection kit is used for detection by ultra-high performance liquid chromatography.

[0029] Furthermore, the kit may include calibrators, quality control samples, diluents, etc.

[0030] Furthermore, the detection of sST2 content is performed using an sST2 assay kit and an immunoassay method.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] This invention is the first to use total PA, TTR tetramer, misfolded protein, protein misfolding rate, and sST2 as biomarkers. The inventors have found that the levels of total PA and TTR tetramer in the blood are significantly lower in high-risk ATTR-CA individuals than in non-high-risk individuals, while the calculated values ​​of misfolded protein, protein misfolding rate, and sST2 levels are significantly higher in high-risk ATTR-CA individuals. More specifically, the inventors have found that the combined use of TTR tetramer, protein misfolding rate, and sST2 has higher accuracy, specificity, and sensitivity for diagnosing high-risk ATTR-CA individuals, and shows high consistency in the validation population. Furthermore, no imaging examinations or invasive tissue biopsies are required. Therefore, this combination of biomarkers can be used for the identification, early screening, and diagnosis of high-risk ATTR-CA individuals, which is of great significance for improving the diagnostic rate of high-risk ATTR-CA individuals, enabling early intervention and treatment, and reducing mortality. It also provides new ideas and strategies for the early diagnosis and treatment of ATTR-CA. Attached Figure Description

[0033] Figure 1The differences in the levels of total PA, TTR tetramer, misfolded protein, protein misfolding rate, and sST2 in the serum of non-high-risk and high-risk ATTR-CA individuals in Example 1 are shown.

[0034] Figure 2 ROC curve analysis of the ATTR-CA high-risk population diagnostic model constructed from the biomarker combination in Example 2;

[0035] Figure 3 The difference in the levels of TTR tetramer, protein misfolding rate, and sST2 in the serum of non-high-risk and high-risk ATTR-CA individuals in Example 3 is shown. Detailed Implementation

[0036] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] In this invention, "ATTR-CA" is also called "ATTR-CM", and the two names can be used interchangeably.

[0038] Example 1: Analysis of differences in serum levels of total PA, TTR tetramer, misfolded protein, protein misfolding rate, and sST2 between non-high-risk and high-risk ATTR-CA populations.

[0039] 1. Experimental subjects and grouping:

[0040] Serum samples were collected from 70 patients diagnosed with heart disease at the First Hospital of Jilin University. Among all enrolled patients, 35 were diagnosed as high-risk for ATTR-CA as the disease group (21 males and 14 females), and 35 were diagnosed as non-high-risk for ATTR-CA as the disease control group (19 males and 16 females). This study was approved by the Ethics Committee of the First Hospital of Jilin University.

[0041] Inclusion criteria for high-risk groups for ATTR-CA:

[0042] For patients exhibiting one or more of these characteristics, cardiac amyloidosis (CA), particularly ATTRCA, should be considered:

[0043] (1) Elderly patients with heart failure (LVEF≥40%), with no left ventricular enlargement but unexplained left ventricular hypertrophy (LVH);

[0044] (2) Echocardiography showed LVH, but electrocardiogram showed no QRS high voltage;

[0045] (3) Troponin levels remained elevated at a low level;

[0046] (4) Elderly patients with low pressure gradient and low flow velocity aortic valve stenosis, accompanied by right ventricular hypertrophy;

[0047] (5) Intolerance to angiotensin system inhibitors and / or beta-blockers due to hypotension (especially orthostatic hypotension);

[0048] (6) Multiple peripheral neuropathy (PN), especially with autonomic dysfunction (diarrhea and constipation of unknown cause, orthostatic hypotension, urinary retention, urinary incontinence, etc.);

[0049] (7) Familial PN;

[0050] (8) Bilateral carpal tunnel syndrome and / or lumbar spinal stenosis in the elderly;

[0051] (9) Recurrent bilateral cataracts.

[0052] Patients with one or more of the following imaging features may also be indicative of ATTR-CA:

[0053] (1) Electrocardiogram (ECG): The typical ECG presentation of left ventricular hypertrophy on echocardiography and cardiac arrest (CA) in the limbs and / or precordial leads is low voltage QRS waves and pseudo-infarction signs of Q or T waves. Low voltage is highly specific for CA. Patients with ATTR-CA are more likely to have conduction block and / or atrial fibrillation. CA should be considered in patients with unexplained left ventricular hypertrophy complicated by atrial fibrillation and high-degree atrioventricular block.

[0054] (2) Echocardiography: The simultaneous presence of seemingly nonspecific ultrasound imaging features strongly suggests the possibility of cardiac arrest (CA), such as biventricular enlargement, thickening of the ventricular wall (both left and right ventricular walls) without ventricular cavity enlargement, granular hyperechoic areas in the myocardium, thickening of the atrioventricular valves, thickening of the atrial septum, small amount of pericardial effusion, and restrictive diastolic dysfunction. Left ventricular ejection fraction is basically normal or slightly decreased. The sensitivity of left ventricular ejection fraction in evaluating systolic function in ATTR-CA patients is poor, while strain imaging (showing a decrease in the absolute value of the peak longitudinal strain) can detect systolic dysfunction earlier, especially when a characteristic "apex-preserving" pattern appears, i.e., the longitudinal strain (LS) in the base and mid-segment of the left ventricle is reduced while the apex is normal [apical longitudinal strain / (average of mid-segment + base longitudinal strain) > 1.0] helps to differentiate CA from left ventricular hypertrophy caused by other reasons. Inferolateral wall thickness ≥ 14 mm.

[0055] (3) Cardiac magnetic resonance imaging (CMR): ATTR-CA showed asymmetric ventricular septal hypertrophy (79%), symmetric LVH (18%), or normal left ventricular geometry (3%), often accompanied by right ventricular hypertrophy. The typical "delayed enhancement of amyloid (LGE) pattern" is extensive subendocardial LGE, inconsistent with the regional distribution of coronary artery blood supply. It can also present as diffuse transmural or intramyocardial patchy LGE. T1 mapping (which can be used in patients with renal dysfunction who cannot receive gadolinium injection) showed elevated native T1 values, accompanied by elevated extracellular volume fraction (ECV).

[0056] (4) Radionuclide bone scintillation scan: 99Tcm phosphate derivative radionuclide scan shows myocardial uptake of grade 2-3, which may indicate ATTR CA. Grade 2 means that myocardial uptake is equal to rib uptake, and grade 3 means that myocardial uptake is greater than rib uptake, accompanied by significant reduction in rib uptake / no rib uptake.

[0057] Or, compare the values ​​of total PA, TTR tetramer, misfolded protein, or protein misfolding rate in a patient's serum sample with the reference range; for individuals whose values ​​are outside the reference range.

[0058] Exclusion criteria:

[0059] (1) Subjects in the active phase of tumor, severe infection, acute inflammation, renal failure, as well as patients who have experienced acute coronary syndrome, unstable angina, stroke, transient ischemic attack, coronary revascularization, cardiac device implantation, heart valve repair or major surgery due to worsening heart failure within 1 month.

[0060] (2) Subjects who have previously received liver and / or heart transplants;

[0061] (3) Subjects with primary (light chain) or secondary amyloidosis.

[0062] Reference indicators for differentiating immunoglobulin light chain cardiac amyloidosis (AL-CA) include an abnormal serum free light chain ratio (κ / λ < 0.26 or > 1.65), abnormal monoclonal immunoglobulins detected on serum / urine protein immunofixation electrophoresis, and elderly AL-CA patients with concurrent monoclonal immunoglobulinemia (MGUS) or elevated serum free light chains due to renal insufficiency may be misdiagnosed as ALCA. In such cases, 99Tcm phosphate derivative radionuclide imaging is helpful for CA typing; a negative result suggests ALCA, while a positive result requires tissue biopsy, especially endocardial myocardial biopsy with amyloid identification, to definitively diagnose the condition. If the risks associated with biopsy outweigh the benefits, it is advisable to discuss any differing results with a local hematologist, researcher, and healthcare supervisor.

[0063] ATTR-CA Inclusion Criteria for Non-High-Risk Groups:

[0064] Heart disease patients for whom no clear indications of ATTR-CA high risk were found.

[0065] 2. Experimental methods:

[0066] The total PA content in serum samples was detected using a PA detection kit (immunoturbidimetric assay); the TTR tetramer content in serum samples was detected using a TTR tetramer detection kit (ultra-high performance liquid chromatography); and the sST2 content in serum samples was detected using an sST2 detection kit (immunoassay). The misfolded protein content and protein misfolding rate were calculated based on the total PA and TTR tetramer content, i.e., misfolded protein content = total PA content - TTR tetramer content; protein misfolding rate (%) = misfolded protein content / total PA content.

[0067] 3. Experimental Results:

[0068] The results are as follows Figure 1 As shown in the figure, compared with the non-high-risk ATTR-CA population, the levels of total PA (p<0.0001) and TTR tetramer (p<0.0001) were significantly lower in the blood of the high-risk ATTR-CA population, while the levels of misfolded protein (p<0.0001), protein misfolding rate (p<0.0001), and sST2 (p<0.0001) were significantly higher, and all differences were statistically significant.

[0069] Example 2: Comparative Analysis of the Diagnostic Performance of Single Biomarkers

[0070] The individual diagnostic performance of total PA, TTR tetramer, misfolded protein, protein misfolding rate, and sST2 was analyzed, and their AUC, cutoff value, specificity, and sensitivity were calculated. The results are shown in Table 1.

[0071] Table 1: Performance analysis of single biomarkers in diagnosing high-risk populations for ATTR-CA

[0072]

[0073]

[0074] As shown in Table 1, the AUC values ​​of the five biomarkers used alone to diagnose high-risk ATTR-CA populations all reached 0.858 or higher, indicating high sensitivity and specificity. This suggests that these five biomarkers can be used alone to diagnose high-risk ATTR-CA populations.

[0075] Example 3: Establishment of a diagnostic model for high-risk populations using ATTR-CA

[0076] To facilitate early screening and diagnosis of high-risk individuals for ATTR-CA, a diagnostic model was established, employing a combined approach of multiple biomarkers. Using SPSS software, binary logistic regression analysis was performed, taking into account multicollinearity and clinical significance among the biomarkers. The final model incorporated TTR tetramer, protein misfolding rate, sST2, and a constant, and the model equation was determined as: Logit(p) = 2.067 - 1.843 × [TTR tetramer level] + 0.178 × [protein misfolding rate] + 0.035 × [sST2 level]. The receiver operating characteristic (ROC) curve was used to assess the sensitivity, specificity, and diagnostic efficacy of the diagnosis. The area under the ROC curve (AUC) of the established model was 0.962. The maximum Youden index was determined as the diagnostic cutoff value, which was 0.62. When p > 0.62, the patient was diagnosed as being in the high-risk group for ATTR-CA; otherwise, the patient was diagnosed as being in the non-high-risk group for ATTR-CA. The diagnostic method had a specificity of 97.1% and a sensitivity of 88.6%. Figure 2 .

[0077] Example 4: Validation of the ATTR-CA Diagnostic Model for High-Risk Populations

[0078] Based on the same criteria as the discovery cohort, 36 high-risk ATTR-CA patients and 34 non-high-risk ATTR-CA patients were re-collected at the First Hospital of Jilin University as a validation cohort. Total PA levels in serum samples were detected using a PA detection kit (immunoturbidimetric assay); TTR tetramer levels in serum samples were detected using a TTR tetramer detection kit (ultra-high performance liquid chromatography); and sST2 levels in serum samples were detected using an sST2 detection kit (immunoassay). Protein misfolding rates were calculated based on the total PA and TTR tetramer levels. The TTR tetramer levels in the validation serum samples were significantly lower in the high-risk ATTR-CA group than in the non-high-risk group, while the protein misfolding rates and sST2 levels were significantly higher in the high-risk ATTR-CA group than in the non-high-risk group (p < 0.001 for all). Figure 3 .

[0079] The model established in Example 3 was used for the diagnosis of high-risk ATTR-CA populations. The TTR tetramer, protein misfolding rate, and sST2 values ​​from the validation samples were substituted into the calculations. The training set samples were divided into predicted positive and predicted negative groups using a cutoff value (>0.62). The actual state of high-risk ATTR-CA populations was designated as the "true positive group," and the actual state of non-high-risk ATTR-CA populations was designated as the "true negative group." Sensitivity is the "true positive rate," which is the probability of correctly identifying high-risk ATTR-CA populations; specificity is the "true negative rate," which is the probability of correctly identifying non-high-risk ATTR-CA populations. Based on the four-fold table calculations, the model's sensitivity for ATTR-CA high-risk populations was 94.4%, and its specificity was 97.1% (Table 2).

[0080] Table 2: Validation of the diagnostic efficacy of the diagnostic model

[0081] Diagnostic model ATTR-CA high-risk groups ATTR-CA Non-high-risk groups total Predicted positive (>0.62) 34 1 35 Predicted negative (≤0.62) 2 33 35 total 36 34 70

[0082] Therefore, the data from both the training and validation sets show that the combination of TTR tetramer, protein misfolding rate, and sST2 as biomarkers for early screening and diagnosis of high-risk ATTR-CA populations has good accuracy, specificity, and sensitivity.

[0083] It should be noted that the above embodiments are only specific and clear descriptions of the technical solutions and features of this application. Solutions or features that are prior art or common knowledge to those skilled in the art will not be described in detail in the above embodiments.

[0084] Furthermore, the technical solutions of this application are not limited to the above-described embodiments. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art. In addition, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims of this invention.

Claims

1. The application of a combination of biomarkers for early screening and diagnosis of transthyretin-cardiac amyloidosis (ATTR-CA) in the preparation of products for early screening and diagnosis of ATTR-CA high-risk populations, characterized in that, The biomarker combination consists of total PA, TTR tetramer, misfolded protein, protein misfolding rate, and sST2. The ATTR-CA high-risk population is a group of people with ATTR-CA warning signs or whose biomarker values ​​are not within the reference range when compared with the reference range. The product includes an ATTR-CA high-risk population diagnostic model. The model uses the detected or calculated values ​​of biomarkers as input variables to construct the diagnostic model. The model uses the equation: Logit (p) = 2.067 – 1.843 × [TTR tetramer content] + 0.178 × [protein misfolding rate] + 0.035 × [sST2 content]. The cutoff value for evaluating the ATTR-CA high-risk population by the model diagnostic results is 0.

62. When p > 0.62, the person is diagnosed as a high-risk population for ATTR-CA. Otherwise, the diagnosis is that the individual is not at high risk for ATTR-CA.

2. The application according to claim 1, characterized in that, The product is a kit containing reagents for detecting total PA, reagents for detecting TTR tetramer, and / or reagents for detecting sST2.

3. The application according to claim 2, characterized in that, In the kit, total PA is detected by immunoturbidimetry, TTR tetramer is detected by ultra-high performance liquid chromatography, and / or sST2 is detected by immunoassay.

4. The application according to claim 2 or claim 3, characterized in that, The sample from the test subject in the kit is serum or plasma.

5. The application according to claim 2, characterized in that, The control samples for the kit were obtained from non-high-risk individuals with ATTR-CA, and the samples were serum or plasma.

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