WO2018043822A1 - Diagnostic method and diagnostic kit for diagnosing development or not of post-acute coronary syndrome depression using interleukin 1β - Google Patents

Abstract

The present invention relates to a method for diagnosing the development or not of depression, and more specifically, to a diagnostic method and a diagnostic kit for predicting the development or not of post-acute coronary syndrome depression by analyzing the concentration and genotype of a particular biomarker in the blood of an acute coronary syndrome patient.

Description

Diagnosis and Diagnostic Kit of Acute Coronary Syndrome after Interleukin 1β

The present invention relates to a method for diagnosing depression, and more specifically, a diagnostic method for predicting depression after acute coronary syndrome by analyzing the concentration and genotype of a specific biomarker in the blood of a patient with acute coronary syndrome. For diagnostic kits.

Depression is often accompanied by acute coronary syndrome (ACS). In addition, depression with ACS predicts a poor prognosis (Lichtman et al. 2014). Corresponding mechanisms for the link between depression and ACS were investigated, and the inflammatory hypothesis was highlighted. Inflammation is associated with serotonin disorders and hypothalamic-pituitary-adrenal axis hyperactivity, and the inflammatory process is involved in the pathophysiology of depression (Maes et al., 2011A, Lee et al., 2013). In addition, the role of inflammation in the etiology and risk of onset of ACS is well documented, and the magnitude of the inflammatory response during ACS predicts the prognosis of heart disease (BERTON et al., 2009). That is, inflammation is not only associated with depression and ACS separately (Maes et al. 2011B) but also with poor prognosis of ACS (Poole et al., 2011). In this context, previous studies have suggested a significant association between depression and inflammatory markers including C-reactive protein and cytokines in ACS (Wilkowska et al., 2015).

Interleukin (IL) -1β, an important proinflammatory cytokine that induces the production of other cytokines, is associated with poor prognosis of ACS by contributing to the development of atherosclerosis. It has been shown that depression / burnout patients in ACS have higher IL-1β concentrations (Appels et al., 2000), and escitalopram has been shown to lower IL-1β concentrations in ACS model mice (Bah et al., 2011). Recent in vitro studies have shown that IL-1β signaling is involved in the inflammatory cascade in the development of depression in ACS patients, and that statins act as anti-inflammatory agents by down-regulating IL-1β (Ma et al., 2016). However, it is difficult to draw conclusions about this association due to the limited number of subjects, cross-sectional design, and small number of clinical studies. In addition, IL-1β secretion is regulated by IL-1β gene polymorphism: individuals with -511T or + 3953T secreted higher concentrations of IL-1β than individuals with -511C or + 3953C polymorphisms.

Therefore, considering the two IL-1β polymorphisms -511T or + 3953T, it is necessary to investigate the effect of serum IL-1β concentration on ACS depression after ACS depression.

The present inventors have completed the present invention by revealing that the IL-1β blood concentration and gene polymorphism have a significant correlation with the onset of depression after acute coronary syndrome.

Therefore, the objective of the present invention is to analyze the acute coronary syndrome in patients with acute coronary syndrome and acute coronary syndrome through the analysis of IL-1β concentration and IL-1β gene polymorphism. It is to provide a method for diagnosing depression after arterial syndrome.

Another object of the present invention is to measure the IL-1β concentration and IL-1β polymorphism in the blood of patients with acute coronary syndrome, so that the diagnosis of acute stage depression after acute coronary syndrome can be predicted and diagnosed. It is to provide a diagnostic kit for depression after acute coronary syndrome, which can prevent depression in advance.

The objects of the present invention are not limited to the above-mentioned objects, and other objects that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object of the present invention, the present invention comprises a measurement step of measuring the IL-1β concentration in the blood of patients with acute coronary syndrome; Genotyping step of investigating IL-1β-511C / T polymorphism of the patient; And a diagnosis step of determining whether acute depression is developed according to the measured IL-1β concentration and the analyzed IL-1β -511C / T genotype.

In a preferred embodiment, the first condition wherein the measured IL-1β concentration is greater than or equal to the reference concentration in the diagnosis step, and the second condition wherein the IL-1β -511C / T genotype of the analyzed patient is ?? 511T / T genotype. It is judged that the acute depression is developed only when all are satisfied.

In a preferred embodiment, the incidence of acute depression is assessed to be 58% or greater when the first and second conditions are met.

In a preferred embodiment, a 56% chance of developing acute depression is not diagnosed if the first and second conditions are not met.

In a preferred embodiment, the baseline concentration is 3.5 pg per ml of serum.

In a preferred embodiment, the genotyping step comprises the steps of: separating the DNA comprising the 511th base of the IL-1β gene isolated from the patient; Amplifying the separated DNA using a sense primer and an antisense primer; And examining the presence of IL-1β -511TT genotype by examining the amplified DNA using a restriction enzyme capable of recognizing a 511C → T mutation.

In addition, the present invention provides a measurement means for measuring the serum IL-1β concentration in the blood of the patient; And genotyping means for analyzing the IL-1β -511C / T gene polymorphism of the patient and analyzing whether or not it possesses the -511TT genotype; and providing a diagnosis kit for acute coronary syndrome after depression.

The present invention has the following effects.

First, according to the method for diagnosing depression of acute coronary syndrome according to the present invention, the presence of acute coronary syndrome predicts acute stage of depression in patients with acute coronary syndrome through analysis of IL-1β concentration and IL-1β gene polymorphism. Can be diagnosed.

In addition, the diagnostic kit for acute coronary syndrome syndrome according to the present invention is to determine the incidence of acute stage depression after acute coronary syndrome simply by measuring the IL-1β concentration and IL-1β gene polymorphism in the blood of patients with acute coronary syndrome. Because it can be predicted and diagnosed, it is possible to prevent the depression of the patient preemptively and has clinical usefulness.

This technical effect of the present invention is not limited to the above-mentioned range, and even if not explicitly mentioned, the effects of the invention that can be recognized by those skilled in the art from the description of the specific contents for the practice of the invention to be described later also Of course included.

1 is a graph showing serum concentrations of interleukin-1β (IL-1β) due to two gene polymorphisms and depressive disorder states at baseline and follow-up after acute coronary syndrome (ACS).

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, the terms "comprise" or "having" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described in the specification, and that one or more other features It should be understood that it does not exclude in advance the possibility of the presence or addition of numbers, steps, actions, components, parts or combinations thereof.

Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art.

In interpreting a component, it is interpreted to include an error range even if there is no separate description.

In the case of a description of a temporal relationship, for example, if the temporal after-degree relationship is described as 'after', 'following', 'after', 'before', etc. This includes non-consecutive cases unless' is used.

The characteristic parts of each of the various embodiments of the present invention can be combined or combined with each other, partly or wholly, and technically various interlocking and driving are possible, and each of the embodiments can be implemented independently or in relation to each other. It can also be done together.

Hereinafter, with reference to the accompanying drawings and preferred embodiments will be described in detail the technical configuration of the present invention.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.

The technical features of the present invention reveal that IL-1β blood concentration and IL-1β-511C / T gene polymorphism are significantly associated with the development of depression after acute coronary syndrome. The present invention provides a method and a diagnostic kit for diagnosing depression after acute coronary syndrome using a blood marker of IL-1β and IL-1β-511C / T polymorphism as a biomarker to predict and / or diagnose the disease. .

Accordingly, the method for diagnosing depression after acute coronary syndrome according to the present invention includes measuring a concentration of IL-1β in the blood of a patient having acute coronary syndrome; Genotyping step of investigating IL-1β-511C / T polymorphism of the patient; And a diagnosis step of determining whether acute depression is developed according to the measured IL-1β concentration and the analyzed IL-1β -511C / T genotype.

Here, the reference concentration may be defined as a concentration of 3.5pg per 1ml serum, and the acute phase means a period within two weeks from the time of the occurrence of acute coronary syndrome, acute depression is the point when the acute coronary syndrome occurs Depression occurs onset within two weeks.

More specifically, only when the first condition where the IL-1β concentration measured at the diagnosis stage is higher than the reference concentration and the second condition where the IL-1β -511C / T genotype of the analyzed patient is -511T / T genotype are satisfied are met. Acute depression can be considered to occur, and when the first and second conditions are met, the likelihood of developing acute depression is more than 58%. On the other hand, when the first and second conditions were not met, the probability of developing acute depression was more than 56%.

At this time, genotyping may include separating the DNA comprising the 511 th base of the IL-1β gene isolated from the patient; Amplifying the separated DNA using a sense primer and an antisense primer; And examining the presence of the IL-1β -511TT genotype by examining the amplified DNA using a restriction enzyme capable of recognizing a 511C → T mutation.

Next, the acute coronary syndrome syndrome depression kit of the present invention is a diagnostic means for measuring the serum IL-1β concentration in the blood of the patient; And genotyping means for analyzing the patient's IL-1β -511C / T gene polymorphism to analyze whether or not it possesses the -511TT genotype.

As described above, the present invention is one or more of IL-1β concentration and IL-1β-511C / T polymorphism of the patient after acute coronary syndrome affects the depression after acute coronary syndrome, in particular blood IL- above the reference concentration We predicted the onset of acute depression associated with acute coronary syndrome by proving that the interaction between 1β concentration and IL-1β -511T / T genotype is significantly related to the development of acute depression after acute coronary syndrome. It has significant clinical advantages by providing diagnostic methods and diagnostic kits that can be used to treat depression that worsens the prognosis of acute coronary syndrome. That is, by using the present invention it is possible to facilitate the prevention and treatment by selecting a patient prone to depression associated with acute coronary syndrome with a large disease burden.

Example

1. Research subject

The data in this study were drawn for analysis from a larger study called the Korean Depression (K-DEPACS) study in acute coronary syndrome (ACS). The K-DEPACS study was conducted in 2006 to investigate the epidemiology of depression in ACS using an observational predictive design. Participants were sampled at baseline from 969 patients who were admitted to ACS in Chonnam National University Hospital (N = 4,809) who met eligibility criteria and agreed to both participation and blood sampling. Evaluation at baseline was done within two weeks of ACS to assess the acute phase of ACS. A total of 711 baseline patients were followed up to one year later at the outpatient clinic. The study psychiatrist evaluated using a mini-international neuropsychiatric interview (MINI, Sheehan et al., 1998) to identify participants' major or minor depressive disorder. All participants provided a written consent form for K-DEPACS, which has been approved by Ethics Committee of Biomedical Research Ethics Committee of Chonnam National University Hospital.

2. Evaluation of Depression

Diagnosis of depressive disorders was performed using a structured DSM-IV mental and diagnostic interview defining MINI, a major or minor depressive disorder. Interviews have been translated into Korean and standardized (Yoo et al., 2006). Since ACS patients are usually discharged within two weeks of intensive care, they did not require a duration of symptoms longer than two weeks at baseline as a diagnostic criterion for depressive disorder. However, a standard two-week symptom duration was needed for follow-up diagnosis. Since there were not many patients with major depression that could be analyzed separately, patients with major and minor depression were included in the study in the same category. Baseline and one-year follow-up assessments were used to assess depression in the acute and chronic phases of ACS.

3. Measurement of Serum IL-1β Level

Serum IL-1β concentrations were analyzed using a solid-phase sandwich enzyme immunoassay kit (Invitrogen, Camaro, CA, USA).

4. IL-1β Gene Polymorphism Analysis

IL-1β genotype was investigated using polymerase chain reaction (PCR) and PCR-based restriction fragment length polymorphism analysis, considering -511C / T or + 3953C / T. Taking into account the infrequency of the + 3953T / T genotype, they divided into two groups (C / C or C / T).

DNA isolation was isolated from leukocytes extracted from the patient's blood using a DNA extraction kit (QIAmp blood kit, Qiagen) according to the manufacturer's protocol. The isolated DNA samples were amplified with a GeneAmp PCR machine (Perkin Elmer 9600) using a primer set (sense primer (5'-TGAAGGAGAAGGTGTCTGCGGGA-3 ') and antisense primer (5'-AGGACGGTGCGGTGAGAGTG-3'). In order to amplify the 198 bp product generated by the PCR amplification as described above, after denaturation at 95 ° C. for 60 seconds, the primer was annealed at 62 ° C. for 90 seconds, and then the primer extension reaction was performed at 72 ° C. for 60 seconds. The procedure was repeated 35 cycles. The amplified fragments were digested at 37 ° C. for 4 hours with a restriction enzyme Hin f1 (10 unit / reaction mixture, purchased from MBI Fermentas) that can recognize 511C → T mutations in the amplified fragments. The fragments treated with Hin f1 were then electrophoresed with polyacrylamide gel and stained with EtBr (ethidium bromide) to observe the mutated state.

5. Demographic and Clinical Covariates

* At baseline, characteristics that act as confounding or mediating associations between depression and ACS were evaluated. Data were obtained on age, gender, education, residency (whether alone or not), accommodation (owned or rented), current occupation (whether or not employed), and prior and family history of depression. The following cardiovascular risk factors were identified: previous and family history of ACS, diagnosed hypertension and diabetes, hypercholesterolemia by fasting serum total cholesterol concentration (200 mg / dL), obesity as measured by body mass index (BMI), and Current smoking status reported. To measure the current heart condition, the degree of ACS was estimated by Killip classification, left ventricular ejection fraction (LVEF) was estimated using ultrasound, and serum for troponin I and creatine kinase-MB (CK-MB). Cardiac biomarkers were measured.

6. Statistical Analysis

Baseline variables, including demographic data, depression characteristics, cardiovascular risk factors, and current heart condition, were compared according to depression at baseline using the t-test or χ ² test. Then, features with significant relevance (P <0.05) were considered as covariates in multivariate analysis. Depending on the presence of depression at baseline and at follow-up one year after baseline, IL-1β concentration and two genotypes were compared using the t-test and the χ ² test. To determine the effect of potential interactions, the association between depression status and IL-1β concentration was initially analyzed according to the two genotypes. The interaction between IL-1β concentration and each genotype was then analyzed using a multivariate logistic regression model after appropriate covariate adjustments. In addition, the sensitivity analysis was performed using the same statistical method except for participants with a history of depression. All analyzes were performed using SPSS 18.0.

7. Results

(1) Demographic and clinical characteristics of all participants

969 (84.1%) of the total K-DEPACS samples (N = 1,152) agreed with the blood samples. There was no significant difference in characteristics at baseline in those who agreed or disagreed with blood samples (P> 0.15 for all). Depressive disorder (major + minor) was diagnosed in 378 patients (39.8%) at baseline and was followed up in 183 (25.7%). Follow-up evaluation was performed after one year in 711 (73%) of the initial 969 participants. Untracked group (N = 258) correlated with significantly increased age and higher Killip class (P <0.05 for both).

 Demographic and clinical characteristics affecting acute depression were investigated at baseline and the results are shown in Table 1 below. As can be seen from Table 1, female gender, low education level, living alone, rental housing, current unemployment status, the presence of hypertension and diabetes, and current smoking status were significantly associated with acute depression (all for P <0.05). ).

	No depressive disorder (N = 591)	Depressive disorder (N = 378)	Statistical coefficient	P-value *
Socio-demographic characteristics
Age, mean (SD) years	57.7 (11.3)	59.0 (10.8)	t = -1.860	0.063
Gender, N (%) female	118 (20.0)	151 (39.9)	χ 2 = 45.90	<0.001
Education, mean (SD) years	10.2 (4.8)	9.3 (4.4)	t = + 3.013	0.003
Living alone, N (%)	47 (8.0)	45 (11.9)	χ 2 = 4.191	0.041
Housing, N (%) rented	73 (12.4)	77 (20.4)	χ 2 = 11.33	0.001
Currently unemployed, N (%)	192 (32.5)	176 (46.6)	χ 2 = 19.39	<0.001
Depression characteristics
Previous depression, N (%)	17 (2.9)	17 (4.5)	χ 2 = 1.789	0.181
Family history of depression, N (%)	11 (1.9)	12 (3.2)	χ 2 = 1.716	0.190
HAMD, mean (SD) score	3.6 (2.7)	14.2 (5.0)	t = -38.09	<0.001
Cardiac risk factors, N (%)
Previous ACS	20 (3.4)	19 (5.0)	χ 2 = 1.610	0.205
Family history of ACS	15 (2.5)	16 (4.2)	χ 2 = 2.138	0.144
Hypertension	252 (42.6)	206 (54.5)	χ 2 = 13.01	<0.001
Diabetes mellitus	93 (15.7)	98 (25.9)	χ 2 = 15.13	<0.001
Hypercholesterolemia	296 (50.1)	190 (50.3)	χ 2 = 0.003	0.956
Obesity	259 (43.8)	156 (41.3)	χ 2 = 0.614	0.433
Current smoker	249 (42.1)	117 (31.0)	χ 2 = 12.26	<0.001
Current cardiac status
Killip class> 1, N (%)	97 (16.4)	71 (18.8)	χ 2 = 0.904	0.342
LVEF, mean (SD)%	61.4 (11.2)	60.8 (11.4)	t = + 0.772	0.440
Troponin I, mean (SD) mg / dL	9.9 (16.6)	9.9 (11.8)	t = + 0.063	0.949
CK-MB, mean (SD) mg / dL	17.6 (41.1)	17.1 (30.4)	t = + 0.178	0.858
Other factors
Creatinine, mean (SD) mg / dl	0.9 (0.3)	0.9 (0.3)	t = + 1.013	0.462
Vitamin supplement, N (%)	13 (2.2)	10 (2.6)	χ 2 = 0.198	0.657
* P-values using t-tests or χ 2 testsasappropriate.HAMD, Hamilton Depression Rating Scale; ACS, acute coronary syndrome; LVEF, left ventricular ejection fraction; CK-MB, Creatine kinase-MB.

(2) Analysis of the relationship between IL-1β concentration, IL-1β polymorphism and depressive disorder status according to the observation result of baseline and follow-up

To analyze the association between IL-1β concentration and IL-1β gene polymorphism and acute depression status, serum IL-1β and IL-1β gene polymorphisms were analyzed from the blood of ACS patients only at baseline. The categorized into acute depression, which is diagnosed at baseline, and chronic depression, which was diagnosed at follow-up, and analyzed the association between the IL-1β genotype and the measured serum IL-1β concentration according to each depressive disorder. The results are shown in Table 2.

		Depressive disorder at 2 weeks after ACS			Depressive disorder at 1 year after ACS
		Absent (N = 591)	Present (N = 378)	p-value	Absent (N = 528)	Present (N = 183)	p-value
Plasma concentrations, mean (SD) pg / ml		3.5 (1.4)	3.8 (2.0)	0.028	3.6 (1.4)	3.8 (2.1)	0.138
IL-1β -511C / T	C / C	205 (34.7)	105 (27.8)	<0.001	157 (29.7)	41 (22.4)
genotype, N (%)	C / T	289 (48.9)	161 (42.6)		248 (47.0)	90 (49.2)	0.122
	T / T	97 (16.4)	112 (29.6)		123 (23.3)	52 (28.4)
IL-1β + 3953C / T	C / C	528 (89.3)	332 (87.8)	0.468	459 (86.9)	158 (86.3)	0.838
genotype, N (%)	C / T	63 (10.7)	46 (12.2)		69 (13.1)	25 (13.7)
p-value using t-tests or χ 2 tests.

Table 2 shows that ACS patients with acute depression, or depression at baseline, have significantly higher IL-1β concentrations and more -511T alleles. In contrast, no association with the + 3953T / T genotype was found. At follow-up, depression was not related to IL-1β concentration or genotype. All genotypes were at Hardy-Wineberg equilibrium (all P> 0.05).

(3) Analysis of the effect of interaction between IL-1β concentration and IL-1β polymorphism according to depressive disorder status

Looking at the interaction between IL-1β concentration and IL-1β genotype according to the depressed state, C / C, C / T and T / T as shown in FIG. Serum IL-1β concentrations for each genotype were significantly associated with the -511C / T genotype with mean values of 3.3, 3.6 and 3.9 pg / ml (F = 9.344, P <0.001). However, there was no association with the + 3953C / T genotype (F = 0.943, P = 0.332), with mean values of 3.6 and 3.7 pg / ml for the C / C and C / T genotypes, respectively. In particular, ACS patients with acute depression had significantly higher concentrations of IL-1β in the presence of the -511T allele. This association did not appear in chronic depression diagnosed at follow-up. After calibrating baseline covariates, in terms of interaction effects in the logistic regression model, a significant bidirectional interaction between serum IL-1β concentration and -511C / T genotype was found in acute depression (depression diagnosed at baseline). And chronic depression (depression diagnosed at the time of follow-up) was not found.

These results indicate that depression in the acute phase of ACS is significantly associated with IL-1β concentration and the -511T allele. In the presence of the -511T allele, the interaction between acute depression and IL-1β concentration was also significant. However, no association with depression was found in the chronic phase of ACS. The + 3953C / T genotype was not associated with depression in either the acute or the chronic phase.

Therefore, serum IL-1β concentration and IL-1β -511C / T genotyping may be clinically useful because screening to identify groups at risk of depressive disorder in the acute phase of ACS is possible and appropriate therapeutic intervention is possible. Can be.

In addition, depression accompanied by ACS leads to very high disease burden and makes treatment difficult. According to the present invention, only by analyzing IL-1β concentration and IL-1β -511C / T genotype at baseline, May enable more focused interventions in the prevention and / or management of depression associated with ACS. This is because vulnerable patients with genetic predisposition at high IL-1β levels are more likely to experience ACS-associated depression. Thus, the usefulness of the present invention is that if a doctor is aware of the effects of IL-1β concentration and -511C / T genotype on ACS post-ACS, it may be more focused on depression during ACS, leading to acute depression after ACS. Prevent or preemptively treat the disease early onset.

Although the present invention has been shown and described with reference to the preferred embodiments as described above, it is not limited to the above embodiments and those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

Claims (7)

1. Measuring the level of IL-1β in the blood of a patient with acute coronary syndrome;

   Genotyping step of investigating IL-1β-511C / T polymorphism of the patient; And

   Determining the onset of acute depression according to the measured IL-1β concentration and the analyzed IL-1β -511C / T genotype; diagnostic method comprising acute coronary syndrome after depression.
2. The method of claim 1,

   In the diagnosing step, the acute phase is met only when both the first condition in which the measured IL-1β concentration is higher than the reference concentration and the second condition in which the analyzed IL-1β -511C / T genotype is -511T / T genotype are satisfied. A method for diagnosing depression after acute coronary syndrome, characterized in that it is determined that the depression has occurred.
3. The method of claim 2,

   When the first and second conditions are met, the incidence of acute depression is diagnosed to be 58% or more, characterized in that the diagnosis of depression after acute coronary syndrome.
4. The method of claim 2,

   When the first condition and the second condition is not satisfied, the probability of developing acute depression is diagnosed as 56%, characterized in that the diagnosis of depression after acute coronary syndrome.
5. The method according to any one of claims 2 to 4,

   The baseline concentration is 3.5pg per 1ml serum, characterized in that acute coronary syndrome after the onset of depression.
6. The method according to any one of claims 1 to 4,

   Said genotyping step comprises the steps of: separating the DNA comprising the 511 base of the IL-1β gene isolated from the patient; Amplifying the separated DNA using a sense primer and an antisense primer; Determining the presence of IL-1β -511TT genotype by examining the amplified DNA using a restriction enzyme capable of recognizing a 511C → T mutation; depression after acute coronary syndrome How to diagnose.
7. Measuring means for measuring serum IL-1β concentration in the blood of the patient; And

   Kit for diagnosing depression after acute coronary syndrome, comprising genotyping means for investigating the IL-1β -511C / T gene polymorphism of the patient and analyzing whether the patient has a -511TT genotype.

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