KR20240086727A - Biomarker for diagnosing depression and uses thereof - Google Patents
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Abstract
본 발명은 CDH12(Cadherin 12) mRNA 또는 단백질을 포함하는 우울증 진단용 마커 조성물 및 이를 활용한 우울증의 진단용 조성물, 진단용 키트, 진단을 위한 정보 제공 방법에 관한 것이다. 본 발명은 상기 마커를 활용함으로써 간편하고 높은 정확도로의 우울증 진단이 가능하다.The present invention relates to a marker composition for diagnosing depression containing CDH12 (Cadherin 12) mRNA or protein, a composition for diagnosing depression using the same, a diagnostic kit, and a method of providing information for diagnosis. The present invention enables simple and highly accurate diagnosis of depression by utilizing the above marker.
Description
본 발명은 우울증의 진단용 마커 및 이의 활용에 관한 것이다.The present invention relates to diagnostic markers for depression and their use.
우울증은 의욕 저하와 우울감을 주요 증상으로 하여 다양한 인지 및 정신 신체적 증상을 일으켜 일상 기능의 저하를 가져오는 질환이다. 주요 증상으로는 우울감, 무기력감, 불안, 흥미의 저하, 식욕장애, 수면장애, 자살 충동, 무가치감, 부적절한 죄책감, 집중력, 및 기억력 감퇴 등이 있다. 또한, 우울증은 비만, 심혈관질환, 퇴행성 신경질환 등 많은 합병증을 수반하는 매우 복잡한 장애로서, 근본적인 발병 메커니즘은 여전히 불분명하다.Depression is a disease that causes a variety of cognitive and psychosomatic symptoms with decreased motivation and depression as the main symptoms, leading to a decline in daily functions. Main symptoms include depression, helplessness, anxiety, loss of interest, appetite disturbance, sleep disturbance, suicidal thoughts, feelings of worthlessness, inappropriate guilt, concentration, and memory loss. In addition, depression is a very complex disorder that involves many complications such as obesity, cardiovascular disease, and neurodegenerative disease, and the underlying pathogenesis mechanism is still unclear.
우울증의 유병률은 현재 날로 증가하는 추세이며, 세계보건기구(WHO)는 21세기에 인류를 괴롭히는 10대 질병 중 하나로 우울증을 언급했으며, 2020년 세계 5대 부담 질병에 우울증을 포함시켰다. 전세계적으로 1억명 이상의 환자들이 우울증을 앓고 있다는 보고가 있으며, 우울증은 막대한 사회경제적 비용을 초래하는 정신질환으로서, 특히 장애와 자살을 유발하는 주요 원인인바 향후에는 암, 치매 등보다도 높은 사회경제적 비용을 초래하는 질환이 될 것으로 예측된다.The prevalence of depression is currently increasing day by day, and the World Health Organization (WHO) has mentioned depression as one of the top 10 diseases afflicting humanity in the 21st century, and included depression in the top 5 burdensome diseases in the world in 2020. It has been reported that more than 100 million patients worldwide suffer from depression, and depression is a mental illness that causes enormous socioeconomic costs. In particular, it is a major cause of disability and suicide, so in the future, the socioeconomic costs will be higher than those of cancer and dementia. It is predicted that it will be a disease that causes.
우리나라의 자살률은 2014년도 기준인구 10만명당 27.3명으로 OECD 회원 국가의 평균 자살률의 약 2.5배로 압도적인 1위이며, 자살 증가율 역시 최고 수준으로, 자살의 주요한 원인인 우울증에 대한 진단 및 치료가 중요한 실정이다. 그러나, 이러한 현실에도 불구하고 우울증과 관련된 정신건강검진은 객관적인 진단 기술의 부재로 체계화되지 못하고 있다.Korea's suicide rate is 27.3 per 100,000 people in 2014, which is about 2.5 times the average suicide rate of OECD member countries, ranking first. The increase in suicide is also at the highest level, making it important to diagnose and treat depression, a major cause of suicide. am. However, despite this reality, mental health screening related to depression has not been systemized due to the lack of objective diagnostic technology.
기존의 우울증 진단은 임상가에 의한 정신과적 면담, 병력 청취 및 정신상태검사(mental status examination)에 의존하였으며, 이로 인해 임상가 간의 우울증 진단이 상이하거나 진단의 신뢰성 및 타당성에 의구심을 가지는 경우가 많았다.The existing diagnosis of depression depended on a psychiatric interview, history taking, and mental status examination by a clinician, which often resulted in differences in depression diagnoses among clinicians or doubts about the reliability and validity of the diagnosis.
이에, 본 발명자들은 객관적이고 정확성이 높은 우울증 진단방법에 대해 연구하여 우울증 환자에서 정상 대조군과 비교하여 발현량에 유의미한 차이가 있는 단백질 바이오마커를 선별함으로써 본 발명을 완성하였다.Accordingly, the present inventors studied an objective and highly accurate method for diagnosing depression and completed the present invention by selecting protein biomarkers with significant differences in expression levels in patients with depression compared to normal controls.
본 발명은 간편하고 높은 정확도로 우울증의 진단이 가능하도록 하는 마커 조성물을 제공하는 것을 목적으로 한다.The purpose of the present invention is to provide a marker composition that enables the diagnosis of depression easily and with high accuracy.
본 발명은 상기 마커 조성물을 활용한 우울증의 진단용 조성물, 우울증의 진단을 위한 정보 제공 방법을 제공하는 것을 목적으로 한다.The purpose of the present invention is to provide a composition for diagnosing depression using the marker composition and a method for providing information for diagnosing depression.
1. CDH12(Cadherin 12) mRNA 또는 단백질을 포함하는 우울증 진단용 마커 조성물.1. A marker composition for diagnosing depression containing CDH12 (Cadherin 12) mRNA or protein.
2. 위 1에 있어서, C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질을 더 포함하는 우울증 진단용 마커 조성물.2. The marker composition for diagnosing depression according to 1 above, further comprising C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein.
3. 위 1에 있어서, 상기 우울증은 주요 우울 장애(major depressive disorder)인 우울증 진단용 마커 조성물.3. The marker composition for diagnosing depression according to 1 above, wherein the depression is major depressive disorder.
4. CDH12(Cadherin 12) mRNA 또는 단백질의 발현을 검출하는 제제를 포함하는 우울증의 진단용 조성물.4. A composition for diagnosing depression comprising an agent that detects the expression of CDH12 (Cadherin 12) mRNA or protein.
5. 위 4에 있어서, 상기 제제는 항체, 압타머, DNA, RNA, PNA, 단백질 또는 폴리펩티드인 우울증의 진단용 조성물.5. The composition for diagnosing depression according to item 4 above, wherein the agent is an antibody, aptamer, DNA, RNA, PNA, protein, or polypeptide.
6. 위 4에 있어서, C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현을 검출하는 제제를 더 포함하는 우울증의 진단용 조성물.6. The composition for diagnosing depression according to item 4 above, further comprising an agent for detecting the expression of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein.
7. 위 4에 있어서, 상기 우울증은 주요 우울 장애(major depressive disorder)인 우울증의 진단용 조성물.7. The composition for diagnosing depression according to item 4 above, wherein the depression is major depressive disorder.
8. 위 4 내지 7 중 어느 하나의 우울증의 진단용 조성물을 포함하는 우울증의 진단용 키트.8. A kit for diagnosing depression containing the composition for diagnosing depression according to any one of items 4 to 7 above.
9. 개체로부터 분리된 시료에서 CDH12(Cadherin 12) mRNA 또는 단백질의 발현을 검출하는 단계를 포함하는 우울증의 진단을 위한 정보 제공 방법.9. A method of providing information for the diagnosis of depression, including the step of detecting the expression of CDH12 (Cadherin 12) mRNA or protein in a sample isolated from an individual.
10. 위 9에 있어서, 상기 시료는 말초혈액인 우울증의 진단을 위한 정보 제공 방법.10. The method of providing information for the diagnosis of depression in item 9 above, wherein the sample is peripheral blood.
11. 위 9에 있어서, 상기 CDH12(Cadherin 12) mRNA 또는 단백질의 발현량이 대조군 대비 적으면, 상기 개체가 대조군 대비 우울증에 걸렸을 가능성이 더 높다는 정보를 제공하는 우울증의 진단을 위한 정보 제공 방법.11. The method of providing information for the diagnosis of depression in item 9 above, wherein if the expression level of CDH12 (Cadherin 12) mRNA or protein is lower than that of the control group, the individual is more likely to have depression compared to the control group.
12. 위 9에 있어서, 상기 시료에서 C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현을 검출하는 단계를 더 포함하는 우울증의 진단을 위한 정보 제공 방법.12. The method of providing information for the diagnosis of depression according to item 9 above, further comprising the step of detecting the expression of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein in the sample.
13. 위 12에 있어서, 상기 시료는 말초혈액이고, C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현량이 대조군 대비 적으면, 상기 개체가 대조군 대비 우울증에 걸렸을 가능성이 더 높다는 정보를 제공하는 우울증의 진단을 위한 정보 제공 방법.13. In item 12 above, the sample is peripheral blood, and if the expression level of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein is lower than the control group, it provides information that the individual is more likely to have depression compared to the control group. How to provide information for the diagnosis of depression.
14. 위 9에 있어서, 상기 우울증은 주요 우울 장애(major depressive disorder)인 우울증의 진단을 위한 정보 제공 방법.14. A method of providing information for the diagnosis of depression in item 9 above, wherein the depression is major depressive disorder.
본 발명은 간편하고 비침습적인 방법으로 우울증의 진단이 가능하도록 한다.The present invention enables diagnosis of depression in a simple and non-invasive manner.
본 발명은 높은 정확도로 우울증을 진단할 수 있다.The present invention can diagnose depression with high accuracy.
도 1 내지 3은 우울증 환자군에서 말초 혈액에서의 3종의 마커 mRNA(ADCY9, CDH12, C9orf24)의 대조군 대비 발현 차이를 나타낸 것이다.Figures 1 to 3 show the differences in expression of three marker mRNAs (ADCY9, CDH12, C9orf24) in peripheral blood in the depression patient group compared to the control group.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 우울증 진단용 마커 조성물에 관한 것이다.The present invention relates to a marker composition for diagnosing depression.
본 발명의 조성물은 CDH12(Cadherin 12) mRNA 또는 단백질을 우울증 진단용 마커로서 활용한다.The composition of the present invention utilizes CDH12 (Cadherin 12) mRNA or protein as a marker for diagnosing depression.
본 발명에서 진단 대상인 우울증은 의욕 저하와 우울감을 주요 증상으로 하여 다양한 인지 및 정신 신체적 증상을 일으켜 일상 기능의 저하를 가져오는 질환을 말한다. 주요우울장애(Major depression disorder)는 최소 2주 이상, 하루 중 대부분의 시간 동안 우울한 기분, 흥미저하, 식욕 및 체중의 변화, 수면장애, 무가치감, 피로, 자살사고 등이 동반될 때 진단되는 것으로, 이러한 우울증의 가장 대표적인 유형으로, 본 발명은 이를 포함한 우울증을 진단하는데 활용될 수 있다.Depression, which is the subject of diagnosis in the present invention, refers to a disease that causes various cognitive and psychosomatic symptoms with low motivation and depression as the main symptoms, leading to a decline in daily functions. Major depressive disorder is diagnosed when a person is accompanied by depressed mood, loss of interest, changes in appetite and weight, sleep disturbance, feelings of worthlessness, fatigue, and suicidal thoughts for most of the day for at least two weeks. This is the most representative type of depression, and the present invention can be used to diagnose depression including this.
본 발명은 본 발명자들이 우울증 환자의 시료에서의 CDH12(Cadherin 12) mRNA 또는 단백질의 발현 수준이 정상 대조군과 유의한 차이가 있음을 확인한 것에 착안한 것이다. 즉, CDH12(Cadherin 12) mRNA 또는 단백질을 우울증 진단용 마커로서 활용할 수 있다.The present invention is based on the fact that the present inventors confirmed that the expression level of CDH12 (Cadherin 12) mRNA or protein in samples from patients with depression was significantly different from that of normal controls. In other words, CDH12 (Cadherin 12) mRNA or protein can be used as a marker for diagnosing depression.
본 발명의 마커 조성물은 C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질을 더 포함할 수 있다.The marker composition of the present invention may further include C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein.
C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질도 그 발현 수준이 우울증 환자에서 정상 대조군 대비 유의한 차이를 보이는 것으로서, CDH12(Cadherin 12) 뿐만 아니라 이들을 마커로서 활용하는 경우에 보다 정확한 우울증 진단이 가능하다.The expression level of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein also shows a significant difference in depression patients compared to normal controls, so more accurate diagnosis of depression is possible when these, as well as CDH12 (Cadherin 12), are used as markers. do.
본 발명의 조성물의 마커들은 개체의 시료 내에 존재하는 것일 수 있다. 시료는 예를 들면 조직, 세포, 혈액, 혈청, 혈장, 타액 또는 뇨 등일 수 있으며, 비침습적이고 간편한 시료 획득의 측면에서 시료는 말초혈액일 수 있다.Markers of the composition of the present invention may be present in a sample of an individual. The sample may be, for example, tissue, cells, blood, serum, plasma, saliva, or urine, and in terms of non-invasive and easy sample acquisition, the sample may be peripheral blood.
또한, 본 발명은 우울증의 진단용 조성물에 대한 것이다.Additionally, the present invention relates to a composition for diagnosing depression.
본 발명의 우울증의 진단용 조성물은 CDH12(Cadherin 12) mRNA 또는 단백질의 발현을 검출하는 제제를 포함한다.The composition for diagnosing depression of the present invention includes an agent that detects the expression of CDH12 (Cadherin 12) mRNA or protein.
전술한 바와 같이, 우울증 환자의 시료에서의 CDH12(Cadherin 12) mRNA 또는 단백질의 발현 수준이 정상 대조군과 유의한 차이가 있으므로, 이를 검출함으로써 우울증의 진단이 가능하다.As described above, since the expression level of CDH12 (Cadherin 12) mRNA or protein in samples from patients with depression is significantly different from that of normal controls, it is possible to diagnose depression by detecting this.
본 발명의 진단 대상인 우울증은 전술한 바와 같다.Depression, the diagnosis subject of the present invention, is as described above.
CDH12(Cadherin 12) mRNA 또는 단백질의 발현을 검출하는 제제는 예를 들면 항체, 압타머, DNA, RNA, PNA, 단백질, 폴리펩티드 등일 수 있다.Agents that detect the expression of CDH12 (Cadherin 12) mRNA or protein may be, for example, antibodies, aptamers, DNA, RNA, PNA, proteins, polypeptides, etc.
또한, 본 발명의 우울증의 진단용 조성물은 C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현을 검출하는 제제를 더 포함할 수 있다.In addition, the composition for diagnosing depression of the present invention may further include an agent for detecting the expression of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein.
전술한 바와 같이, 우울증 환자의 시료에서의 C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현 수준이 정상 대조군과 유의한 차이가 있으므로, 이들을 검출함으로써 보다 정확한 우울증의 진단이 가능하다.As described above, since the expression level of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein in samples from patients with depression is significantly different from that of normal controls, a more accurate diagnosis of depression is possible by detecting these.
C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현을 검출하는 제제는 예를 들면 항체, 압타머, DNA, RNA, PNA, 단백질, 폴리펩티드 등일 수 있다.Agents that detect the expression of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein may be, for example, antibodies, aptamers, DNA, RNA, PNA, proteins, polypeptides, etc.
또한, 본 발명은 우울증의 진단용 키트에 관한 것이다.Additionally, the present invention relates to a kit for diagnosing depression.
본 발명의 키트는 전술한 우울증의 진단용 조성물을 포함한다. 그 외에 그 키트가 이용하는 전술한 마커의 발현량을 측정하는 분석방법에 적합한 하나 이상의 다른 구성 성분 조성물, 용액 또는 장치를 포함할 수 있다.The kit of the present invention includes the composition for diagnosing depression described above. In addition, the kit may include one or more other components, compositions, solutions, or devices suitable for the analysis method used to measure the expression level of the above-mentioned marker.
상기 키트는 상기 마커의 발현량을 측정하기 위한 키트일 경우, RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. RT-PCR 키트는 마커 유전자의 mRNA에 대한 특이적인 각각의 프라이머 쌍 이외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시리보뉴클레오티드(dNTPs), Taq-폴리머라제 및 역전사효소와 같은 효소, DNase, RNase 억제제, DEPC-수(dEPC water), 멸균수 등을 포함할 수 있다. 또한, 정량 대조군으로 사용되는 유전자에 특이적인 프라이머 쌍을 포함할 수 있다.When the kit is for measuring the expression level of the marker, it may be a kit containing the essential elements necessary to perform RT-PCR. In addition to each pair of primers specific for the mRNA of the marker gene, the RT-PCR kit also contains test tubes or other suitable containers, reaction buffer, deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, and RNase. It may include inhibitors, DEPC-water (dEPC water), sterilized water, etc. Additionally, it may include a pair of primers specific to the gene used as a quantitative control.
상기 키트는 상기 마커의 뉴클레오티드 서열, 상기 뉴클레오티드 서열에 상보적인 서열, 상기 뉴클레오티드의 단편 또는 상기 뉴클레오티드 서열에 의해 코딩되는 단백질에 특이적으로 결합하는 물질의 면역학적 검출을 위하여 기질, 적합한 완충용액, 발색 효소 또는 형광물질로 표지된 2차 항체, 발색 기질을 포함할 수 있다. 상기 기질은 니트로셀룰로오스 막, 폴리비닐 수지로 합성된 96 웰 플레이트, 폴리스티렌 수지로 합성된 96 웰 플레이트 및 유리 슬라이드 글라스 등이 이용될 수 있고, 발색효소는 퍼옥시다아제(peroxidase), 알칼라인 포스파타아제(alkaline phosphatase)가 사용될 수 있고, 형광물질은 FITC, RITC 등이 사용될 수 있고, 발색 기질은 2,2'-아지노-비스(3-에틸벤조티아졸린-6-설폰산)(ABTS) 또는 o-페닐렌디아민(OPD), 테트라메틸 벤지딘(TMB) 등이 사용될 수 있다.The kit includes a substrate, a suitable buffer solution, and a color development for the immunological detection of a substance that specifically binds to the nucleotide sequence of the marker, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide, or a protein encoded by the nucleotide sequence. It may include a secondary antibody labeled with an enzyme or a fluorescent substance, or a chromogenic substrate. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass, and the coloring enzyme may be peroxidase, alkaline phosphatase ( alkaline phosphatase) can be used, the fluorescent substance can be FITC, RITC, etc., and the chromogenic substrate is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or o- Phenylenediamine (OPD), tetramethyl benzidine (TMB), etc. can be used.
상기 키트는 상기 마커의 발현량을 측정할 수 있는 마이크로어레이일 수 있다. 상기 마이크로어레이는 상기 지표인자를 이용하여 당해 기술분야에 공지된 방법에 따라 당업자가 용이하게 제조할 수 있으며, 일 구체예에 따르면 상기 마커 mRNA 또는 그의 단편에 해당하는 서열의 cDNA가 프로브로서 기판에 부착되어 있는 마이크로어레이일 수 있다.The kit may be a microarray capable of measuring the expression level of the marker. The microarray can be easily manufactured by a person skilled in the art according to methods known in the art using the indicator factors, and according to one embodiment, cDNA of a sequence corresponding to the marker mRNA or a fragment thereof is applied to the substrate as a probe. It may be an attached microarray.
또한, 본원의 키트는 마커 성분에 특이적으로 결합하는 항체, 기질과의 반응에 의해서 발색하는 표지체가 접합된 2차 항체 접합체(conjugate), 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 효소반응 정지용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있으나, 이에 제한되지 않을 수 있다.In addition, the kit of the present application includes an antibody that specifically binds to the marker component, a secondary antibody conjugate conjugated with a label that develops color by reaction with the substrate, a color-generating substrate solution for color development with the label, a washing solution, and an enzyme. It may contain a reaction stopping solution, etc., and may be manufactured into a number of separate packaging or compartments containing the reagent components used, but may not be limited thereto.
본원의 키트는 환자 시료 내 상기 마커의 발현 수준을 측정할 수 있는 제제뿐만 아니라, 발현 수준 분석에 적합한 한 종류 이상의 조성물, 용액 또는 장치를 포함할 수 있다. 예를 들어, 상기 키트는 항체의 면역학적 검출을 위하여 기질, 적당한 완충용액, 검출 라벨로 표지된 2차 항체, 및 발색 기질 등을 포함할 수 있으나, 이에 제한되지 않을 수 있다.The kit herein may include not only an agent capable of measuring the expression level of the marker in a patient sample, but also one or more types of compositions, solutions, or devices suitable for expression level analysis. For example, the kit may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a detection label, and a chromogenic substrate for immunological detection of an antibody, but may not be limited thereto.
구체적인 일례로, 상기 키트는 ELISA 키트, 샌드위치 ELISA등 다양한 ELISA 방법을 구현하기 위하여, ELISA를 수행하기 위해 필요한 필수 요소를 포함하는 것을 특징으로 하는 키트일 수 있다. 이러한 ELISA 키트는 상기 단백질들에 대한 특이적인 항체를 포함한다. 항체는 상기 마커에 대한 특이성 및 친화성이 높고 다른 단백질에 교차 반응성이 거의 없는 항체로, 모노클로날 항체, 폴리클로날 항체 또는 재조합 항체일 수 있다. 또한 ELISA 키트는 대조군 단백질에 특이적인 항체를 포함할 수 있다. 그 외 ELISA 키트는 결합된 항체를 검출할 수 있는 시약, 예를 들면, 표지된 2차 항체, 발색단(chromopores), 효소 및 그의 기질 또는 항체와 결합할 수 있는 다른물질 등을 포함할 수 있으나, 이에 제한되지 않을 수 있다.As a specific example, the kit may be a kit characterized by including essential elements required to perform ELISA in order to implement various ELISA methods such as ELISA kit and sandwich ELISA. These ELISA kits contain specific antibodies against the above proteins. The antibody has high specificity and affinity for the marker and has little cross-reactivity to other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. Additionally, ELISA kits may include antibodies specific for control proteins. In addition, ELISA kits may include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes and their substrates, or other substances that can bind to antibodies. It may not be limited to this.
이 외에도, 상기 키트는 웨스턴 블롯, 면역침전분석법, 보체 고정 분석법, 유세포분석, 또는 단백질 칩 등을 구현하기 위한 키트일 수 있으며, 각 분석 방법에 적합한 부가적인 구성을 추가로 포함할 수 있다. 이 분석 방법들을 통하여, 항원-항체 복합체 형성량을 비교함으로써 우울증을 진단할 수 있다.In addition, the kit may be a kit for implementing Western blot, immunoprecipitation analysis, complement fixation analysis, flow cytometry, or protein chip, and may further include additional components suitable for each analysis method. Through these analysis methods, depression can be diagnosed by comparing the amount of antigen-antibody complex formation.
또한, 본 발명은 우울증의 진단을 위한 정보 제공 방법에 관한 것이다.Additionally, the present invention relates to a method of providing information for diagnosing depression.
본 발명의 우울증의 진단을 위한 정보 제공 방법은 개체로부터 분리된 시료에서 CDH12(Cadherin 12) mRNA 또는 단백질의 발현을 검출하는 단계를 포함한다.The method of providing information for diagnosing depression of the present invention includes detecting the expression of CDH12 (Cadherin 12) mRNA or protein in a sample isolated from an individual.
개체는 우울증의 보유 여부의 확인이 필요한 개체이다. 이는 인간을 포함한 포유류일 수 있고, 인간일 수 있다. The entity is an entity that requires confirmation of whether or not it has depression. This may be a mammal, including humans, or it may be a human.
시료는 예를 들면 조직, 세포, 혈액, 혈청, 혈장, 타액 또는 뇨 등일 수 있고, 비침습적인 획득의 용이성 측면에서 시료는 말초혈액 일 수 있다.The sample may be, for example, tissue, cells, blood, serum, plasma, saliva, or urine, and in terms of ease of non-invasive acquisition, the sample may be peripheral blood.
mRNA 또는 단백질 발현의 검출은 당 분야에 공지된 방법에 의할 수 있다.Detection of mRNA or protein expression can be done by methods known in the art.
예를 들면 상기 mRNA의 시료 내 농도를 측정하는 방법으로서 역전사효소 중합효소반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(Competitive RT-PCR), 실시간 역전사효소 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블롯팅 (Northern blotting) 및 DNA 칩 등이 있으나, 이에 제한되는 것은 아니다. 상기 단백질의 시료 내 농도를 측정하는 방법으로서 상기 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 단백질의 양을 확인할 수 있다. 이를 위한 분석 방법으로는 면역탁본검사, 샌드위치 측정법(sandwich assay), ELISA(enzyme linked immunosorbent assay), 방사선면역분석(RIA: Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(rocket) 면역전기영동, 조직면역염색(immunohistochemistry), 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), FACS(fluorescence-activated cell sorting), 웨스턴 블롯팅, 유체 세포 측정법(flow cytometry), 효소기질발색법, 항원-항체 응집법 및 단백질 칩(protein chip) 등이 있고, 바람직하게는 ELISA(enzyme linked immunosorbent assay)를 이용할 수 있으나, 이에 제한되는 것은 아니다.For example, methods for measuring the concentration of the mRNA in the sample include reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (Competitive RT-PCR), and real-time reverse transcriptase polymerase reaction (Real-time RT- PCR), RNase protection assay (RPA), Northern blotting and DNA chip, etc., but are not limited thereto. As a method of measuring the concentration of the protein in a sample, the amount of the protein can be confirmed using an antibody that specifically binds to the protein. Analysis methods for this include immunorubbing test, sandwich assay, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, and Ouchterlony immunodiffusion method. , rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting (FACS), Western blotting, fluid cytometry ( flow cytometry, enzyme substrate color development method, antigen-antibody agglutination method, and protein chip, etc. Preferably, ELISA (enzyme linked immunosorbent assay) can be used, but is not limited to this.
본 발명의 방법은 CDH12(Cadherin 12) mRNA 또는 단백질의 발현량이 대조군 대비 유의한 차이가 있으면, 상기 개체가 대조군 대비 우울증에 걸렸을 가능성이 더 높다는 정보를 제공하는 단계를 더 포함할 수 있다.The method of the present invention may further include providing information that if there is a significant difference in the expression level of CDH12 (Cadherin 12) mRNA or protein compared to the control group, the subject is more likely to have depression compared to the control group.
대조군은 예를 들면 우울증을 보유하지 않는 대조군(정상 대조군)일 수 있다. 그 비교가 되는 값은 대조군 1 개체에서의 동일 시료에서의 발현량, 대조군 복수개체의 동일 시료에서의 평균값 등이 될 수 잇으나, 대조군에서의 발현량을 대표할 수 있는 값이라면 제한되지 않고 사용할 수 있다.The control group may be, for example, a control group that does not have depression (normal control group). The value to be compared can be the expression level in the same sample from one control subject, the average value in the same sample from multiple control subjects, etc., but can be used without limitation as long as it is a value that can represent the expression level in the control group. You can.
우울증 환자에서의 CDH12(Cadherin 12) mRNA 또는 단백질의 발현량은 대조군과 유의한 차이가 난다. 예를 들면 시료가 말초혈액인 경우, CDH12(Cadherin 12) mRNA 또는 단백질의 발현량이 대조군 대비 적으면, 상기 개체가 대조군 대비 우울증에 걸렸을 가능성이 더 높다는 정보를 제공할 수 있다.The expression level of CDH12 (Cadherin 12) mRNA or protein in patients with depression is significantly different from that in the control group. For example, when the sample is peripheral blood, if the expression level of CDH12 (Cadherin 12) mRNA or protein is lower than that of the control group, information may be provided that the individual is more likely to have depression compared to the control group.
본 발명의 방법은 상기 시료에서 C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현을 검출하는 단계를 더 포함할 수 있다.The method of the present invention may further include the step of detecting the expression of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein in the sample.
C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현의 검출은 앞서 예시한 방법대로 수행될 수 있다.Detection of expression of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein can be performed according to the method exemplified above.
본 발명의 방법은 C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질의 발현량이 대조군 대비 유의한 차이가 있으면, 상기 개체가 대조군 대비 우울증에 걸렸을 가능성이 더 높다는 정보를 제공하는 단계를 더 포함할 수 있다.The method of the present invention may further include providing information that if there is a significant difference in the expression level of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein compared to the control group, the subject is more likely to have depression compared to the control group. there is.
우울증 환자에서의 C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질은 그 발현량이 대조군과 유의한 차이가 난다. 예를 들면 시료가 말초혈액인 경우, C9orf24(Chromosome 9 Open Reading Frame 24) mRNA 또는 단백질이 대조군 대비 적으면, 상기 개체가 대조군 대비 우울증에 걸렸을 가능성이 더 높다는 정보를 제공할 수 있다.The expression level of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein in patients with depression is significantly different from that in the control group. For example, if the sample is peripheral blood, if C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein is low compared to the control group, it can provide information that the individual is more likely to have depression compared to the control group.
실시예. 우울증 환자의 말초 혈액에서의 3종 마커의 대조군과의 발현량 차이Example. Differences in expression levels of three types of markers in the peripheral blood of patients with depression compared to the control group
화순전남대병원 정신건강의학과에서 건강한 성인 15명(control)과 우울증(Major Depression Disorder, MDD)으로 확인된 15명의 말초혈액에서 RNA를 추출하여 3종의 마커 mRNA의 발현 수준의 차이를 조사하였다.At the Department of Psychiatry at Chonnam National University Hwasun Hospital, RNA was extracted from the peripheral blood of 15 healthy adults (control) and 15 people confirmed to have depression (Major Depression Disorder, MDD), and differences in the expression levels of three types of marker mRNA were investigated.
실험에 사용된 샘플은 설문조사를 통해 선별된 대조군 15명, 우울증 환자군 15명의 말초혈액으로 화순 전남대학교병원 정신건강의학과로부터 제공받았다. 말초혈액은 EDTA 진공 채혈관에 담겨 운반되었고, 약 1 mL씩 EP tube에 분주하여 실험 전까지 -80℃에 보관하였다. 표 1은 대조군 샘플 목록, 표 2는 우울증 환자 샘플 목록이다.The samples used in the experiment were the peripheral blood of 15 control subjects and 15 depression patients selected through a survey and were provided by the Department of Psychiatry at Chonnam National University Hospital in Hwasun. Peripheral blood was transported in an EDTA vacuum collection tube, and approximately 1 mL was dispensed into EP tubes and stored at -80°C until the experiment. Table 1 is a list of control samples, and Table 2 is a list of depression patient samples.
대조군 (n=15) 및 우울증 환자군 (n=15)의 전혈로부터 RNA를 추출하였다. 실험 방법은 다음과 같다.RNA was extracted from whole blood of the control group (n=15) and the depression patient group (n=15). The experimental method is as follows.
1) 300 μL의 전혈에 TRIzol® LS Reagent (Invitrogen, CA, USA) 700 μL를 분주하고 파이펫팅을 통해 용액이 섞이도록 한다.1) Dispense 700 μL of TRIzol® LS Reagent (Invitrogen, CA, USA) into 300 μL of whole blood and mix the solutions through pipetting.
2) Chloroform (Sigma-Aldrich, St. Louis, MO, US) 200 μL를 분주하고 약 10초 간 voltexing 후 실온에서 3분간 incubation 해준다.2) Dispense 200 μL of Chloroform (Sigma-Aldrich, St. Louis, MO, US), voltex for about 10 seconds, and incubate for 3 minutes at room temperature.
3) 원심분리 (12,000 xg, 15분, 4℃)3) Centrifugation (12,000 xg, 15 minutes, 4℃)
4) 상층의 투명층을 새로운 EP tube에 옮겨 담는다. 4) Transfer the upper transparent layer to a new EP tube.
5) Isopropanol (Sigma-Aldrich, St. Louis, MO, US)을 투명층과 동량 넣어주고 파이펫팅을 통해 용액이 잘 섞이도록 한다.5) Add the same amount of Isopropanol (Sigma-Aldrich, St. Louis, MO, US) as the transparent layer and mix the solution well through pipetting.
6) 실온에서 30분간 incubation6) Incubation for 30 minutes at room temperature
7) 원심분리 (12,000 xg, 15분, 4℃)7) Centrifugation (12,000 xg, 15 minutes, 4℃)
8) Pellet의 생성을 확인하고, 상층액을 제거한다.8) Confirm the formation of pellets and remove the supernatant.
9) 70 % 에탄올을 1 mL 넣어주고 파이펫팅 해준다.9) Add 1 mL of 70% ethanol and pipette.
10) 원심분리 (7,500 xg, 10분, 4℃)10) Centrifugation (7,500 xg, 10 minutes, 4℃)
11) 상층액을 완전히 제거해주고, 클린 벤치에서 약 15~20분간 pellet을 말려준다.11) Completely remove the supernatant and dry the pellet on a clean bench for about 15 to 20 minutes.
12) RNase free water를 20 μL 분주하여 파이펫팅을 통해 잘 섞어준다. 12) Dispense 20 μL of RNase free water and mix well by pipetting.
추출된 RNA는 NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) 기기를 사용하여 농도를 측정하였다.The concentration of extracted RNA was measured using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
200 ng의 total RNA 로부터 GoScript™ Reverse Transcription System (Promega, Madison, USA)키트를 사용하여 cDNA를 합성하였고, 타겟 유전자인 ADCY9, CDH12, C9orf24를 대상으로 SYBR Green assay를 사용하여 QuantStudio 5 Real-Time PCR Instrument(Thermo Scientific, Wilmington, DE, USA) 기기로 실험을 진행하였다 (in duplicate). Multiple plate들의 Threshold amplification cycle number data는 QuantStudio Design and Analysis Software (v1.2.x) and the delta-Ct method를 사용하여 결과 값을 모아 분석하였다.cDNA was synthesized from 200 ng of total RNA using the GoScript™ Reverse Transcription System (Promega, Madison, USA) kit, and QuantStudio 5 Real-Time PCR was performed using SYBR Green assay for the target genes ADCY9, CDH12, and C9orf24. The experiment was conducted with Instrument (Thermo Scientific, Wilmington, DE, USA) (in duplicate). Threshold amplification cycle number data from multiple plates were collected and analyzed using QuantStudio Design and Analysis Software (v1.2.x) and the delta-Ct method.
실험에 사용된 프라이머의 시퀀스는 아래 표 3과 같다.The sequences of primers used in the experiment are shown in Table 3 below.
통계적 분석은 GraphPad Prism software (v8.0.1)를 사용하였다. 그룹 별 비교를 위해 Unpaired t-test를 사용하였고, P-value < 0.05 를 통계적으로 유의한 것이라고 고려하였다Statistical analysis was performed using GraphPad Prism software (v8.0.1). Unpaired t-test was used to compare groups, and P-value < 0.05 was considered statistically significant.
도 1 내지 3을 참고하면, 우울증 환자군에서 3종의 마커 mRNA의 발현이 대조군 대비 유의하게 낮은 것을 확인할 수 있다.Referring to Figures 1 to 3, it can be seen that the expression of three types of marker mRNA in the depression patient group was significantly lower than that in the control group.
서열목록 전자파일 첨부Sequence list electronic file attached
Claims (14)
A marker composition for diagnosing depression comprising CDH12 (Cadherin 12) mRNA or protein.
The marker composition for diagnosing depression according to claim 1, further comprising C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein.
The marker composition for diagnosing depression according to claim 1, wherein the depression is major depressive disorder.
A composition for diagnosing depression comprising an agent for detecting the expression of CDH12 (Cadherin 12) mRNA or protein.
The composition for diagnosing depression according to claim 4, wherein the agent is an antibody, aptamer, DNA, RNA, PNA, protein, or polypeptide.
The composition for diagnosing depression according to claim 4, further comprising an agent for detecting the expression of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein.
The composition for diagnosing depression according to claim 4, wherein the depression is major depressive disorder.
A kit for diagnosing depression, comprising the composition for diagnosing depression according to any one of claims 4 to 7.
A method of providing information for the diagnosis of depression, comprising the step of detecting the expression of CDH12 (Cadherin 12) mRNA or protein in a sample isolated from an individual.
The method of claim 9, wherein the sample is peripheral blood.
The method of claim 9, wherein when the expression level of CDH12 (Cadherin 12) mRNA or protein is low compared to the control group, the method provides information that the individual is more likely to have depression compared to the control group.
The method of claim 9, further comprising detecting the expression of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein in the sample.
The method of claim 12, wherein the sample is peripheral blood, and if the expression level of C9orf24 (Chromosome 9 Open Reading Frame 24) mRNA or protein is low compared to the control group, the sample is a depression test that provides information that the individual is more likely to have depression compared to the control group. How to provide information for diagnosis.
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