KR20230101768A - Asessment methods and diagnostic kit for depressive disorders in women using genetic biomarkers comprising the rs62640397 region of the FDX1L gene - Google Patents

Abstract

The present invention relates to a technology for diagnosing depression using biomarkers, and more specifically, using the rs62640397 region of the FDX1L gene, which was developed on the basis that mutations in a specific gene increase the risk of final non-response in a state of poor early treatment response to antidepressants. A biomarker for predicting the risk of depression in women, a method for providing information for diagnosing the risk of depression in women using the biomarker, and a diagnostic kit.

Description

A biomarker for predicting the risk of depression in women using the rs62640397 region of the FDX1L gene, and a method and diagnostic kit for diagnosing the risk of depression in women using the biomarker {Assessment methods and diagnostic kit for depressive disorders in women using genetic biomarkers comprising the rs62640397 region of the FDX1L gene}

The present invention relates to a technology for diagnosing depression using biomarkers, and more specifically, a biomarker for predicting the risk of depression in women using the rs62640397 region of the FDX1L gene developed on the basis that mutations in a specific gene appearing in women increase the risk of depression. A marker, a method for providing information for diagnosing women's risk of depression using the biomarker, and a diagnostic kit.

Depression is a major contributor to the global burden of disease. Epidemiologic studies have shown that depression is more prevalent in women worldwide, including in Asian countries, where it occurs about two to three times more often in women than in men, although the difference is small. In addition, the clinical characteristics and treatment outcomes of depressive disorder in women are different from those in men. Great efforts have been made to understand the mechanisms underlying the gender differences observed in depressive disorders. However, no clear mechanism has been reported.

The effect of sex on the heritability of depressive disorders was investigated based on a significant genetic contribution (35–40%), although no firm conclusions were reached. Previous association analyzes and genome-wide association analyzes (GWAS) have reported gender-specific genetic associations, but the results of these studies have rarely been replicated, except for associations with PCLO. Moreover, some studies reported a stronger genetic effect on depression in men, while others found a more pronounced genetic effect in women, or reported no detectable differences in genetic effects between the sexes. This discrepancy may be due to age differences, different diagnostic methods, or limitations of methods used to detect genetic differences associated with depression between the sexes.

To identify gender differences in the genetic contribution to depression, an alternative genetic study design is needed to compensate for the limitations of previous genetic studies. As a result, this will increase our understanding of the pathogenesis of depressive disorders and lead to the development of new therapeutic targets.

Large-scale whole exome sequencing (WES) data combined with sufficient clinical information have the potential to address the limitations of previous studies regarding gender differences in genetic influences on depression. Although previous GWAS have been successful in identifying indirect genetic associations that could potentially contribute to depressive disorders, the method of examining only these common and low-impact genetic variants is insufficient to explain the entire genetic background of depressive disorders. The same is true even when using meta-analysis methods to increase statistical power.

Also, previous GWAS results are overwhelmingly biased towards studies of European populations and cannot be generalized. As such, in order to discover the genetic vulnerability of depressive disorders that have not yet been understood, recent studies are conducting analyzes by applying WES. WES has the advantage of being able to capture interactions between common and rare genetic variations in protein coding regions. However, gender differences in genetic vulnerability to depressive disorders have never been investigated using large-scale WES data.

Therefore, it is necessary to develop new genetic markers that predict women's risk of depression based on gender differences observed in depressive disorders.

Korean Registered Patent No. 10-2047479

The present inventors have completed the present invention by revealing a biomarker for predicting the risk of depression in women, which is significantly associated with a higher risk of depression in women than in men, as a result of a number of studies.

Therefore, an object of the present invention is to confirm that a mutation at a specific site in one or more of the PDE4A, FDX1L, and MYO15B genes can predict the risk of depression in women, so that the higher prevalence of depressive disorders in women than in men may be due to inherited mutations. It is to provide a biomarker composition including a biomarker capable of relatively accurately predicting a woman's risk of depression as well as presenting genetic evidence that there is.

Another object of the present invention is to detect a biomarker for predicting the risk of depression in women, thereby providing strong evidence that the risk of depression may vary depending on the gender difference of the patient, so that the doctor can take into account the gender difference of the patient It can contribute to determining appropriate prevention and treatment strategies for patients with gender and genetic risk factors, thereby providing information on the diagnosis of women's depression risk, which can reduce the disease burden of depressed patients, especially female depressed patients. .

Another object of the present invention is to identify a mutation site of a biomarker for predicting a woman's risk of depression from a biological sample isolated from an individual, so that a doctor can predict a female patient at risk of developing depression, and thus preemptively prevent the patient's depression symptoms from deepening. It is to provide a diagnostic kit and microarray for predicting the risk of depression in women having clinical usefulness because the treatment strategy can be changed.

The 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 description below.

In order to achieve the above-described object of the present invention, the present invention is a polynucleotide of 10 to 100 nucleotides in length comprising the rs62640397 (T>C) region of the FDX1L gene or a polynucleotide complementary thereto as a control for depression in women It provides a biomarker composition for risk prediction.

In addition, the present invention provides a diagnostic kit for predicting the risk of depression in women, including a detection means capable of detecting the above-described biomarker composition.

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 pair of primers capable of amplifying the polynucleotide, and cDNA of the polynucleotide.

In addition, the present invention provides a microarray for predicting women's depression risk including the above-described biomarker composition.

In addition, the present invention comprises the steps of obtaining DNA or RNA of the FDX1L gene in the test subject's biological sample; And confirming the base of the rs62640397 region contained in the biomarker composition of claim 1 from the obtained DNA or RNA; provides a method for providing information on the diagnosis of women's depression risk including.

In a preferred embodiment, when the base of the rs62640397 region is identified as C, it can be diagnosed that the risk of depression increases 2.3 times compared to a control group in which the base of the rs62640397 region is T.

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

First, according to the biomarker composition of the present invention, by confirming that mutations at specific sites in one or more of the PDE4A, FDX1L and MYO15B genes can predict the risk of depression in women, a higher prevalence of depressive disorders in women than in men is inherited In addition to providing genetic evidence that this may be due to a mutation, it is also able to determine a woman's risk of depression relatively accurately.

In addition, according to the information providing method for diagnosing the risk of depression in women of the present invention, by detecting biomarkers for predicting the risk of depression in women, it is possible to provide strong evidence that the risk of depression may vary depending on the gender of the patient, so that doctors can Considering the gender difference of patients, it can contribute to determining appropriate prevention and treatment strategies for patients with gender genetic risk factors for depressive diseases, thereby reducing the disease burden of depressed patients, especially female depressed patients.

In addition, according to the diagnostic kit and microarray of the present invention, female patients with genetic risk factors for depression can be predicted by identifying the mutation site of the biomarker for predicting the risk of depression in women from biological samples isolated from the subject. It has clinical usefulness as it can preemptively change the treatment strategy before the depressive 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 description below.

1 is a flow chart showing steps for genetic analysis for predicting women's depression risk. As in-silico analysis tools, the following five programs, SIFT, PolyPhen2, CADD, LRT, and MutationTaster, were selected to predict deleterious mutations.
Figure 2 shows an overview of the permutation test.
Figure 3a is a graph of the genetic burden compared between men and women in depressed patients [depressive disorder patients (n = 1000)], Figure 3b is a graph of the genetic burden compared between men and women in the general population [1KGP CHB and healthy controls in the JPT population (n = 207)]. Comparison of the genetic variation in each category (point) indicates a high genetic burden in males (relative genetic burden >1) or a high genetic burden in females (relative genetic burden <1). The red dashed line represents the significance threshold (0.05).
4a and 4b are graphs comparing genetic burdens between males and females in depressed patients and the general population using a modified rare variant definition (allele frequency <1%).
5 is a graph of permutation test results for differences in genetic burden according to gender. A permutation test p-value was presented comparing the Wilcoxon rank sum test value using 10,000 permutations of random male and female labels and the test value analyzed in depressed patients. Black and blue solid lines are comparison values with depressed patients, solid lines represent variation-level analysis and dotted lines represent gene-level analysis.
6A to 6D are graphs comparing male and female multigene risk scores (PRS) in depressed patients and the general population.

Terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as "comprise" or "having" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the description of the invention, but one or more other It should be understood that it does not preclude the possibility of addition or existence of features, numbers, steps, operations, 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. These terms are only used for the purpose of distinguishing one component from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention.

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 a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present invention, they should not be interpreted in an ideal or excessively formal meaning. don't

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

In the case of a description of a temporal relationship, for example, when a temporal precedence relationship is described as 'after', 'continue to', 'after ~', 'before', etc., 'immediately' or 'directly' Including non-consecutive cases unless ' is used.

The human genome genetically shows more than 0.1% diversity, and this difference can be used as a tool for genetic fingerprinting. Single nucleotide polymorphisms (SNPs) can be used as individual genetic fingerprint recognition patterns, and more than 10 million variations are widely known by race and country by the human genome project. These SNPs can be detected simply by sequencing, and this diversity is rare, discrete and specific. However, there may be very frequent variations in some parts. The present inventors have found that women's risk of depression can be simply diagnosed by using the characteristics of a single nucleotide polymorphism (SNP) as described above in a specific gene.

As used herein, the term “diagnosis” means confirming the presence or character of a pathological condition. In the context of the present invention, “diagnosis” means determining a woman's risk of depression based on an ex vivo analysis of bodily fluids.

The term "rs_id" used in the present invention refers to an rs-ID, an independent marker assigned to all SNPs initially registered by NCBI, which began accumulating SNP information in 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 that can indicate a disease state. In the context of the present invention concerning the diagnosis of women's risk of depression, "biomarker" means a specific SNP site of any one of the PDE4A gene, the FDX1L gene and the MYO15B gene.

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 sample types obtained from an individual and may also be used in diagnostic or monitoring assays. Biological fluid samples include blood, cerebrospinal fluid (CSF), urine and other liquid samples of biological origin. If necessary, the sample may be pre-processed, for example for concentration and separation.

As used herein, the term “subject” refers to a mammal, preferably a human, and the terms subject, patient, or subject may be used interchangeably herein.

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 throughout to describe the invention indicate like elements.

The technical feature of the present invention is to confirm that mutations in one or more specific regions of the PDE4A, FDX1L, and MYO15B genes can predict the risk of depression in women, and based on this, the higher prevalence of depression in women than in men may be due to inherited mutations predicts women's risk of depression relatively accurately, allowing physicians to determine appropriate prevention and treatment strategies for patients with gender-specific genetic risk factors for depressive disorders, taking into account gender differences in patients. And / or to provide a diagnostic kit for women's depression risk and a method for providing information on women's depression risk, including a configuration for detecting any one or more specific SNPs among the diagnosable PDE4A gene, FDX1L gene, and MYO15B gene.

Therefore, the biomarker composition for predicting the risk of depression in women of the present invention includes a polynucleotide of 10 to 100 nucleotides in length including the rs201432982 region (C > A, T) of the PDE4A gene or a polynucleotide complementary thereto as a control, or , a polynucleotide of 10 to 100 nucleotides in length containing the rs62640397 (T>C) region or rs79442975 (G>A) region of the FDX1L gene or a polynucleotide complementary thereto as a control, or rs820182 (A> C) or a polynucleotide of 10 to 100 nucleotides in length including the rs820148 (A>G) region or a polynucleotide complementary thereto may be included as a control.

This is because, as will be described later, the present invention clearly confirmed the effect of a mutation at a specific site in one or more of the PDE4A, FDX1L, and MYO15B genes to increase the risk of depression in women.

That is, the specific SNP polynucleotide obtained from one or more of the PDE4A, FDX1L and MYO15B genes included in the biomarker composition of the present invention was used in 1000 patients with depressive disorder (70.7% female) to evaluate gender differences in the genetic structure of depression. ) by performing full-length exome sequencing on a sample to investigate whether a single nucleotide polymorphism (SNP) in a specific gene could be a biomarker related to the diagnosis of depression risk in women.

Qualitative analysis revealed three variants (rs201432982 in PDE4A, rs62640397 and rs79442975 in FDX1L) that mapped to chromosome 19p13.2 and two novel variants (rs820182 and rs820148) in MYO15B at the chromosome 17p25.1 locus for depression in women. We identified five genetic markers potentially associated with increased risk of disability. Specifically, depressed patients who were homozygous for these variants exhibited more severe depressive symptoms and higher suicide rates than patients who were not homozygous (ie, heterozygous and homozygous for the non-linked allele). In addition, quantitative analysis showed that the genetic burden of protein truncation and deleterious mutations was higher in females than in males, even after permutation testing. Here, healthy individuals without mental disorders (n = 72, 26.4% female) and East Asian subpopulation 1000 Genome Project data (n = 207, 50.7% female) were used as control data, and genetic variation between males and females was qualitatively and quantitatively Direct comparisons were made using the study design.

The PDE4A gene is a key regulator of cAMP second messenger signaling, which is widely expressed in brain regions that control memory and mood, such as the prefrontal cortex, hippocampus, and amygdala, and is considered a potential target for depression. In addition, in PDE4A-deficient mice, not only anxiety-inducing behavior and impairment of emotion-related memory were confirmed in association with increased urinary corticosterone, but also chronic administration of antidepressants increased PDE4A expression. Thus, this gene may be associated with synaptic plasticity affected by antidepressants, and mutations in PDE4A reduce neuronal firing through the hypothalamic-pituitary-adrenal axis, resulting in impairment of the negative feedback mechanism, leading individuals to depressive disorders. can make you vulnerable. The fact that PDE4A was found only in females can be supported by previous studies, in which expression of PDE4-related enzymes has been reported at high levels in the ovaries and that PDE4 plays a role in steroidogenesis and regulation of inflammatory responses. FDX1L (also known as FDX2) is known to contribute to mitochondrial myopathy and/or neurological symptoms, but no previous studies have reported an association between this gene and depressive disorders. Nevertheless, the two mutations of FDX1L are located downstream of the ribonucleoprotein, PTB Binding 1 (RAVER1) previously reported to be associated with depressive disorders. It has been suggested to be discovered as a replacement for other important genes in . . Mutations in FDX2 and/or RAVER1 are associated with mitochondrial dysfunction, which is known to contribute to accelerated oxidative stress and apoptosis, release of related neurotransmitters, and particularly increased levels of stress hormones in a sex-specific manner, contributing to the development of depression. can contribute Little is known about the role of MYO15B mapping to chromosome 17q25.1. However, this gene is known to be associated with white matter hyperintensity, which has been reported to be closely related to cognitive dysfunction, increased risk of dementia, and depression.

On the other hand, the female depression risk diagnosis kit of the present invention includes rs201432982 of the PDE4A gene (C>A, T), rs62640397 of the FDX1L gene (T>C), and rs79442975 of the FDX1L gene (G>A). ) region, rs820182 (A>C) region of MYO15B gene, rs820148 (A>G) region of MYO15B gene, or a detection means capable of detecting any one or more polynucleotides or their complementary polynucleotides. Here, the detection means may include 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.

Of course, if necessary, it can be implemented as a microarray containing a biomarker composition for diagnosing women's depression risk.

In addition, the method for providing information on the diagnosis of depression risk in women of the present invention comprises the steps of obtaining any one of DNA or RNA of the PDE4A gene, DNA or RNA of the FDX1L gene, and DNA or RNA of the MYO15B gene in a biological sample of the test subject; And confirming any one or more of the rs201432982 base, rs62640397 base, rs79442975 base, rs820182 base, and rs820148 base contained in the biomarker composition from the obtained DNA or RNA. do. Here, as in the investigation results described later, if the base at the rs201432982 site is identified as either A or T, it can be diagnosed that the risk of depression increases 2.6 times compared to the control group in which the base at the rs201432982 site is C, and the base at the rs62640397 site If is confirmed as C, it can be diagnosed that the risk of depression increases 2.3 times compared to the control group in which the base at the rs62640397 site is T. By comparison, it can be diagnosed that the risk of depression is increased by 2.3 times, and if the base at the rs820182 site is identified as C, the risk of depression can be diagnosed as increased by 1.9 times compared to the control group in which the base at the rs820182 site is A, and at the rs820148 site If the base of is identified as G, it can be diagnosed that the risk of depression increases 1.8 times compared to the control group in which the base of the rs820148 region is A.

Example

1. Research method

1.1. Participants and Datasets

The Depression (Depressive Disorder) dataset comes from the MAKE Biomarker discovery for Enhancing Antidepressant Treatment Effect and Response (MAKE BETTER) study, details of which have been previously published. The MAKE BETTER study was designed to investigate biomarkers for treatment response using a prospective design. The current study used baseline data from all participants who agreed to provide blood samples for genetic testing. Patients with major depressive disorder, dysthymic disorder, and depressive disorder not otherwise specified who recently visited the Department of Psychiatry at Chonnam National University Hospital were consecutively recruited. Detailed eligibility criteria are described in the Supplementary Methods. Depressive disorder was assessed by the study psychiatrist using the Mini-International Neuropsychiatric Interview (MINI), a structured diagnostic psychiatric interview based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria.

For the MAKE BETTER study, the inclusion criteria were: i) 7 years of age or older; ii) diagnosed with major depressive disorder, dysthymic disorder, or depressive disorder not otherwise classified (NOS), as confirmed using the Mini-International Neuropsychiatric Interview1; iii) Hamilton Depression Rating Scale score ≥ 14; iv) Able to complete the questionnaire, understand the purpose of the study, and sign the informed consent form. The exclusion criteria are as follows. i) an unstable or uncontrollable medical condition; ii) inability to complete psychiatric evaluation or adhere to pharmacotherapy due to serious physical illness; iii) current or lifetime DSM-IV diagnosis of bipolar disorder, schizophrenia, schizoaffective disorder, schizophreniform disorder, psychotic disorder not elsewhere classified, or other psychotic disorder; iv) history of organic psychosis, epilepsy or seizure disorder; v) history of anticonvulsant medication; vi) Hospitalization for all psychiatric diagnoses except for depressive disorders (eg alcohol/drug dependence) vii) Electroconvulsive therapy for current depressive episode; viii) Pregnancy or breastfeeding.

Two control data sets were used in this study. The first is a biomarker-based diagnostic algorithm study for post-traumatic syndrome, which investigated biomarkers predictive of post-traumatic syndrome, including depression, anxiety, or post-traumatic stress disorder (PTSD), in a 2-year prospective study in patients with severe traumatic body injury ( BioPTS) was used for some of the participants. Of the 141 participants in the BioPTS study, 72 were free of psychiatric disorders (e.g., depression, anxiety, or post-traumatic stress disorder) during the 2-year follow-up, even after serious physical injury, and who agreed to provide blood samples for the present analysis. was included as a control in The absence of a psychiatric disorder was diagnosed by the study psychiatrist using the Clinician-Administered PTSD Scale-5 (CAPS-5) modified by MINI and DSM-5 criteria.

For the BioPTS study, the inclusion criteria were: i) Hospitalization for 24 hours or more after serious physical injury (injury severity score ≥ 9) 3; ii) > 18 years of age; iii) Native Korean speakers. To maintain representativeness, the following exclusion criteria were applied. i) moderate or severe brain injury (Glasgow coma scale <10) 4; ii) a primary clinical diagnosis of a psychiatric disorder not associated with post-traumatic stress disorder that may affect post-traumatic stress processes, or a current diagnosis of psychosis or bipolar disorder, or suicide attempt; iii) Significant underlying cognitive disorders such as organic psychiatric disorders or neurocognitive disorders.

A second control data set was obtained from the 1000 Genomes Project, Phase 3 (1KGP) public database and consisted of data from individuals who declared themselves healthy without a specific clinical phenotype at the time of sample collection. Of the 26 populations in 1KGP, data on Han Chinese from Beijing, China (CHB) and Japanese from Tokyo, Japan (JPT), both within a fairly narrow geographic range and genetically similar to Koreans, were used as additional control groups in the present investigation.

All participants in the MAKE BETTER study and BioPTS provided written informed consent. Both studies were conducted in accordance with the institutional guidelines and the 1964 Declaration of Helsinki and were approved by the Institutional Review Board of Chonnam National University Hospital.

1.2. Full-length exome sequencing

DNA was extracted from venous blood samples from MAKE BETTER and BioPTS study participants who consented to genetic testing. WES was performed as follows to screen coding sequence regions across the entire genome using Illumina HiSeq. WES screening for coding sequence regions across the entire genome was performed using an Illumina HiSeq 2500 sequencer (Illumina, Inc., San Diego, CA) according to standard protocols as described in the manufacturer's instructions. The SureSelect Human All Exon V5 + UTR probe set was used, covering 359,555 exons of 21,522 genes and with a total target region of 75 Mb. To generate standard exome capture libraries, the Agilent SureSelect Target Enrichment protocol for Illumina paired-end sequencing libraries (version B.3, June 2015) was used with 3 μg of input gDNA. DNA quantity and quality were measured using PicoGreen reagent and a Nanodrop spectrophotometer. Genomic DNA aliquots (1 μg) were fragmented using the adaptive focused acoustics technique (Covaris). Fragmented DNA was repaired, the 'A' residue was ligated to the 3' end and then an Agilent adapter was ligated to the fragment. Once ligation was evaluated, the adapter ligation products were amplified by PCR and the final purified products were quantified using qPCR based on the qPCR quantification protocol guide and quality assessed using the Caliper LabChip High Sensitivity DNA Kit (PerkinElmer, Inc. Hopkinton, MA) did For exome capture, 250 ng of the DNA library was mixed with hybridization buffer, blocking mix, RNase block and 5 μl of the SureSelect all exon capture library according to the standard Agilent SureSelect Target Enrichment protocol. Hybridization was performed at 65 °C using a thermocycler lid option heated at 105 °C for 24 h in a PCR machine. Captured DNA was amplified and final purified products were quantified by qPCR using the qPCR quantification protocol guide and quality assessed using TapeStation DNA screen tape (Agilent). Next, sequencing was performed on a HiSeq™ 2500 platform (Illumina, Inc., San Diego, CA). The reads were mapped to the human genome reference sequence (hg19/GRCh37) using BWA-MEM (v0.7.7). Aligned reads were classified and indexed using SAMTools (v0.1.19) 7 and PCR duplicates were marked using MarkDuplicates from the Picard toolkit (http://broadinstitute.github.io/picard). The Genome Analysis Toolkit (GATK, v2.8.1) was used for local rearrangements and base recalibration (dbSNP137 for single nucleotide variants (SNVs), Mills at site hg19 for Indels and 1000 Genome Project standard InDels were used). SNVs and short insertions/deletions (InDels) were identified using HaplotypeCaller in GATK. Following the recommendations listed in GATK's Best Practice Variant Detection document, filtering was performed by applying the following conditions: "QD <2.0", "MQ <40.0", "FS> 60.0", "MQRankSum <12.5", "For SNPs ReadPosRankSum <-8.0"; "QD < 2.0", "ReadPosRankSum < -20.0", "FS > 200.0" for InDels. Variants assigned by GATK as SNPCluster (clusterSize = 3, clusterWindowSize = 35) were filtered to reduce the possibility of false positive calls. Protein coding gene regions were defined using SnpEff 4.1 (build 2015-01-07). Variants that were putatively likely to have functional effects were selected based on the following variant types: missense, stop gain, stop loss and start loss. To estimate the pathogenicity of variants, they were annotated using three in silico variant hazard prediction scores: Sorting Intolerant From Tolerant (SIFT), PolyPhen2 (PP2), and Combined Annotation Dependent Depletion (CADD). For analysis comparable to WES data, only mutations in the same target region were extracted from 1KGP whole-genome sequencing data and used for downstream analysis.

1.3. Demographic and clinical characteristics of depressed patients.

The current analysis considered demographic and clinical characteristics potentially associated with depressive disorders in men and women. Demographic data included age, years of education, employment status and number of chronic physical disabilities. Clinical features of depressive disorder were also evaluated as follows. diagnosis of depressive disorder; number of depressive episodes; age of onset; duration of the current episode; family history of depressive disorder; past history of suicide attempts; severity of depressive symptoms according to the Hamilton Rating Scale for Depression score; severity of anxiety symptoms according to the anxiety subscale score of the Hospital Anxiety Depression Scale; The severity of suicidal ideation according to the suicide-related items of the Brief Psychiatric Rating Scale; and subdiagnostic types of depression including melancholia, atypical and psychotic features according to DSM-IV criteria.

1.4. statistical analysis.

The analysis was conducted using a qualitative and quantitative analysis design to compare the genetic effects of depressive disorders between men and women in terms of prevalence, which was used in previous studies of other psychiatric disorders that showed gender-biased prevalence. The qualitative analysis design was based on the qualitative hypothesis that specific variants contribute differently to depressive disorders according to gender. The quantitative analysis design assumes that a greater genetic burden is required for male individuals to develop depressive disorders, following the quantitative hypothesis that the predominance of depressive disorders in women may be due to a genetic protective effect against depression in men.

In the qualitative analysis design, the summarized analysis scheme shown in Figure 1 was used to estimate the genetic effects of gender status. Variation frequencies were compared between males and females for all functional variants (missing, stop-gain, stop-loss, and start-loss categories) using Fisher's exact test.

Assuming that the combined mutation rates between males and females in healthy controls are not significantly different, the minimum P-value obtained from the control data (P = 5.12E-05) was considered the threshold for empirical significance and therefore of minimal clinical significance. indicates Differences between male and female groups. To assess bias, the statistical significance of gender differences between depressed patients was compared with controls (controls without mental illness after severe injury, 1KGP CHB and JPT combined groups). Variations below the threshold in the depressive disorder group were considered to be significantly associated with gender-specific genetic structure. Single variance analysis was performed using Fisher's exact test based on allele frequency. We tested the robustness of the results by performing three additional tests of association, including Fisher's exact test and Cochran-Armitage trend test based on genotype frequencies in both dominant and recessive models. The independent effects of the variance on depressive disorder in both genders were potentially significant demographic and clinical findings identified by comparisons between men and women with depressive disorder as appropriate using t-, χ2 or Fisher's exact test. It was estimated using a multivariate logistic regression model and the Cochran-Mantel-Haenszel test after adjusting for the trait (P < 0.1, “negative” confounding identified by comparison of men and women with depression). To assess the clinical impact of a sex-specific variant in both sexes and the overall population with depressive disorder, Wilcoxon rank-sum or chi-square tests, as appropriate, were used to compare clinical characteristics of homozygous depressed patients to heterozygous or heterozygous for carriers of the relevant variant. Compared with the clinical characteristics of non-carriers. In addition, haplotype analysis was performed on the five identified variants. Haplotypes were inferred using PHASE v.2.040. Clinical characteristics of depressed patients with haplotypes consisting of five alternative variants in both alleles (homozygous) were compared with clinical characteristics of heterozygous or non-carriers using the Wilcoxon rank sum test. Consistent with previous studies, the severity of depressive disorder was determined by age of onset, frequency of recurrent episodes, family history of depressive disorder, overall severity of depression according to the baseline Hamilton Depression Rating Scale score, and suicide item score (≥ 4) of the Brief Psychiatric Rating Scale. It was defined as the presence or absence of anxiety according to the high suicidal tendency and the anxiety subscale score of the Hospital Anxiety Depression Scale. Bonferroni correction was used to correct the overall Type I error rate to 0.05 for 7 comparisons (0.05/7 = 0.007) in multiple comparisons, i.e. clinical effects analysis.

In the quantitative analysis design, to estimate the genetic effect according to gender status, the variants were annotated and grouped into three annotation categories as shown in Table 1.

i) allele frequency (‘rare’ or other variant if minor allele frequency <0.1% in 1 KGP); ii) function (miss sense, splice site, frame shift, stop loss, stop gain, stop hold hold, start loss or 'functional' if applicable from any of the InDels categories within a frame, or splice site, frame shift, stop loss; 'Protein truncation variant (PTV)' if included in the stop-gain, sustained-hold or start-loss categories); and iii) hazardous (‘harmful’ if SIFT ≤ 0.05 or CADD ≥ 15 or other variant).

Group Description Rare 1KGP MAF < 0.1% Functional Missense, splice site, frameshift, stop lost, stop gain, stop retained, start lost, inframe InDels Protein Truncating Variants (PTVs) Splice site, frameshift, stop lost, stop gain, stop retained, start lost Deleterious SIFT ≤ 0.05 or CADD ≥ 15 1KGP, 1000 Genomes Project, EAS, East Asian population, AF, Allele Frequency

In addition, using these annotation categories for genetic analysis, eight category combinations (rare functional, functional, rare functional hazardous, functional hazardous, rare PTV, PTV, rare PTV hazardous, and PTV hazardous) contained 17,512 variants with at least 1 mutation. It was generated for 967 autosomal protein-coding genes in dogs and for 967 autosomal genes reported to be genetically associated with MDD in the MK4MDD database. For each category combination, the genetic burden of individual subjects was estimated by counting the number of variants or genes overlapping the defined variant categories, and results were compared between males and females using the Wilcoxon rank sum test.

To test the frequency at which the model occurs by chance, a permutation test was performed using randomly permuted gender-labeled patient data as shown in Figure 2 and as follows. (i) Under the null hypothesis (i.e., no significant difference in variance or genetic burden between males and females), gender labels in patient data are permuted so that males and females assigned in random order are replaced by non-sequential data (i.e., female 707 and 293 men). ii) Genetic burden between data assigned as ‘male’ and ‘female’ in this permuted data set was compared using the Wilcoxon rank sum test. iii) Recorded the test statistic 'W' obtained using the Wilcoxon rank-sum test. iv) Steps i) to iii) were repeated 10,000 times (T = 10,000). The permuted P-value (C/T) was derived by calculating how many times the W value obtained from the analysis of the permuted data set was greater than the W value obtained using the observed data set (C). Based on recent PGC-MDD summary statistics (https://www.med.unc.edu/pgc/download-results/mdd/) obtained from European populations to compare men and women using the Polygenic Risk Score (PRS). PRS was constructed through PLINK and PRSice-2 with SNP weights. Common SNPs (MAF > 0.01) identified in the 1KGP European population were aggregated using LD parameters of r > 0.1 in a 250 kb window. PRSs were obtained using LD-clumped independent SNPs with p-values associated with associations below eight thresholds (P < 10-4, 0.001, 0.01, 0.05, 0.1, 0.2, 0.5 and 1). We compared PRS between men and women using the Wilcoxon rank-sum test in depressed patients and healthy controls in the BioPTS study.

All statistical analyzes were performed using R (v3.2.3; http://www.r-project.org/).

2. Results

2.1. subject of study

All samples included in the analysis are summarized in Table 2 below.

Of the 1265 depressed patients who participated in the MAKE BETTER study, 1000 consented to blood sampling for genotyping, and these constituted the depressive disorder sample in the current investigation. Baseline characteristics of blood draw participants and non-consent participants were not significantly different except for the number of depressive episodes, suicidal ideation severity, and melancholia pattern (all P > 0.066).

Phenotype Ethnicity Samples Females Males Major Depressive Disorder Korean 1000 707 293 Control from BioPTS Korean 72 19 53 (No psychiatric disorders even after severe physical injury) Control from general population
(healthy normal) Han Chinese in Beijing, China (CHB) and Japanese in Tokyo, Japan (JPT) 207 105 102 All the mixed population of the 1000 Genomes Project (1KGP) 2504 1271 1233 BioPTS, Biomarker-Based Diagnostic Algorithm for posttraumatic syndrome after physical injury

Patients who refused to provide blood samples were more likely to have episodes of depression (P < 0.001), were less likely to be jobless (P = 0.003), had less severe suicidal ideation (P = 0.039), and were less likely to have melancholy features. more (P = 0.037). Of 1000 patients with depression, 707 (70.7%) were female. The characteristics of female and male depressed patients are described in Table 3. Female patients were less educated, more likely to be unemployed, more likely to experience recurrent depression, and more likely to have more severe depressive symptoms.

Table 3 presents the demographic and clinical characteristics of depressed patients, analyzed using t-test, χ2 test or Fisher's exact test (where applicable), values in bold indicate wider significance cutoffs (P < 0.1).

The first control data set consisted of 72 patients from BioPTS who did not experience any psychiatric disorder during the 2-year follow-up period even after suffering serious physical injury. Unlike the depression group, the BioPTS group had more males (73.6%) than females (26.4%). A second control data set included CHB and JPT population data (n = 207) that were part of 1KGP (n = 2504). About half (50.7%) of the CHB and JPT combined population were female.

2.2. Gender-specific genetic heterogeneity associated with depression determined by qualitative analysis

The frequency distributions of 236,274 functional variants identified in 1000 patients with depressive disorder were compared between males and females using Fisher's exact test as shown in FIG. 1 . As shown in Table 4, five variants with clinically significant differences between males and females with depressive disorder were found (Table 4; P < 5.12E-05), and these variants were almost twice as frequent in females as in males. did.

Table 4 presents gender-specific variances detected in depressive disorders compared to the general population, with abbreviations defined as; SIFT, Sorting Intolerant From Tolerant; PP2, PolyPhen2; CADD, Combined Annotation Dependent Depletion; HGVS.p, Human Genome Variation Society Protein Reference Sequence; MAF, minor allele frequency; REF, reference allele; ALT, alternative allele; Fisher's p, Fishers exact test p-value; OR, odd ratio; CATT p, Cochran-Armitage trend test p-value; MDD, major depressive disorder; CHBJPT, Chinese Han in Beijing, Japanese in Tokyo, Japan; BioPTS, Biomarker-Based Diagnostic Algorithm Study in Post Traumatic Syndrome; NA, not applicable.

On the other hand, none of the five variants shown in Table 4 had different frequencies in males and females in the control group. Three of the five mutations mapped to the phosphodiesterase 4A (PDE4A) (rs201432982) and ferredoxin 1-like (FDX1L) (rs62640397 or rs79442975) loci were in the same chromosomal cytoband (19p13.2), whereas the other two The dog mutations (rs820182, rs820148) were in the Myosin-XVB (MYO15B) gene located on chromosome 17p25.1. Both mutations in the FDX1L gene were in complete linkage disequilibrium (D' = 1) in the entire 1KGP subpopulation. Odds ratios calculated using dominant and recessive model analyzes for all five variants indicate that they are more frequent in depressed women than in men with depressive disorders.

The independent effects of variance on depressive disorder by sex status, determined using a multivariate logistic regression model and the Cochran-Mantel-Haenszel test after adjustment for potential demographic and clinical characteristics, are summarized in Table 5.

Gene Variant SIFT CADD PP2 Exon HGVS.p Type Multi-variate logistic regression test Cochran-Mantel-Haenszel test Statistical
coeffients (β) OR
(95% CI) p-value Statistical
coeffients (χ2) p-value PDE4A rs201432982(C > A, T) 0.04 23.5 NA 1/15 p.Arg57Trp Missense 0.962 2.62
(1.6-4.4) 1.74E-04 27.82 0.023 FDX1L
(RAVER1 downstream ) rs62640397 (T>C) 0.00 0.017 0.069 1/5 p.Arg29Gly Missense 0.850 2.34
(1.5-3.7) 1.31E-04 32.44 5.61E-03 rs79442975 (G>A) NA 9.108 NA 1/4 NA Splice region variant MYO15B rs820182 (A>C) NA 5.116 NA 48/62 NA Splice region variant 0.614 1.85
(1.4-2.5) 8.89E-05 29.71 0.013 rs820148 (A>G) NA 6.033 NA 45/62 NA Splice region variant 0.575 1.78
(1.3-2.5) 3.87E-04 28.97 0.016 Data were adjusted for age, education, unemployment status, diagnosis of depression, recurrent depression, family history of depression, and Hamilton Depression Rating Scale baseline score
SIFT, Sorting Intolerant From Tolerant; PP2, PolyPhen2; CADD, combined annotation dependent depletion; HGVS.p, Human genome variation society -protein reference sequence; OR, Odds Ratio, CI, Confidence Interval, LR, Logistic Regression

Even after correction, the P-value remained close to the significance level (P = 1E-04 and P = 2E-02, respectively).

To assess the clinical relevance of these variants, clinical characteristics indicative of depression severity were compared between carriers homozygous for the relevant SNP and homozygotes for either heterozygous or non-carriers. Under the hypothesis that homozygosity for both variants would cause more severe symptoms, comparisons were made using the Wilcoxon rank-sum and chi-square test, as appropriate. As shown in Tables 6 to 10, which describe the clinical characteristics of depressed patients with or without gender-specific genetic mutations, the greater the number of mutations, the more serious symptoms can be caused.

In Tables 6 to 10, P-values were analyzed using the Wilcoxon rank sum or chi-square test where appropriate. Values in bold show statistical significance after Bonferroni correction. HAMD, Hamilton Depression Rating Scale; HADS, Hospital Anxiety Depression Scale; BPRS, Brief Psychiatric Rating Scale; means NA, not applicable;

Depressed patients who were homozygous for one or more variants had more severe depressive symptoms, including higher baseline depression scores and more suicidal ideation, even after Bonferroni correction. When the sample was separated into males and females, only the female group showed a similarly significant association with severe depressive symptoms after Bonferroni correction, but rather improved statistical significance. On the other hand, in males, the clinical phenotypes of homozygous carriers and heterozygous or non-carriers were not significantly different, except for values that could not be calculated due to lack of data. Analysis of individual variances also revealed similar trends, with statistical significance only maintained for higher baseline depression scores in MYO15B carriers after Bonferroni correction. In particular, in 32 depressed patients, a haplotype consisting of five alternative variants (T-C-A for PDE4A-FDX1L and C-G for MYO15B) in which two alleles were homozygous was more prominent in female patients, even in these haplotype carriers. A similar clinical pattern was identified, ie higher baseline depression scores and greater severity of suicidal ideation (see Tables 11-13).

In Tables 11 to 13, P-values were analyzed using either the Wilcoxon rank sum or chi-squared test, depending on the context. Values in bold show statistical significance after Bonferroni correction. HAMD, Hamilton Depression Rating Scale; HADS, Hospital Anxiety Depression Scale; BPRS, Brief Psychiatric Rating Scale; means NA, not applicable;

However, due to the limited number of variant carriers in the current study, future studies may be needed to confirm the association between the five variants and their haplotypes and clinical outcomes. Nonetheless, our results show that these five variants, which are more frequent in women with depression, could potentially contribute to gender heterogeneity in clinical features of depression.

2.3. Male protective effect in depression confirmed by quantitative analysis

The number of variants and genes overlapping the eight defined annotation categories (rare functional, functional, rare functional hazard, functional hazard, rare protein truncation variant (PTV), PTV, rare PTV hazard, and PTV hazard) at the variant- and gene-level. defined as genetic burden. We compared the genetic burden between males and females for genes associated with depression in the MK4MDD (Multi-Level Knowledge Base and Analysis Platform for Major Depressive Disorder) database and for autosomal protein-coding genes whose associations have not yet been confirmed. As shown in Figures 3A and 3B, Wilcoxon rank sum test 78% (25 /32) showed a higher genetic burden in men than in women. Random distribution was observed in the depressive disorder group and the healthy control group. 59.4% (19/32) showed a higher genetic burden in males. In the depressive disorder group, the genetic burden at the level of variants overlapping deleterious PTVs in autosomal protein-coding genes was significantly higher in males (Fig. 3a; P = 0.021), and the genetic burden at the gene level was borderline significant in the same category. slightly significant (P = 0.051). When analyzing differences in genetic burden according to gender for the genes reported in the MK4MDD database, no category reached statistical significance. In healthy controls, as expected, there was no significant difference in genetic burden between males and females (Fig. 3b), and a similar trend was observed when the same analysis was performed with modified rare variant definition (allele frequency <1%) (Fig. 3b). 4a and 4b).

To get a rough estimate of the true distribution of genetic burden, we shuffled the sex labels for our data set 10,000 times and performed a permutation test using these random permutation sets to compare the genetic burden between the assigned sex labels. The deleterious PTVs remained significantly enriched in males after permutation analysis ( FIG. 5 ; variance-level permutations, P = 0.011 and gene-level permutations, P = 0.026). This indicates that the fact that male depressive disorder requires a greater genetic burden is not an accidental finding.

In the present invention, PRS analysis based on the recent PGC-MDD summary statistics of the European population was additionally applied to verify the male protective effect against depression using WES data (see Figs. 6a to 6d). Using p < 0.01 as the p-value threshold, male patients with depressive disorder had a significantly higher PRS than female patients with depressive disorder (p = 0.045 by Wilcoxon rank sum test). However, in the BioPTS control group, PRS was not significantly different between men and women at all p-value threshold levels.

The main result of the genetic study according to the present invention using WES data as described above is that five mutations in the PDE4A, FDX1L, and MYO15B genes are associated with an increased risk of depressive disorder in women, and depressed patients who are homozygous for these mutations They are more likely to experience severe depressive symptoms. And we found that a higher genetic burden, especially in protein truncations and deleterious mutations, is required for males to develop depressive disorders, which may contribute to higher resilience to depressive disorders in males. Overall, these findings provide evidence for a genetic factor for female predominance in depressive disorders.

In particular, patients who were homozygous for the five variants in PDE4A, FDX1L and MYO15B (which did not differ significantly between the sexes of the control group) associated with an increased risk of depressive disorder in women found based on qualitative analysis had more severe depressive symptoms and higher rates of depressive symptoms. suffered from higher suicide rates, suggesting that these five variants have clinical significance as patients with more variant alleles experienced more severe depressive symptoms. In addition, no male-specific risk variants were identified by qualitative analysis. This result is consistent with previous genetic studies that reported that specific genetic factors associated with depressive disorders were more common in women than in men.

Although the present invention has been shown and described with preferred embodiments as described above, it is not limited to the above embodiments, and to those skilled in the art within the scope of not departing from the spirit of the present invention Various changes and modifications will be possible.

Claims (7)

A biomarker composition for predicting the risk of depression in women comprising a polynucleotide of 10 to 100 nucleotides in length or a polynucleotide complementary thereto containing the rs62640397 (T>C) region of the FDX1L gene as a control.
A diagnostic kit for predicting women's risk of depression comprising a detection means capable of detecting the biomarker composition of claim 1.
According to claim 2,
Depression in women, characterized in that 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 Diagnostic kit for risk prediction.
A microarray for predicting the risk of depression in women comprising the biomarker composition of claim 1. Obtaining DNA or RNA of the FDX1L gene from a biological sample of a test subject; and
A method for providing information on diagnosing a woman's risk of depression, comprising: confirming the base of the rs62640397 region contained in the biomarker composition of claim 1 from the obtained DNA or RNA.
According to claim 5,
When the base of the rs62640397 region is identified as C, the risk of depression in women can be diagnosed as 2.3 times higher compared to a control group in which the base of the rs62640397 region is T. Method for providing information for diagnosing depression risk.
According to claim 5 or 6,
The biological sample is a method for providing information for diagnosing women's depression risk, characterized in that selected from bodily fluid or tissue containing blood.

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