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

Abstract

The invention provides application of a biomarker combination for early screening and diagnosis of transthyretin cardiac amyloidosis (ATTR-CA) high-risk groups in preparation of a product for early screening and diagnosis of the ATTR-CA high-risk groups. In general, one or more biomarkers, such as total prealbumin (total PA), transthyretin (TTR) tetramer, misfolded protein, protein misfolding rate, and soluble growth stimulus expression gene 2 protein (sST2). In ATTR-CA high-risk groups and non-high-risk groups, the biomarkers show significant difference, which prompts that the biomarkers are related to ATTR-CA. When a plurality of biomarkers are combined for use, the biomarkers have relatively high sensitivity and specificity in early screening and diagnosis of ATTR-CA high-risk groups, have relatively high diagnosis efficiency, and provide a new thought and strategy for early diagnosis and treatment of ATTR-CA.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to the use of a biomarker combination for early screening and diagnosis of a high-risk population for transthyretin cardiac amyloidosis (ATTR-CA), and in the preparation of a product for early screening and diagnosis of a high-risk population for ATTR-CA. Background Art

[0002] Transthyretin cardiac amyloidosis (ATTR-CA) is an infiltrative cardiomyopathy, also known as transthyretin amyloid cardiomyopathy (ATTR-CM). It is the most common form 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 fibrils that deposit in the myocardial interstitium. Based on the presence or absence of TTR gene mutations, the disease is divided into hereditary / mutant (ATTRm) and wild-type (ATTRwt).

[0003] ATTR-CA is a progressive, debilitating and fatal disease, with patients suffering from poor quality of life, low survival rate and poor prognosis. In the past, it has been considered a rare disease, but is now recognized as a more common and important cause of heart failure and death worldwide. Due to the lack of understanding of the disease and the heterogeneity of symptoms, the disease remains underdiagnosed, resulting in an underestimated incidence and easy missed diagnosis and misdiagnosis. At the same time, the lack of specific clinical manifestations in the early stages of ATTR-CA also poses a great challenge to early screening and diagnosis. Therefore, it is crucial to focus on screening of high-risk populations and clinically identify and diagnose ATTR-CA in the early stages of the disease, so as to provide patients with a wider range of treatment options, thereby improving survival rates and preventing consequences that may lead to irreversible loss of physical function and decreased quality of life.

[0004] With the deepening of understanding of ATTR-CA disease and the development of endomyocardial biopsy, radionuclide bone scintigraphy ( 99 Tc mThe development of diagnostic technologies such as PYP and genetic testing has made early diagnosis of ATTR-CA possible. However, existing examination methods still have limitations. Endomyocardial biopsy is the "gold standard" for ATTR-CA diagnosis, but myocardial biopsy is an invasive procedure that requires a professional medical team and equipment, and the cost of surgery and pathological analysis is high. 99 Tc m -PYP can be used to diagnose ATTR-CA non-invasively. Although the positive test rate is high, it is associated with radiation exposure, high cost, poor accessibility, and a strong dependence on technology and interpretation. Genetic testing is the gold standard for distinguishing ATTRm from ATTRwt, but the cost and technical threshold are high. Conventional imaging examinations such as echocardiography and cardiac magnetic resonance (CMR) can show characteristic changes of myocardial amyloidosis but may not be sensitive in the early stages. Imaging features, such as myocardial thickness and signal abnormalities, may be helpful in the diagnosis of ATTR-CA but may also be seen in other cardiomyopathies. 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. In addition, there is currently a lack of clinical methods that can directly detect TTR in the blood.

[0005] Therefore, there is an urgent need for some new biomarkers with high sensitivity and specificity for the identification, early screening and diagnosis of high-risk groups for ATTR-CA without the need for imaging examinations and invasive tissue biopsies. Summary of the Invention

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

[0007] To achieve the above object, the present invention provides the following technical solutions:

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

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

[0010] A further preferred embodiment: the product comprises a diagnostic model for ATTR-CA high-risk population.

[0011] A further preferred embodiment: the model uses the biomarker detection value or calculated value as an input variable 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 embodiment: the cutoff value of the model diagnostic results for evaluating the ATTR-CA high-risk population is 0.62. When p>0.62, the patient is diagnosed as a high-risk ATTR-CA population; otherwise, the patient is diagnosed as a non-high-risk ATTR-CA population.

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

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

[0015] In a further preferred embodiment, the sample of the test subject in the kit is serum or plasma.

[0016] A further preferred embodiment: the control sample of the kit is derived from a non-high-risk group for ATTR-CA, and the sample is serum or plasma.

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

[0018] Step 1: Collect samples from the test subjects and control samples;

[0019] Step 2: Detecting the total PA content of the test subject sample and the control sample;

[0020] Step 3: Detecting the content of TTR tetramer in the test subject sample and the control sample;

[0021] Step 4: Detecting the sST2 content in the test subject sample and the control sample;

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

[0023] Step 6: Substitute the biomarker test value or calculated value into the model equation to obtain the calculation result;

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

[0025] As used herein, the subject is a human.

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

[0027] As used herein, the method for detecting the total PA content level is a PA determination kit that uses immunoturbidimetry for detection.

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

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

[0030] Furthermore, the sST2 content is detected using an sST2 determination kit and an immunoassay is used for detection.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention is the first to use total PA, TTR tetramers, misfolded proteins, protein misfolding rate, and sST2 as biomarkers. The inventors have found that the levels of total PA and TTR tetramers in the blood of individuals at high risk for ATTR-CA are significantly lower than those at low risk, while the levels of misfolded proteins, calculated protein misfolding rate, and sST2 are significantly higher in individuals at high risk for ATTR-CA than those at low risk. More specifically, the inventors have found that the combined use of TTR tetramers, protein misfolding rate, and sST2 has higher accuracy, specificity, and sensitivity for diagnosing individuals at high risk for ATTR-CA, and exhibits high consistency in validation populations. More particularly, this combination eliminates the need for imaging studies or invasive tissue biopsies. Therefore, this combination of biomarkers can be used for identifying, early screening, and diagnosing individuals at high risk for ATTR-CA. This combination is of great significance for improving the diagnosis rate, enabling early intervention, and reducing mortality in these individuals. It also provides new insights and strategies for the early diagnosis and treatment of ATTR-CA. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1The difference in the levels of total PA, TTR tetramer, misfolded protein, protein misfolding rate, and sST2 in the serum of ATTR-CA non-high-risk population and ATTR-CA high-risk population in Example 1;

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

[0035] Figure 3 The differences in the levels of TTR tetramer, protein misfolding rate and sST2 in the serum of ATTR-CA non-high-risk population and ATTR-CA high-risk population in Example 3 are shown. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and beneficial effects of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention and the corresponding drawings. It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the claimed invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0037] In the present invention, "ATTR-CA" is also referred to as "ATTR-CM", and the two names can be used interchangeably.

[0038] Example 1: Analysis of the differences in the levels of total PA, TTR tetramers, misfolded proteins, protein misfolding rate, and sST2 in the serum of ATTR-CA non-high-risk groups and ATTR-CA high-risk groups

[0039] 1. Experimental subjects and groups:

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

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

[0042] Cardiac amyloidosis (CA), particularly ATTRCA, should be considered in patients with 1 or more of the following features:

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

[0044] (2) echocardiography shows LVH but the electrocardiogram does not show high QRS voltage;

[0045] (3) persistent low-level elevation of troponin;

[0046] (4) Elderly people with low-gradient, low-flow aortic stenosis and 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 (unexplained diarrhea and constipation, 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 cataracts in both eyes.

[0052] Patients with 1 or more of the following imaging features may also be suspected of ATTR-CA:

[0053] (1) Electrocardiogram: The typical electrocardiographic manifestation of CA in limb and / or precordial leads when echocardiography reveals left ventricular hypertrophy is low QRS voltage 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. The possibility of CA should be considered in patients with unexplained left ventricular hypertrophy combined with atrial fibrillation and high-degree atrioventricular block.

[0054] (2) Echocardiography: If some seemingly non-specific ultrasound imaging manifestations coexist, they are highly suggestive of CA, such as biatrial enlargement, ventricular wall thickening (both left and right ventricular walls) without ventricular cavity enlargement, intramyocardial granular hyperechoicity, atrioventricular valve thickening, atrial septal thickening, a small amount of pericardial effusion, and restrictive diastolic dysfunction. The left ventricular ejection fraction is basically normal or slightly decreased. The left ventricular ejection fraction has poor sensitivity in evaluating the systolic function of ATTR-CA patients, while strain imaging (manifested by a decrease in the absolute value of the longitudinal strain peak) can detect systolic dysfunction earlier, especially the characteristic "apical sparing" pattern, that is, the longitudinal strain (LS) of the left ventricular base and mid-segment is reduced while the apex is normal [apical longitudinal strain / (average longitudinal strain of the mid + base)>1.0], which helps to distinguish CA from left ventricular hypertrophy caused by other reasons. Inferolateral wall thickness ≥14mm.

[0055] (3) Cardiac magnetic resonance imaging (CMR): ATTR-CA presents with asymmetric ventricular septal hypertrophy (79%), symmetric LVH (18%), or normal left ventricular geometry (3%), often with right ventricular hypertrophy. The typical "late amyloid enhancement (LGE) pattern" is extensive subendocardial LGE that does not conform to the regional distribution of coronary artery blood supply. It can also manifest as diffuse transmural or intramyocardial patchy LGE. T1 mapping (which can be used for patients with renal dysfunction who cannot receive gadolinium injection) measures an elevated native T1 value and an elevated extracellular volume fraction (ECV).

[0056] (4) Radionuclide bone scintigraphy: 99Tcm phosphate derivative radionuclide scanning shows myocardial uptake of 2 to 3, which is considered to be a possible 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 significantly reduced rib uptake / no rib uptake.

[0057] Or the values ​​of total PA, TTR tetramer, misfolded protein or protein misfolding rate in the patient's serum sample are compared with the reference interval, and the value is not within the reference interval.

[0058] Exclusion criteria:

[0059] (1) Subjects with active tumors, severe infections, acute inflammation, renal failure, and patients who have undergone acute coronary syndrome, unstable angina, stroke, transient ischemic attack, coronary artery revascularization, cardiac device implantation, heart valve repair, or major surgery due to worsening heart failure within 1 month;

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

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

[0062] Reference criteria for distinguishing immunoglobulin light chain cardiac amyloidosis (AL-CA) include an abnormal serum free light chain ratio (κ / λ <0.26 or >1.65) and the presence of an abnormal monoclonal immunoglobulin on serum / urine protein immunofixation electrophoresis. Elderly patients with ATTRCA who have 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 testing can assist in CA typing. A negative result is considered highly likely to be ALCA, while a positive result requires tissue biopsy, particularly endomyocardial biopsy with amyloid identification, for a definitive diagnosis. If the risks associated with a biopsy outweigh the benefits, alternative results should be discussed with local hematologists, investigators, and medical supervisors.

[0063] Inclusion criteria for ATTR-CA non-high-risk population:

[0064] Patients with heart disease who do not have clear indications of being at high risk for ATTR-CA.

[0065] 2. Experimental methods:

[0066] The total PA content in serum samples was detected using a PA detection kit (immunoturbidimetric method); the TTR tetramer content in serum samples was detected using a TTR tetramer detection kit (ultra-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 contents, 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, it can be seen that compared with the non-high-risk group for ATTR-CA, the levels of total PA (p<0.0001) and TTR tetramer (p<0.0001) in the blood of the high-risk group for ATTR-CA were significantly reduced, while the levels of misfolded protein (p<0.0001), protein misfolding rate (p<0.0001) and sST2 (p<0.0001) were significantly increased, and the differences were statistically significant.

[0069] Example 2: Comparative analysis of diagnostic performance of single biomarkers

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

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

[0072]

[0073]

[0074] As can be seen from Table 1, the AUC values ​​of the five biomarkers used alone to diagnose the ATTR-CA high-risk population all reached 0.858 or above, with high sensitivity and specificity, indicating that these five biomarkers can be used alone to diagnose the ATTR-CA high-risk population.

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

[0076] To screen and diagnose high-risk individuals for ATTR-CA early, a diagnostic model was established that combined multiple biomarkers for diagnosis. Using SPSS software, binary logistic regression analysis was performed. Taking into account multicollinearity among biomarkers and clinical significance, the final model included TTR tetramers, protein misfolding rate, sST2, and a constant. 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 evaluate 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 ATTR-CA high-risk group was diagnosed; otherwise, the ATTR-CA non-high-risk group was diagnosed, with a specificity of 97.1% and a sensitivity of 88.6%. Figure 2 .

[0077] Example 4: Validation of the ATTR-CA high-risk population diagnostic model

[0078] Based on the same criteria as the discovery cohort, 36 cases of ATTR-CA high-risk population and 34 cases of ATTR-CA non-high-risk population were re-collected in the First Hospital of Jilin University as the validation cohort. The PA detection kit (immunoturbidimetric method) was used to detect the content of total PA in serum samples; the TTR tetramer detection kit (ultra-performance liquid chromatography) was used to detect the content of TTR tetramer in serum samples; the sST2 detection kit (immunoassay) was used to detect the content of sST2 in serum samples; the protein misfolding rate was calculated based on the content of total PA and TTR tetramer. The TTR tetramer content in the serum of the validation samples was significantly lower in the ATTR-CA high-risk population than in the ATTR-CA non-high-risk population, while the protein misfolding rate and sST2 in the ATTR-CA high-risk population were significantly higher than those in the ATTR-CA non-high-risk population (p values ​​< 0.001). Figure 3 .

[0079] The model established in Example 3 was used to diagnose ATTR-CA high-risk populations, and the TTR tetramer, protein misfolding rate and sST2 of the validation sample were substituted for calculation. The training set samples were divided into a predicted positive group and a predicted negative group by the cutoff value (>0.62). The actual status of the ATTR-CA high-risk population was the "true positive group", and the actual status of the ATTR-CA non-high-risk population was the "true negative group". Sensitivity is the "true positive rate", which is the probability of correctly judging the ATTR-CA high-risk population; specificity is the "true negative rate", which is the probability of correctly judging the ATTR-CA non-high-risk population. According to the four-cell table, it can be calculated that the diagnostic sensitivity of the model for the ATTR-CA high-risk population is 94.4%, and the specificity is 97.1% (Table 2).

[0080] Table 2: Diagnostic efficacy validation of diagnostic models

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

[0082] It can be seen from this that the data from the training set and the validation set of the present invention show that the combination of biomarkers based on TTR tetramer, protein misfolding rate and sST2 for early screening and diagnosis of ATTR-CA high-risk population 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 technical features of the present application. For those skilled in the art, solutions or features that belong to the prior art or common knowledge will not be described in detail in the above embodiments.

[0084] In addition, the technical solutions of the present application are not limited to the above-mentioned embodiments. Those skilled in the art should regard the specification as a whole. 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. Furthermore, for those skilled in the art, improvements or changes can be made based on the above description, and all these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A use of a biomarker combination for early screening and diagnosis of a high-risk population for transthyretin cardiac amyloidosis (ATTR-CA) in the preparation of a product for early screening and diagnosis of a high-risk population for ATTR-CA, characterized in that: The biomarkers include total PA, TTR tetramer, misfolded protein, protein misfolding rate and sST2.

2. The use according to claim 1, characterized in that The biomarker panel consists of TTR tetramer, protein misfolding rate, and sST2.

3. The use according to claim 1 or claim 2, characterized in that The ATTR-CA high-risk population is a group of people who have ATTR-CA warning signs or whose biomarker values ​​are compared with the reference interval and are not within the reference interval.

4. The use according to any one of claims 1 to 3, characterized in that The product includes a diagnostic model for ATTR-CA high-risk populations. The model uses biomarker detection values ​​or calculated values ​​as input variables to construct a 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].

5. The use according to claim 4, characterized in that The cutoff value of the model diagnostic results for evaluating the ATTR-CA high-risk population is 0.

62. When p>0.62, the population is diagnosed as the ATTR-CA high-risk population; Otherwise, they were diagnosed as non-high-risk group for ATTR-CA.

6. The use according to any one of claims 1 to 5, characterized in that The product is a kit comprising a reagent for detecting total PA, a reagent for detecting TTR tetramers and / or a reagent for detecting sST2.

7. The use according to claim 6, 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.

8. The use according to claim 6 or claim 7, characterized in that The sample of the subject to be tested in the kit is serum or plasma.

9. The use according to any one of claims 6 to 8, characterized in that The control samples of the kit are derived from a non-high-risk population for ATTR-CA, and the samples are serum or plasma.

Images