KR102314971B1 - Method for providing information of prediction and diagnosis of obesity using methylation level of LZTS3 gene and composition therefor - Google Patents

Method for providing information of prediction and diagnosis of obesity using methylation level of LZTS3 gene and composition therefor Download PDF

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KR102314971B1
KR102314971B1 KR1020200009645A KR20200009645A KR102314971B1 KR 102314971 B1 KR102314971 B1 KR 102314971B1 KR 1020200009645 A KR1020200009645 A KR 1020200009645A KR 20200009645 A KR20200009645 A KR 20200009645A KR 102314971 B1 KR102314971 B1 KR 102314971B1
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황진택
최효경
정상원
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Abstract

본 발명의 비만 특이적 마커 유전자의 메틸화를 검출함으로써 비만의 발병을 조기에 예측 및 진단할 수 있다. 본 발명의 LZTS3 메틸화 바이오마커는 비만의 위험성 평가, 진단 및 치료 타겟의 선정에도 활용될 수 있다.By detecting the methylation of the obesity-specific marker gene of the present invention, the onset of obesity can be predicted and diagnosed early. The LZTS3 methylation biomarker of the present invention can also be utilized for risk assessment of obesity, diagnosis, and selection of a treatment target.

Description

LZTS3 유전자의 메틸화 수준을 이용한 비만의 예측 또는 진단을 위한 정보제공방법 및 이를 위한 조성물{Method for providing information of prediction and diagnosis of obesity using methylation level of LZTS3 gene and composition therefor}TECHNICAL FIELD [0001] Method for providing information of prediction and diagnosis of obesity using methylation level of LZTS3 gene and composition therefor

본 발명은 LZTS3 (leucine zipper tumor suppressor family member 3) 유전자의 메틸화 수준을 이용한 비만의 예측 또는 진단을 위한 정보제공방법 및 이를 위한 조성물에 관한 것으로, 구체적으로는 LZTS3 유전자의 메틸화 수준을 이용한 비만의 예측 또는 진단을 위한 정보제공방법, 비만의 예측 또는 진단용 조성물 및 키트에 관한 것이다.The present invention relates to a method for predicting or diagnosing obesity using the methylation level of the leucine zipper tumor suppressor family member 3 ( LZTS3 ) gene, and to a composition therefor, and more particularly, prediction of obesity using the methylation level of the LZTS3 gene Or it relates to a method for providing information for diagnosis, a composition and kit for predicting or diagnosing obesity.

비만은 그 자체의 위험성보다 비만으로 인해 유발될 수 있는 여러 합병증 때문에 그 심각성이 더욱 크게 인식되고 있다. 비만은 고혈압, 고지혈증, 당뇨병 등의 대사증후군, 지방간, 관절 이상, 및 암 발병 위험을 증가시킨다고 알려져 있다. 2010년 세계보건기구에서 발표한 바에 따르면, 정상 체중인 사람에 비해 비만인 사람에게서 고혈압, 당뇨병, 이상지질혈증이 동반될 위험이 2배 이상(고혈압 2.5배, 당뇨병 2배, 고콜레스테롤혈증 2.3배, 고중성지질혈증 2.4배) 높게 나타났다. 상기 질환들 이외에도 여성의 경우 체지방이 과다하면 성호르몬의 균형이 깨져 심한 경우 불임증을 초래할 수 있고, 자궁내막암과 유방암의 위험성이 높아진다고 알려져 있다. 더불어 비만은 신체적인 질환뿐 만 아니라 사회적 고립감이나 소외, 자신감 결여, 및 우울감과 같은 정신적 질환을 유발할 수 있으므로 비만의 예방 및 치료에 대한 필요성은 매우 중요하게 인식되고 있다.The seriousness of obesity is being recognized more because of the various complications that can be caused by obesity than the risk itself. Obesity is known to increase the risk of high blood pressure, hyperlipidemia, metabolic syndrome such as diabetes, fatty liver, joint abnormalities, and cancer. According to the World Health Organization (WHO) report in 2010, the risk of high blood pressure, diabetes, and dyslipidemia is more than doubled in obese people compared to people of normal weight (hypertension 2.5 times, diabetes 2 times, hypercholesterolemia 2.3 times, hypertriglyceridemia 2.4 times). In addition to the above diseases, it is known that excessive body fat in women can cause infertility in severe cases due to the imbalance of sex hormones, and increase the risk of endometrial cancer and breast cancer. In addition, since obesity can cause not only physical diseases but also mental diseases such as social isolation or alienation, lack of self-confidence, and depression, the need for prevention and treatment of obesity is recognized as very important.

비만의 발병은 유전적 요인, 환경적 요인, 그리고 식이요인이 복합적으로 작용하여 일어나는데, 식이요인 중에서는 고지방식이(high-fat diet, HFD)로 대표되는 고열량식이가 비만의 주요 원인으로 손꼽힌다. 잘못된 식습관으로 인해 비만해지면 내당불내성, 고혈압, 고지혈증과 같은 증상으로 대표되는 대사증후군이 생길 가능성이 높아지며, 비알코올성 지방간의 발병 위험도 함께 증가한다.The onset of obesity is caused by a combination of genetic factors, environmental factors, and dietary factors. . If you become obese due to bad eating habits, you are more likely to develop metabolic syndrome, which is represented by symptoms such as glucose intolerance, high blood pressure, and hyperlipidemia, and the risk of developing nonalcoholic fatty liver also increases.

대사증후군과 비알코올성 간질환은 일반적으로 인슐린 저항성(insulin resistance)과 연관이 있는 것으로 알려져 있지만 아직 명확한 원인 기전은 밝혀지지 않았다. 특히 간은 탄수화물·지질·단백질의 합성, 분해, 저장 등 대사의 중심이 되는 기관으로, 비만한 경우 간 조직에서 인슐린 저항성이 나타나고 간의 탄수화물 및 지질대사가 변화하며, 결과적으로 비알코올성 지방간의 위험이 높아진다고 알려져 있다. 뿐만 아니라 간에 지방이 축적되면 간Metabolic syndrome and nonalcoholic liver disease are generally known to be associated with insulin resistance, but the exact etiological mechanism has not yet been elucidated. In particular, the liver is an organ that is central to metabolism, such as synthesis, breakdown, and storage of carbohydrates, lipids, and proteins. In the case of obesity, insulin resistance appears in the liver tissue, and the carbohydrate and lipid metabolism of the liver changes, and consequently, the risk of nonalcoholic fatty liver is increased. is known to increase. In addition, when fat accumulates in the liver, the liver

조직에서 염증성 지표들이 높아진다고 보고되고 있다.It has been reported that inflammatory markers are elevated in tissues.

최근에는 후성유전학적 연구 방법 및 기술이 발전함에 따라, 비만으로 인해 간 조직에서 대사 교란이 일어나고 염증반응이 증가하는 원인을 후성유전학 차원에서 규명하고자 하는 연구들이 증가하고 있다. 후성유전적 변화는 DNA 염기 서열의 변화 없이 유전자 발현을 조절할 수 있으며, 가역적이고 대를 이어 유전되는 특징을 가진다. 특히, DNA 메틸화는 후성유전학적 변화의 주요 기전으로서, 식이요인이 건강에 미치는 영향을 밝히기 위해 많은 연구자들이 이에 관심을 가지고 연구하고 있다. 고지방식이가 DNA 메틸화 패턴 변화에 미치는 영향에 대한 연구는 간 조직을 비롯하여 지방조직과 근육 조직을 대상으로 이루어지고 있다. 고지방식이가 DNA 메틸화 변화에 미치는 영향을 개별 유전자 수준에서 분석한 연구가 대다수이며 high-throughput 방법을 통한 전장 유전체 수준(genome-wide)의 연구는 매우 부족한 실정이다. Recently, with the development of epigenetic research methods and technologies, studies are increasing in epigenetics to identify the cause of metabolic disturbance and increased inflammatory response in liver tissue due to obesity. Epigenetic changes can regulate gene expression without changing the DNA base sequence, and have reversible and inherited characteristics. In particular, DNA methylation is a major mechanism of epigenetic change, and many researchers are studying it with interest to elucidate the effect of dietary factors on health. Studies on the effect of a high-fat diet on changes in DNA methylation patterns are being conducted on adipose tissue and muscle tissue as well as liver tissue. Most studies have analyzed the effect of a high-fat diet on DNA methylation changes at the level of individual genes, and studies at the genome-wide level through high-throughput methods are very scarce.

임신 기간부터 시작하여 성인기까지 지속적으로 고지방식이를 섭취한 경우의 DNA 메틸화 패턴 변화를 전장 유전체 수준에서 분석한 연구가 있지만, DNA 메틸화 패턴은 임신/수유 기간뿐만 아니라 성인기에도 영향을 받을 수 있기 때문에 성인기에 고지방식이를 섭취하는 것이 전장 유전체 차원의 DNA 메틸화 패턴 변화에 미치는 영향 역시 연구가 필요하다.Although there are studies that analyzed changes in DNA methylation pattern at the whole genome level when a high-fat diet is consumed continuously from pregnancy to adulthood, DNA methylation pattern can be affected not only during pregnancy/lactation but also in adulthood. The effect of high-fat diet intake in adulthood on changes in DNA methylation patterns at the genome-wide level also needs to be studied.

본 발명자들은 비만의 예측 또는 진단을 위한 특이적 메틸화 바이오마커를 발굴하기 위해 노력하였다. 그 결과, 정상군과 비만군의 시료에 대한 유전체 메틸화 분석을 수행하여, 두 그룹 간에 메틸화 수준에 현저한 차이가 있는 LZTS3 (leucine zipper tumor suppressor family member 3) 유전자의 엑손 부위를 확인하고, 해당 부위에 대한 DNA 메틸화 시퀀싱을 수행하여 비만군에서 상기 부위의 메틸화 수준 감소가 있음을 규명함으로써 본 발명을 완성하였다. The present inventors tried to discover specific methylation biomarkers for the prediction or diagnosis of obesity. As a result, by performing genome methylation analysis on samples from the normal group and the obese group, the exon region of the leucine zipper tumor suppressor family member 3 ( LZTS3) gene, which has a marked difference in methylation level between the two groups, was identified, and the By performing DNA methylation sequencing, the present invention was completed by confirming that there was a decrease in the methylation level of the site in the obese group.

따라서, 본 발명의 목적은 비만의 예측 또는 진단용 조성물을 제공하는데 있다. Accordingly, an object of the present invention is to provide a composition for predicting or diagnosing obesity.

본 발명의 다른 목적은 비만의 예측 또는 진단용 키트를 제공하는데 있다. Another object of the present invention is to provide a kit for prediction or diagnosis of obesity.

본 발명의 또 다른 목적은 비만의 예측 또는 진단을 위한 정보를 제공하는 방법을 제공하는데 있다.Another object of the present invention is to provide a method for providing information for prediction or diagnosis of obesity.

본 발명의 일 양태에 따르면 LZTS3 (leucine zipper tumor suppressor family member 3) 유전자의 메틸화 여부를 검출할 수 있는 물질을 포함하는 비만의 예측 또는 진단용 조성물에 관한 것이다. According to one aspect of the present invention, there is provided a composition for predicting or diagnosing obesity, which includes a substance capable of detecting whether or not methylation of a leucine zipper tumor suppressor family member 3 ( LZTS3) gene is present.

본 발명자들은 비만의 예측 또는 진단을 위한 특이적 메틸화 바이오마커를 발굴하기 위해 노력하였고 그 결과, 정상군과 비만군의 시료에 대한 유전체 메틸화 분석을 수행하여, 두 그룹 간에 메틸화 수준에 현저한 차이가 있는 LZTS3 유전자의 엑손 부위를 확인하고, 해당 부위에 대한 DNA 메틸화 시퀀싱을 수행하여 비만군에서 상기 부위의 메틸화 수준 감소가 있음을 규명하였다. The present inventors tried to discover a specific methylation biomarker for prediction or diagnosis of obesity, and as a result, genome methylation analysis was performed on samples from the normal group and the obese group, and LZTS3 with a significant difference in methylation level between the two groups The exon region of the gene was identified, and DNA methylation sequencing was performed on the region to confirm that there was a decrease in the methylation level of the region in the obese group.

본 발명의 비만 특이적 메틸화 마커 유전자는 비만 위험성 평가, 예측, 진단 및 치료 타겟의 선정에도 사용될 수 있다. The obesity-specific methylation marker gene of the present invention may also be used for obesity risk assessment, prediction, diagnosis, and selection of a treatment target.

본 발명에 있어서, 상기 메틸화는 LZTS3 유전자의 프로모터 부위, 5' 비전사 영역(5' untranslated region), 인트론 및 엑손 중 어느 한 부위에 위치하는 CpG의 메틸화를 측정하는 것일 수 있고, 예를 들어, 서열번호 1로 표시되는 영역의 CpG 의 메틸화를 측정하는 것일 수 있다.In the present invention, the methylation may be to measure the methylation of CpG located in any one of the promoter region, 5' untranslated region, intron and exon of the LZTS3 gene, for example, It may be to measure CpG methylation in the region represented by SEQ ID NO: 1.

포유류 세포의 게놈 DNA에는 A, C, G, T 외에 시토신 환의 5번째 탄소에 메틸기가 붙은 5-메틸시토신 (5-mC)이 있다. 5-mC는 항상 CG 다이뉴클레오타이드의 C에만 오며 (5'-mCG-3'), 이러한 CG를 흔히 CpG라고 표시한다. CpG의 C는 대부분이 메틸기가 붙어서 메틸화 되어있다. 이러한 CpG의 메틸화는 알루 (alu)나 전이인자 (transposon)와 같이 게놈 내에 반복되는 염기서열 (repetitive sequence)이 발현되지 못하도록 억제하며, 포유류 세포에서 유전자 외 변화가 가장 흔히 나타나는 부위이다. In the genomic DNA of mammalian cells, in addition to A, C, G, and T, there is 5-methylcytosine (5-mC) with a methyl group attached to the 5th carbon of the cytosine ring. 5-mC always occurs only at C of a CG dinucleotide (5'-mCG-3'), and this CG is often denoted as CpG. Most of C in CpG is methylated with a methyl group attached to it. This methylation of CpG inhibits the expression of repetitive sequences in the genome, such as alu or transposon, and is the site where extragenetic changes most frequently occur in mammalian cells.

본 발명에서, 상기 메틸화 여부를 검출할 수 있는 물질은 상기 메틸화된 부위를 포함하는 단편을 증폭할 수 있는 프라이머 쌍, 상기 메틸화된 부위와 혼성화할 수 있는 프로브, 상기 메틸화된 부위와 결합할 수 있는 메틸화 특이적 결합 단백질, 메틸화 특이적 결합 항체 또는 압타머, 시퀀싱 프라이머, 시퀀싱 바이 신세시스 프라이머 및 시퀀싱 바이 라이게이션 프라이머로 구성된 군에서 선택된 어느 하나 이상일 수 있다. In the present invention, the substance capable of detecting whether the methylation is present is a primer pair capable of amplifying a fragment including the methylated site, a probe capable of hybridizing with the methylated site, and a substance capable of binding to the methylated site It may be any one or more selected from the group consisting of a methylation specific binding protein, a methylation specific binding antibody or aptamer, a sequencing primer, a sequencing-by-synthesis primer, and a sequencing-by-ligation primer.

상기 메틸화는 바이설파이트 시퀀싱, 시퀀싱 바이 신세시스 (sequencing by synthesis), 시퀀싱 바이 라이게이션 (sequencing by ligation), 메틸화 특이 PCR (methylation specific PCR), 실시간 메틸화 특이 PCR (real time methylation specific PCR), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 메틸화 DNA 특이적 결합 항체 또는 압타머를 이용한 PCR 또는 핵산 칩 등의 방법에 의하여 검출되는 것일 수 있으나, 이에 제한되지 않는다.The methylation may be performed by bisulfite sequencing, sequencing by synthesis, sequencing by ligation, methylation specific PCR, real time methylation specific PCR, or methylated DNA. It may be detected by a method such as PCR using a specific binding protein, PCR using a methylated DNA specific binding antibody or an aptamer, or a nucleic acid chip, but is not limited thereto.

바이설파이트 시퀀싱 방법은 메틸화 CpG를 함유한 핵산을 검출하는 방법으로, 핵산을 함유한 시료를 비메틸화 시토신을 변형시키는 제제와 접촉시키는 단계 및 메틸화-비의존적 올리고뉴클레오티드 프라이머를 사용하여 시료의 CpG-함유 핵산을 증폭시키는 단계를 포함한다. 상기 비메틸화 시토신을 변형시키는 제제는 바이설파이트일 수 있다. 바이설파이트 (bisulfite)가 처리된 DNA 상의 비메틸화된 (unmethylated) 시토신 (C) 염기는 탈아민화 (deamination)되어 우라실(U) 염기로 바뀌는 반면, 메틸화된 시토신 염기는 그대로 시토신으로 남아있게 된다. 즉, 바이설파이트 처리에 의해 DNA 상에서 시토신 염기의 메틸화 유무에 따라 서로 구별할 수 있도록 다른 염기로 표지된다. 상기 올리고뉴클레오티드 프라이머는 변형된 메틸화 및 비메틸화 핵산을 구별하지 않고 핵산을 증폭하는 것을 특징으로 할 수 있다. 상기 증폭된 산물을 시퀀싱 프라이머를 이용하여 Sanger 방법으로 시퀀싱하거나 차세대 시퀀싱 (next generation sequencing, NGS) 방법으로 분석하여 메틸화 핵산을 검출할 수 있다.The bisulfite sequencing method is a method for detecting a nucleic acid containing methylated CpG, comprising the steps of contacting a sample containing the nucleic acid with an agent that modifies unmethylated cytosine and using a methylation-independent oligonucleotide primer to obtain CpG- and amplifying the containing nucleic acid. The agent that modifies the unmethylated cytosine may be a bisulfite. The unmethylated (unmethylated) cytosine (C) base on the bisulfite-treated DNA is deamination to change to the uracil (U) base, while the methylated cytosine base remains as cytosine. That is, they are labeled with different bases so that they can be distinguished from each other according to the presence or absence of methylation of cytosine bases on DNA by bisulfite treatment. The oligonucleotide primer may be characterized in that it does not discriminate between modified methylated and unmethylated nucleic acids and amplifies nucleic acids. The methylated nucleic acid can be detected by sequencing the amplified product by the Sanger method using a sequencing primer or by analyzing the amplified product by a next generation sequencing (NGS) method.

상기 차세대 시퀀싱 방법은 시퀀싱 바이 신세시스 (Sequencing by synthesis)와 시퀀싱 바이 라이게이션 (Sequencing by ligation) 방법으로 하는 것을 특징으로 할 수 있다. 이 방법의 특징은 bacterial clone을 만드는 대신 단일 DNA 단편을 공간적으로 분리하여 in situ로 증폭하고 (clonal amplification), 시퀀싱을 해낸다는 것이다. 이때, 수십 만개의 단편을 동시에 읽어내기 때문에 매시브 페러럴 시퀀싱 (massive parallel sequencing) 방법으로 불리기도 한다. 기본적으로는 시퀀싱 바이 신세시스 방법이며, 모노 혹은 디뉴클레오티드를 순차적으로 붙여가면서 시그널을 얻는 방법을 사용하는데 파이로시퀀싱, ion torrent, Solexa 방법들이 여기에 해당한다.The next-generation sequencing method may be characterized by performing a sequencing by synthesis method and a sequencing by ligation method. A characteristic of this method is that, instead of creating a bacterial clone, a single DNA fragment is spatially separated, amplified in situ (clonal amplification), and sequencing is performed. At this time, since hundreds of thousands of fragments are simultaneously read, it is also called a massive parallel sequencing method. Basically, it is a sequencing-by-synthesis method, and a method of obtaining a signal by sequentially attaching mono or dinucleotides is used, and pyrosequencing, ion torrent, and Solexa methods fall into this category.

시퀀싱 바이 신세시스에 기반하는 NGS 장비로는 로슈 (Roche)사의 454 플랫폼, 일루미나 (Illumina)사의 HiSeq 플랫폼, 라이프테크놀로지 (Life Technology)사의 Ion PGM 플랫폼, 마지막으로 퍼시픽바이오사이언스 (Pacific BioSciences)사의 PacBio 플랫폼이 있다. 454와 Ion PGM은 클로날증폭 (clonal amplification)방법으로 emulsion PCR을 사용하며, HiSeq은 브릿지 증폭 (Bridge amplification)을 사용한다. 시퀀싱 바이 신세시스 방법은 한 개의 뉴클레오티드를 순차적으로 붙여가며 DNA를 합성시켜 나갈 때 발생되는 인산(phosphate), 수소이온, 혹은 미리 붙여 놓은 형광을 검출하여 서열을 읽어 나간다. 서열을 검출하는 방법에 있어, 454는 인산을 이용하는 파이로시퀀싱 (pyroseqeuncing) 방법을 사용하며, Ion PGM은 수소이온 검출을 이용한다. HiSeq과 PacBio는 형광을 검출하여 서열을 해독한다.As NGS equipment based on Sequencing by Synthesis, Roche's 454 platform, Illumina's HiSeq platform, Life Technology's Ion PGM platform, and finally Pacific BioSciences' PacBio platform are have. 454 and Ion PGM use emulsion PCR as a clonal amplification method, and HiSeq uses bridge amplification. The sequencing-by-synthesis method reads the sequence by detecting phosphate, hydrogen ion, or pre-attached fluorescence generated when DNA is synthesized by sequentially attaching one nucleotide. In the method for detecting the sequence, 454 uses a pyrosequencing method using phosphoric acid, and Ion PGM uses hydrogen ion detection. HiSeq and PacBio detect fluorescence to decode the sequence.

시퀀싱 바이 라이게이션은 DNA ligase를 이용하는 시퀀싱 기술로 DNA 염기서열에 존재하는 특정 위치의 뉴클레오티드를 확인하는 기술이다. 대부분의 시퀀싱 기술이 중합효소를 사용하는 것과 달리 중합효소를 사용하지 않으며 DNA ligase가 미스매치 서열을 ligation하지 않는 특징을 이용한다. SOLiD 시스템이 여기에 해당한다. 이 기법에서는 간격을 두면서 두 개씩 염기를 읽는데, 프라이머 리셋 (primer reset)을 통해 독립적으로 다섯 번을 반복하기 때문에, 최종적으로는 각 염기를 두 번씩 중복하여 읽어서 정확도를 높인다.Sequencing-by-ligation is a sequencing technology using DNA ligase, and is a technology to identify nucleotides at a specific position in a DNA base sequence. Unlike most sequencing technologies that use a polymerase, it does not use a polymerase and uses the feature that DNA ligase does not ligate mismatched sequences. SOLiD systems fall into this category. In this technique, two bases are read at intervals, and since each base is independently repeated five times through primer reset, the accuracy is improved by reading each base twice in the end.

메틸화 특이 PCR, 실시간 메틸화 특이 PCR, 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 메틸화 DNA 특이적 결합 항체 또는 압타머를 이용한 PCR은 PCR을 기반으로 메틸화를 검출하는 방법이다. Methylation-specific PCR, real-time methylation-specific PCR, PCR using methylated DNA-specific binding protein, and PCR using methylated DNA-specific binding antibody or aptamer are methods for detecting methylation based on PCR.

메틸화 특이 PCR 방법을 상세히 설명하면 다음과 같다. 지노믹 DNA에 바이설파이트를 처리하면 5'-CpG'-3 부위의 시토신이 메틸화된 경우에는 그대로 시토신으로 남아 있고, 비메틸화된 경우에는 우라실로 변하게 된다. 따라서, 바이설파이트 처리 후 변형된 염기서열을 대상으로 5'-CpG-3' 염기서열이 존재하는 부위에 해당하는 PCR 프라이머를 제작하고 이때 메틸화된 경우에 해당되는 PCR 프라이머와 비메틸화된 경우에 해당하는 두 종류의 프라이머를 제작한다. 지노믹 DNA를 바이설파이트로 변형시킨 다음, 상기 두 종류의 프라이머를 이용하여 PCR을 하면 메틸화된 경우에는 메틸화된 염기서열에 해당되는 프라이머를 사용한 것에서 PCR 산물이 만들어지게 되고, 반대로 비메틸화인 경우에는 비메틸화에 해당되는 프라이머를 이용한 것에서 PCR 산물이 만들어진다. 메틸화 여부는 아가로즈겔 전기영동방법으로 정성적으로 확인할 수 있다.A detailed description of the methylation-specific PCR method is as follows. When genomic DNA is treated with bisulfite, when the cytosine of the 5'-CpG'-3 site is methylated, it remains as cytosine as it is, and when it is unmethylated, it is changed to uracil. Therefore, for the modified nucleotide sequence after bisulfite treatment, a PCR primer corresponding to the region where the 5'-CpG-3' nucleotide sequence exists, and the PCR primer corresponding to the methylated case and the unmethylated case Two types of primers are prepared. If genomic DNA is transformed with bisulfite and then PCR is performed using the above two types of primers, a PCR product is produced from the primers corresponding to the methylated base sequence in the case of methylation, and conversely, in the case of non-methylation PCR products are made from those using primers corresponding to unmethylation. Whether methylation or not can be qualitatively confirmed by an agarose gel electrophoresis method.

실시간 메틸화 특이 PCR은 메틸화 특이 PCR 방법을 실시간 측정방법으로 전환한 것으로, 지노믹 DNA에 바이설파이트를 처리한 후, 메틸화된 경우에 해당하는 PCR 프라이머를 디자인하고, 이들 프라이머를 이용하여 실시간 PCR을 수행하는 것이다. 이때, 증폭된 염기서열과 상보적인 TaqMan 프로브를 이용하여 검출하는 방법과 Sybr green을 이용하여 검출하는 두 가지 방법이 있다. 따라서, 실시간 메틸화 특이 PCR은 메틸화된 DNA만을 선택적으로 정량 분석할 수 있다. 이때, in vitro methylated DNA 샘플을 이용하여 표준곡선을 작성하고, 표준화를 위하여 염기서열내에 5'-CpG-3' 서열이 없는 유전자를 음성대조군으로 함께 증폭하여 메틸화 정도를 정량 분석한다.Real-time methylation-specific PCR is a conversion of a methylation-specific PCR method to a real-time measurement method. After genomic DNA is treated with bisulfite, PCR primers corresponding to methylation are designed, and real-time PCR is performed using these primers. is to perform At this time, there are two methods: a method of detecting using a TaqMan probe complementary to the amplified nucleotide sequence and a method of detecting using Sybr green. Therefore, real-time methylation-specific PCR can selectively quantitatively analyze only methylated DNA. At this time, a standard curve is prepared using an in vitro methylated DNA sample, and for standardization, a gene without a 5'-CpG-3' sequence is amplified together as a negative control group, and the degree of methylation is quantitatively analyzed.

메틸화 DNA 특이적 결합 단백질을 이용한 PCR 또는 DNA 칩 방법은 메틸화 DNA에만 특이적으로 결합하는 단백질을 DNA와 혼합할 경우, 메틸화 DNA에만 특이적으로 단백질이 결합하기 때문에 메틸화 DNA만을 선택적으로 분리할 수 있는 특징을 이용한 것이다. 지노믹 DNA를 메틸화 DNA 특이적 결합 단백질과 섞어준 후, 메틸화된 DNA만을 선택적으로 분리할 수 있다. 이들 분리된 DNA를 엑손 부위에 해당하는 PCR 프라이머를 이용하여 증폭한 후, 아가로즈 전기영동으로 메틸화 여부를 측정한다.The PCR or DNA chip method using a methylated DNA-specific binding protein can selectively separate only methylated DNA because the protein specifically binds only to methylated DNA when a protein that specifically binds only methylated DNA is mixed with DNA. feature is used. After mixing the genomic DNA with the methylated DNA-specific binding protein, only the methylated DNA can be selectively isolated. After the isolated DNA is amplified using a PCR primer corresponding to the exon region, methylation is measured by agarose electrophoresis.

또한, 정량 PCR 방법으로도 메틸화 여부를 측정할 수 있으며, 메틸화 DNA 특이적 결합 단백질로 분리한 메틸화 DNA를 형광 염료로 표지하여 상보적인 프로브가 집적된 DNA칩에 하이브리디제이션시킴으로써 메틸화 여부를 측정할 수 있다. In addition, methylation can be measured by quantitative PCR, and methylation can be measured by hybridizing methylated DNA separated with a methylated DNA-specific binding protein with a fluorescent dye to a DNA chip integrated with a complementary probe. can

본 발명의 다른 일 양태에 따르면, 본 발명은 본 발명에 따른 조성물을 포함하는 비만의 예측 또는 진단용 키트에 관한 것이다. According to another aspect of the present invention, the present invention relates to a kit for predicting or diagnosing obesity comprising the composition according to the present invention.

본 발명의 비만의 예측 또는 진단용 키트는, LZTS3 유전자의 메틸화 여부를 검출할 수 있는 물질을 포함하는 조성물을 유효성분으로 포함하고 있어, 생물학적 시료로부터 분리된 게놈 DNA 시료를 대상으로 LZTS3 유전자의 메틸화를 검출하여 비만을 예측 또는 진단할 수 있다.Predictive or diagnostic kit of the invention Obesity is, it contains a composition comprising a substance which can detect methylation if the LZTS3 gene as an active ingredient, the methylation of LZTS3 gene targeting genomic DNA sample isolated from the biological sample It can be detected to predict or diagnose obesity.

상기 게놈 DNA 시료는 CpG를 함유하는 것이 바람직하다. 통상적으로, CpG-함유 핵산은 DNA이나, 이에 한정되지 않으며, DNA 또는 DNA와 mRNA를 포함하는 RNA를 함유하는 시료를 포함할 수 있다. 여기서 DNA 또는 RNA는 단일가닥 또는 이중가닥일 수 있으며, 또는 DNA-RNA 하이브리드를 함유할 수 있다.The genomic DNA sample preferably contains CpG. Typically, the CpG-containing nucleic acid is DNA, but is not limited thereto, and may include a sample containing DNA or RNA including DNA and mRNA. Here, the DNA or RNA may be single-stranded or double-stranded, or may contain a DNA-RNA hybrid.

본 발명의 키트는 샘플을 담는 구획된 캐리어 수단, 비메틸화 시토신을 민감하게 절단하는 제제를 함유하는 첫 번째 용기, CpG 함유 핵산을 증폭하기 위한 프라이머를 함유하는 두 번째 용기 및 절단된 또는 절단되지 않은 핵산의 존재를 검출하는 수단이 함유된 세 번째 용기를 포함하는 하나 이상의 용기를 포함할 수 있다. 일 구현예에서, 상기 프라이머는 게놈상에서 메틸화가 일어났는지 여부를 검출하기 위한 PCR, 메틸화 특이 PCR, 실시간 메틸화 특이 PCR, 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 메틸화 DNA 특이적 결합 항체 또는 압타머를 이용한 PCR, 정량 PCR, 핵산 칩, 시퀀싱, 시퀀싱 바이 신세시스 또는 시퀀싱 바이 라이게이션에 사용하기 위한 프라이머일 수 있다. The kit of the present invention comprises a compartmentalized carrier means containing a sample, a first container containing an agent that sensitively cleaves unmethylated cytosine, a second container containing primers for amplifying CpG-containing nucleic acids, and cleaved or uncleaved It may comprise one or more containers comprising a third container containing means for detecting the presence of the nucleic acid. In one embodiment, the primer is PCR for detecting whether methylation has occurred on the genome, methylation-specific PCR, real-time methylation-specific PCR, PCR using methylated DNA-specific binding protein, methylated DNA-specific binding antibody or aptamer It may be a primer for use in PCR, quantitative PCR, nucleic acid chip, sequencing, sequencing-by-synthesis, or sequencing-by-ligation.

본 발명의 다른 일 양태에 따르면, 본 발명은 분리된 생물학적 시료로부터 LZTS3 유전자의 메틸화 여부를 측정하는 단계를 포함하는 비만의 예측 또는 진단을 위한 정보를 제공하는 방법에 관한 것이다. According to another aspect of the present invention, the present invention relates to a method of providing information for predicting or diagnosing obesity, comprising measuring whether the LZTS3 gene is methylated from an isolated biological sample.

본 발명에 있어서, 상기 메틸화 여부 측정은 LZTS3 유전자의 프로모터, 5'UTR(Untranslated region), 인트론 및 엑손 중 어느 한 부위에 위치하는 CpG의 메틸화를 측정하는 것일 수 있고, 예를 들어, 서열번호 1로 표시되는 영역의 CpG 의 메틸화를 측정하는 것일 수 있다. In the present invention, the determination of whether methylation may be performed may be to measure the methylation of CpG located at any one site among the promoter, 5'UTR (untranslated region), intron, and exon of the LZTS3 gene, for example, SEQ ID NO: 1 It may be to measure CpG methylation in the region indicated by .

상기 메틸화 여부를 측정하는 단계는 PCR, 메틸화 특이 PCR (methylation specific PCR), 실시간 메틸화 특이 PCR (real time methylation specific PCR), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 정량 PCR, DNA 칩, 파이로시퀀싱 및 바이설파이트 시퀀싱으로 구성된 군에서 선택되는 방법에 의하여 수행될 수 있다. The step of measuring the methylation is PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, pyrosequencing and bisulfite sequencing.

상기 생물학적 시료는 비만 의심 또는 진단 대상 개체 유래의 세포, 조직, 생검, 파라핀조직, 혈액, 혈청, 혈장 및 소변으로 이루어진 군으로부터 선택되는 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.The biological sample may be one or more selected from the group consisting of cells, tissues, biopsies, paraffin tissues, blood, serum, plasma and urine derived from a subject suspected of obesity or diagnosis, but is not limited thereto.

본 발명에 따르면, 상기 방법은 정상 대조군의 LZTS3 유전자의 메틸화와 비교하여 LZTS3 유전자의 메틸화 수준이 감소하면 비만으로 결정하는 단계를 추가적으로 포함할 수 있다.According to the present invention, the method may further include the step of determining obesity when the methylation level of the LZTS3 gene is decreased compared to the methylation of the LZTS3 gene of the normal control group.

본 발명의 비만 특이적 마커 유전자의 메틸화를 검출함으로써 비만의 발병을 조기에 예측 및 진단할 수 있다. 본 발명의 LZTS3 메틸화 바이오마커는 비만의 위험성 평가, 진단 및 치료 타겟의 선정에도 활용될 수 있다.By detecting the methylation of the obesity-specific marker gene of the present invention, the onset of obesity can be predicted and diagnosed early. The LZTS3 methylation biomarker of the present invention can also be utilized for risk assessment of obesity, diagnosis, and selection of a treatment target.

도 1은 고지방고당 식이에 따른 마우스의 체중 증가를 측정한 결과이다.
도 2a는 고지방고당 식이를 12주 동안 공급한 후 마우스의 간 무게를 측정한 결과이다.
도 2b는 고지방고당 식이를 12주 동안 공급한 후 마우스의 부고환 지방 무게를 측정한 결과이다.
도 2c는 고지방고당 식이를 24주 동안 공급한 후 마우스의 간 무게를 측정한 결과이다.
도 2d는 고지방고당 식이를 24주 동안 공급한 후 마우스의 부고환 지방 무게를 측정한 결과이다.
도 3은 고지방고당 식이를 12주, 24주 동안 공급한 마우스에서 Lzts3 유전자의 DNA 메틸화 수준을 측정한 결과이다.
도 4는 고지방고당 식이를 12주 동안 공급한 마우스의 간에 대하여 메틸화 시퀀싱을 실시한 결과이다.
도 5는 고지방고당 식이를 24주 동안 공급한 후 마우스의 간에서 Lzts3 유전자의 발현을 분석한 결과이다.1 is a result of measuring the weight gain of mice according to a high-fat, high-sugar diet.
Figure 2a is a result of measuring the liver weight of the mouse after supplying a high-fat, high-sugar diet for 12 weeks.
Figure 2b is a result of measuring the weight of the epididymal fat of the mouse after supplying a high-fat, high-sugar diet for 12 weeks.
Figure 2c is a result of measuring the liver weight of the mouse after feeding a high-fat, high-sugar diet for 24 weeks.
Figure 2d is a result of measuring the epididymal fat weight of the mouse after feeding a high-fat, high-sugar diet for 24 weeks.
3 is a result of measuring the DNA methylation level of the Lzts3 gene in mice fed a high-fat, high-sugar diet for 12 weeks and 24 weeks.
4 is a result of methylation sequencing of the liver of mice fed a high-fat, high-sugar diet for 12 weeks.
5 is a result of analyzing the expression of the Lzts3 gene in the liver of mice after supplying a high-fat, high-sugar diet for 24 weeks.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시 예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

실시예 1. 비만 동물 모델 구축Example 1. Construction of an obese animal model

8주령의 C57BL/6N 수컷 마우스를 중앙실험동물(서울, 대한민국)에서 구입하여, 1주 동안 일반사료 98052602(Research Diets, Inc., NJ, USA)를 제공하여 안정화 시켰다. 고지방고당 식이(high-fat and high-sucrose diet, D12079B, Research Diets, Inc.)와 일반사료 98052602(Research Diets, Inc.)를 제공하면서 몸무게 기록 후 12주, 24주째에 간 및 지방 조직을 분리하였다. 8-week-old C57BL/6N male mice were purchased from Central Laboratory Animals (Seoul, Korea), and were stabilized by providing regular feed 98052602 (Research Diets, Inc., NJ, USA) for 1 week. Liver and adipose tissue was isolated at 12 and 24 weeks after weight recording while providing a high-fat and high-sucrose diet (D12079B, Research Diets, Inc.) and a regular diet 98052602 (Research Diets, Inc.) did.

대조군과 비교하여 고지방고당 식이를 공급한 마우스에서 몸무게와 간 및 부고환 지방 무게가 현저히 증가하였으며, 이를 통해 비만이 유도되었음을 확인하였다 (도 1, 도 2a 내지 2d). Compared with the control group, body weight and liver and epididymal fat weights were significantly increased in mice fed a high-fat, high-sugar diet, and it was confirmed that obesity was induced through this ( FIGS. 1 and 2a to 2d ).

실시예 2. 메틸화 분석Example 2. Methylation analysis

2-1. RRBS 라이브러리 구축2-1. Building the RRBS library

MspI 및 ApeKI을 포함하는 RRBS 라이브러리를 구축하기 위해 지노믹 DNA 500 ng을 MspI(NEB, Ipswich, MA, USA)과 37℃에서 7시간 동안 반응시켰다. 그 다음, ApeKI(NEB)을 첨가하여 75℃에서 16 내지 20시간 동안 반응시켰다. 제한효소로 절단된 산물은 MiniElute PCR Purification Kit(Qiagen, Venlo, Netherlands)로 정제하였다. 정제 후 dA를 첨가하고, 메틸화된 어댑터를 연결시켰다. 전기영동을 실시하고 아가로스 겔에서 160-420 bp 크기의 슬라이스를 분리하였다. 제조사의 지시에 따라 ZYMO EZ DNA Methylation-Gold Kit(ZYMO Research, Irvine, CA, USA)를 이용하여 바이설파이트 전환을 수행하였다. To construct an RRBS library containing MspI and ApeKI, 500 ng of genomic DNA was reacted with MspI (NEB, Ipswich, MA, USA) at 37°C for 7 hours. Then, ApeKI (NEB) was added and reacted at 75° C. for 16 to 20 hours. The restriction enzyme digested product was purified with MiniElute PCR Purification Kit (Qiagen, Venlo, Netherlands). After purification, dA was added and the methylated adapter was ligated. After electrophoresis, slices having a size of 160-420 bp were separated on an agarose gel. Bisulfite conversion was performed using the ZYMO EZ DNA Methylation-Gold Kit (ZYMO Research, Irvine, CA, USA) according to the manufacturer's instructions.

PfuTurbo Cx Hotstart DNA polymerase(Agilent technologies, Santa Clara, CA, USA)를 이용하여 PCR을 수행하여 최종 라이브러리를 얻었다. RRBS 라이브러리는 Agilent 2100 Bioanalyzer(Agilent Technologies)로 분석하였다.　샘플을 시퀀싱하기 전에 시퀀싱 가능한 라이브러리 단편의 양을 qPCR을 통해 결정하였다. 이어서, 샘플은 용출 버퍼(QIAGEN)를 이용하여 10 nM로 희석하였다. RRBS 라이브러리는 NextSeq500(Illumina, San Diego, CA, USA)로 시퀀싱하였다. PCR was performed using PfuTurbo Cx Hotstart DNA polymerase (Agilent technologies, Santa Clara, CA, USA) to obtain the final library. RRBS libraries were analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Before sequencing the samples, the amount of sequencingable library fragments was determined via qPCR. The samples were then diluted to 10 nM using elution buffer (QIAGEN). The RRBS library was sequenced with NextSeq500 (Illumina, San Diego, CA, USA).

2-2. RRBS 데이터 분석2-2. RRBS data analysis

Raw reads의 퀄리티를 조절하기 위해 FastQC v0.11.2 (www.bioinformatics.babraham.ac.uk/projects/fastqc/)를 실시하고, trim galore v0.4.1(www.bioinformatics.babraham.ac.uk/projects/trim_galore/)를 이용하여 어댑터 시퀀싱을 절단하였다. 절단된 서열은 BS-seeker2 v2.0.10 (Guo et al., 2013)를 이용하여 쥣과의 레퍼런스 게놈(mm10)에 얼라인하였다. RRBS 라이브러리의 MspI 및 ApeKI 단편을 커버하기 위해 in silico에서 30 내지 500 bp 길이 범위의 이중 효소 MspI(CCGG) 및 ApeKI(GCWGC) 단편을 구축하였다. 리드(read) 당 4개의 불일치를 허용하도록 하여 로컬 얼라인먼트 모드에서 Bowtie2로 리드를 얼라인하였다. 맵핑 비율을 향상시키기 위해 맵핑되지 않은 리드를 페어드-엔드(paried-end) 모드에서 재맵핑하였다. 두 개의 페어드-엔드 메이트가 겹치는 경우, 하나의 메이트를 제거한 후 각 CpG 사이트의 메틸화 수준을 측정하였다. To control the quality of raw reads, FastQC v0.11.2 (www.bioinformatics.babraham.ac.uk/projects/fastqc/) was performed, and trim galore v0.4.1 (www.bioinformatics.babraham.ac.uk/projects/ trim_galore/) was used to cut adapter sequencing. The cleaved sequence was aligned to the murine reference genome (mm10) using BS-seeker2 v2.0.10 (Guo et al., 2013). To cover the MspI and ApeKI fragments of the RRBS library, double enzyme MspI (CCGG) and ApeKI (GCWGC) fragments were constructed in silico ranging in length from 30 to 500 bp. Reads were aligned with Bowtie2 in local alignment mode, allowing 4 mismatches per read. To improve the mapping ratio, unmapped reads were remapped in paired-end mode. When two paired-end mates overlapped, the methylation level of each CpG site was measured after removing one mate.

12주, 24주째에 고지방고당식이, 대조식이 군에서 각 3마리씩 선별(몸무게 기준)하여 간 및 지방조직에서 DNA 추출 후 RRBS 방법으로 전장 유전체의 DNA 메틸화 변화를 분석하여, 12주, 24주째의 간 조직에서 Lzts3 유전자 엑손에 위치한 CpGs에서 고지방고당 식이에 의한 메틸화 감소를 확인하였다(q < 0.05, 20% 이상 감소)(도 3).At 12 weeks and 24 weeks, three animals were selected (based on weight) from the high-fat, high-sugar diet and control diet groups, and DNA was extracted from the liver and adipose tissue. In liver tissue, CpGs located in the Lzts3 gene exon reduced methylation by a high-fat high-sugar diet ( q < 0.05, decreased by more than 20%) ( FIG. 3 ).

2-3. 통계처리를 통한 2-3. through statistical processing Lzts3Lzts3 메틸화 부위 선발 Methylation site selection

그룹간 DMR(100 bp)를 식별하기 위해 커스텀 Perl 스크립트를 사용하였다. 간단히 설명하면, 게놈상의 DNA 메틸화 수준은 레퍼런스 게놈(mm10)을 통해 고정된 크기 윈도우(100 bp)를 50 bp 단위로 슬라이딩시킴으로써 프로파일링하였다. 주어진 윈도우에서 모든 CpG 부위의 DNA 메틸화 비율(0 내지 1)을 Mann-Whitney U 테스트(q < 0.05)를 사용하여 두 그룹(12주 및 24주에 HFHS 대 대조군) 사이에서 비교하였다. 신뢰할 수 없는 DMR 후보를 걸러내기 위해 10개 미만의 판독 값으로 커버되거나 그룹간 평균 차이가 < 0.2를 나타내는 영역은 제거하였다. 확인된 DMR은 UCSC 레퍼런스 유전자 주석(mm10)과 함께 HOMER(v5.7)를 사용하여 주석을 달았다. A custom Perl script was used to identify intergroup DMRs (100 bp). Briefly, DNA methylation levels on the genome were profiled by sliding a fixed size window (100 bp) through a reference genome (mm10) in increments of 50 bp. The DNA methylation rates (0 to 1) of all CpG sites in a given window were compared between the two groups (HFHS versus control at 12 and 24 weeks) using the Mann-Whitney U test (q < 0.05). To filter out unreliable DMR candidates, areas covered by less than 10 readings or with mean differences between groups <0.2 were removed. The identified DMRs were annotated using HOMER (v5.7) with UCSC reference gene annotation (mm10).

선별 부위의 개별 CpG 메틸화 변화를 BSAS(bisulfite amplicon sequencing) 방법으로 분석하였다. 분석에 사용된 프라이머 세트는 표 1과 같다. Changes in individual CpG methylation at the selection sites were analyzed by bisulfite amplicon sequencing (BSAS). The primer sets used for the analysis are shown in Table 1.

서열번호SEQ ID NO: 명명denomination 서열(5'-3')sequence (5'-3') 33 Lzts3 BSAS-FLzts3 BSAS-F TAGGTTATTAGGATTTGGGGATTTTTAGGTTATTAGGATTTGGGGATTTT 44 Lzts3 BSAS-RLzts3 BSAS-R ACCACCTACTACAACTTCCCACTACACCACCTACTACAACTTCCCACTAC

분석 결과, 고지방고당 식이를 공급한 마우스 간에서 16개 CpGs의 DNA 메틸화 감소가 유도됨을 확인하였다(도 4, 표 2). As a result of the analysis, it was confirmed that a decrease in DNA methylation of 16 CpGs was induced in the mouse liver fed a high-fat, high-sugar diet (FIG. 4, Table 2).

PositionPosition GeneGene Tissuetissue Genomic regiongenomic region StrandStrand #CpG#CpG ChrChr LociLoci CpG_1CpG_1 22 130,635,994 130,635,994 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_2CpG_2 22 130,636,001 130,636,001 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_3CpG_3 22 130,636,012 130,636,012 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_4CpG_4 22 130,636,030 130,636,030 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_5CpG_5 22 130,636,061 130,636,061 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_6CpG_6 22 130,636,078 130,636,078 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_7CpG_7 22 130,636,081 130,636,081 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_8CpG_8 22 130,636,117 130,636,117 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_9CpG_9 22 130,636,140 130,636,140 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_10CpG_10 22 130,636,140 130,636,140 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_11CpG_11 22 130,636,149 130,636,149 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_12CpG_12 22 130,636,190 130,636,190 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_13CpG_13 22 130,636,199 130,636,199 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_14CpG_14 22 130,636,219 130,636,219 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_15CpG_15 22 130,636,236 130,636,236 Lzts3Lzts3 LiverLiver ExonExon (+)(+) CpG_16CpG_16 22 130,636,258 130,636,258 Lzts3Lzts3 LiverLiver ExonExon (+)(+)

표 2의 마우스 CpG 서열은 인간 서열과 75% 이상의 상동성을 나타내는 것으로 확인되었다(표 3). It was confirmed that the mouse CpG sequence in Table 2 exhibits at least 75% homology with the human sequence (Table 3).

PositionPosition GeneGene Tissuetissue Genomic regiongenomic region StrandStrand #CpG#CpG ChrChr LociLoci CpG_1CpG_1 2020 3,165,7033,165,703 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_2CpG_2 2020 3,165,7193,165,719 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_3CpG_3 2020 3,165,7243,165,724 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_4CpG_4 2020 3,165,7353,165,735 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_5CpG_5 2020 3,165,7433,165,743 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_6CpG_6 2020 3,165,7533,165,753 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_7CpG_7 2020 3,165,7793,165,779 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_8CpG_8 2020 3,165,7843,165,784 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_9CpG_9 2020 3,165,8013,165,801 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_10CpG_10 2020 3,165,8043,165,804 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_11CpG_11 2020 3,165,8403,165,840 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_12CpG_12 2020 3,165,8783,165,878 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_13CpG_13 2020 3,165,8903,165,890 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_14CpG_14 2020 3,165,8923,165,892 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_15CpG_15 2020 3,165,9133,165,913 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_16CpG_16 2020 3,165,9423,165,942 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_17CpG_17 2020 3,165,9563,165,956 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_18CpG_18 2020 3,165,9593,165,959 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_19CpG_19 2020 3,165,9813,165,981 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_20CpG_20 2020 3,165,9913,165,991 LZTS3LZTS3 LiverLiver ExonExon (-)(-) CpG_21CpG_21 2020 3,166,0163,166,016 LZTS3LZTS3 LiverLiver ExonExon (-)(-)

2-4. Real-time RT-PCR 활용 유전자 발현 분석 2-4. Gene expression analysis using real-time RT-PCR

제조사의 지시에 따라 RNeasy Mini kit(Qiagen)를 이용하여 세포로부터 RNA를 분리하였다. 500 ng의 총 RNA와 역전사 효소(TOYOBO, Osaka, Japan)를 30℃에서 10분, 42℃에서 20분 및 99℃에서 5분 동안 반응시켜 cDNA를 합성하였다. SYBR green super mix(TOYOBO) 및 thermal cycler(Bio-Rad, Hercules, CA, USA)를 이용하여 95℃에서 10초, 58℃에서 10초, 72℃에서 20초의 반응을 40회 반복하고 최종 멜팅 커브 스텝을 실시하여 유전자를 증폭시켰다.RNA was isolated from the cells using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized by reacting 500 ng of total RNA with reverse transcriptase (TOYOBO, Osaka, Japan) at 30° C. for 10 minutes, 42° C. for 20 minutes, and 99° C. for 5 minutes. Using SYBR green super mix (TOYOBO) and a thermal cycler (Bio-Rad, Hercules, CA, USA), the reaction was repeated 40 times at 95°C for 10 seconds, at 58°C for 10 seconds, and at 72°C for 20 seconds, followed by a final melting curve. step was performed to amplify the gene.

유전자 증폭에 사용된 프라이머 서열은 표 4와 같다. The primer sequences used for gene amplification are shown in Table 4.

서열번호SEQ ID NO: 명명denomination 서열(5'-3')sequence (5'-3') 55 Lzts3-FLzts3-F CAAGGCTCCACCCCAGTATCAAGGCTCCACCCCAGTAT 66 Lzts3-RLzts3-R TGGTCTGTGGAGGACACAAGTGGTCTGTGGAGGACACAAG

비만 동물모델의 간 조직에서 Lzts3 유전자 발현분석을 실시하여 DNA 메틸화 감소에 의해 Lzts3 발현이 감소함을 확인하였다(도 5). Lzts3 gene expression analysis was performed in the liver tissue of an obese animal model, and it was confirmed that Lzts3 expression was reduced by DNA methylation reduction (FIG. 5).

<110> KOREA FOOD RESEARCH INSTITUTE <120> Method for providing information of prediction and diagnosis of obesity using methylation level of LZTS3 gene and composition therefor <130> PN190390 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 4196 <212> DNA <213> Homo sapiens <400> 1 gcagcgcgcc ccgccgggcg tgcttcgggc tgcgccagca agcggggccg gtggcgcccg 60 tgtcggagac cccgcgccgg accctgaggc agcgaaggag aaaactgcag tccgggatgg 120 ctcagtcggc ccctccaaag tcgcgcagct ggttcgggcc gagccccgac tgcgagagtg 180 aggcacatgg cccctgcaga ccgggcctcg gagggtccca ggcttgagga cccgtcggcc 240 cctcaacccc ttggaaaggc aaggagagaa gcctgcgctg gtagtgagct tccacttctg 300 ccctgactgc aacacttagt gcccccctgg cttagtcatg gcgaagctgg agacgctgcc 360 tgtgcgcgct gacccagggc gggatcctct cctggccttt gccccacggc cctccgagct 420 tggacccccg gacccccgcc tggccatggg cagcgtgggc agtggggtgg cccatgccca 480 ggagtttgcc atgaagagcg tgggtacccg cacagggggt gggggcagcc agggcagttt 540 ccctggcccc cgaggcagtg gcagtggggc cagcagggag aggccgggcc gctacccctc 600 agaggacaag ggtctcgcca actccctcta cctcaatggt gagctgcggg gcagtgacca 660 caccgatgtc tgtggcaacg tggttggcag cagcggaggc agcagcagca gtggtggcag 720 tgacaaagcc ccaccgcagt atcgtgagcc cagccaccca cccaagctcc tggccacctc 780 tggcaagcta gaccagtgct cagaaccact agttcggccg tcggccttca agcctgtcgt 840 acccaagaat ttccactcca tgcagaattt gtgccccccg cagaccaatg ggactcctga 900 gggacggcag ggccctggtg gcctcaaagg cggactggac aagtctcgga ccatgactcc 960 agcgggtggg agtgggagtg gcctctcaga ctcaggccgg aactccctca caagcctgcc 1020 cacctacagc tccagctaca gccagcacct ggcacccctc agtgcctcca ccagccacat 1080 taaccgcatt ggcactgcca gctatggtag tggtagtggc ggcagcagcg gtggggggtc 1140 gggctaccag gacctgggga cctccgatag tggacgggcc tccagcaaga gtgggtcgtc 1200 gtcatctatg gggcggccag gccacctggg ctctggggag ggcggaggtg gaggcctgcc 1260 tttcgcggcc tgctcaccgc cctcccccag tgcactcatc caggagctgg aggagcggct 1320 gtgggagaag gagcaggagg tggcagctct gcggcgcagc ctggagcaga gcgaggcggc 1380 tgtggcccag gtactggagg agcggcagaa ggcgtgggag cgggagctgg ccgagctgcg 1440 gcagggctgc agcgggaagc tacagcaggt ggcccgacgt gcccagcgcg cccagcaggg 1500 cctacagctg caggtgttgc ggctgcagca ggacaagaag cagctgcagg aggaggcggc 1560 ccggctgatg cggcagcggg aagagctgga ggacaaggtg gccgcctgcc agaaggagca 1620 ggccgacttc ctgccccgga tagaggaaac taagtgggag gtgtgccaga aggctggcga 1680 gatctccctc ctgaagcagc agctgaagga ctcgcaggcg gatgtgtcgc agaagttgag 1740 tgagatcgtg ggactgcgct cgcagctgcg ggagggccgg gcttcgctgc gggagaagga 1800 ggagcagctg ctcagcctgc gggactcctt cagcagcaag caggccagcc tggagctggg 1860 cgaaggcgag ctgcctgccg cctgcctcaa gccggcgctg acccccgtgg acccggccga 1920 gccacaggat gctctggcca cctgcgagag cgacgaggct aagatgcgcc gtcaggccgg 1980 ggtggccgct gccgcctcct tggtttccgt ggacggggag gcggaggctg gcggggagag 2040 cgggacgcgg gccctgcggc gggaggtggg gcggctgcag gccgagctgg cggctgagcg 2100 gcgggcccgg gagcgccagg gtgccagctt cgccgaggag cgccgcgtgt ggctggagga 2160 gaaggagaag gtgatcgagt accagaagca gctgcagctg agctacgtgg agatgtacca 2220 gcgcaaccag cagctggagc gcaggctgcg ggagcgcggg gccgcagggg gtgcaagcac 2280 gcccactccc cagcatggcg aggagaagaa ggcctggacc ccctcccgcc tcgagcgcat 2340 tgagtccaca gaaatctgat cgacctgggc actcggcatt ttgacacatg tcctgtcaaa 2400 aggccagagt ccccagtgtc ccctcccctc catctctctt ccccatagac cccataaccc 2460 cagaccaaag aggttctcta agcagctgtg accaggttcc tccctcccca cctgccctcc 2520 tagctccagc actgcccccg tggcagccca cttggacccc cctaaaagga gggaatagga 2580 ggagggcagg gtgagtgggg gcaatcctag gtggtggggg agtcatgctc cctttctcgg 2640 cacccccttg ttggagatgg aggcagcaga cgtgcagtgc cataaggtgc cccagtcctt 2700 ctggaggcct gggctgctac tgttggccac cctgtgtcta gtgatgctct ctgtgctcac 2760 ctcctaggcc atggagcctg agggggcctg caccaggttt gctgaaactg acagagcctg 2820 ggctccagac ctctctccct cctacagtgc tctccctccc tgggcagatt ggcaggacaa 2880 gtgggagcag atggcctgcc tttggctgag agggctacct gcccagcccc tcccccaaca 2940 agatctcttg gactcaggcc tcagagcctg gcctggttgt gagtgtgtgt ccctgtgtgt 3000 gtgttgcggg aggggaggac tggggctgga agtccagcac ccagggaaga tctgtcctcc 3060 tgttcttggg aagcgttgcc tgacggcttc tcggctctac cctcaccctt ctggccagga 3120 tcccgcaggg caacagcccc atctgcttgg ctgaccccac acccaggacc actgtccggc 3180 tctaacacag ctattaagtg ctacctgcct ctcaggcact ctcctcgccc agtttctgag 3240 gtcagacgag tgtctgcgat gtcttcccgc actctattcc cccagcctct ttctgctttc 3300 atgctcagca catcatcttc ctaggcagtc tcttccccaa agtctcacct tttcttccaa 3360 tagaaaattc cgcttgacct ttggtgcact gcccacttcc cagctccact ggcccaagtc 3420 tgagccggag gcccttgttt tgggggcggg gggagagttg gatgtgattg cccttgaaga 3480 acaaggctga cctgagaggt tcctggcgcc ctgaggtggc tcagcacctg cccagggtag 3540 gcctggcatg aggggttagg tcagccaatg tcagctgctt ctcttggggc cctctcagag 3600 tctatctccc caagacagga agggaaaagc aaatttctaa ttcaccagca ataaaaattg 3660 gaggaggctt ggccctcagc ccttatatct ctctcttttt cactctcttc ctcccacccc 3720 caagactgag ttttgggggg caaggtggag agagctggca actactgtga gcaagtcccc 3780 tagcccctga ccagcctcct cccatgactg gtgactgttt aatgagctgt gcatccccca 3840 caaaaacatg agtgcccctc tgtgtggcct ctaaccctct gcacagccct tctgggtggt 3900 cctcaccagg tctctgagct gggtgggagg ccatcctggc gaccactgcc cattccattc 3960 acccctcact gtacctgccc tagaacctgg gcctaggcca caggggcagg gagaagagaa 4020 ggcattagta agaaaaaaat agaaaaaaat atgaacagac tcagctttgg gacgtccaac 4080 cacaaaagaa attatatata aatatatata aatatatatc tctaccatat gtgatggaaa 4140 gactttttgt tttcctttcc caaagaaata aaacggaaaa agcctcttga gtggta 4196 <210> 2 <211> 4319 <212> DNA <213> Mus musculus <400> 2 gggaggggcc aagagcagag cccggccgcg ccgcgcgccc tgtggggcgg gcttcgggct 60 gcgccagaga gcgggacagc tagcgccggt gtctgagcct ctgcgccgga ctctgaggca 120 gtgaaggaga aagctgtagt acgggatggc ttggttggct agtccaaagt cgcgcagctg 180 cttagggccg agcctggatt tcgagaaaga gccatatggc ccctgcagac ctggcctcgg 240 agggtcccaa gcttgaggac ccgccggccc cccacctctt tggaaagtgc ccttctggct 300 taatcatggc gaagctggag acgttacctg tgcgcgctga tccagggagg gatcctctcc 360 tggcctttgc cccccggcct tctgagcttg gacccccaga tccccgcctg actatgggca 420 gtgtgggtag tggggtgacc catgctcagg agtttcctat gaaaagcgtg ggcactcgaa 480 cagggggtgg gggcaaccaa ggcagtttcc ctggtccaag aagcggtggt agcggggcca 540 acagggagag acctggtcgc tacccatcag aggacaaggt tctcgccaac tctctctacc 600 tcaatggcga gctgcggggc agtgaccaca cagatgtctg tggtaatgtg gtgggcagca 660 gtgggggcag cagcagcagt gggggcagcg acaaggctcc accccagtat cgcgaaccca 720 atcacccacc caagctcctg accacctctg gcaagctaga ccagtgctcg gagccactag 780 ttcggccatc agccttcaag cctgttgtac ccaagaactt tcattccatg cagaacttgt 840 gtcctccaca gaccaatggg acccctgagg gacgacaggg ccccgcgggc ctcaaaggcg 900 gactggacaa gtctcggacc atgaccccag ctggtgggag tgggggcggc ctctcagact 960 caggccggaa ctcactcaca agcttgccca cctatagctc cagctatagc cagcatctgg 1020 cacccctcag tgcttccacc agccatatca accgtattgg caccgctggc tatagtagtg 1080 gcagcagcgg tggggggtca ggctaccagg atctggggac ctctgacagt gggcgggctt 1140 ccagtaagag tgggtcgtca tcatccatgg ggcggtcagg ccacctggga tccggggagg 1200 gcggaaatgg gggcctgcca tttgcagcct gttcaccacc ctcgcccagt gcgctgatcc 1260 aggagctgga ggagcggctg tgggagaagg agcaggaggt ggcagctctg cggcgcagcc 1320 tggagcagag cgaggcagcc gtggcccagg tgttggagga gcggcagaag gcatgggagc 1380 gggagctagc cgagctccgg cagggctgca gtgggaagct gcagcaggtg gcccgccgtg 1440 cccagcgtgc ccagcagggc ctacagctgc aggtgctgcg gttacagcag gacaagaaac 1500 agctgcagga ggaggcggcc cagctgataa ggcaacggga agagctggag gacaaggtgg 1560 ccgtctgtca gaaggagcag gccgacttcc tgccccggat ggaggaaact aagtgggagg 1620 tgtgccagaa ggccggtgag atttccctcc tgaagcaaca actgaaggac tctcaggccg 1680 atgtgtcgca gaagttgagt gagatcgtgg ggctgcgctc acagcttcgg gagggtaggg 1740 cctcgctccg ggagaaggag gagcagctgc tcagcttgag ggactccttc ggcagcaaac 1800 aggccagcct ggagctgagt gaaggcgagc tgccccctgc ctgcctcaag cctgcgctga 1860 cccccgtgga cctggtcgag ccacaggagg cgctggcctc ctgtgagagt gacgaggcca 1920 agatgcgccg gcaggctggc gtggcggctg ccgcttctct ggtttctgtt gatggggagg 1980 tggaggctgg aggagagggt gggacacggg ccctacgcag ggaggtgggg agattgcagg 2040 ctgagctggc tgctgagcgg cgtgcccgtg agcgccaggg tgccagcttt gcggaggagc 2100 gccgggtgtg gttggaggag aaggagaagg tcattgagta tcagaaacag ctccagctga 2160 gttatgtgga gatgtatcag cgcaaccagc agctggagcg gcgacttcgg gagcgtgggg 2220 cagcaggggg gtccagcacg cctactcccc agcatggaga ggaaaagaaa gcctggaccc 2280 cctcccgcct ggagcgcatc gagtccactg aaatctgatt ctctacgtgc acaaccttct 2340 gacacgtgcc ctgtctgaag gtcggagtca ccaatgtccc ttccattcat ctcccttccc 2400 cacgtgtgct gcacatcccc agaccaaaga ggttctcgaa gcagctgtga ccagcccctc 2460 cccccacttg tcccccgtcc ttgcagctcc agcactgcct ttgtgggtga ccactccaga 2520 ccctcctgcc aaggagagca tgggagaggg cagagtacaa tggggtgagg tcccaggcgc 2580 atggagcaag gctccctttc tggggaagcc tcgtgttgga gatagaggca gcaggcctgc 2640 gttgccaggg agtaccccag cccctttccc ttcttgggct gctaccgccg ccaccaccgt 2700 caccaccatc tgtccagtgc tactgtctgt gccctcctag gccacacagc ctgagggagc 2760 ttgcatgggt ctgcccgtgt cgacagaccc taggctccca gtctctctcc ttcatgtgct 2820 attctccttc cccaggcaga ttggcagggg aagtgggagc agatggcctg cctgtggttg 2880 agagggctac ctgcccgacc cctcccccac caaggtatct tgggctccag cctcagagcc 2940 tggactagtt ctgagcatgt gtccctctgt gtatagtgag gaagtgagag cagaggctgg 3000 aaactcaatc ccaaggggag atctgccttc ctaccctcgg gaagggtcac ctagaggctt 3060 ctcagcccca ccctcaccct ctggccagga tcctacaagg ctacatcccc tgctgcttgg 3120 ttgacccccc caaatccagg gtcactgctc agctctgctt atagctatac ctgcctctta 3180 ggtcttctgt ttgcccgttt ctgaggtcag atggatggat gtctgatgtc ttcctgtgtt 3240 ttccctcgat ctctcccctt ggcctccttt cgcactcaca tcttcccagg ctgccttttc 3300 ttccaagttt cacctttcct cccagcagaa agctctgctt ggcttttggt gcactgctca 3360 cccccagctc cactggccca agtctgagcc tggggccctt gctttgaggg gccagtccga 3420 gttggatggg actgccctca aggaacaagg ctaatgtgtg tgtgtatgtt agagagagag 3480 agagagagag agagagagag agagagagag agagagagag agagagagag agagaaagag 3540 agagagagag aaagagagag agggaggact actgaccctt gggttgcttg ccagactctt 3600 gagttggctc atttgtccgg gcagggttgg ctggtatggt catcagtatg ggtcgtgaca 3660 gtcaatgtca cttccttctc taggatctct cagattctat ctccccaaga caggcaggga 3720 aattctaatt tctctcttaa gaaagtaaat ttccacttca ccagcaataa caatcagagg 3780 agacttggcc ctcagtcctt gtatttctct ttttcactct ctcttccacc ccaagatgga 3840 atcttgggga caggagggag agggttggta actactgtga gcaagtcccc caagccctga 3900 ccagcctcct cccatgactg gtgactgttt aacaagctgt gcatccccca caaaaatagg 3960 agtgccctat ttgtagtctc cacctctctg cacagctcct tctgggtggt cctcagcagg 4020 gttttgacct tgattggcgg ctactctggc aaccactgcc cattcttcta atccctcaca 4080 atacgtgatt cagaccctga gcctggtcca cagtgactgg gaaaaggcat taacacaaaa 4140 caaaagagat ggggggagag aaaaatatga acagctttgg aatgtccaac tgcaaaataa 4200 attatatata aatatataaa tatataaata tatatatcta ccatatgtga tggaaagagt 4260 tttcattttt cctttcccaa agaaataaaa tggagaaagc ctcttggatg gcaattctg 4319 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Lzts3 BSAS-F <400> 3 taggttatta ggatttgggg atttt 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Lzts3 BSAS-R <400> 4 accacctact acaacttccc actac 25 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Lzts3-F <400> 5 caaggctcca ccccagtat 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lzts3-R <400> 6 tggtctgtgg aggacacaag 20 <110> KOREA FOOD RESEARCH INSTITUTE <120> Method for providing information of prediction and diagnosis of obesity using methylation level of LZTS3 gene and composition therefor <130> PN190390 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 4196 <212> DNA <213> Homo sapiens <400> 1 gcagcgcgcc ccgccgggcg tgcttcgggc tgcgccagca agcggggccg gtggcgcccg 60 tgtcggagac cccgcgccgg accctgaggc agcgaaggag aaaactgcag tccgggatgg 120 ctcagtcggc ccctccaaag tcgcgcagct ggttcgggcc gagccccgac tgcgagagtg 180 aggcacatgg cccctgcaga ccgggcctcg gagggtccca ggcttgagga cccgtcggcc 240 cctcaacccc ttggaaaggc aaggagagaa gcctgcgctg gtagtgagct tccacttctg 300 ccctgactgc aacacttagt gcccccctgg cttagtcatg gcgaagctgg aacgctgcc 360 tgtgcgcgct gacccagggc gggatcctct cctggccttt gccccacggc cctccgagct 420 tggacccccg gacccccgcc tggccatggg cagcgtgggc agtggggtgg cccatgccca 480 gggattttgcc atgaagagcg tgggtacccg cacagggggt gggggcagcc agggcagttt 540 ccctggcccc cgaggcagtg gcagtggggc cagcagggag aggccgggcc gctacccctc 600 agaggacaag ggtctcgcca actccctcta cctcaatggt gagctgcggg gcagtgacca 660 caccgatgtc tgtggcaacg tggttggcag cagcggaggc agcagcagca gtggtggcag 720 tgacaaagcc ccaccgcagt atcgtgagcc cagccaccca cccaagctcc tggccacctc 780 tggcaagcta gaccagtgct cagaaccact agttcggccg tcggccttca agcctgtcgt 840 acccaagaat ttccactcca tgcagaattt gtgccccccg cagaccaatg ggactcctga 900 gggacggcag ggccctggtg gcctcaaagg cggactggac aagtctcgga ccatgactcc 960 agcgggtggg agtgggagtg gcctctcaga ctcaggccgg aactccctca caagcctgcc 1020 cacctacagc tccagctaca gccagcacct ggcacccctc agtgcctcca ccagccacat 1080 taaccgcatt ggcactgcca gctatggtag tggtagtggc ggcagcagcg gtggggggtc 1140 gggctaccag gacctgggga cctccgatag tggacgggcc tccagcaaga gtgggtcgtc 1200 gtcatctatg gggcggccag gccacctggg ctctggggag ggcggaggtg gaggcctgcc 1260 tttcgcggcc tgctcaccgc cctcccccag tgcactcatc caggagctgg aggagcggct 1320 gtgggagaag gagcaggagg tggcagctct gcggcgcagc ctggagcaga gcgaggcggc 1380 tgtggcccag gtactggagg agcggcagaa ggcgtgggag cgggagctgg ccgagctgcg 1440 gcagggctgc agcgggaagc tacagcaggt ggcccgacgt gcccagcgcg cccagcaggg 1500 cctacagctg caggtgttgc ggctgcagca ggacaagaag cagctgcagg aggaggcggc 1560 ccggctgatg cggcagcggg aagagctgga ggacaaggtg gccgcctgcc agaaggagca 1620 ggccgacttc ctgccccgga tagaggaaac taagtgggag gtgtgccaga aggctggcga 1680 gatctccctc ctgaagcagc agctgaagga ctcgcaggcg gatgtgtcgc agaagttgag 1740 tgagatcgtg ggactgcgct cgcagctgcg ggagggccgg gcttcgctgc gggagaagga 1800 ggagcagctg ctcagcctgc gggactcctt cagcagcaag caggccagcc tggagctggg 1860 cgaaggcgag ctgcctgccg cctgcctcaa gccggcgctg acccccgtgg acccggccga 1920 gccacaggat gctctggcca cctgcgagag cgacgaggct aagatgcgcc gtcaggccgg 1980 ggtggccgct gccgcctcct tggtttccgt ggacggggag gcggaggctg gcggggagag 2040 cgggacgcgg gccctgcggc gggaggtggg gcggctgcag gccgagctgg cggctgagcg 2100 gcgggcccgg gagcgccagg gtgccagctt cgccgaggag cgccgcgtgt ggctggagga 2160 gaaggagaag gtgatcgagt accagaagca gctgcagctg agctacgtgg agatgtacca 2220 gcgcaaccag cagctggagc gcaggctgcg ggagcgcggg gccgcagggg gtgcaagcac 2280 gcccactccc cagcatggcg aggagaagaa ggcctggacc ccctcccgcc tcgagcgcat 2340 tgagtccaca gaaatctgat cgacctgggc actcggcatt ttgacacatg tcctgtcaaa 2400 aggccagagt ccccagtgtc ccctcccctc catctctctt ccccatagac cccataaccc 2460 cagaccaaag aggttctcta agcagctgtg accaggttcc tccctcccca cctgccctcc 2520 tagctccagc actgcccccg tggcagccca cttggacccc cctaaaagga gggaatagga 2580 ggagggcagg gtgagtgggg gcaatcctag gtggtggggg agtcatgctc cctttctcgg 2640 cacccccttg ttggagatgg aggcagcaga cgtgcagtgc cataaggtgc cccagtcctt 2700 ctggaggcct gggctgctac tgttggccac cctgtgtcta gtgatgctct ctgtgctcac 2760 ctcctaggcc atggagcctg agggggcctg caccaggttt gctgaaactg acagagcctg 2820 ggctccagac ctctctccct cctacagtgc tctccctccc tgggcagatt ggcaggacaa 2880 gtgggagcag atggcctgcc tttggctgag agggctacct gcccagcccc tcccccaaca 2940 agatctcttg gactcaggcc tcagagcctg gcctggttgt gagtgtgtgt ccctgtgtgt 3000 gtgttgcggg agggggaggac tggggctgga agtccagcac ccagggaaga tctgtcctcc 3060 tgttcttggg aagcgttgcc tgacggcttc tcggctctac cctcaccctt ctggccagga 3120 tccgcaggg caacagcccc atctgcttgg ctgaccccac acccaggacc actgtccggc 3180 tctaacacag ctataagtg ctacctgcct ctcaggcact ctcctcgccc agtttctgag 3240 gtcagacgag tgtctgcgat gtcttcccgc actctattcc cccagcctct ttctgctttc 3300 atgctcagca catcatcttc ctaggcagtc tcttccccaa agtctcacct tttcttccaa 3360 tagaaaattc cgcttgacct ttggtgcact gcccacttcc cagctccact ggcccaagtc 3420 tgagccggag gcccttgttt tgggggcggg gggagagttg gatgtgattg cccttgaaga 3480 acaaggctga cctgagaggt tcctggcgcc ctgaggtggc tcagcacctg cccagggtag 3540 gcctggcatg aggggttagg tcagccaatg tcagctgctt ctcttggggc cctctcagag 3600 tctatctccc caagacagga agggaaaagc aaatttctaa ttcaccagca ataaaaattg 3660 gaggaggctt ggccctcagc ccttatatct ctctcttttt cactctcttc ctccccacccc 3720 caagactgag ttttgggggg caaggtggag agagctggca actactgtga gcaagtcccc 3780 tagcccctga ccagcctcct cccatgactg gtgactgttt aatgagctgt gcatccccca 3840 caaaaacatg agtgcccctc tgtgtggcct ctaaccctct gcacagccct tctgggtggt 3900 cctcaccagg tctctgagct gggtgggagg ccatcctggc gaccactgcc cattccattc 3960 acccctcact gtacctgccc tagaacctgg gcctaggcca caggggcagg gagaagagaa 4020 ggcattagta agaaaaaaat agaaaaaaat atgaacagac tcagctttgg gacgtccaac 4080 cacaaaagaa attatatata aatatata aatatatatc tctaccatat gtgatggaaa 4140 gactttttgt tttcctttcc caaagaaata aaacggaaaa agcctcttga gtggta 4196 <210> 2 <211> 4319 <212> DNA <213> Mus musculus <400> 2 gggaggggcc aagagcagag cccggccgcg ccgcgcgccc tgtggggcgg gcttcgggct 60 gcgccagaga gcgggacagc tagcgccggt gtctgagcct ctgcgccgga ctctgaggca 120 gtgaaggaga aagctgtagt acgggatggc ttggttggct agtccaaagt cgcgcagctg 180 cttagggccg agcctggatt tcgagaaaga gccatatggc ccctgcagac ctggcctcgg 240 agggtcccaa gcttgaggac ccgccggccc cccacctctt tggaaagtgc ccttctggct 300 taatcatggc gaagctggag acgttacctg tgcgcgctga tccagggagg gatcctctcc 360 tggcctttgc cccccggcct tctgagcttg gacccccaga tccccgcctg actatgggca 420 gtgtgggtag tggggtgacc catgctcagg agtttcctat gaaaagcgtg ggcactcgaa 480 cagggggtgg gggcaaccaa ggcagtttcc ctggtccaag aagcggtggt agcggggcca 540 acagggagag acctggtcgc tacccatcag aggacaaggt tctcgccaac tctctctacc 600 tcaatggcga gctgcggggc agtgaccaca cagatgtctg tggtaatgtg gtgggcagca 660 gtgggggcag cagcagcagt gggggcagcg acaaggctcc accccagtat cgcgaaccca 720 atcacccacc caagctcctg accacctctg gcaagctaga ccagtgctcg gagccactag 780 ttcggccatc agccttcaag cctgttgtac ccaagaactt tcattccatg cagaacttgt 840 gtcctccaca gaccaatggg acccctgagg gacgacaggg ccccgcgggc ctcaaaggcg 900 gactggacaa gtctcggacc atgaccccag ctggtgggag tgggggcggc ctctcagact 960 caggccggaa ctcactcaca agcttgccca cctatagctc cagctatagc cagcatctgg 1020 cacccctcag tgcttccacc agccatatca accgtattgg caccgctggc tatagtagtg 1080 gcagcagcgg tggggggtca ggctaccagg atctggggac ctctgacagt gggcgggctt 1140 ccagtaagag tgggtcgtca tcatccatgg ggcggtcagg ccacctggga tccggggagg 1200 gcggaaatgg gggcctgcca tttgcagcct gttcaccacc ctcgcccagt gcgctgatcc 1260 aggagctgga ggagcggctg tgggagaagg agcaggaggt ggcagctctg cggcgcagcc 1320 tggagcagag cgaggcagcc gtggcccagg tgttggagga gcggcagaag gcatgggagc 1380 gggagctagc cgagctccgg cagggctgca gtgggaagct gcagcaggtg gcccgccgtg 1440 cccagcgtgc ccagcagggc ctacagctgc aggtgctgcg gttacagcag gacaagaaac 1500 agctgcagga ggaggcggcc cagctgataa ggcaacggga agagctggag gacaaggtgg 1560 ccgtctgtca gaaggagcag gccgacttcc tgccccggat ggaggaaact aagtgggagg 1620 tgtgccagaa ggccggtgag atttccctcc tgaagcaaca actgaaggac tctcaggccg 1680 atgtgtcgca gaagttgagt gagatcgtgg ggctgcgctc acagcttcgg gagggtaggg 1740 cctcgctccg ggagaaggag gagcagctgc tcagcttgag ggactccttc ggcagcaaac 1800 aggccagcct ggagctgagt gaaggcgagc tgccccctgc ctgcctcaag cctgcgctga 1860 cccccgtgga cctggtcgag ccacaggagg cgctggcctc ctgtgagagt gacgaggcca 1920 agatgcgccg gcaggctggc gtggcggctg ccgcttctct ggtttctgtt gatggggagg 1980 tggaggctgg aggagagggt gggacacggg ccctacgcag ggaggtgggg agattgcagg 2040 ctgagctggc tgctgagcgg cgtgcccgtg agcgccaggg tgccagcttt gcggaggagc 2100 gccgggtgtg gttggaggag aaggagaagg tcattgagta tcagaaacag ctccagctga 2160 gttatgtgga gatgtatcag cgcaaccagc agctggagcg gcgacttcgg gagcgtgggg 2220 cagcaggggg gtccagcacg cctactcccc agcatggaga ggaaaagaaa gcctggaccc 2280 cctcccgcct ggagcgcatc gagtccactg aaatctgatt ctctacgtgc acaaccttct 2340 gacacgtgcc ctgtctgaag gtcggagtca ccaatgtccc ttccattcat ctcccttccc 2400 cacgtgtgct gcacatcccc agaccaaaga ggttctcgaa gcagctgtga ccagcccctc 2460 cccccacttg tccccccgtcc ttgcagctcc agcactgcct ttgtgggtga ccactccaga 2520 ccctcctgcc aaggagagca tgggagaggg cagagtacaa tggggtgagg tcccaggcgc 2580 atggagcaag gctccctttc tggggaagcc tcgtgttgga gatagaggca gcaggcctgc 2640 gttgccaggg agtaccccag cccctttccc ttcttgggct gctaccgccg ccaccaccgt 2700 caccaccatc tgtccagtgc tactgtctgt gccctcctag gccacacagc ctgagggagc 2760 ttgcatgggt ctgcccgtgt cgacagaccc taggctccca gtctctctcc ttcatgtgct 2820 attctccttc cccaggcaga ttggcagggg aagtgggagc agatggcctg cctgtggttg 2880 agagggctac ctgcccgacc cctcccccac caaggtatct tgggctccag cctcagagcc 2940 tggactagtt ctgagcatgt gtccctctgt gtatagtgag gaagtgagag cagaggctgg 3000 aaactcaatc ccaaggggag atctgccttc ctaccctcgg gaagggtcac ctagaggctt 3060 ctcagcccca ccctcaccct ctggccagga tcctacaagg ctacatcccc tgctgcttgg 3120 ttgacccccc caaatccagg gtcactgctc agctctgctt atagctatac ctgcctctta 3180 ggtcttctgt ttgcccgttt ctgaggtcag atggatggat gtctgatgtc ttcctgtgtt 3240 ttccctcgat ctctcccctt ggcctccttt cgcactcaca tcttcccagg ctgccttttc 3300 ttccaagttt cacctttcct cccagcagaa agctctgctt ggcttttggt gcactgctca 3360 cccccagctc cactggccca agtctgagcc tggggccctt gctttgaggg gccagtccga 3420 gttggatggg actgccctca aggaacaagg ctaatgtgtg tgtgtatgtt agagagagag 3480 3540 agagagagag agagagagag agagagagag agagagagag aaagagagag agggaggact actgaccctt gggttgcttg ccagactctt 3600 gagttggctc atttgtccgg gcagggttgg ctggtatggt catcagtatg ggtcgtgaca 3660 gtcaatgtca cttccttctc taggatctct cagattctat ctccccaaga caggcaggga 3720 aattctaatt tctctcttaa gaaagtaaat ttccacttca ccagcaataa caatcagagg 3780 agacttggcc ctcagtcctt gtatttctct ttttcactct ctcttccacc ccaagatgga 3840 atcttgggga caggagggag agggttggta actactgtga gcaagtcccc caagccctga 3900 ccagcctcct cccatgactg gtgactgttt aacaagctgt gcatccccca caaaaatagg 3960 agtgccctat ttgtagtctc cacctctctg cacagctcct tctgggtggt cctcagcagg 4020 gttttgacct tgattggcgg ctactctggc aaccactgcc cattcttcta atccctcaca 4080 atacgtgatt cagaccctga gcctggtcca cagtgactgg gaaaaggcat taacacaaaa 4140 caaaagagat ggggggagag aaaaatatga acagctttgg aatgtccaac tgcaaaataa 4200 attatatata aatatataaa tatataaata tatatatcta ccatatgtga tggaaagagt 4260 tttcattttt cctttcccaa agaaataaaa tggagaaagc ctcttggatg gcaattctg 4319 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Lzts3 BSAS-F <400> 3 taggttatta ggatttgggg atttt 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Lzts3 BSAS-R <400> 4 accacctact acaacttccc actac 25 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Lzts3-F <400> 5 caaggctcca ccccagtat 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lzts3-R <400> 6 tggtctgtgg aggacacaag 20

Claims (8)

서열번호 2로 표시되는 마우스 LZTS3 (leucine zipper tumor suppressor family member 3) 유전자의 2번 염색체 130,635,994 내지 130,636,258번째 염기 사이에 존재하는 CpG의 메틸화 여부를 검출할 수 있는 물질을 포함하는 비만의 예측 또는 진단용 조성물. A composition for prediction or diagnosis of obesity comprising a substance capable of detecting whether CpG is methylated between the bases 130,635,994 to 130,636,258 of the mouse LZTS3 (leucine zipper tumor suppressor family member 3) gene represented by SEQ ID NO: 2 . 삭제delete 제 1 항에 있어서, 상기 메틸화 여부를 검출할 수 있는 물질은 메틸화된 CpG의 부위를 포함하는 단편을 증폭할 수 있는 프라이머 세트, 메틸화된 CpG의 부위와 혼성화할 수 있는 프로브, 메틸화된 CpG의 부위와 결합할 수 있는 메틸화 특이적 결합단백질, 메틸화 특이적 결합 항체 또는 압타머, 시퀀싱 프라이머, 시퀀싱 바이 신세시스 프라이머 및 시퀀싱 바이 라이게이션 프라이머로 이루어진 군으로부터 선택되는 하나 이상인 것인, 비만의 예측 또는 진단용 조성물.The method of claim 1, wherein the substance capable of detecting methylation is a primer set capable of amplifying a fragment containing a methylated CpG region, a probe capable of hybridizing with a methylated CpG region, and a methylated CpG region. A methylation-specific binding protein capable of binding to, a methylation-specific binding antibody or aptamer, a sequencing primer, a sequencing-by-synthesis primer, and a sequencing-by-ligation primer, which is at least one selected from the group consisting of, the prediction or diagnosis of obesity . 제 1 항 또는 제 3 항 중 어느 한 항의 조성물을 포함하는 비만의 예측 또는 진단용 키트.A kit for prediction or diagnosis of obesity comprising the composition of any one of claims 1 to 3. 분리된 생물학적 시료로부터 서열번호 2로 표시되는 마우스 LZTS3(leucine zipper tumor suppressor family member 3) 유전자의 2번 염색체 130,635,994 내지 130,636,258번째 염기 사이에 존재하는 CpG의 메틸화를 측정하는 단계를 포함하는 비만의 예측 또는 진단을 위한 정보를 제공하는 방법.Prediction of obesity comprising the step of measuring the methylation of CpG present between the bases 130,635,994 to 130,636,258 of chromosome 2 of the mouse LZTS3 (leucine zipper tumor suppressor family member 3) gene represented by SEQ ID NO: 2 from the isolated biological sample How to provide information for diagnosis. 제 5 항에 있어서, 상기 방법은 정상 대조군의 CpG의 메틸화와 비교하여 CpG의 메틸화 수준이 감소하면 비만으로 결정하는 단계를 추가적으로 포함하는 것인, 비만의 예측 또는 진단을 위한 정보를 제공하는 방법. The method of claim 5, wherein the method further comprises determining obesity when the methylation level of CpG is decreased compared to that of CpG in the normal control group. 제 5 항에 있어서, 상기 생물학적 시료는 비만 의심 또는 진단 대상 개체 유래의 세포, 조직, 생검, 파라핀조직, 혈액, 혈청, 혈장 및 소변으로 이루어진 군으로부터 선택되는 하나 이상인 것인, 비만의 예측 또는 진단을 위한 정보를 제공하는 방법. The prediction or diagnosis of obesity according to claim 5, wherein the biological sample is at least one selected from the group consisting of cells, tissues, biopsies, paraffin tissues, blood, serum, plasma and urine derived from individuals suspected of or diagnosed with obesity. How to provide information for 제 5 항에 있어서, 상기 메틸화를 측정하는 단계는 PCR, 메틸화 특이 PCR(methylation specific PCR), 실시간 메틸화 특이 PCR(real time methylation specific PCR), 메틸화 DNA 특이적 결합 단백질을 이용한 PCR, 정량 PCR, DNA 칩, 파이로시퀀싱 및 바이설파이트 시퀀싱으로 이루어진 군으로부터 선택되는 방법에 의하여 수행되는 것인, 비만의 예측 또는 진단을 위한 정보를 제공하는 방법.The method of claim 5, wherein the measuring of the methylation comprises PCR, methylation specific PCR, real time methylation specific PCR, PCR using a methylated DNA-specific binding protein, quantitative PCR, DNA A method of providing information for prediction or diagnosis of obesity, which is performed by a method selected from the group consisting of chip, pyrosequencing and bisulfite sequencing.
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