KR20220099803A - Assessment methods and diagnostic kit for depressive disorders with earlier age at onset using genetic biomarkers - Google Patents

Abstract

The present invention relates to a technology for diagnosing depression using a biomarker, and more specifically, to a biomarker for predicting depression with an early-onset age developed on the basis that mutations in specific genes increase the risk of developing depression with an early-onset age, a method for providing information for diagnosing depression with an early-onset age using the biomarker, and a diagnostic kit.

Description

Genetic biomarkers for predicting depression with early onset age, information providing methods and diagnostic kits for diagnosing depression with early onset age using the biomarkers {Assessment methods and diagnostic kit for depressive disorders with earlier age at onset using genetic biomarkers }

The present invention relates to a depression diagnosis technology using a biomarker, and more specifically, a bio for predicting depression with an early onset age developed on the basis that mutation of a specific gene increases the risk of developing depression at an earlier onset age. It relates to a marker, a method for providing information and a diagnostic kit for the diagnosis of depression with an early onset age using the biomarker.

Depression is a pervasive mental disorder with a high disease burden. Despite substantial evidence for the heritability of depression, the genetic structure of depression remains unclear (Sullivan et al., 2000). Previous genome-wide association studies (GWAS), which failed to identify genetic markers for major depression (Major Depressive Disorder Working Group of the Psychiatric Gwas Consortium et al., 2013), for example, maximize sample size (Howard et al. , 2019; Hyde et al., 2016; Wray et al., 2018; It suggested that various analysis strategies are needed to overcome the

Age at onset (AAO) was considered as a potential indicator of underlying genetic risk for depression, based on findings from familial cohesion and genetic studies suggesting that early onset depression is associated with a family history of the disorder (Kendler et al., 2009; Nienberg et al., 2007; Tozzi et al., 2008). Moreover, the risk of early onset depression in children of parents with disabilities was 13 times greater than in children of controls (Wickramarantne and Weissman et al., 1998). Recently, GWAS suggested that the importance of genetic factors for depression differs between adult depression and early-onset depression, and that early-onset cases show greater genetic overlap with schizophrenia and bipolar disorder (Power et al., 2017). ). This finding was supported by a follow-up study that adolescence-onset depression has a distinct depression-specific genetic profile (based on polygenetic risk scores) for very early-onset depression with genetic overlap with ADHD and schizophrenia (Kwong et al. ., 2019; Rice et al., 2019).

As an example of the study results on AAO-related genetic variation in depression, the TUSC3 gene is associated with early-onset depression in men, whereas the intergenic region of chromosome 3 is associated with adult-onset depression (Power et al., 2012). and 2017). Moreover, previous GWAS looked at only common, low-impact genetic variants that did not fully capture the genetic background of depression. In addition, GWAS did not confirm the association of specific variants with AAO in depression due to the lack of repeat validation of specific variants and unknown functions of the identified variants.

Therefore, the identification of genetic markers of depression with an early onset age (AAO) can reduce the disease burden through genetic susceptibility screening, and thus a novel genetic marker for predicting depression with an early onset age in adults has been developed. need to be

Republic of Korea Patent No. 10-1919753

The present inventors completed the present invention by revealing a biomarker for predicting depression with an early onset age that is significantly correlated with the risk of developing depression before the age of 30 as a result of a number of studies.

Therefore, an object of the present invention is to confirm that a mutation in a specific region in one or more of the CCL14, FYB, GPRASP1 and CTNND2 genes can predict depression with an early onset age before the age of 30, thereby targeting adults 30 years of age. To provide a biomarker composition for predicting depression having an early onset age, including a biomarker for predicting depression having an early onset age that can relatively accurately determine the risk of developing depression in the past.

Another object of the present invention is to identify depressed patients before or early onset of depression through genetic susceptibility screening by detecting a biomarker for predicting depression with an early onset age, and a decision-making process for treatment drugs or treatment methods through early intervention It is to provide an information provision method for the diagnosis of depression with an early onset age that can contribute to the disease burden and reduce the burden of disease.

Another object of the present invention is to predict a patient with a genetic risk factor for early onset depression by identifying a mutation site of a biomarker for predicting depression having an early onset age from a biological sample isolated from an individual, so the patient's depression symptoms It is to provide a diagnostic kit and microarray for predicting depression having an early onset age with clinical usefulness because it can be prevented and/or treated preemptively before this deepening.

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

In order to achieve the object of the present invention described above, the present invention provides a polynucleotide of 10 to 100 nucleotides in length including the rs75238886 (G>A) region of the CCL14 gene or a polynucleotide complementary thereto as a control. It provides a biomarker composition for diagnosing depression with

In addition, the present invention provides a biomarker composition for diagnosing depression having an early onset age comprising a polynucleotide of 10 to 100 nucleotides in length including the rs35384751 (G>T) region of the FYB gene or a complementary polynucleotide thereof as a control. do.

In addition, the present invention provides a biomarker composition for diagnosing depression having an early onset age comprising a polynucleotide of 10 to 100 nucleotides in length including the rs201921260 (T>C) region of the GPRASP1 gene or a complementary polynucleotide thereof as a control. do.

In addition, the present invention provides a biomarker composition for diagnosing depression having an early onset age comprising a polynucleotide of 10 to 100 nucleotides in length including the rs202234398 (G>A) region of the CTNND2 gene or a complementary polynucleotide thereof as a control. do.

In addition, the present invention provides a diagnostic kit for depression having an early onset age including a detection means capable of detecting any one of the above-described biomarker compositions.

In a preferred embodiment, the detection means includes at least one of a probe capable of detecting the polynucleotide included in the biomarker composition, a primer pair capable of amplifying the polynucleotide, and cDNA of the polynucleotide.

In addition, the present invention provides a microarray for diagnosing depression having an early onset age comprising one or more of the biomarker compositions described above.

In addition, the present invention comprises the steps of obtaining DNA or RNA of the CCL14 gene from a biological sample of a subject; and confirming the base of the rs75238886 region included in the biomarker composition of claim 1 from the obtained DNA or RNA; provides an information providing method for diagnosing depression with an early onset age, including.

In a preferred embodiment, when the base of the rs75238886 site is identified as A, it can be diagnosed that the risk of developing depression at an earlier age is increased by 2.2 times compared to the control group having the base of the rs75238886 site G.

In addition, the present invention comprises the steps of obtaining DNA or RNA of the FYB gene from a biological sample of a subject; and identifying the base of the rs35384751 region contained in the biomarker composition of claim 2 from the obtained DNA or RNA; provides an information providing method for diagnosing depression with an early onset age, including.

In a preferred embodiment, when the base of the rs35384751 site is identified as T, it can be diagnosed that the risk of developing depression at an earlier age is increased by 1.53 times compared to the control group having the base of the rs35384751 site G.

In addition, the present invention comprises the steps of obtaining DNA or RNA of the GPRASP1 gene from a biological sample of a subject; and confirming the base of the rs201921260 region included in the biomarker composition of claim 3 from the obtained DNA or RNA; provides an information providing method for diagnosis of depression with an early onset age, including.

In a preferred embodiment, when the base of the rs201921260 site is identified as C, it can be diagnosed that the risk of developing depression at an earlier age is increased by 2.95 times compared to the control group having the base of the rs201921260 site T.

In addition, the present invention comprises the steps of obtaining DNA or RNA of the CTNND2 gene from a biological sample of a subject; and identifying the base of the rs202234398 region contained in the biomarker composition of claim 4 from the obtained DNA or RNA; provides an information providing method for diagnosing depression with an early onset age, including.

In a preferred embodiment, when the base of the rs202234398 site is identified as A, it can be diagnosed that the risk of developing depression at an earlier age increases by 1.94 times compared to the control group having the base of the rs202234398 site G.

In a preferred embodiment, the subject is an adult 20 years or older, and the biological sample is selected from a body fluid or tissue including blood.

First, according to the biomarker composition of the present invention, it was confirmed that a mutation in a specific region in one or more of CCL14, FYB, GPRASP1 and CTNND2 genes can be a biomarker for predicting depression with an early onset age before 30 years of age. By doing so, it is possible to relatively accurately determine the risk of developing depression before the age of 30 in adults.

In addition, according to the information providing method for the diagnosis of depression with an early onset age of the present invention, it is possible to identify patients with depression before or in the early stage of the onset of depression through genetic susceptibility screening using a biomarker for predicting depression with an early onset age. Early intervention by doctors before depression develops can help determine the medication or method of treatment.

In addition, according to the diagnostic kit and microarray of the present invention, it is possible to predict patients with genetic risk factors for early onset depression by identifying the mutation site of a biomarker for predicting depression having an early onset age from a biological sample isolated from an individual. Therefore, it has clinical usefulness because it can prevent and/or treat the patient before the symptoms of depression deepen.

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

1 is a flow chart (VCF: Variant Call Format File, FDR: False Discovery Ratio) showing an overview of the analysis process of the present invention.
Figure 2 is a graph of the Kaplan Meier survival analysis results using the log-rank test for the association between the age of onset of depression and the CCL14 gene mutation for all depressed patients.
3 is a graph of the Kaplan Meier survival analysis results using the log-rank test for the association between the age of onset of depression and the FYB gene mutation for all depressed patients.
4 is a graph of the Kaplan Meier survival analysis results using the log-rank test for the association between the age of onset of depression and the GPRASP1 gene mutation for all depressed patients.
5 is a graph of the Kaplan® Meier survival analysis results using the log-rank test for the association between the age of onset of depression and the CTNND2 gene mutation for all depressed patients.

The terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the description of the invention is present, but one or more other It should be understood that it does not preclude the possibility of addition or presence of features or numbers, steps, operations, components, parts, or combinations thereof.

Terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, a 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 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 to which this invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having meanings consistent with the meanings in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present invention. does not

In interpreting the components, it is interpreted as including an error range even if there is no separate explicit description.

In the case of a description of a temporal relationship, for example, if the temporal relationship is described as 'after', 'following', 'after', 'before', etc., 'immediately' or 'directly' This includes cases that are not continuous unless ' is used.

The human genome shows genetic diversity of more than 0.1%, and this difference can be used as a tool for genetic fingerprint recognition. Single nucleotide polymorphism (SNP) can be used as an individual genetic fingerprint recognition pattern, and more than 10 million diversity is widely known by race and country by the human genome project. These SNPs can be detected by simple sequencing, and this diversity is rare, individual, and specific. However, there may be very frequent mutations in some areas. The present inventors have discovered that depression with an early onset age can be diagnosed simply by using the characteristics of single nucleotide polymorphism (SNP) as described above.

As used herein, the term “diagnosis” refers to confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, "diagnosis" means determining the likelihood of developing depression before the age of 30 in an adult patient based on an ex vivo analysis of body fluids.

As used herein, the term “rs_id” refers to rs-ID, an independent marker given to all SNPs initially registered by the NCBI, which has been accumulating SNP information since 1998. As such, rs_id described in the table means a SNP marker representing the genetic mutation of the present invention.

As used herein, the term "biomarker" refers to a substance capable of indicating a disease state. In the context of the present invention relating to the diagnosis of depression with an early onset age, "biomarker" means a specific SNP site of any one of CCL14 gene, FYB gene, GPRASP1 gene and CTNND2 gene. "Biomarker" may or may not appear in individuals suffering from depression or prone to depression with an early onset age compared to a normal healthy state, depending on the type.

As used herein, the term "blood" includes whole blood, serum and plasma.

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

As used herein, the term “biological sample” includes various types of samples obtained from individuals and may be used in diagnostic or monitoring analysis. Biological fluid samples include blood, cerebrospinal fluid (CSF), urine, and other liquid samples of biological origin. If desired, the sample may be pretreated, for example, for concentration and separation.

As used herein, the term “subject” is a mammal, preferably a human, and the terms individual or subject may be used interchangeably in the present invention.

As used herein, the term "early onset age" refers to the age at which depression occurs, especially in adults over 20 years of age, who developed depression before the age of 30. .

Hereinafter, the technical configuration 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 and may be embodied in other forms. Like reference numbers used to describe the invention throughout the invention refer to like elements.

The technical feature of the present invention confirms that mutations in one or more specific regions of the CCL14, FYB, GPRASP1 and CTNND2 genes can be a biomarker for predicting depression with an early onset age. A diagnostic kit for diagnosing depression having an early onset age comprising a configuration for detecting a specific SNP of any one or more of the CCL14 gene, the FYB gene, the GPRASP1 gene, and the CTNND2 gene for prediction and/or diagnosis of the possibility of developing depression in the past; The purpose of this is to provide an information provision method for the prediction of depression with an early onset age.

Here, the CCCL14 gene is a C-C motif chemokine ligand 14 (CCL14) gene, and CCL14, one of the cytokine gene clusters, is known to regulate immune cell homeostasis and inflammation by attracting and activating leukocytes. (Moelants et al., 2013). The FYB gene is a FYN binding protein (FYB) gene that encodes an adapter protein involved in T cell signaling and mTOR signaling pathway, which activates T cells and produces inflammatory cytokines including IL-2. (da Silva et al., 1997; Griffith et al., 2001; Kotajima-Murakami et al., 2019). Depending on their major roles in inflammation, CCL14 and FYB have been shown to be associated with systemic lupus erythematosus and diabetes (Addobbati et al., 2013, 2015; Vyshkina et al., 2008; Suchy-Dice et al., 2014). Mutations in the CCL14 and FYB genes may increase the risk of depression in early AAO, as inflammation has been identified as one of the etiological mechanisms underlying depressive disorder.

The GPRASP1 gene, a G protein-coupled receptor-associated sorting protein 1 (GPRASP1) gene, is a direct interaction partner of the M2-muscarinic acetylcholine receptor (CHRM2), Considering the effect of iodine on mood (Furey and Drevets, 2006), it can be considered as a genetic risk factor for depressive disorder (Luo et al., 2005; Wang et al. 2004). The CTNND2 gene is a catenin delta 2 (CTNND2) gene, which encodes δ-catenin expressed in cortical and hippocampal neurons (Ho et al., 2000), and during the early development of the cerebrum and maintenance of dendrites (Turner et al., 2000). al., 2015) play an essential role in neuronal migration in the brain. Based on its important role in neuronal maintenance and plasticity, the CTNND2 gene has been shown to be associated with anxiety and major depressive disorders in human GWAS (Nivard) as well as anxiety-related traits in mice (Rat Genome Sequencing Consortium, 2013). et al., 2014).

Therefore, the biomarker composition for diagnosing depression having an early onset age of the present invention contains a polynucleotide of 10 to 100 nucleotides in length including the rs75238886 (G>A) region of the CCL14 gene or a complementary polynucleotide thereof as a control, A polynucleotide having a length of 10 to 100 nucleotides including the rs35384751 (G>T) region of the FYB gene or a polynucleotide complementary thereto as a control, or 10 to 100 including the rs201921260 (T>C) region of the GPRASP1 gene A polynucleotide of canine nucleotide length or its complementary polynucleotide is included as a control, or a polynucleotide of 10 to 100 nucleotides in length including the rs202234398 (G>A) region of the CTNND2 gene or a complementary polynucleotide thereof is included as a control. can do.

As will be described later, the present invention clearly confirms the effect of mutation in a specific region in one or more of the CCL14, FYB, GPRASP1 and CTNND2 genes to increase the risk of developing depression before the age of 30 in adults.

That is, a specific SNP polynucleotide obtained from one or more of the CCL14, FYB, GPRASP1 and CTNND2 genes included in the biomarker composition of the present invention is a Korean depressed patient in order to identify a genetic marker for depression in consideration of AAO, as will be described later. It was obtained by performing full-length exome sequencing of 1,000 samples to investigate whether single nucleotide polymorphism (SNP) of a specific gene could be a biomarker related to the diagnosis of depression with an early onset age. This is because sequencing revealed that four mutations in the CCL14, FYB, GPRASP1 and CTNND2 genes were associated with an increased risk of depressive disorder at an early onset. Full-length exome sequencing analysis had the advantage of capturing both rare and common variants in the exome (protein-coding region).

On the other hand, the diagnostic kit for depression with early onset age of the present invention includes the rs75238886 (G>A) region of the CCL14 gene, the rs35384751 (G>T) region of the FYB gene, and rs201921260 (T>C) of the GPRASP1 gene included in the biomarker composition. ) site, and a detection means capable of detecting a polynucleotide of any one of the rs202234398 (G>A) region of the CTNND2 gene or a polynucleotide complementary thereto. Here, the detection means may include at least one of a probe capable of detecting a polynucleotide included in the biomarker composition, a primer pair capable of amplifying the polynucleotide, and cDNA of the polynucleotide.

Of course, if necessary, it can be implemented as a microarray containing a biomarker composition for the diagnosis of depression with an early onset age.

In addition, the information providing method for the diagnosis of depression with an early onset age of the present invention is the DNA or RNA of the CCL14 gene, the DNA or RNA of the FYB gene, the DNA or RNA of the GPRASP1 gene, the DNA or RNA of the CTNND2 gene in the biological sample of the subject. obtaining any one of the following; And confirming any one of the bases of the rs75238886 site, rs35384751 base, rs201921260 base, and rs202234398 base included in the biomarker composition from the obtained DNA or RNA. Here, if the base of the rs75238886 site is identified as A as shown in the investigation results to be described later, the risk of developing depression at an earlier age is increased by 2.2 times compared to the control group whose base at the rs75238886 site is G. It can be diagnosed, rs35384751 If the base of the site is confirmed as T, it can be diagnosed that the risk of developing depression at an earlier age is increased by 1.53 times compared to the control group whose base is G at the rs35384751 site. It can be diagnosed that the risk of developing depression at an earlier age is increased by 2.95 times compared to the control whose base is T at the rs201921260 site, and when the base at the rs202234398 site is identified as A, the control group in which the base at the rs202234398 site is G It can be diagnosed as a 1.94-fold increase in the risk of developing depression at an earlier age compared to

Example

1. Study outline

Analysis was performed on data from the MAKE BETTER study for antidepressant treatment effect and response improvement, which investigated biomarkers of treatment response using a prospective design. The details of the study have been previously published (Kang et al., 2018). The current study analyzed baseline data from all participants who agreed to provide blood samples for genetic testing. A patient who recently visited the Department of Psychiatry at Chonnam National University Hospital and was diagnosed with major depressive disorder, dysthymic disorder, or other depressive disorder based on the Mini-International Neuropsychiatric Interview (MINI, Diagnostic and Statistical Manual of Mental Illness Fourth Edition [DSM-IV]) (Sheehan et al., 1998) were serially recruited through structured diagnostic interviews). Eligibility criteria are as follows.

<Eligibility criteria>

For the MAKE BETTER study, the inclusion criteria are as follows. i) 7 years of age or older; ii) diagnosed with major depressive disorder, dysthymic disorder, or depressive disorder not otherwise classified (NOS), not otherwise specified, as confirmed using the Mini-International Neuropsychiatric Interview (Sheehan et al., 1998); iii) Hamilton Depression Rating Scale (Hamilton, 1960) score ≥ 14; iv) Fill out the questionnaire, understand the purpose of the study, and be able to sign the informed consent form. Exclusion criteria are as follows. i) an unstable or uncontrollable medical condition; ii) failure to complete a psychiatric evaluation or adherence to drug therapy due to serious physical illness; iii) a current or lifetime DSM-IV diagnosis of bipolar disorder, schizophrenia, schizoaffective disorder, schizophrenia, a psychotic disorder not elsewhere classified or other psychotic disorder; iv) history of organic psychosis, epilepsy or seizure disorders; v) history of anticonvulsant treatment; vi) hospitalization for any psychiatric diagnosis except depressive disorder (eg alcohol/drug dependence) vii) electroconvulsive therapy for current episode of depression; viii) Pregnancy or breastfeeding.

The MAKE BETTER study was approved by the Chonnam National University Hospital Institutional Review Board, and written informed consent was obtained from all participants.

2. Full-length exome sequencing

DNA was extracted from venous blood samples of MAKE BETTER participants who consented to genetic testing. After performing exome capture using the SureSelect Human All Exon V5-UTR kit (Agilent Technologies, Santa Clara, CA, USA), sequencing was performed in either 100-bp or 150-bp reads in paired-end mode ( in paired-end mode) (HiSeq2500; Illumina, San Diego, CA, USA). A bioinformatics pipeline was used to process the captured exome data following the best practice recommendations contained in the Genome Analysis Toolkit (GATK) version 3.3-0 (van der Auwera et al., 2013). BWA 0.7.5a was used to align the read nucleic acid read to the human genome reference sequence (hg19/GRCh37) (Li et al., 2010). Aligned reads were further processed using InDel (short insertions and deletions) realignment, MarkDuplicates package from Picard software (ver. 1.92; http://picard.sourceforge.net) and sequence score recalibration (BQSR).

Single nucleotide variant and InDel detections were performed simultaneously on all samples using Haplotype Caller (GATK) (DePristo et al., 2011; McKenna et al., 2010; van der Auwera et al., 2013). Annotated with SNPEff 4.2 (Cingolani et al., 2012a) and SNPSift 4.2 (Cingolani et al., 2012b) software using variants dbNSFP (ver. 2.9.1) and Ensembl GRCh37.75.

3. Demographic and Clinical Characteristics of Depressed Patients

The demographic and clinical characteristics potentially associated with AAO of depressive disorder were evaluated. Data were obtained on age, years of education, employment status, and number of chronic disabilities. Information on depressive disorder includes depressive disorder diagnosis, number of depressive episodes, AAO of depression, current episode duration, family history of depressive disorder, history of past suicide attempts, severity of depressive symptoms according to the Hamilton Rating Scale (Hamilton, 1960), hospital anxiety depression The severity of anxiety symptoms according to the anxiety subscale of the scale (Zigmond and Snaith, 1983), the severity of suicidal ideation according to the suicide-related items of the Brief Psychiatric Rating scale (Overall and Gorham, 1962), and the DSM-IV criteria (American Psychiatric Association) , 2000), detailed diagnostic features of depression including melancholia, atypical and psychotic features were also obtained.

4. Statistical analysis

Participants were divided into three groups (early onset: 20 ≤ age ≤ 30 years, adult onset: 30 < age < 65 years, late onset: age ≥ 65 years) based on previous studies (Alexopoulos, 2005; Power et al., 2012; Wickramarantne and Weissman et al., 1998). Baseline characteristics, including sociodemographic and clinical factors, were compared between groups through analysis of variance (ANOVA) and Scheff's post hoc test or χ2 test was applied as appropriate. Factors significantly associated with AAO (P < 0.05) were entered as covariates in the corrected analysis performed in the next step.

The analysis procedure is illustrated in FIG. 1 . All nonsynonymous mutations leading to amino acid changes were analyzed. Hazard ratios were obtained using the Cox proportional hazards model to estimate the effect of individual variations on AAO in depressive disorder.

Carriers and non-carriers of individual coding variants were compared in the AAO aspect of depressive disorder, with adjustments for sociodemographic and clinical characteristics. Due to multiple comparisons, FDR (False Discovery Rate) corrected P-values were obtained by the Benjamini-Hochberg method. FDR-corrected P-values <0.05 were considered statistically significant.

The quality of the sequenced nucleic acid reads was assessed during preprocessing by checking the bam files using the IGV viewer (http://software.broadinstitute.org/software/igv/) for possible errors during mechanical filtering. . False positive mutations were eliminated. A final manual review was also performed by three bioinformatics experts to confirm the results. Kaplan Meier analysis and log-rank test were used to compare AAO according to candidate variants and graph the effect of individual variants on AAO in depressive disorder.

The clinical impact of candidate variants was evaluated using the t test, Wilcoxon rank sum test, χ2 test, or Fisher's exact test for each of the individual variants and for any one of all variants identified as heterozygous or homozygous carriers and ratios. It was determined by comparing the holders.

All statistical analyzes were performed using R software (ver. 3.2.3; http://www.rproject.org/).

5. Results

(1) Target

Of the 1,262 patients who participated in the MAKE BETTER study, 1,000 patients with depressive disorder were included in the study population by consenting to genetic testing. The patient characteristics are listed in Table 1 below. Those who did not consent to blood collection had more depressive episodes (P < 0.001), were more likely to have a current job (P = 0.003), and had less severe suicidal thoughts than those who had blood drawn (P < 0.001). P = 0.039) and melancholic patterns (P = 0.037) were many. The mean (SD) AAO of 1,000 patients was 52.0 (16.4) years (range: 8–84 years).

Patients with early onset depression were younger, more educated, more likely to be currently unemployed, more depressive episodes, a family history of depressive disorder and a history of suicide attempts, and more severe anxiety symptoms, suicidal thoughts and atypical features. . They were more likely to be single and less likely to show melancholy than other AAO groups (P <0.05). These important factors were included as covariates in the Cox proportional hazards model.

(2) Genetic variation associated with AAO in depressive disorder

To estimate the effect of individual nonsynonymous variants on AAO in depressive disorder, groups of carriers and non-carriers of the variant were compared using the Cox proportional hazards model after adjustment for sociodemographic and clinical characteristics. FDR-corrected P-values were obtained with the Benjamini-Hochberg procedure as mentioned above.

After confirming the read quality, candidate genes (FDR-corrected P<0.1) were further analyzed to include genes with FDR-corrected P<0.05 in the final analysis. Table 2 shows the number of significant variants and related genes derived with the Cox proportional hazards model with P-values corrected for the false discovery rate (FDR). Table 3 shows the statistically significant genetic variations related to the age of early onset of depressive disorder.

FDR adjusted p-value < 0.1 FDR adjusted p-value < 0.05 N = 33
RNPEP,SNAP47,SNAP47,PRSS38,TET1,TBRG1,CIT,FOXA1,TEKT5,GCSH,HS3ST3B1,LIG3,CCL14,ABCA7,DOCK6,PER2,COL6A1,ELFN2,CASR,ADIPOQ,MFSD8,FAM218A,FAM114A1,CTNAD2, FYB,DST,LANCL2,EXT1,HTRA4,OTUD6B,OR2K2,ORM1,GPRASP1 N = 4
CCL14, CTNND2, FYB, GPRASP1

Gene Position rsID PP2 SIFT CADD Carrier (homozygotes) Wild Type Cox-PH
FDR-adjusted P HR 95% CI 1KGP 1KGP
EAS CCL14 chr17:34313612 G>A rs75238886 0.773 0 12.72 31(0) 969 0.00008 ^‡ 2.2 1.52-3.18 0.02556 0.0466 FYB chr5:39202689 G>T rs35384751 0.459 0 8.0 111(1) 889 0.00008 ^‡ 1.53 1.25-1.87 0.01637 0.0288 GPRASP1 chrX:101909416 T>C rs201921260 0.999 0.51 7.5 10(0) 990 0.00308 ^† 2.95 1.56-5.55 0.00053 0.001 CTNND2 chr5:11364820 G>A rs202234398 0.919 0.16 24.5 14(0) 986 0.02785* 1.94 1.12-3.38 0.0004 0.002

rsID, reference ID; SIFT, =orting Intolerant From Tolerant; PP2, = PolyPhen2; CADD, Combined Annotation Dependent Depletion; Cox-PH P-adjusted for FDR, P-value obtained using Cox proportional hazards model corrected for FDR (False Discovery Rate), HR, hazard ratio, CI, confidence interval, 1KGP, allele from 1000 Genome Project Phase 3 frequency; Allele frequencies in East Asia in 1KGP EAS, 1000 Genome Project Phase 3.

*, p value <0.05; †, p-value <0.01'‡, p-value <0.001

As shown in Table 3, four variants, namely rs75238886 of the CC motif chemokine ligand 14 (CCL14) gene, rs35384751 of the FYN binding protein (FYB) gene, rs201921260 of the G protein-coupled receptor-associated classification protein 1 (GPRASP1) gene and catenin The rs202234398 delta 2 (CTNND2) gene was significantly associated with a high risk of early onset (1.53 ?? 2.95).

AAO of depressive disorder was compared between carriers and non-carriers of each individual variant using Kaplan-Meier survival analysis with log-rank test, and the results are shown in Figures 2-5. As shown, patients with one of the four variants were more likely to suffer from depression with early AAO.

(3) Association between clinical characteristics and genetic variation in depressed patients

To estimate the clinical impact of an identified variant, each variant and carriers and non-carriers of one or more of the four variants were evaluated for clinical characteristics using the t test, Wilcoxon's rank sum test, χ2 test, or Fisher's exactness. were compared and the results are shown in Table 4.

HAMD, Hamilton Depression Rating Scale; HADS, Hospital Anxiety Depression Scale; BPRS, Brief Psychiatric Rating Scale.

As shown in Table 4, depressed patients with one or more of the four variants are younger and more likely to suffer from depression in early AAO. They were also more likely to relapse and experience atypical depression. However, there were no individual mutations related to any clinical characteristics except for early AAO.

A major finding of these full-length exome sequencing analyzes is that four variants of the CCL14, FYB, GPRASP1 and CTNND2 genes are associated with an increased risk of developing depression with an early onset age. There were no individual mutations related to the clinical characteristics of depressed patients except for AAO, but any mutation in any of the four mutations was associated with a younger age, early AAO, relapse and atypical depression. That is, depressed patients with at least one candidate mutation were younger and were more likely to experience recurrent and atypical depression, even though individual mutations were not associated with clinical features.

Consistent with these results, age onset of depressive disorder (AAO) is known to influence symptoms and clinical course. Patients with early onset depression were more likely to have a chronic course and experienced more depressive episodes (Zisook et al., 2007, 2004). Atypical depression, characterized by mood responsiveness and opposite bodily responses (eating and sleeping), is generally a feature of early-onset depression (Novik et al., 2004; Stewart et al., 2009).

The present invention is the first study to identify genetic markers of depressive disorder AAO using full-length exome sequencing. Since GWAS has not been re-validated for specific variants, and even validated variants failed to establish associations of specific variants with AAO in depression due to unknown function, we used full-length exome sequencing, which would capture both common and rare variants. There was an advantage to being able to.

As such, in terms of clinical implications, using the biomarker for screening for depression with an early onset age according to the present invention provides the advantages of non-invasiveness and convenience, and genetic sensitivity screening, that is, genetics of depression with an early onset age. By identifying the marker, it will be able to contribute to the decision-making process for a drug or treatment method, and it will be of great help as a potential tool to preemptively prevent or treat patients' depression.

Although the present invention has been illustrated and described with reference to preferred embodiments as described above, it is not limited to the above-described embodiments and is intended for those of ordinary skill in the art to which the invention pertains within the scope not departing from the spirit of the present invention. Various changes and modifications will be possible.

Claims (16)

A biomarker composition for diagnosing depression having an early onset age comprising a polynucleotide of 10 to 100 nucleotides in length or a complementary polynucleotide thereof including the rs75238886 (G>A) region of the CCL14 gene as a control.
A biomarker composition for diagnosing depression having an early onset age comprising a polynucleotide of 10 to 100 nucleotides in length or a complementary polynucleotide thereof including the rs35384751 (G>T) region of the FYB gene as a control.
A biomarker composition for diagnosing depression with an early onset age comprising a polynucleotide of 10 to 100 nucleotides in length or a complementary polynucleotide thereof including the rs201921260 (T>C) region of the GPRASP1 gene as a control.
A biomarker composition for diagnosing depression having an early onset age comprising a polynucleotide of 10 to 100 nucleotides in length or a complementary polynucleotide thereof including the rs202234398 (G>A) region of the CTNND2 gene as a control. [Claim 5] A diagnostic kit for depression having an early onset age comprising a detection means capable of detecting the biomarker composition of any one of claims 1 to 4.
6. The method of claim 5,
The detection means includes at least one of a probe capable of detecting the polynucleotide included in the biomarker composition, a primer pair capable of amplifying the polynucleotide, and cDNA of the polynucleotide. Depression diagnostic kit with
A microarray for diagnosing depression with an early onset age comprising the biomarker composition of any one of claims 1 to 4.
obtaining DNA or RNA of the CCL14 gene from a biological sample of the subject; and
A method for diagnosing depression with an early onset age, comprising: confirming the base of the rs75238886 region included in the biomarker composition of claim 1 from the obtained DNA or RNA.
9. The method of claim 8,
When the base of the rs75238886 site is identified as A, the risk of developing depression at an earlier age is increased by 2.2 times compared to the control group having the base of the rs75238886 site G. A method of providing information on the diagnosis of depression.
obtaining DNA or RNA of the FYB gene from a biological sample of the subject; and
A method for diagnosing depression with an early onset age, comprising: identifying the base of the rs35384751 region contained in the biomarker composition of claim 2 from the obtained DNA or RNA.
11. The method of claim 10,
When the base of the rs35384751 site is confirmed to be T, the risk of developing depression at an earlier age is increased by 1.53 times compared to the control group having the base of the rs35384751 site G, which can be diagnosed as early onset age, characterized in that A method of providing information on the diagnosis of depression.
obtaining DNA or RNA of the GPRASP1 gene from a biological sample of the subject; and
A method for diagnosing depression with an early onset age, comprising: confirming the base of the rs201921260 region included in the biomarker composition of claim 3 from the obtained DNA or RNA.
13. The method of claim 12,
When the base of the rs201921260 site is identified as C, the risk of developing depression at an earlier age is 2.95 times higher than the control group having the base of the rs201921260 site T, which can be diagnosed as early onset age, characterized in that A method of providing information on the diagnosis of depression.
obtaining DNA or RNA of the CTNND2 gene from a biological sample of the subject; and
A method for diagnosing depression with an early onset age, comprising: confirming the base of the rs202234398 region included in the biomarker composition of claim 4 from the obtained DNA or RNA.
15. The method of claim 14,
When the base of the rs202234398 site is identified as A, the risk of developing depression at an earlier age is increased by 1.94 times compared to the control group having the base of the rs202234398 site G, which can be diagnosed as early onset age A method of providing information on the diagnosis of depression.
16. The method according to any one of claims 8 to 15,
The test subject is an adult 20 years or older, and the biological sample is an information providing method for diagnosing depression with an early onset age, characterized in that it is selected from a body fluid or tissue containing blood.

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