KR20190090269A - Assessment method for diagnosing and predicting late-life depression and diagnostic kits comprising thereof - Google Patents

Abstract

The present invention relates to a method for diagnosing depression. More particularly, the present invention relates to a diagnostic kit and a detection method for late-life depression, capable of detecting the presence and/or the risk of development of late-life depression by analyzing the concentration of a specific biomarker in the body fluid or tissue of patients. The detection method includes a step of measuring the level or amount of the biomarker of late-life depression contained in a biological sample of the patients; and a step of determining the presence or risk of development of late-life depression based on the level or amount of the biomarker of late-life depression.

Description

[0001] The present invention relates to a method for diagnosing depression in an elderly person,

The present invention relates to a method of diagnosing depression, and more particularly, to a method and apparatus for detecting depression in the elderly which can detect the presence and / or risk of future depression by analyzing the concentration of a specific biomarker in a body fluid or tissue of a patient, Lt; / RTI >

Depression is a major concern for public health and 17% of the general population experience this condition at least once during their lifetime (Flint and Kendler, 2014). Depression also has the risk of suicide, the most serious consequence of depressive disorder. In addition, depression is expected to be a major contributor to the burden of disease in 2030 (WHO, 2008). Thus, considerable efforts have been made to identify biomarkers to understand the pathophysiology of this disorder and to improve diagnostic and therapeutic outcomes (Lopresti et al., 2014; Pae et al., 2008). These efforts have proven that genetic and environmental factors are involved in the onset of depression. In particular, genetic factors contribute up to 40% of the risk of depression, and environmental factors, including early life adversity, contribute to the remainder (Kendler et al., 2006).

In this context, the hypothalamus-pituitary-adrenal (HPA) axis regulation disorder was a major research focus because of its main role in regulating the stress response (Zunszain et al., 2011). HPA axis control imbalance has been consistently associated with increased risk of depression through modified stress responses (Nemeroff, 2004; Parinate and Lightman, 2008, Radke et al., 2011). The HPA axis is regulated by a negative feedback loop through the interaction of glucocorticoids with glucocorticoid receptors (GR) (Alt et al., 2010). Previous studies investigating HPA axis control disorders that may be the cause of depression have therefore explored the abnormal function of GR and have found that reduced peripheral GR levels and reduced GR mRNA are associated with this condition (Yehuda et al Webster et al., 2002; O'Kane et al., 2012). Expression of GR is regulated by a variety of genetic polymorphisms of the GR gene (Spijker and van Rossum 2012) and by the posterior genetic variation (de Kloet et al., 2005). A posteriori genetic variation refers to a change in gene expression that does not affect the nucleotide sequence of DNA that normally occurs through interaction with environmental stressors, and a postmortem change, including DNA methylation in the GR gene, (Champagne, 2010). It has also been suggested that the role of the neurotransmitter in neurodegenerative diseases is important.

Methylation at the CpG site in the GR gene region, known as the nuclear receptor subfamily 3, group C, member 1 (nuclear receptor subfamily 3, group C, member 1; NR3C1), is one of the most studied epigenetic mutations. The more methylation of the NR3C1 gene is associated with a decrease in the expression of the GR gene, similar to the typical DNA methylation of other genes (Moore et al., 2013; Weber et al., 2007). It has been shown that hypermethylation of NR3C1 is associated with early life adversity known as a risk factor for depression (Oberlander et al., 2008; Perroud et al., 2011; Tyrka et al., 2015) Hill et al., 2001 ). Based on this, it can be assumed that the change in methylation of NR3C1, which is associated with early life adversity, can contribute to the vulnerability of individuals to the onset of depression.

However, few studies have investigated the association between depression and NR3C1 methylation and the results were inconsistent. More NR3C1 methylation was found in children and adolescents with depression / internalization problems (Dadd et al., 2015; van der Knapp et al., 2015). However, in adults, some have reported that increased NR3C1 methylation is associated with depression, while others have reported that NR3C1 methylation reduction is associated with depression.

In addition, of the previous studies, a patient-controlled study design was used except for the study by van der Knapp et al. (2015) (Dadd et al., 2015; Nantharat et al., 2016; Roy et al., 2017; Na et al., 2016; Bustamante et al., 2016; Tyrka et al., 2016). This implies that the metaphysical link between depression and NR3C1 methylation has been extensively studied. Only one study in adolescents investigated the relationship between NR3C1 methylation and internalization disability through a three-year longitudinal study. NR3C1 methylation at 16 years predicted the onset of internalization disorders after three years (van der Knapp et al., 2015). However, given the lack of consistent research results and lack of longitudinal studies, there is a clear limit to generalize these findings as depression in old age. This limitation is evident in that most studies have been performed in adolescents who are most strongly affected by NR3C1 methylation associated with childhood trauma following relatively short periods of onset of trauma.

The present inventors have accomplished the present invention by finding that the increase in NR3C1 methylation is significantly associated with the risk of the onset and / or onset of depression in the elderly.

Therefore, it is an object of the present invention to provide a method for diagnosing early onset depression and / or predicting the onset risk of geriatric depression by confirming that methylation of a specific region of the NR3C1 gene can be a biomarker of senile depression, And to provide a method for detecting depression in old age that can contribute to the decision process.

Another object of the present invention is to measure the degree of methylation of NR3C1 in the blood of an old patient so that the risk of the onset of the disease can be predicted as well as the existence of the old age depression, so that the depression can be preemptively prevented in the old age patient, And to provide a diagnostic kit for detecting depression in old age.

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

In order to achieve the objects of the present invention described above, the present invention provides a method for measuring the level or amount of the age-depressed biomarker contained in a biological sample of a patient; And a determining step of determining the presence or risk of developing senile depression based on the level or amount of the age-related depression biomarker.

In a preferred embodiment, the age-depressed biomarker is methylation of a CpG region contained in the nucleotide region -3132 of the NR3C1 gene at the 5'terminal -3187 of the NR3C1 gene, and the increase in the degree of methylation of the CpG region is indicative of the presence or the onset of senile depression It represents a danger.

In a preferred embodiment, the determining step is performed by comparing the level or amount of the senile depression biomarker in the sample with a reference level or amount of the senile depression biomarker, wherein the reference level is obtained from a population not suffering from senile depression A level of sensitivity and specificity that distinguishes the depressed patient group based on the methylation level obtained from the group of individuals including the methylation level and the old depression patients, and the level or amount of the depression biomarker of the old age is The absolute cutoff level or above indicates the presence or risk of developing senile depression.

In a preferred embodiment, the CpG region is at least one of CpG1, CpG2 and CpG3.

In a preferred embodiment, if the methylation of the CpG2 is above 10.5% in the determining step, the sensitivity to determine the presence of old age depression is greater than 69% and the specificity is greater than 55%.

In a preferred embodiment, if the methylation of the CpG3 is 6.5% or more in the determining step, the sensitivity to determine the presence of old age depression is 62% or more and the specificity is 56% or more.

In a preferred embodiment, when the mean methylation value of CpG1, CpG2 and CpG3 is 7.8% or more in the determination step, the sensitivity to determine the presence of old age depression is 70% or more and the specificity is 54% or more.

In a preferred embodiment, the greater the methylation of the CpG2 by 1%, the greater the likelihood of the presence of depression in the elderly by 8%.

In a preferred embodiment, the greater the methylation of the CpG3 by 1%, the greater the likelihood of the presence of depression in the elderly by 7%.

In a preferred embodiment, the 1% increase in the methylation mean value of CpG1, CpG2, and CpG3 increases the likelihood of the presence of depression in the elderly by 9%.

In a preferred embodiment, if the methylation of the CpG2 is greater than 10.5% in the determination step, the sensitivity to determine the likelihood of developing age-related depression within two years is at least 73% and the specificity is at least 57%.

In a preferred embodiment, the 1% increase in methylation of the CpG2 in the determining step increases the likelihood of developing depression in the elderly by 8%.

In a preferred embodiment, the biological sample is selected from bodily fluids or tissues comprising blood.

The present invention also includes means for measuring old age depression biomarkers that measure the methylation of the CpG region contained in the 5'-terminal -3187 to -3132 nucleotide region of the NR3C1 gene for use in determining the presence or risk of developing senile depression The present invention provides a diagnostic kit for detecting depression in the elderly.

In a preferred embodiment, the measuring means uses a polymerase chain reaction (PCR) with sodium bisulfite or a monoclonal antibody to 5-methylcytosine.

In a preferred embodiment, the CpG region is at least one of CpG1, CpG2 and CpG3.

In a preferred embodiment, the diagnostic kit is a microarray.

The present invention has the following excellent effects.

First, the present invention can provide a biomarker capable of accurately determining old age depression by confirming that methylation of a specific region of the NR3C1 gene can be a biomarker of old age depression.

In addition, according to the method for detecting depression in the elderly of the present invention, it is possible to diagnose depression early in life and / or to predict an onset risk, thereby contributing to a decision making process for a therapeutic drug or a treatment method.

Further, according to the diagnostic kit for detecting depression in the elderly of the present invention, the presence of the depression in the elderly can be predicted only by measuring the degree of methylation of the specific region of NR3C1 in the blood of the patient in the old age, And thus have clinical utility.

Although the present invention has been fully described by way of example with reference to the accompanying drawings, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course.

FIG. 1 shows a schematic diagram and sequence for analysis of the methylation status of the NR3C1 gene.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprising" or "having ", and the like, are intended to specify the presence of stated features, integers, steps, operations, elements, parts, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, parts, or combinations thereof.

The terms first, second, etc. may be used to describe various elements, but the elements 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 a second component, and similarly, the second component may also be referred to as a first component.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the meaning of the context in the relevant art and, unless expressly defined in the present invention, are to be interpreted as an ideal or overly formal sense Do not.

The term "diagnosis" as used herein means to identify the presence or characteristic of a pathological condition. For purposes of the present invention, "diagnosis" means determining the presence and risk of developing senile depression based on in vitro analysis of body fluids.

The term "biomarker " as used herein refers to a material capable of exhibiting a disease state. In the context of the present invention relating to the diagnosis of depression in old age, "biomarker" means indicating the degree of methylation of a particular region of the NR3C1 gene. The level or amount of a "biomarker" increases in patients who are suffering from depression in the elderly or who are susceptible to depression in the elderly compared to the normal healthy state.

The term "blood" as used herein includes whole blood, serum and plasma.

The term "antibody" as used herein means an immunoglobulin that specifically binds to an antigenic region of a biomolecule. In the context of the present invention, an antibody specifically binds to a biomarker and also includes a polyclonal antibody, a monoclonal antibody, and a recombinant antibody.

The term "cut-off level" as used herein refers to a relative level or absolute level determined to be able to distinguish individuals who are suffering from or at risk of developing senile depression. The cutoff level is given as a fold difference in the case of the relative level or as a characteristic value expressed as% in the case of the absolute value. As indicated herein, values above the cutoff level are considered to enable the determination of the presence of old age depression and / or the risk of developing senile depression.

As used herein, the term " predicting "means finding that an individual is significantly more likely to develop a biological disorder.

The term "biological sample " as used herein includes various sample types obtained from an individual and can also be used in diagnostic or monitoring analysis. Biological fluid samples include blood, cerebrospinal fluid (CSF), urine and other liquid samples from biological sources. If necessary, the sample can be pre-treated, for example, for concentration and separation.

An "individual" is a mammal, preferably a human, and the terms individual or entity may be used interchangeably herein.

As used herein, the term "reference value" refers to a reference level or a value having an absolute value, a relative value, an upper limit and / or a lower limit, a range of values, an average value, a median, May be a numerical value compared to a particular control or baseline value.

The baseline value may be calculated from the individual sample values, for example, the values obtained from the samples obtained from the subject to be examined at an earlier time point, or the values obtained from the samples of patients of old age depression other than the subject being examined, or the "normal" And may be based on values obtained from samples from non-diagnosed subjects. The reference value may be based on a sample obtained from a plurality of samples, for example from a plurality of old-age depressed patients or normal individuals, and may be based on a sample group containing the sample to be tested.

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

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

The technical feature of the present invention is to confirm that methylation of a specific region of the NR3C1 gene can be used as a biomarker for senile depression that can predict the early diagnosis and / or risk of developing depression in the elderly, Or a diagnostic kit for determining the degree of methylation of a specific region of the NR3C1 gene in order to predict and / or diagnose the risk of onset.

Therefore, the method for detecting old age depression of the present invention comprises: a measurement step of measuring the level or amount of the old age depression biomarker included in the biological sample of the patient; And a determination step of determining the presence or risk of developing senile depression based on the level or amount of the old age depression biomarker.

More specifically, the aging depression biomarker measured in the measurement step is the methylation of the CpG region contained in the nucleotide region of -3132 at the 5'-terminal end of the NR3C1 gene, and the increase in the degree of methylation of the CpG region is associated with the depression Presence or risk of onset. Here, the CpG region is at least one of CpG1, CpG2, and CpG3, and as shown in FIG. 1, the 5'-terminal 3F7 -3132 nucleotide region of the 5'-terminal 1F exon region of the NR3C1 gene, -3167) to (-3146 to -3132). The measurement method used in the measurement step may be a known method useful for determining the level or amount of the age depression biomarker and for determining or diagnosing using the measurement level or amount. In the present invention, the detection method can be carried out both in vitro and / or in vivo, but preferably the detection method of the present invention can be an in vitro method based on a sample obtained from an individual and provided in vitro.

In addition, the determination step is carried out by comparing the level or amount of the old age depression biomarker in the sample with the reference level or amount of the old age depression biomarker, wherein the reference level includes the methylation level obtained from the group not suffering from the old age depression and the old age depression And the level or amount of the age-depressed biomarker is a level at which the sensitivity and specificity are different from each other in the depression group based on the methylation level obtained from the group of the depressed individuals, Or the risk of onset.

The risk of developing depression in the elderly in the present and at least two years in the future can be diagnosed or predicted by measuring the biomarkers of depression in the elderly at the current time measured as described below.

That is, if the measured methylation of CpG2 was greater than or equal to 10.5% cutoff, the sensitivity to determine the presence of old age depression was greater than 69% and the specificity was greater than 55%. In addition, if the methylation of CpG3 was> 6.5% cut-off level, the sensitivity to determine the presence of depression in the elderly was greater than 62% and the specificity was greater than 56%. In particular, the mean values of CpG1, CpG2 and CpG3 methylation were also measured, and the mean value of CpG1, CpG2 and CpG3 methylation was significantly higher than the cutoff level of 7.8% 70% or more and specificity was 54% or more. In addition, the 1% increase in methylation of CpG2 increased the likelihood of depression to be 8%, and the 1% increase in CpG3 methylation increased the likelihood of depression to be 7%, and the mean methylation of CpG1, CpG2 and CpG3 was 1 %, It is predicted that the possibility of depression in the elderly will increase by 9%.

CpG2, CpG2, and CpG2 were significantly associated with the risk of developing depression in the future at least two years in the future. If the methylation of CpG2 is above 10.5% cut-off level, The sensitivity to determine was greater than 73% and the specificity was greater than 57%. In addition, the 1% increase in methylation of CpG2 was predicted to increase the likelihood of developing depression in the elderly by 8%.

Next, the diagnostic kit for detecting senile depression of the present invention is for use in determining the risk of the onset or the onset of old age depression, wherein a biological sample selected from body fluids or tissues containing blood is used at the 5 ' end of the NR3C1 gene -3132 < / RTI > locus nucleotide region, and means for measuring the methylation of the CpG region contained in the nucleotide region of interest. Here, the measuring means may be any known technical structure capable of measuring the degree of methylation of DNA, for example, using sodium bisulfite and polymerase chain reaction (PCR) -methylcytosine monoclonal antibody may be used. It goes without saying that the diagnostic kit may also be implemented as a microarray.

Example

1. Research subjects

Data are derived from a prospective community-based study of Korea's late-life psychiatric morbidity (Kim et al., 2013). Local residents aged 65 years or older who agreed to live and participate in two geographical watershed areas (one city, one rural) identified in the national registration list constituted reference samples. Follow-up with all baseline participants occurred after 2.4 years (mean (SD) interval, 2.4 (0.3) years.) Depression was assessed at both baseline and follow-up. All data were obtained at baseline. All participants provided written consent at each trial, which was approved by the Chonnam National University Hospital Institutional Review Board.

2. Depression Assessment

Depression was assessed by the Geriatric Mental State Examination, which is used in conjunction with the Diagnostic Algorithm, Automated Geriatric Examination for Computer Assisted Taxonomy (AGECAT). These are fully structured diagnostic tools for assessing mood disorders and are widely applied to international geriatric epidemiology surveys. The possibility of depression was measured in the range of 0 to 5 according to the computer algorithm (AGECAT). As in previous studies (Prina et al., 2011), the AGECAT score, which is considered to be equivalent to the level of depression requiring clinical intervention for depression, was defined as 3 or more. The GMS AGECAT depression criteria includes both moderate and severe symptoms, thus indicating a broader spectrum than that defined in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (Major Depression) (American Psychiatric Association, 2000). GMS B3 was translated into Korean according to the official standardization process (Kim et al., 2003) and managed by a trained investigator. Participants who were already depressed at the estimated baseline (prevalent depression) and those who did not have depression at baseline were classified as participants who were found to have depression at follow-up (incident depression).

3. NR3C1 methylation

DNA methylation status of the NR3C1 gene was examined using a blood sample from a participant who consented to blood collection. DNA was extracted from venous blood using standard procedures. Most previous studies have focused on exon 1F, a homologue of exon 17 in the rat that contains a binding site for nerve growth factor-inducible protein A (NGFI-A) Muller, 2005; Farrell and Okay, 2016), a protein reported to be highly expressed in the hippocampus of the offspring of mothers (McCormick et al., 2000). Thus, human studies conducted so far have focused on this area, which may be related to early life adversity (Oberlander et al., 2008; Perroud et al., 2011; Tyrka et al., 2015). Following previous studies, the methylation pattern of the NR3C1 (GeneBank #AY 436590) exon 1F region was investigated in the present invention. In particular, three CpG sites were surveyed in the first to third CpG sites (2011) described by Perroud et al. And Exxon 1F (Fig. 1) corresponding to the 6th and 8th CpG sites (2008) described by Oberlander et al. Respectively. From the start of exon 2, the CpG-rich region of the NR3C1 exon 1F sequence located between -3187 and -3132 at the translation initiation site (1+) located at 13 nucleotides was analyzed and the three CpGs contained in this region were analyzed. This detail was chosen because of an extensive study of early-life events and methylation changes at these sites in patients with borderline personality disorder, depression, and post-traumatic stress disorder. In a recent review paper, it is reported that this part is relatively consistent with methylation and early life adversity (Daskalakis and Yehuda, 2014).

Genomic DNA (1 μg) was extracted from leukocytes using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, Calif., USA) according to the manufacturer's proposed protocol. The DNA was then bisulfite treated using EpiTech Bisulfite Kit (Qiagen) according to the manufacturer's protocol. The 421b fragment of NR3C1 gene was amplified by polymerase chain reaction (PCR) from DNA treated with bisulfite using the forward and reverse primers designated in FIG. The PCR conditions were a final extension of 94 ° C for 15 minutes followed by 30 seconds at 94 ° C, 30 seconds at 58 ° C, 40 seconds at 72 ° C and 10 minutes at 72 ° C. The PCR products were sequenced using the PSQ 96M pirosequencing system (Biotage) according to the manufacturer ' s protocol using the sequencing primers shown in Fig. The percent methylation of each CpG region was quantified using Pyro Q-CpG software version 1.0.9 (Biotage). Individual methylation percentages and their mean values in the three CpG sites CpG1, CpG2 and CpG3 were used for the analysis.

4. Demographic and clinical covariance

Potentially depressive factors were considered. Data on demographic and clinical characteristics, including age, sex, duration of education, number of chronic disabilities, number of stressful life events (SLEs), cognitive function and disability, . We examined the presence of eleven common and chronic health problems (Lindesay, 1990), including: arthritis or rheumatism; Vision problems; Hearing loss or hearing loss; Persistent cough; Apnea, difficulty breathing, asthma; High blood pressure; Heart disease or angina; Stomach or intestinal problems; Syncope or loss of consciousness; Paralysis, weakness or loss of strength of one leg or arm; Skin inflammation, leg ulcers or severe burns. The total score reflecting the existence of each of these conditions was derived. The number of SLEs was calculated using a list of threat events (Brugha et al., 1985) designed to identify events that lead to long-term sequelae affecting the onset of depression. Participants were asked about nine SLE experiences, including serious illness (oneself), serious illness (close relative), bereavement (immediate family), bereavement (relative or close friend), marital divorce, Problems with your best friend or relative, theft or loss. The positive reactions were summed to obtain the total SLE count. Mini-Mental State Examination was used to assess cognitive function (Folstein et al., 1975). To assess disability, the Korean version of the World Health Organization Disability Assessment Schedule II (Kim et al., 2005) was used.

5. Statistical Analysis

The outcome variables were prevalence of depression (prevalence of depression at baseline) and incidence of depression (at baseline, no depression at baseline, but depressed at follow-up) . The sociodemographic and clinical characteristics of the elderly with or without depression were adequately compared by t-test or χ2 test. Factors significantly associated with outcome (p <0.05) were treated as covariates in the calibrated analysis. Age was also entered as a covariate, taking into account the known association between aging and methylation (Florath et al., 2014). The relationship between NR3C1 methylation percentages and mean values of three CpG sites (CpG1, CpG2 and CpG3) and depression was compared using t-test. A multivariate logistic regression model was applied to assess the effect of NR3C1 methylation on depression. Bonferroni calibrations included multiple comparisons in the NR3C1 methylation percentage analysis, ie, four comparisons (three CpG sites (CpG1, CpG2 and CpG3) and a mean value; 0.05 / 4 = 0.0125]. &Lt; / RTI &gt; Statistical analysis was performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA).

6. Results

(1) Demographic and clinical characteristics of all participants

The basic characteristics of participants participating in this study are shown in Table 1 below.

Participants at baseline Analyzed participants at follow-up
(N = 521) Total
(N = 732) No depression (N = 631) Depression (N = 101) Statistical coefficients P-value ^* Age, mean (SD) years 72.8 (5.8) 72.7 (5.8) 73.7 (6.3) t = -1.672 0.095 72.5 (5.5) Gender, N (%) women 432 (59.0) 359 (56.9) 73 (72.3) χ ² = 8.518 0.004 287 (55.1) Education, mean (SD) years 3.4 (4.2) 3.5 (4.3) 3.1 (4.0) t = + 0.864 0.388 3.5 (4.3) Chronic physical disorders, mean (SD) number 1.7 (1.0) 1.6 (1.0) 2.2 (0.9) t = -5.875 <0.001 1.6 (1.0) Stressful life events, mean (SD) scores 1.2 (0.9) 1.1 (0.9) 1.6 (0.9) t = -4.887 <0.001 1.1 (0.9) MMSE, mean (SD) scores 23.3 (5.0) 23.5 (4.9) 22.1 (5.5) t = + 2.770 0.006 23.8 (4.7) WHODAS II, mean (SD) scores 7.1 (11.1) 5.8 (9.3) 15.1 (17.2) t = -8.017 <0.001 5.3 (8.6) MMSE: Mini-Mental State Examination; WHODAS II: World Health Organization Disability Assessment Scale II.
^* t-test or χ ² testasappropriate.

Table 1 shows the following facts. Of the 732 participants, 101 (13.8%) had depression at baseline. Respondents with chronic depression were more likely to be female, more likely to have chronic disability, more SLE, lower cognitive function, and severe disability (p <0.05). Along with age, these important factors have been chosen as covariates for later calibrated analysis because they are associated with DNA methylation. At the 2 - year follow - up, 521 (82.6%) of the 631 participants without depression were evaluated. In a follow-up evaluation, 86 (16.5%) respondents were reported to be depressed. The remaining 110 were not followed; We could not be reached because of the following reasons. 58 (52%) and 23 (21%) died, 21 (19%) refused to participate, and 8 (7%) were not well. There was no significant difference found between follow-up participants and non-follow-up participants for any trait (p> 0.05).

(2) Cross-sectional association analysis between NR3C1 methylation percentage and depression

At baseline, the cross-sectional association between NR3C1 methylation and baseline point depression is shown in the left column of Table 2.

As shown in Table 2, all methylation of CpG1, CpG2 and CpG3 as well as the mean methylation of the three sites were associated with baseline depression. Furthermore, even after applying Bonferroni correction, baseline depression prevalence was significantly associated with high methylation and high mean methylation rates in CpG2. Independent cross-sectional associations are shown in the left column of Table 3. After covariate correction, baseline depression rates are significantly associated with high NR3C1 methylation and high average methylation percentages at all CpG sites (CpG1, CpG2, and CpG3). After applying the Bonferroni correction, there was a significant correlation between baseline depression and high methylation rate and high mean methylation rate in CpG2 and CpG3 among the irradiated CpG regions.

Methylation site Prevalent depression at baseline Incident depression at 2-year follow-up No depression
(N = 631) Depression
(N = 101) Statistical coefficients P-value ^a No depression (N = 458) Depression
(N = 63) Statistical coefficients P-value ^a CpG 1 7.4 (3.8) 8.7 (5.2) t = -2.262 0.026 7.3 (3.8) 7.8 (3.4) t = -1.033 0.302 CpG 2 11.4 (4.2) 13.2 (5.1) t = -3.695 <0.001 11.2 (4.1) 13.0 (4.3) t = -3.216 0.001 CpG 3 7.5 (3.8) 8.8 (5.4) t = -2.312 0.023 7.4 (3.6) 8.2 (4.5) t = -1.342 0.184 CpG average 8.8 (3.5) 10.2 (4.8) t = -2.834 0.005 8.6 (3.4) 9.7 (3.8) t = -2.253 0.025 NR3C1: nuclear receptor subfamily 3, group C, member 1.
^a p-value using t-tests.
Values in bold type show statistical significance after Bonferroni correction.

(3) The relationship between NR3C1 methylation percentage and depression

NR3C1 methylation and follow-up at 2-year follow-up The end-to-end association between onset and depression is shown in the right column of Table 2. The incidence of depression at follow-up examined at 2 years follow-up was significantly associated with increased methylation of NR3C1 and higher mean methylation rates, especially in CpG2, among CpG regions. After Bonferroni correction, the relationship between follow-up time of onset depression and high NR3C1 methylation level in CpG2 was still significant. The independent end-to-end association between covariate corrected NR3C1 methylation and depression is shown in the right column of Table 3. After Bonferroni correction, the follow-up time of onset depression examined in the 2-year follow-up was found to be independent of the higher methylation of NR3C1, especially in CpG2.

Methylation site Prevalent depression at baseline Incident depression at 2-year follow-up Wald OR (95% CI) Wald OR (95% CI) CpG1 5.187 1.06 (1.01-1.11) ^* 0.867 1.03 (0.97-1.09) CpG2 9.852 1.08 (1.03-1.14) ^† 7.623 1.08 (1.02-1.13) ^† CpG3 7.242 1.07 (1.02-1.13) ^† 0.178 1.01 (0.96-1.07) CpG average 8.972 1.09 (1.03-1.15) ^† 2.464 1.05 (0.99-1.11) All analyses are adjusted for age, gender, number of chronic medical illnesses, cognitive function, disability, number of stressful life events.
NR3C1: nuclear receptor subfamily 3, group C, member 1.
^* p-value &lt;0.05; ^† p-value &lt;0.01; ^‡ p-value <0.001.
Values in bold type show statistical significance after Bonferroni correction.

(4) Association between NR3C1 methylation and depression in old age

These results show a longitudinal study of the elderly population in the community. The main finding was that the prevalence of baseline depression at baseline, followed by follow-up at 2 years follow-up, was significantly associated with the incidence of depression and high NR3C1 methylation. This association was independent of age, sex, number of chronic disabilities, number of SLEs, potential covariates including cognitive function and disability.

Through this association, it was confirmed that the methylation of the CpG region contained in the nucleotide region from -3187 to -3132 of the NR3C1 gene shown in FIG. 1 was an old age depression biomarker. Especially, the increase of the degree of methylation of the CpG region caused the depression Or the risk of onset.

Receiver operating characterisc (ROC) analysis was performed to determine the baseline levels of old age depression biomarkers to compare the level or amount of old age depression biomarkers among the measured biological samples. The methylation cutoff level was calculated from the methylation levels of the depressed and non - depressed elderly patients, and the sensitivity and the specificity of the discrimination to distinguish the depressed group from the depressed group.

In addition, the methylation cutoff level of the measured CpG2 was calculated by ROC analysis. As a result, the methylation cutoff level of CpG2 was 10.5%. If the CpG2 methylation value measured in the biological sample obtained from the subject was above the cutoff level, the sensitivity to determine the presence of old age depression was 69% or more and the specificity was 55% or more. Here, the sensitivity means that the probability of diagnosing a patient with depression in the old age is 69%, and the specificity means that the probability of diagnosing the absence of depression in the old age is 55%.

 The methylation cutoff level of CpG3 was calculated to be 6.5% in the same manner as CpG2. If the measured CpG3 methylation value is above the cutoff level, the sensitivity to determine the presence of old age depression is 62% or more and the specificity is 56% or more Respectively.

In addition, the methylation of CpG1, CpG2 and CpG3 was measured, and their average value was calculated. The cutoff level of the methylation average value of CpG1, CpG2 and CpG3 was determined by ROC analysis. If the cut-off level of the calculated mean was above 7.8%, the sensitivity to determine the presence of old age depression was greater than 70% and the specificity was greater than 54%.

In addition, to calculate the probability of predicting the presence of depression in NR3C1 methylation, the multivariate analysis using various factors that affect methylation and depression showed that the methylation of CpG2 increased by 1% There was a 7% increase in the likelihood of depression in the elderly with a 1% increase in methylation of CpG3, a 1% increase in the mean value of CpG1, CpG2, and CpG3, %, Respectively.

CpG2, CpG2, and CpG3 were significantly associated with the risk of developing depression in the future for at least two years. If the methylation cut-off level of CpG2 was> 10.5%, ROC analysis showed that depression in the elderly Sensitivity was more than 73%, and specificity was more than 57%. In addition, multivariate analysis showed that as the methylation of CpG2 increased by 1%, the likelihood of developing depression in the elderly was predicted to increase by 8%.

Thus, the present invention can support the role of post-sexual factors associated with the HPA axis in geriatric depression, and in particular, can serve as a biomarker for the geriatric depression of methylation of the NR3C1 gene. In terms of clinical implications, the use of NR3C1 gene methylation as an aging depression biomarker provides the advantages of non-invasiveness and convenience, so that the present invention can be used as a potential tool to identify elderly people at risk of current and future depression It could be helpful.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications will be possible.

Claims (17)

A measurement step of measuring a level or an amount of an old age depression biomarker included in a biological sample of a patient; And
And determining the presence or risk of developing senile depression based on the level or amount of said senile depression biomarker.
The method according to claim 1,
The aging depression biomarker is characterized by the methylation of the CpG region contained in the nucleotide region -3132 of the NR3C1 gene at the 5'-terminal end of -3187, and the increase in the degree of methylation of the CpG region is indicative of the presence or risk of developing senile depression Detection of depression in old age.
3. The method of claim 2,
The determining step is performed by comparing the level or amount of the old age depression biomarker in the sample with the reference level or amount of the old age depression biomarker,
The reference level is a level balanced between sensitivity and specificity to discriminate depressive patients based on the methylation level obtained from a group not suffering from old age depression and the methylation level obtained from a group of individuals including old age depression patients,
Wherein the level or amount of the age-depressed biomarker is indicative of the presence or risk of developing senile depression at a predetermined relative or absolute cutoff level or greater, respectively.
The method of claim 3,
Wherein the CpG region is at least one of CpG1, CpG2, and CpG3.
5. The method of claim 4,
Wherein the sensitivity of determining the presence of depression in the elderly is greater than or equal to 69% and the specificity is greater than or equal to 55% when the methylation of CpG2 is greater than or equal to 10.5% in the determining step.
5. The method of claim 4,
Wherein when the methylation of the CpG3 is 6.5% or more in the determining step, the sensitivity is 62% or more and the specificity is 56% or more, which determines the presence of old age depression.
5. The method of claim 4,
Wherein when the methylation average value of the CpG1, CpG2, and CpG3 is 7.8% or more in the determining step, the sensitivity is 70% or more and the specificity is 54% or more to determine the presence of depression in the elderly.
8. The method according to any one of claims 5 to 7,
Wherein the CpG2 methylation is increased by 1%, and the likelihood of the presence of depression in the elderly is increased by 8%.
8. The method according to any one of claims 5 to 7,
Wherein the CpG3 methylation is increased by 1%, and the possibility of the presence of depression in the elderly is increased by 7%.
8. The method according to any one of claims 5 to 7,
The method of claim 1, wherein the CpG1, CpG2, and CpG3 methylation values are increased by 1%, and the likelihood of an aging depression is increased by 9%.
5. The method of claim 4,
Wherein the CpG2 methylation of the CpG2 is greater than or equal to 10.5% in the determination step, the sensitivity to determine the possibility of developing senile depression within two years is 73% or more, and the specificity is 57% or more.
12. The method of claim 11,
Wherein the probability of developing depression in the elderly is increased by 8% as the methylation of the CpG2 is increased by 1% in the determining step.
13. The method according to any one of claims 1 to 7, 11, or 12,
Wherein the biological sample is selected from body fluids or tissues comprising blood.
Old age depression detection, including means of measuring old age depression biomarkers that measure the methylation of the CpG region contained in the 5'terminal -3187 to 32 position nucleotide region of the NR3C1 gene for use in determining the presence or risk of developing senile depression Diagnostic kit for.
15. The method of claim 14,
The method for detecting depression in the elderly is characterized in that the measurement means uses a polymerase chain reaction (PCR) with sodium bisulfite or a monoclonal antibody to 5-methylcytosine Kits.
15. The method of claim 14,
Wherein the CpG region is at least one of CpG1, CpG2, and CpG3.
17. The method according to any one of claims 14 to 16,
Wherein the diagnostic kit is a microarray.

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