JP2022022709A - miRNA DIAGNOSIS BIOMARKER FOR SEVERE DRUG ERUPTION - Google Patents
miRNA DIAGNOSIS BIOMARKER FOR SEVERE DRUG ERUPTION Download PDFInfo
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Abstract
Description
本発明は、重症薬疹の発症や病勢の診断を補助するための方法に関する。 The present invention relates to a method for assisting in the diagnosis of the onset and pathological condition of severe drug eruption.
薬疹とは、医薬品及びその代謝物により誘発される皮膚反応の総称であり、そのうち最も重篤なものとして、Stevens-Johnson症候群(SJS)や中毒性表皮壊死融解症(toxic epidermal necrolysis; TEN)、薬剤性過敏症症候群(drug-induced hypersensitivity syndrome; DIHS)等が知られている。これら重症薬疹は、罹患率が少ないものの、致死性が高く、回復後も重篤な後遺症が残ることがあるため、これら重症薬疹が疑われる患者を早期に診断し、適切な治療を速やかに開始することが重要である。そのため、SJS/TENやDIHSを早期に診断できるバイオマーカーの開発が嘱望されている。 Drug eruption is a general term for skin reactions induced by drugs and their metabolites, the most serious of which are Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). , Drug-induced hypersensitivity syndrome (DIHS), etc. are known. Although these severe drug eruptions have a low morbidity rate, they are highly lethal and may have serious sequelae after recovery. Therefore, patients suspected of having these severe drug eruptions should be diagnosed early and appropriate treatment should be promptly performed. It is important to start with. Therefore, the development of biomarkers that can diagnose SJS / TEN and DIHS at an early stage is desired.
重症薬疹の発症と免疫系の異常な活性化との関連はこれまでの報告において強く示唆されている。そのため、免疫系に作用するサイトカインやケモカイン等が重症薬疹の血液中タンパク質バイオマーカーとして多く報告されている。その代表的な例としては、TARC (ケモカインCCL17) 、グラニュライシン(Granulysin、GNLY)、FAS-L、インターロイキン6、IP-10 (ケモカインCXCL10)、およびそれらの組み合わせがなど挙げられる(非特許文献1, 2, 3)。このうち、TARCはアトピー性皮膚炎の重症度を反映するバイオマーカーとして既に保険収載されている(非特許文献4)。
Previous reports have strongly suggested a link between the development of severe drug eruption and abnormal activation of the immune system. Therefore, many cytokines and chemokines that act on the immune system have been reported as blood protein biomarkers for severe drug eruption. Typical examples include TARC (chemokine CCL17), granulysin (GNLY), FAS-L,
MicroRNA (miRNA)は、約22塩基長の機能性微小核酸であり、RNA干渉により生体中の遺伝子発現を制御する役割を担うことが知られている。これまでに、種々のmiRNAsが創傷修復における上皮再形成や血管新生を制御することが報告されており、また強皮症、乾癬、および皮膚がん等の皮膚疾患の病態形成に深く関与することが知られている(非特許文献5)。しかしながら、血液中に存在するmiRNAsの発現量と、重症薬疹の発症や重症化との関連についてはほとんど報告されていない。 MicroRNA (miRNA) is a functional micronucleic acid with a length of about 22 bases, and is known to play a role in controlling gene expression in a living body by RNA interference. It has been reported that various miRNAs control epithelial remodeling and angiogenesis in wound repair, and are deeply involved in the pathogenesis of skin diseases such as scleroderma, psoriasis, and skin cancer. Is known (Non-Patent Document 5). However, few reports have been made on the relationship between the expression level of miRNAs present in blood and the onset or aggravation of severe drug eruption.
本発明は、Stevens-Johnson症候群/中毒性表皮壊死症(SJS/TEN)や薬剤性過敏症症候群(DIHS)を高い精度で診断することを補助する方法、これらの治療奏効性を客観的に評価する方法、また重症化せずに治癒する軽症薬疹症例と重症化する症例の鑑別を補助する方法を提供することを課題とする。 The present invention is a method for assisting in highly accurate diagnosis of Stevens-Johnson syndrome / toxic epidermal necrolysis (SJS / TEN) and drug-induced hypersensitivity syndrome (DIHS), and objectively evaluates the therapeutic response of these methods. It is an object of the present invention to provide a method for treating mild drug eruption that heals without becoming severe and a method for assisting in the distinction between a case of mild drug eruption and a case of becoming severe.
752種のmiRNAに対するLNA-enhanced Probe/Primersを用いたmiRCURY LNA microRNA PCR法により、Stevens-Johnson症候群/中毒性表皮壊死症(SJS/TEN)や薬剤性過敏症症候群(DIHS)の重症化、あるいは病勢変化の診断に有用と考えられるバイオマーカーmiRNAsを探索した。まず、ボルケーノプロット、ROC曲線(Receiver Operating Characteristic curve)及び多重比較統計試験を用いて、探索用血清検体セットに対するmiRCURY LNA microRNA PCR測定で定量値が入手可能であった502種のmiRNAsのうち、重症薬疹の急性期と回復期で顕著な発現変動を示すmiRNAsを17種同定した。その後、異なる検体セットの血清をTaqman Advanced miRNA Assay法で測定した検証試験において、回復期例と重症薬疹の急性期との間における顕著な発現変動の再現性が確認できたmiRNAsを4種(hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p, hsa-miR-222-3p)同定した。 Stevens-Johnson syndrome / toxic epidermal necrolysis (SJS / TEN) or drug reaction with eosin syndrome (DIHS) is aggravated by miRCURY LNA microRNA PCR method using LNA-enhanced Probe / Primers for 752 miRNAs. We searched for biomarker miRNAs that may be useful in diagnosing disease changes. First, of the 502 miRNAs for which quantitative values were available for miRCURY LNA microRNA PCR measurements on exploratory serum sample sets using volcano plots, ROC curves (Receiver Operating Characteristic curves) and multiplex comparative statistical tests. We identified 17 miRNAs that showed marked fluctuations in expression during the acute and convalescent stages of drug eruption. After that, in a verification test in which sera of different sample sets were measured by the Taqman Advanced miRNA Assay method, 4 types of miRNAs were confirmed to have reproducibility of remarkable expression fluctuation between convalescent cases and acute stage of severe drug eruption (4 types of miRNAs. hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p, hsa-miR-222-3p) Identified.
探索群におけるhsa-miR-205-5p、hsa-miR-21-5p、hsa-miR-21-3p及びhsa-miR-222-3pの補正サイクル値(ΔCq値)を用いたROC曲線解析の結果、hsa-miR-205-5pはSJS/TENとDIHSの急性期症例と回復期例を高い精度で(area under the ROC curve: AUROC 0.85以上)で鑑別した。特に、SJS/TENにおける本miRNA分子の病勢診断能は既報マーカーであるGNLY(AUROC 0.54)より顕著に高かった。一方、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pについて、DIHSの病勢診断において有意な診断能が認められた。さらに、hsa-miR-205-5p、hsa-miR-21-5p及びhsa-miR-21-3pを組み合わせた重症薬疹診断モデルは、SJS/TENやDIHSにおける病勢診断能を向上させ、かつSJS/TENへと病状が進行することが知られる多型紅斑重症型(EM major)の病勢予測に関しても、既報マーカーとより高い精度で実現可能であると示した。 Results of ROC curve analysis using correction cycle values (ΔCq values) of hsa-miR-205-5p, hsa-miR-21-5p, hsa-miR-21-3p and hsa-miR-222-3p in the search group , Hsa-miR-205-5p differentiated between acute and convalescent cases of SJS / TEN and DIHS with high accuracy (area under the ROC curve: AUROC 0.85 or higher). In particular, the disease diagnostic ability of this miRNA molecule in SJS / TEN was significantly higher than that of the previously reported marker GNLY (AUROC 0.54). On the other hand, for hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p, significant diagnostic ability was observed in the diagnosis of DIHS disease. In addition, a severe drug eruption diagnostic model combining hsa-miR-205-5p, hsa-miR-21-5p and hsa-miR-21-3p improves the ability to diagnose disease in SJS / TEN and DIHS, and also SJS. It was also shown that it is possible to predict the condition of erythema multiforme (EM major), which is known to progress to / TEN, with higher accuracy than the previously reported markers.
検証群における上記4種のmiRNAsの病勢診断能並びに重症化診断能についてROC曲線解析したところ、いずれのmiRNAも重症薬疹の病勢診断において、高い診断能を示した。また、miRCURY LNA microRNA PCR法で得られた結果と同様、hsa-miR-205-5p、hsa-miR-21-5p及びhsa-miR-21-3pを組み合わせた重症薬疹診断モデルは解析したすべての薬疹病型において高い病勢診断能を示した。一方、今回解析した4種miRNAsのすべてがSJS/TENまたはDIHSのいずれか、あるいはその両病型の重症化診断において有意な診断能を示したものの、個別のmiRNA分子の重症化予測能については重症薬疹の病型間で差異が認められた。解析の結果、SJS/TENに対してはhsa-miR-205-5p、DIHSに対してはhsa-miR-21-3pが最も有望な重症化予測バイオマーカーとなりうることが示された。3種のmiRNAsを組み合わせることにより、それぞれのmiRNAバイオマーカーの重症化予測能を低下させることなく、SJS/TEN(AUROC 0.86)及びDIHS(AUROC 0.93)の両方の重症化予測に適応でき、その予測精度は既存マーカーのTARC(AUROC 0.83)より高いことが示された。 ROC curve analysis was performed on the pathological diagnosis ability and aggravation diagnostic ability of the above four types of miRNAs in the verification group, and all miRNAs showed high diagnostic ability in the pathological diagnosis of severe drug eruption. In addition, as with the results obtained by the miRCURY LNA microRNA PCR method, all the severe drug eruption diagnostic models combined with hsa-miR-205-5p, hsa-miR-21-5p and hsa-miR-21-3p were analyzed. It showed a high ability to diagnose the disease in the drug eruption type. On the other hand, although all of the four miRNAs analyzed this time showed significant diagnostic ability in the diagnosis of aggravation of either SJS / TEN or DIHS, or both types, the ability to predict the aggravation of individual miRNA molecules was described. Differences were observed among the types of severe drug eruption. Analysis showed that hsa-miR-205-5p for SJS / TEN and hsa-miR-21-3p for DIHS could be the most promising predictive biomarkers for aggravation. By combining three types of miRNAs, it is possible to adapt to and predict the severity of both SJS / TEN (AUROC 0.86) and DIHS (AUROC 0.93) without deteriorating the severity prediction ability of each miRNA biomarker. The accuracy was shown to be higher than the existing marker TARC (AUROC 0.83).
hsa-miR-205-5p、hsa-miR-21-5p、hsa-miR-21-3p及びhsa-miR-222-3p について、重症薬疹と他の皮膚疾患との診断能を評価した結果、SJS/TENとアトピー性皮膚炎、乾癬、水痘、中毒疹との鑑別においてhsa-miR-205-5pは高い診断能(AUROC>0.85)を示すことが認められた。一方、hsa-miR-21-5p及びhsa-miR-21-3pはSJS/TENと自己免疫性水疱症との間において高い診断能を示した。これらの診断能は、いずれも既存マーカーと比べ、著しく高いことが示された。次に、これらmiRNAsを用いた、DIHSとその他の皮膚疾患の診断能を評価したところ、単独利用の場合、hsa-miR-21-3pが中毒疹を除くすべての対照皮膚疾患との鑑別において、AUROC 0.90以上の極めて高い診断能を示した。また、hsa-miR-205-5p、 hsa-miR-21-5p及びhsa-miR-21-3pを組み合わせた重症薬疹診断モデルについて同様の評価を行ったところ、中毒疹を含むすべての皮膚疾患と良好な診断能を示した。さらに、本診断モデルに既報マーカーであるTARCを組み合わせることにより、鑑別診断能の向上が確認でき、DIHSと全ての対照皮膚疾患をAUROC 0.89以上で鑑別できることが示された。以上より、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3p単体、あるいはそれらを組み合わせた診断モデルが、重症薬疹の病勢診断、重症化予測、さらに他の皮膚疾患との鑑別に有用なバイオマーカーとなることが示された。 As a result of evaluating the diagnostic ability of hsa-miR-205-5p, hsa-miR-21-5p, hsa-miR-21-3p and hsa-miR-222-3p for severe drug eruption and other skin diseases, In the differentiation of SJS / TEN from atopic dermatitis, psoriasis, chickenpox and drug eruption, hsa-miR-205-5p was found to show high diagnostic ability (AUROC> 0.85). On the other hand, hsa-miR-21-5p and hsa-miR-21-3p showed high diagnostic ability between SJS / TEN and autoimmune vesicular disease. All of these diagnostic abilities were shown to be significantly higher than those of existing markers. Next, when the diagnostic ability of DIHS and other skin diseases using these miRNAs was evaluated, hsa-miR-21-3p was differentiated from all control skin diseases except toxic eruption when used alone. It showed extremely high diagnostic ability with AUROC 0.90 or higher. In addition, a similar evaluation was performed on a severe drug eruption diagnosis model combining hsa-miR-205-5p, hsa-miR-21-5p and hsa-miR-21-3p, and all skin diseases including toxic eruption were evaluated. It showed good diagnostic ability. Furthermore, by combining this diagnostic model with the previously reported marker TARC, it was confirmed that the differential diagnosis ability was improved, and it was shown that DIHS and all control skin diseases can be differentiated with AUROC 0.89 or higher. Based on the above, the diagnostic model of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p alone or a combination thereof is the pathological condition of severe drug eruption. It has been shown to be a useful biomarker for diagnosis, prediction of aggravation, and differentiation from other skin diseases.
本発明は、これらの知見に基づいて、完成されたものである。本発明の要旨は以下の通りである。
(１)hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における遺伝子発現を測定することを含む、重症薬疹の検査方法。
(２)発現を測定するmiRNAが、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１種のmiRNAであり、測定値が重症薬疹の病勢診断を補助する(１)記載の方法。
(３)発現を測定するmiRNAが、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１種のmiRNAであり、測定値が重症薬疹の重症化予測を補助する(１)に記載の方法。
(４)発現を測定するmiRNAが、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１種のmiRNAであり、測定値が重症薬疹と他の皮膚疾患との鑑別を補助する(１)に記載の方法。
(５)発現を測定するmiRNAが、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3p発現の組み合わせからなる群から選択される少なくとも一組のmiRNAの組み合わせである(２)に記載の方法。
(６)発現を測定するmiRNAが、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3p発現の組み合わせからなる群から選択される少なくとも一組のmiRNAの組み合わせである(３)に記載の方法。
(７)発現を測定するmiRNAが、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3p発現の組み合わせからなる群から選択される少なくとも一組のmiRNAの組み合わせである(４)に記載の方法。
(８)hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における遺伝子発現を測定することができる試薬を含む、重症薬疹の検査のためのキット。
The present invention has been completed based on these findings. The gist of the present invention is as follows.
(1) Subject-derived at least one miRNA selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. A method for testing for severe drug eruption, which comprises measuring gene expression in a sample of.
(2) At least one miRNA whose expression is to be measured is selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. The method according to (1), which is a species of miRNA and whose measured value assists in the diagnosis of severe drug eruption.
(3) At least one miRNA whose expression is to be measured is selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. The method according to (1), which is a species of miRNA and whose measured value assists in predicting the severity of severe drug eruption.
(4) At least one miRNA whose expression is to be measured is selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. The method according to (1), which is a species of miRNA and whose measured value assists in the differentiation of severe drug eruption from other skin diseases.
(5) The miRNA whose expression is to be measured is selected from the group consisting of a combination of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p expression. The method according to (2), which is a combination of at least one set of miRNAs.
(6) The miRNA whose expression is to be measured is selected from the group consisting of a combination of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p expression. The method according to (3), which is a combination of at least one set of miRNAs.
(7) The miRNA whose expression is to be measured is selected from the group consisting of a combination of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p expression. The method according to (4), which is a combination of at least one set of miRNAs.
(8) Subject-derived at least one miRNA selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. A kit for testing for severe drug eruption, which contains reagents that can measure gene expression in a sample of.
本発明により見いだされたmiRNAの発現量を、単独、あるいは複数項目を測定することにより、重症薬疹疑いの患者と非薬剤性皮膚疾患患者を鑑別することが可能である。加えて、本発明は、重症薬疹を発症した患者の病勢変化や重症化リスクを高い精度で診断することができる。 By measuring the expression level of miRNA found by the present invention alone or by measuring a plurality of items, it is possible to distinguish a patient suspected of having severe drug eruption from a patient with a non-drug skin disease. In addition, the present invention can diagnose the disease state change and the risk of aggravation of a patient who has developed severe drug eruption with high accuracy.
以下、本発明の実施の形態を詳細に説明する。 Hereinafter, embodiments of the present invention will be described in detail.
本発明は、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における遺伝子発現を測定することを含む、重症薬疹の検査方法を提供する。 The present invention is a subject for at least one miRNA selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. Provided are methods of testing for severe drug eruption, including measuring gene expression in a sample of origin.
薬疹とは、薬物に反応して生じる発疹であり、医薬品による副作用の一種である。一般的に、薬剤服用後１～２週間後に発症することが多く、原因薬物を中止することで軽快するため、診断は容易である。しかし、重篤な薬疹である重症薬疹は、原因薬剤の中止のみでは軽快しない場合も多く、適切な治療を行わなければ、生命を脅かすため、早期診断が重要である。 Drug eruption is a rash that occurs in response to a drug and is a type of side effect caused by a drug. In general, it often develops 1 to 2 weeks after taking the drug, and it is easy to diagnose because it is relieved by discontinuing the causative drug. However, severe drug eruption, which is a serious drug eruption, is often not relieved only by discontinuation of the causative drug, and if appropriate treatment is not performed, it is life-threatening, so early diagnosis is important.
重症薬疹の代表例である、Stevens-Johnson症候群(SJS)および中毒性表皮壊死融解症(toxic epidermal necrolysis; TEN)は、致死率が高く、失明や呼吸器障害などの後遺症を残す難治性の疾患である。SJSとTENは同じスペクトラムの病態であると考えられており、TENの症状はSJSと類似するものの、より重篤であり、TENはSJSから進行する場合が多い。SJSでは、発熱を伴う***、眼結膜、外陰部などの皮膚粘膜移行部における広範囲な粘膜病変がみられ、皮膚の汎発性の紅斑に伴って表皮の壊死性障害に基づくびらん・水疱を特徴とする。その面積は体表面積の10%未満である。一方、TENは、広範囲な紅斑と、全身の10％以上の水疱、表皮剥離・びらんなどの顕著な表皮の壊死性障害を認め、高熱と粘膜疹を伴い、医薬品による皮膚障害の中で最も重篤とされている。SJS及びTENの発生頻度は、人口100万人当たりそれぞれ年間1～6人及び0.4～1.3人とされる。 Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), which are typical examples of severe drug eruption, are highly lethal and intractable with sequelae such as blindness and respiratory disorders. It is a disease. SJS and TEN are thought to be pathological conditions of the same spectrum, and although the symptoms of TEN are similar to SJS, they are more severe and TEN often progresses from SJS. SJS has extensive mucosal lesions in the mucosal transitions such as the lips, ocular conjunctiva, and genitals with fever, and is characterized by erosions and blisters due to necrotizing disorders of the epidermis with generalized erythema of the skin. And. Its area is less than 10% of the body surface area. On the other hand, TEN has extensive erythema and marked epidermal necrolysis such as blisters of 10% or more of the whole body, epidermal detachment and erosion, accompanied by high fever and enanthem, and is the most severe skin disorder caused by drugs. It is said to be serious. The frequency of SJS and TEN is 1 to 6 and 0.4 to 1.3 per 1 million population, respectively.
薬剤性過敏性症候群(drug-induced hypersensitivity syndrome; DIHS)もまた、SJS/TENと並ぶ重症型の薬疹である。38度以上の高熱を伴う紅斑丘疹や多形紅斑が全身にみられ、進行すると紅皮症となる。通常粘膜疹は伴わないか軽度であるが、ときに口腔粘膜のびらんを認める。全身のリンパ節腫脹、肝機能障害をはじめとする臓器障害、末梢白血球異常(白血球増多、好酸球増多、異型リンパ球の出現)がみられる。経過中にヒトヘルペスウイルス (HHV)－６の再活性化が起きるのが特徴であり、リンパ節腫脹又はHHV-6の再活性化が証明できない場合は、非典型DIHSとして診断される。通常の薬疹とは異なり、原因医薬品の投与後すぐには発症せずに2週間以上経ってから発症することが多く、原因薬物の中止後も症状は続き、軽快するまで1ヶ月以上の経過を要することがしばしば認められる。 Drug-induced hypersensitivity syndrome (DIHS) is also a severe drug eruption along with SJS / TEN. Erythema multiforme and erythema multiforme with high fever of 38 degrees or higher are seen throughout the body, and when it progresses, it becomes erythroderma. Usually with no or mild enanthem, but sometimes erosions of the oral mucosa are present. Systemic lymphadenopathy, organ disorders such as liver dysfunction, and peripheral leukocytosis (leukocytosis, eosinophilia, appearance of atypical lymphocytes) are observed. It is characterized by reactivation of human herpesvirus (HHV) -6 during the course and is diagnosed as atypical DIHS if lymphadenopathy or reactivation of HHV-6 cannot be demonstrated. Unlike normal drug eruption, it does not develop immediately after administration of the causative drug, but often develops after 2 weeks or more, and the symptoms continue even after discontinuation of the causative drug, and it takes more than 1 month to improve. Is often found to require.
これらの重症薬疹は、早期に診断し、適切な治療を速やかに開始することが重要とされているが、その初期症状は軽症薬疹の症状と類似しているため、重症化する患者を早期に識別、あるいは予測することは現状困難である。また、重症化患者に対して適切な治療を提供するためには、病勢(病状の活動性)を把握することが重要である。 It is important to diagnose these severe drug eruptions at an early stage and start appropriate treatment promptly, but since the initial symptoms are similar to those of mild drug eruptions, patients who become severe are treated. It is currently difficult to identify or predict at an early stage. In addition, it is important to understand the pathological condition (activity of the medical condition) in order to provide appropriate treatment to severely ill patients.
これまで、アトピー性皮膚炎の重症度評価バイオマーカーとして知られるTARC (ケモカインCCL17) がDIHSの検証に、あるいはSJS/TENの検出に対して、グラニュライシンやFasリガンド等が、重症薬疹の血液中タンパク質マーカーとして提案されている。一方、本発明では、これまでに血液試料を用いた重症薬疹の診断では利用されていないmiRNAsについて着目し、それらの発現量(ΔCq値)を単独あるいは組み合わせることにより、重症薬疹の病勢診断や重症化予測に応用することを示す。 Until now, TARC (chemocain CCL17), which is known as a biomarker for evaluating the severity of atopic dermatitis, has been used for DIHS verification, or for SJS / TEN detection, granuricin, Fas ligand, etc. have been used for the blood of severe drug eruption. It has been proposed as a medium protein marker. On the other hand, the present invention focuses on miRNAs that have not been used in the diagnosis of severe drug eruption using blood samples, and diagnoses the disease state of severe drug eruption by using their expression levels (ΔCq values) alone or in combination. It is shown to be applied to the prediction of aggravation.
hsa-miR-205-5p(miRBase ID: MIMAT0000266)は、皮膚組織に高発現することが報告されているmiRNAである。これまでに、本遺伝子の発現が新生児期における重層上皮形成や毛包幹細胞の増殖に必要不可欠であることが報告されている(Nat Cell Biol. 2013 Oct;15(10):1153-63)。また、本遺伝子が皮膚の創傷治癒における再上皮化に寄与することも知られている(Biochim Biophys Acta. 2016 Aug;1862(8):1443-52)。さらに、本分子は皮膚がんの上皮間葉転換に関与する遺伝子の発現を抑制することにより、がん細胞の転移や成長を抑える役割を担うことも報告されている(Mol Carcinog. 2016 Aug;55(8):1229-42)。hsa-miR-205-5pの塩基配列を配列番号１に示す。 hsa-miR-205-5p (miRBase ID: MIMAT0000266) is a miRNA that has been reported to be highly expressed in skin tissue. So far, it has been reported that the expression of this gene is essential for stratified epithelium formation and hair follicle stem cell proliferation in the neonatal period (Nat Cell Biol. 2013 Oct; 15 (10): 1153-63). It is also known that this gene contributes to re-epithelialization in skin wound healing (Biochim Biophys Acta. 2016 Aug; 1862 (8): 1443-52). Furthermore, it has been reported that this molecule plays a role in suppressing the metastasis and growth of cancer cells by suppressing the expression of genes involved in epithelial-mesenchymal transition in skin cancer (Mol Carcinog. 2016 Aug; 55 (8): 1229-42). The base sequence of hsa-miR-205-5p is shown in SEQ ID NO: 1.
hsa-miR-21-3p(miRBase ID: MIMAT0004494)は、種々の細胞に発現することがしられているが、ヒト正常組織においては筋膜、動脈、神経、くも膜において高発現する。また、皮膚における本miRNAの発現も確認されている。本miRNAはMIR21遺伝子のよりコードされるmiR-21前駆体塩基配列の3’末端側に存在し、複数の酵素によるプロセシングを経て産生される。本miRNAは紫外線に起因する皮膚炎症を抑制する機能を有することが報告されている(EMBO Mol Med. 2016 Aug 1;8(8):919-36.)。一方で、本miRNAは血管新生能及び線維芽細胞の遊走能の向上を介した創傷治癒に寄与することも知られている(Theranostics. 2018 Jan 1;8(1):169-184.)。hsa-miR-21-3pの塩基配列を配列番号２に示す。
hsa-miR-21-3p (miRBase ID: MIMAT0004494) has been shown to be expressed in various cells, but is highly expressed in fascia, arteries, nerves, and arachnoid in normal human tissues. The expression of this miRNA in the skin has also been confirmed. This miRNA is located on the 3'end side of the miR-21 precursor base sequence encoded by the MIR21 gene and is produced through processing by multiple enzymes. It has been reported that this miRNA has a function of suppressing skin inflammation caused by ultraviolet rays (EMBO Mol Med. 2016
hsa-miR-21-5p(miRBase ID: MIMAT0000076)は肺、甲状腺、皮膚、脾臓等において多く発現することが報告されている。本miRNAは、hsa-miR-21-3pと同じ遺伝子(MIR21)によりコードされており、そのトランスクリプトの5’末端側に存在し、複数の酵素によるプロセシングを経て産生される。本分子は、乳がん、大腸がん、グリオーマ等の種々の固形がん細胞において、がん遺伝子として機能する (Int J Biol Sci. 2011;7(5):685-90)。一方で、本miRNAはT細胞のT細胞受容体を介したシグナル伝達に関与する遺伝子の発現を抑制することにより、その活性化を負に制御することも報告されている(Biochimie. 2014 Dec;107 Pt B:319-26)。また、本miRNAはケラチノサイトにおいて、線維化刺激により発現上昇し、細胞遊走能を向上させ、皮膚の創傷修復を促進する役割を担うことが報告されている(Int J Biol Sci. 2011;7(5):685-90.)。hsa-miR-21-5pの塩基配列を配列番号３に示す。 It has been reported that hsa-miR-21-5p (miRBase ID: MIMAT0000076) is highly expressed in lungs, thyroid gland, skin, spleen and the like. This miRNA is encoded by the same gene (MIR21) as hsa-miR-21-3p, is located on the 5'end of the transcript, and is produced through processing by multiple enzymes. This molecule functions as an oncogene in various solid cancer cells such as breast cancer, colon cancer, and glioma (Int J Biol Sci. 2011; 7 (5): 685-90). On the other hand, it has also been reported that this miRNA negatively regulates its activation by suppressing the expression of genes involved in T cell receptor-mediated signal transduction (Biochimie. 2014 Dec; 107 Pt B: 319-26). In addition, it has been reported that this miRNA is upregulated in keratinocytes by stimulation of fibrosis, improves cell migration ability, and promotes skin wound repair (Int J Biol Sci. 2011; 7 (5). ): 685-90.). The base sequence of hsa-miR-21-5p is shown in SEQ ID NO: 3.
hsa-miR-222-3p(miRBase ID: MIMAT0000279)は、正常組織において、前立腺、膀胱、皮膚、甲状腺、肺等の組織に高く発現することが確認されている。本miRNAはこれまでに、乾癬や肥厚性瘢痕の皮膚組織において正常皮膚組織と比べ、高発現することが知られている(Medicine (Baltimore). 2015 Feb;94(7):e458., J Dermatol Sci. 2010 Jun;58(3):177-85)。また、本遺伝子はケラチノサイトにおけるマトリックス分解酵素の発現を制御することが報告されている(J Dermatol Sci. 2010 Jun;58(3):177-85.)。さらに、本遺伝子は破骨細胞形成を抑制することが明らかとなっている(Int J Mol Sci. 2016 Feb; 17(2): 240.)。また、本miRNAは種々のがん細胞において、細胞増殖や細胞遊走/浸潤能の促進に寄与し、その増悪に関与することが知られている(PLoS One. 2014 Jan 31;9(1):e87563., Technol Cancer Res Treat. Jan-Dec 2019;18:1533033819892256., Cell Signal. 2019 Nov;63:109386.)。hsa-miR-222-3pの塩基配列を配列番号４に示す。 It has been confirmed that hsa-miR-222-3p (miRBase ID: MIMAT0000279) is highly expressed in tissues such as prostate, bladder, skin, thyroid, and lung in normal tissues. This miRNA has been known to be highly expressed in the skin tissue of psoriasis and hypertrophic scars compared to normal skin tissue (Medicine (Baltimore). 2015 Feb; 94 (7): e458., J Dermatol. Sci. 2010 Jun; 58 (3): 177-85). It has also been reported that this gene regulates the expression of matrix-degrading enzymes in keratinocytes (J Dermatol Sci. 2010 Jun; 58 (3): 177-85.). Furthermore, this gene has been shown to suppress osteoclast formation (Int J Mol Sci. 2016 Feb; 17 (2): 240.). In addition, this miRNA is known to contribute to the promotion of cell proliferation and cell migration / infiltration in various cancer cells and to be involved in its exacerbation (PLoS One. 2014 Jan 31; 9 (1): e87563., Technol Cancer Res Treat. Jan-Dec 2019; 18: 1533033819892256., Cell Signal. 2019 Nov; 63: 109386.). The base sequence of hsa-miR-222-3p is shown in SEQ ID NO: 4.
これらmiRNAsは、重症薬疹のバイオマーカーとして新規であり、かつ有用である。 These miRNAs are novel and useful as biomarkers for severe drug eruption.
本発明において、被験者は、重症薬疹の発症が疑われる哺乳動物であるが、発症の危険性が考えられるすべての哺乳動物を対象としてもよい。哺乳動物は、典型的にはヒトである。被験者由来の試料としては、被験者から得た細胞、組織、体液など、具体的には、被験者の血液(例えば、全血、血清、血漿、血漿交換外液など)等を例示することができる。通常の血液検査(臨床検査)で得られる全血、血清あるいは血漿を血液サンプルとして使用するとよい。 In the present invention, the subject is a mammal suspected of developing severe drug eruption, but may be targeted at all mammals at risk of developing severe drug eruption. Mammals are typically humans. Examples of the sample derived from the subject include cells, tissues, body fluids, etc. obtained from the subject, specifically, the blood of the subject (for example, whole blood, serum, plasma, plasma exchange external fluid, etc.) and the like. Whole blood, serum or plasma obtained by a normal blood test (clinical test) may be used as a blood sample.
本発明の方法において、被験者由来の試料における発現の測定は、試料中の上記miRNAの存在量を測定すればよい。測定する手段としては、特に限定されることなく公知の方法を用いればよく、ノーザンブロット法、RT-PCR法、リアルタイムPRC法、デジタルPCR法、miRNAマイクロアレイ法、Small RNA Sequencingなどを公知の方法として挙げることができる。 In the method of the present invention, the expression in a sample derived from a subject may be measured by measuring the abundance of the above miRNA in the sample. As the means for measurement, a known method may be used without particular limitation, and known methods include Northern blotting, RT-PCR, real-time PRC, digital PCR, miRNA microarray, and Small RNA Sequencing. Can be mentioned.
上記miRNAの発現を測定するためには、上記miRNAと特異的にハイブリダイズできる核酸プローブを用いるとよい(ノーザンブロット法で測定する場合)。あるいはまた、上記miRNAを鋳型として合成されるcDNAを特異的に増幅できる少なくとも１対の核酸プライマーを用いてもよい(RT-PCR法で測定する場合)。さらに、上述の核酸プローブとプライマーセットの組み合わせを用いてもよい(リアルタイムPCR法・デジタルPCR法で測定する場合)。核酸プローブ及び核酸プライマーは、上記miRNAの遺伝子情報(上述)に基づいて設計することができる。核酸プローブは、通常、約１５～１５００塩基のものが適当である。核酸プローブは、放射性元素、蛍光色素、酵素などで標識するとよい。核酸プライマーは、通常、約１５～３０塩基のものが適当である。核酸プライマーを放射性元素、蛍光色素、酵素などで標識してもよい。 In order to measure the expression of the above miRNA, it is preferable to use a nucleic acid probe that can specifically hybridize with the above miRNA (when measuring by Northern blotting). Alternatively, at least one pair of nucleic acid primers capable of specifically amplifying the cDNA synthesized using the above miRNA as a template may be used (when measured by the RT-PCR method). Further, the combination of the above-mentioned nucleic acid probe and primer set may be used (when measuring by real-time PCR method / digital PCR method). Nucleic acid probes and nucleic acid primers can be designed based on the above-mentioned genetic information of miRNA (described above). A nucleic acid probe of about 15 to 1500 bases is usually suitable. The nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme or the like. As the nucleic acid primer, one having about 15 to 30 bases is usually suitable. Nucleic acid primers may be labeled with radioactive elements, fluorescent dyes, enzymes and the like.
発現を測定するmiRNAは１種類でもよいし、複数種類であってもよい。複数のmiRNA発現データを参照することにより、より正確な評価が可能となりうる。代表的な組み合わせとして、表2及び表3(hsa-miR-205-5p, hsa-miR-21-5p, hsa-miR-21-3p)などを例示できるが、これらに限定されるわけではない。複数のmiRNA発現を同時に検出するためには、マルチプレックスリアルタイムPCR(複数種の蛍光プローブを使用)、ＤＮＡアレイ(プローブを基板に固定)(Nat Rev Drug Discov. 2002, 1:951-960)、small RNA Sequencing等の検出法を用いてもよい。 The expression of miRNA may be one type or a plurality of types. By referring to multiple miRNA expression data, more accurate evaluation may be possible. Typical combinations include, but are not limited to, Table 2 and Table 3 (hsa-miR-205-5p, hsa-miR-21-5p, hsa-miR-21-3p). .. For simultaneous detection of multiple miRNA expressions, multiplex real-time PCR (using multiple fluorescent probes), DNA array (probeing on substrate) (Nat Rev Drug Discov. 2002, 1: 951-960), A detection method such as small RNA Sequencing may be used.
miRNAsからなる群より選択される少なくとも１個のmiRNAについて、被験者由来の試料における発現を測定し、その発現レベルが高い場合(例えばΔCq値が低い)に、重症薬疹を発症している可能性が高いと判定し、前記レベルが低い場合(例えばΔCq値が高い)に、重症薬疹を発症している可能性が低いと判定することができる。 For at least one miRNA selected from the group consisting of miRNAs, the expression in the sample derived from the subject is measured, and if the expression level is high (for example, the ΔCq value is low), it is possible that severe drug eruption has occurred. Is high, and when the level is low (for example, the ΔCq value is high), it can be determined that the possibility of developing severe drug eruption is low.
よって、本発明の方法は、重症薬疹の診断(重症薬疹の発症の有無の判定)を補助することができる。 Therefore, the method of the present invention can assist in the diagnosis of severe drug eruption (determination of the presence or absence of the onset of severe drug eruption).
本発明の一つの例として、重症薬疹の診断は、以下のような基準で行うことができる。被験者から採取した血清における上記miRNAsの少なくとも１種の発現量を測定し、発現解析から得られるΔCq値、または複数種miRNAsの組み合わせや診断モデルに個々のΔCq値を代入して得られる診断スコアに対する予め設定されたカットオフ値や基準値よりも低い値が得られた場合、被験者は重症薬疹を発症していると評価する。この予め設定するカットオフ値は、当業者が適宜設定することができる。例えば、重症薬疹を発症していない健常者の定量値の95％信頼区間を基準値としたり、ROC曲線からカットオフ値を設定したりすることができる。あるいは、過去の測定値と比較して、上記miRNAsの発現量のうち少なくとも一つが上昇の傾向を辿った場合、重症薬疹の発症の可能性を疑う。 As one example of the present invention, the diagnosis of severe drug eruption can be made according to the following criteria. For the diagnostic score obtained by measuring the expression level of at least one of the above miRNAs in the serum collected from the subject and substituting the ΔCq value obtained from the expression analysis, or the combination of multiple miRNAs or the individual ΔCq value into the diagnostic model. If a value lower than the preset cut-off value or reference value is obtained, the subject is evaluated as having severe drug eruption. This preset cutoff value can be appropriately set by those skilled in the art. For example, a 95% confidence interval of the quantitative value of a healthy person who has not developed severe drug eruption can be used as a reference value, or a cutoff value can be set from the ROC curve. Alternatively, if at least one of the above-mentioned miRNAs expression levels tends to increase as compared with the past measured values, the possibility of developing severe drug eruption is suspected.
本発明の方法は、重症薬疹の重症化診断にも利用できる。本明細書において、「重症化診断」とは、SJS/TENやDIHSなどの重症薬疹と、播種状紅斑丘疹型皮疹や多形紅斑などの軽症薬疹の判別を意味する。hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１個のmiRNAについて、被験者由来の試料における発現レベルが高い場合に、今後重症薬疹を発症する、あるいは発症している可能性が高いと判定することができる。 The method of the present invention can also be used for aggravation diagnosis of severe drug eruption. As used herein, the term "severe diagnosis" means the distinction between severe drug eruptions such as SJS / TEN and DIHS and mild drug eruptions such as disseminated erythema multiforme-type eruption and erythema multiforme. At least one miRNA selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p in a sample derived from the subject. When the expression level is high, it can be determined that severe drug eruption will develop or is likely to develop in the future.
また、本発明の方法は、重症薬疹の病勢診断に利用できる。本明細書において、「病勢診断」とは、SJS/TENやDIHSなどの重症薬疹の急性期と回復期の識別を意味する。hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１個のタンパク質について、被験者由来の試料における発現レベルが高い場合に、重症薬疹の急性期にある可能性が高いと判定することができる。 In addition, the method of the present invention can be used for pathological diagnosis of severe drug eruption. As used herein, the term "diagnosis of disease" means the distinction between the acute phase and the convalescent phase of severe drug eruption such as SJS / TEN and DIHS. At least one protein selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p in a sample derived from the subject. When the expression level is high, it can be determined that there is a high possibility that the patient is in the acute phase of severe drug eruption.
hsa-miR-205-5pはSJS/TENに対して、hsa-miR-21-3p、hsa-miR-21-5p、hsa-miR-222-3pはDIHSに対して、特に検出性能が高い。 hsa-miR-205-5p has particularly high detection performance for SJS / TEN, and hsa-miR-21-3p, hsa-miR-21-5p, and hsa-miR-222-3p have particularly high detection performance for DIHS.
本発明の方法は、重症薬疹と対照皮膚疾患の鑑別診断に利用できる。本明細書において、「鑑別診断」とは、重症薬疹と対照皮膚疾患の判別を意味する。hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１個のmiRNAについて、被験者由来の試料における発現レベルがカットオフ値あるいは基準値より高い場合に、被験者がアトピー性皮膚炎、乾癬、自己免疫性水疱症、水痘、中毒疹ではなく、SJS/TENやDIHS等の重症薬疹の急性期にある可能性が高いと判定することができる。重症薬疹の鑑別診断のために、バイオマーカーmiRNAを単独で用いてもよいし、またSJS/TENやDIHSの鑑別性能の向上のために、複数のmiRNAの組み合わせ診断モデルとして用いてもよい。代表的な組み合わせの例として、hsa-miR-205-5p、hsa-miR-21-5p及びhsa-miR-21-3pを後述の実施例に記載したが、これに限らず本発明で示す4種miRNAを用いたいずれの組み合わせも使用してもよい。また、これらmiRNAsの組み合わせに既存マーカーであるTARCを追加してもよい。血清中におけるTARCの発現量は、酵素免疫測定法や化学発光酵素免疫測定法等の免疫血清学的検査法により、測定することができる。 The method of the present invention can be used for the differential diagnosis of severe drug eruption and control skin disease. As used herein, "differential diagnosis" means discrimination between severe drug eruption and control skin disease. At least one miRNA selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p in a sample derived from the subject. When the expression level is higher than the cut-off value or the reference value, the subject is not in atopic dermatitis, psoriasis, autoimmune vesicular disease, varicella, toxic eruption, but in the acute phase of severe drug eruption such as SJS / TEN or DIHS. It can be determined that there is a high possibility. The biomarker miRNA may be used alone for the differential diagnosis of severe drug eruption, or may be used as a combined diagnostic model of multiple miRNAs for improving the differential performance of SJS / TEN and DIHS. As examples of typical combinations, hsa-miR-205-5p, hsa-miR-21-5p and hsa-miR-21-3p are described in Examples described later, but the present invention is not limited to this. Any combination with the species miRNA may be used. In addition, TARC, which is an existing marker, may be added to the combination of these miRNAs. The expression level of TARC in serum can be measured by an immunoserologic test method such as an enzyme immunoassay method or a chemiluminescent enzyme immunoassay method.
本発明の方法は、重症薬疹の病勢診断の他、予後の検査、治療効果の確認にも利用できる。例えば、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１個のmiRNAについて、重症薬疹の急性期にある可能性が高いと判定された被験者由来の試料における発現を１回又は異なる時期に複数回測定し、発現レベルがカットオフ値もしくは基準値に近いレベルにまで低下した場合に、治療により重症薬疹から回復したと判定し、前記レベルが高いあるいは低下しない場合に、治療により重症薬疹から回復していない、あるいは、回復が不十分であると判定することができる。 The method of the present invention can be used not only for diagnosing the pathology of severe drug eruption, but also for prognosis examination and confirmation of therapeutic effect. For example, severe drug eruption for at least one miRNA selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. When the expression in a sample derived from a subject determined to be likely to be in the acute phase is measured once or multiple times at different times, and the expression level drops to a cutoff value or a level close to the reference value. It can be determined that the treatment has recovered from the severe drug eruption, and if the level is high or does not decrease, it can be determined that the treatment has not recovered from the severe drug eruption or the recovery is insufficient.
被験者が重症薬疹を発症する、またはすでに発症している可能性が高いと判断された場合には、被疑薬の服用を中止し、患者の病勢や病型の各症状に応じて、適切な治療を開始する。DIHSの薬物療法としては、ステロイド全身投与(プレドニゾロン換算で 0.5～1 mg/kg/日から開始し、適宜漸減)が有効であり、HHV-6 の再活性化による症状の再燃に注意して減量する。SJS/TENの場合は、重症度に応じて用量の異なるステロイド全身投与(プレドニゾロン換算で、中等症は 0.5～1 mg/kg/日、重症例は1～2 mg/kg/日、最重症例はメチルプレドニゾロン 1 g/日3 日間から開始し、症状に応じて適宜漸減)が推奨されており、この他に、高用量ヒト免疫グロブリン(IVIG)静注療法(400 mg/kg/日を5日間連続投与、原則として1コースのみ)、血漿交換療法、眼病変に対する眼表面炎症(ベタメタゾンあるいはデキサメタゾンの点眼(1 日 4 回程度))、瞼球癒着を抑えて眼表面上皮の温存、眼表面の感染症予防などを行うことが挙げられる。 If the subject develops or is likely to have severe drug eruption, discontinue taking the suspected drug and take appropriate measures according to the patient's condition and type of symptoms. Start treatment. As a drug therapy for DIHS, systemic steroid administration (starting from 0.5 to 1 mg / kg / day in terms of prednisolone and gradually decreasing as appropriate) is effective, and the dose should be reduced by paying attention to the relapse of symptoms due to reactivation of HHV-6. do. In the case of SJS / TEN, systemic administration of steroids at different doses depending on the severity (prednisolone equivalent, 0.5 to 1 mg / kg / day for moderate disease, 1 to 2 mg / kg / day for severe cases, most severe cases) Is recommended to start with methylprednisolone 1 g / day for 3 days and taper as appropriate depending on the symptoms), as well as high-dose human immunoglobulin (IVIG) intravenous therapy (400 mg / kg / day 5). Continuous administration for daily days, as a general rule, only one course), plasma exchange therapy, ocular surface inflammation for eye lesions (betamethasone or dexamethasone instillation (about 4 times a day)), suppression of symblepharon adhesion, preservation of ocular surface epithelium, ocular surface It is possible to prevent infectious diseases of.
本発明は、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１個のmiRNAについて、被験者由来の試料における発現を測定すること、その測定値に基づき、被験者が重症薬疹を発症している可能性が高いと評価された場合には、その被験者に治療を施すことを含む、重症薬疹の治療方法も包含する。 The present invention is the subject of at least one miRNA selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. Severe drugs, including measuring expression in a sample of origin and treating the subject if the subject is assessed to be more likely to develop severe drug eruption based on the measurements. It also includes methods for treating eruptions.
本発明は、hsa-miR-205-5p、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pからなる群より選択される少なくとも１個のmiRNAについて、被験者由来の試料における発現を測定することができる試薬を含む、重症薬疹の検査のためのキットも提供する。 The present invention is the subject of at least one miRNA selected from the group consisting of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p. Kits for testing for severe drug eruptions are also provided, including reagents that can measure expression in the sample of origin.
一つの例として、本発明のキットは、上記miRNAと特異的にハイブリダイズできる核酸プローブを試薬として含む。核酸プローブは基板に固定されていてもよい。キットには、さらに、生体試料を採取するための器具、血液凝固剤、被験者由来の試料からRNAを抽出するための試薬類、RNAを検出するための試薬類、取扱説明書などが含まれてもよい。取扱説明書には、キットの使用方法の他、重症薬疹の急性増悪症例の評価及び／又は鑑別基準なども記載しておくとよい。 As an example, the kit of the present invention contains a nucleic acid probe that can specifically hybridize with the above miRNA as a reagent. The nucleic acid probe may be immobilized on the substrate. The kit also includes equipment for collecting biological samples, blood coagulants, reagents for extracting RNA from subject-derived samples, reagents for detecting RNA, instruction manuals, etc. May be good. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or differentiation criteria for acute exacerbation cases of severe drug eruption.
さらに別の一例として、本発明のキットは上記miRNAを鋳型として合成されるcDNAを特異的に増幅できる少なくとも１対の核酸プライマーを試薬として含む。キットには、さらに、被験者由来の試料を採取するための器具、血液凝固剤、被験者由来の試料からRNAを抽出するための試薬類、抽出RNAを逆転写するための試薬、標的cDNAを検出するためのPCR反応試薬、取扱説明書などが含まれるとよい。取扱説明書には、キットの使用方法の他、重症薬疹の急性増悪症例の評価及び／又は鑑別基準なども記載しておくとよい。 As yet another example, the kit of the present invention contains at least one pair of nucleic acid primers as reagents capable of specifically amplifying cDNA synthesized using the above miRNA as a template. The kit also detects instruments for collecting subject-derived samples, blood coagulants, reagents for extracting RNA from subject-derived samples, reagents for reverse transcribing the extracted RNA, and target cDNA. It is preferable to include PCR reaction reagents for this purpose, instruction manuals, and the like. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or differentiation criteria for acute exacerbation cases of severe drug eruption.
本発明のキットには、この他に、標的miRNA発現量の補正に使用する外部スパイクコントロールのmiRNAの標準品、陽性コントロールmiRNA、バッファー、反応停止液、洗浄液、反応容器などを含めてもよい。
本発明のキットは、さらに、TARCを特異的に認識できる抗体あるいは核酸アプタマーを含んでもよい。抗体及びアプタマーはマイクロタイタープレートや磁気ビーズ、セルロース膜や基板に固定されていてもよい。あるいは、TARCのmRNAと特異的にハイブリダイズできる核酸プローブを含んでもよい。核酸プローブは基板に固定されていてもよい。
In addition, the kit of the present invention may include a standard product of an external spike control miRNA used for correcting the expression level of the target miRNA, a positive control miRNA, a buffer, a reaction terminator, a washing solution, a reaction vessel, and the like.
The kit of the present invention may further contain an antibody or nucleic acid aptamer capable of specifically recognizing TARC. Antibodies and aptamers may be immobilized on microtiter plates, magnetic beads, cellulose membranes or substrates. Alternatively, a nucleic acid probe capable of specifically hybridizing with TARC mRNA may be included. The nucleic acid probe may be immobilized on the substrate.
以下、実施例により本発明を更に詳細に説明する。
〔実施例１〕
(１)検体
解析に用いた薬疹並びに関連皮膚疾患検体ついては、各拠点病院(横浜市立大学、島根大学、市立島田市民病院、磐田市立総合病院、奈良県立医科大学、新潟大学)、国立医薬品食品衛生研究所、木原財団、アステラス製薬、及び第一三共の各研究倫理委員会の承認を得て収集した。一方、健常成人検体は、北里大学及び国立医薬品食品衛生研究所の研究倫理委員会の承認を得て収集した。
Hereinafter, the present invention will be described in more detail by way of examples.
[Example 1]
(1) For drug rash and related skin disease specimens used for sample analysis, each base hospital (Yokohama City University, Shimane University, Shimada Municipal Hospital, Iwata City General Hospital, Nara Medical University, Niigata University), National Pharmaceutical Foods Collected with the approval of the Research Ethics Committees of the Institute of Health, Kihara Foundation, Astellas Pharmaceuticals, and Daiichi Sankyo. On the other hand, healthy adult specimens were collected with the approval of the Research Ethics Committee of Kitasato University and the National Institute of Health Sciences.
医薬品による軽症薬疹(多形紅斑、播種状紅斑丘疹型皮疹、湿疹型皮疹等)の発症が疑われた患者、Stevens-Johnson症候群(SJS)・中毒性表皮壊死融解症(toxic epidermal necrolysis; TEN)・薬剤性過敏症症候群(drug-induced hypersensitivity syndrome; DIHS)、多形紅斑重症型（EM major）等の重症薬疹患者の急性期(増悪期付近)、並びにそれら患者の回復期に採血を上記の拠点病院にて、患者の同意のもと行った。薬疹以外の皮膚疾患患者(アトピー性皮膚炎、乾癬、自己免疫性水疱症、水痘、中毒疹(医薬品を起因としない))からの採血も同様に行った。採血には、血清採取用の7 mLの血液凝固促進剤入り採血管を用いた。採血後の検体は室温で30分放置した後、3,000 rpm×10分(15℃～20℃)遠心分離を行い、血清画分を回収した。本試験で使用した血清は-80℃にて凍結保存し、各測定試験実施直前に氷上で再融解を行った。 Patients suspected of developing mild drug eruptions (erythema multiforme, disseminated erythema multiforme, eczema-type eruptions, etc.), Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis; TEN ) ・ Collect blood during the acute phase (near the exacerbation phase) of patients with severe drug rash such as drug-induced hypersensitivity syndrome (DIHS) and EM major, and during the recovery phase of those patients. The procedure was performed at the above-mentioned base hospital with the consent of the patient. Blood was collected from patients with skin diseases other than drug eruption (atopic dermatitis, psoriasis, autoimmune vesicular disease, varicella, toxic eruption (not caused by drugs)). For blood collection, a 7 mL blood collection tube containing a blood coagulation promoter for serum collection was used. The sample after blood collection was left at room temperature for 30 minutes, then centrifuged at 3,000 rpm × 10 minutes (15 ° C to 20 ° C), and the serum fraction was collected. The serum used in this test was cryopreserved at -80 ° C and rethawed on ice immediately before each measurement test.
miRCURY LNA microRNA PCRを用いた重症薬疹バイオマーカー候補miRNAsのスクリーニング解析には、軽症薬疹血清検体32例、重症薬疹検体の急性期血清検体29例、並びに回復期血清検体18例を供した(探索用検体セット)。重症薬疹検体の内訳としては、SJS/TEN9例、DIHS15例、多型紅斑重症型(EM major)5例であった。 For screening analysis of miRNAs, which are candidates for severe drug eruption biomarkers using miRCURY LNA microRNA PCR, 32 mild drug eruption serum samples, 29 acute drug eruption sample samples, and 18 convalescent serum samples were provided. (Sample set for search). The breakdown of severe drug eruption specimens was SJS / TEN in 9 cases, DIHS in 15 cases, and erythema multiforme severe type (EM major) in 5 cases.
Taqman Advanced miRNA Assay法を用いたバイオマーカー候補miRNAsの検証試験では、軽症薬疹の血清検体51例、重症薬疹急性期の血清検体43例 (SJS/TEN13例、DIHS15例、EM major15例) ならびにそれらの回復期検体32例を測定に供した。また、非薬疹の対照皮膚疾患由来血清検体として、アトピー性皮膚炎(16例)、乾癬(16例)、自己免疫性水疱症 (16例)、水痘(16例)及び中毒疹(32例)、加えて健常成人由来血清検体(30例)を測定に供した(検証用検体セット)。
In a validation study of biomarker candidate miRNAs using the Taqman Advanced miRNA Assay method, 51 serum samples of mild drug eruption, 43 serum samples in the acute phase of severe drug eruption (SJS /
(２)RNA抽出及びcDNA合成
血清検体からのtotal RNA抽出は、miRNeasy(登録商標) Mini Kit (Qiagen)を用いて行った。miRCURY LNA microRNA PCR測定用のcDNAの合成には、Universal cDNA Synthesis Kit II(Qiagen)を使用した。一方、Taqman Advanced miRNA Assay測定用のcDNAの合成には、TaqmanTM Advanced miRNA cDNA synthesis kit (Thermo Fisher Scientific)を用いた。なお、Taqman Advanced miRNA Assayの測定用試料調製では、外因性スパイクコントロールとして、cel-miR-54-3p (0.2 fmol)を各血清試料に添加した後に、total RNA抽出及びcDNA合成を行った。
(2) RNA extraction and cDNA synthesis Total RNA extraction from serum samples was performed using miRNeasy® Mini Kit (Qiagen). Universal cDNA Synthesis Kit II (Qiagen) was used to synthesize cDNA for miRCURY LNA microRNA PCR measurement. On the other hand, the Taqman TM Advanced miRNA cDNA synthesis kit (Thermo Fisher Scientific) was used for the synthesis of cDNA for Taqman Advanced miRNA Assay measurement. In the sample preparation for measurement of Taqman Advanced miRNA Assay, total RNA extraction and cDNA synthesis were performed after adding cel-miR-54-3p (0.2 fmol) to each serum sample as an extrinsic spike control.
(３)測定手法並びに補正手法
miRNAの網羅的発現解析には、QUIAGEN社のmiRCURY LNA microRNA PCR システムを用いた。本システムは、Locked Nucleic Acid (RNA修飾核酸)プライマーにより、752種のmiRNAを特異的に検出する測定法である。測定データの解析には、GenExソフトウェア(Multid)を用いて、[1]Interplate calibration(IPC)、[2]Cut off(Cq>37を除外)、[3]Call rate(10％未満を除外)、[4]Missing data(各プローブの最大Cq値＋１で非検出検体を補完)、[5]Normfinder Normalizationという手順により、下記の式で定義されるΔCqを算出した。ΔCq値は標的miRNAの内標遺伝子(リファレンスプローブ)に対する相対発現量を表しており、本値が小さいほど発現量が相対的に高いことを意味する。
For the comprehensive expression analysis of miRNA, QUIAGEN's miRCURY LNA microRNA PCR system was used. This system is a measurement method that specifically detects 752 types of miRNA using Locked Nucleic Acid (RNA-modified nucleic acid) primers. For analysis of measurement data, use GenEx software (Multid), [1] Interplate calibration (IPC), [2] Cut off (excluding Cq> 37), [3] Call rate (excluding less than 10%). , [4] Missing data (maximum Cq value of each probe + 1 complements the undetected sample), [5] Normfinder Normalization, and ΔCq defined by the following formula was calculated. The ΔCq value represents the relative expression level of the target miRNA with respect to the internal standard gene (reference probe), and the smaller this value is, the higher the expression level is.
一方、選定した重症薬疹バイオマーカー候補miRNA (hsa-miR-205-5p; MIMAT0000266, UCCUUCAUUCCACCGGAGUCUG(配列番号１), hsa-miR-21-3p; MIMAT0004494, CAACACCAGUCGAUGGGCUGU(配列番号２), hsa-miR-21-5p; MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA(配列番号３)), hsa-miR-222-3p; MIMAT0000279, AGCUACAUCUGGCUACUGGGU(配列番号４))の検証試験では、各標的miRNAに特異的なプローブ並びにプライマーを含むTaqman Advanced miRNA Assay(Thermo Fisher Scientific社, hsa-miR-205-5p; 477967_mir, hsa-miR-21-3p; 477973_mir, hsa-miR-21-5p; 477975_mir, hsa-miR-222-3p; 477982_mir, cel-miR-54-3p; 478410_mir)を用いた。本測定における各miRNAの発現量(ΔCq値)は、Cq(標的miRNA)からCq(外部スパイクコントロール;cel-miR-54-3p, MIMAT0000025, UACCCGUAAUCUUCAUAAUCCGAG(配列番号１０))を差し引くことにより求めた。なお、miRCURY LNA microRNA PCR システムの解析と同様、算出されたΔCq値が小さいほど、標的miRNAの発現量が相対的に高いことを表す。 On the other hand, selected severe drug rash biomarker candidates miRNA (hsa-miR-205-5p; MIMAT0000266, UCCUUCAUUCCACCGGAGUCUG (SEQ ID NO: 1), hsa-miR-21-3p; MIMAT0004494, CAACACCAGUCGAUGGGCUGU (SEQ ID NO: 2), hsa-miR- In the validation test of 21-5p; MIMAT0000076, UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO: 3)), hsa-miR-222-3p; MIMAT0000279, AGCUACAUCUGGCUACUGGGU (SEQ ID NO: 4)), Taqman Advanced containing probes and primers specific for each target miRNA. miRNA Assay (Thermo Fisher Scientific, hsa-miR-205-5p; 477967_mir, hsa-miR-21-3p; 477973_mir, hsa-miR-21-5p; 477975_mir, hsa-miR-222-3p; 477982_mir, cel- miR-54-3p; 478410_mir) was used. The expression level (ΔCq value) of each miRNA in this measurement was determined by subtracting Cq (external spike control; cel-miR-54-3p, MIMAT0000025, UACCCGUAAUCUUCAUAAUCCGAG (SEQ ID NO: 10)) from Cq (target miRNA). As in the analysis of the miRCURY LNA microRNA PCR system, the smaller the calculated ΔCq value, the higher the expression level of the target miRNA.
(４)統計計算
バイオマーカーmiRNA探索のためのボルケーノプロット解析には統計ソフトウェアRのEnhancedVolcanoパッケージを使用した。ボルケーノプロット作成には二群間におけるStudent’s t-testから算出されたp値の常用対数を使用した。三群以上の群間におけるmiRNA発現量の比較には、one-way ANOVA Dunn’s testを利用した。また、各種miRNAバイオマーカーの診断能の評価は、ROC曲線(receiver Operating Characteristic Curve)から算出されるarea under the ROC curve (AUROC)値を用いて行った。これら統計計算は医療統計ソフトGraphPad Prism8(GraphPad Software)を使用した。重症薬疹バイオマーカー候補miRNAsの診断能向上を目的とした組み合わせ診断モデルは、多重ロジスティック回帰分析により構築した。診断モデルの構築には、統計ソフトウェアJMP12(SAS Institute)を用いた。
(4) Statistical calculation The Enhanced Volcano package of statistical software R was used for volcano plot analysis for biomarker miRNA search. The common logarithm of the p-value calculated from Student's t-test between the two groups was used to create the volcano plot. The one-way ANOVA Dunn's test was used to compare miRNA expression levels among three or more groups. In addition, the diagnostic ability of various miRNA biomarkers was evaluated using the area under the ROC curve (AUROC) value calculated from the ROC curve (receiver operating characteristic curve). The medical statistics software GraphPad Prism8 (GraphPad Software) was used for these statistical calculations. A combined diagnostic model aimed at improving the diagnostic ability of the severe drug eruption biomarker candidate miRNAs was constructed by multiple logistic regression analysis. Statistical software JMP12 (SAS Institute) was used to construct the diagnostic model.
(５)重症薬疹バイオマーカー候補miRNAsの選定
miRCURY LNA microRNA PCRにて測定値が入手可能であった502種のmiRNAsの各ΔCq値から算出されるSJS/TEN(9例)と回復期(18例)の間の発現量比及びDIHS(15例)と回復期(18例)の間の発現量比の絶対値が1.5以上、かつ二群間のStudent’s t-testで算出されたp値が0.05以下であるmiRNAsをボルケーノプロットにより抽出したところ、SJS/TEN急性期において13分子(図1A)、DIHS急性期において41分子(図1B)同定された(選別条件1、図1C)。重症薬疹バイオマーカーとなりうるmiRNAを絞りこむため、重症薬疹の急性期に有意な発現変動を示した全miRNAsについて、各重症薬疹病型と回復期の診断能を算出し、そのAUROC値が0.75以上を示すmiRNA分子(SJS/TEN 6種、DIHS 18種)を抽出した(選別条件2、図1C)。さらに、回復期例とSJS/TEN、DIHS、EM majorにおける各種miRNAの発現量をそれぞれ比較した多重比較検定(one-way ANOVA Dunn’s test)において、いずれか重症薬疹病型と回復期間において統計的有意差を示すmiRNAsを抽出した(SJS/TEN 5種、DIHS 17種、選別条件3、図1C)。上述の選別基準1-3を充たすmiRNAsのうち、重症薬疹バイオマーカー候補miRNAとして有望な4分子(hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p, hsa-miR-222-3p)を選定した。これらmiRNA分子は探索用検体セットにおいて、SJS/TEN、DIHS、あるいはその両方において、回復期と比べ有意に高く発現することが示された(表1)。SJS/TENにおいて特に高い発現変動を示したのはhsa-miR-205-5pであり、一方、DIHSにおいて比較的高い発現変動を示すmiRNAはhsa-miR-21-3p, hsa-miR-21-5p及び, hsa-miR-222-3pであった(表1)。
(5) Selection of candidate miRNAs for severe drug eruption biomarkers
Expression ratio and DIHS (15) between SJS / TEN (9 cases) and convalescent period (18 cases) calculated from each ΔCq value of 502 miRNAs for which measurements were available by miRCURY LNA microRNA PCR. Example) and miRNAs with an absolute expression level ratio between the recovery phase (18 cases) of 1.5 or more and a p value of 0.05 or less calculated by Student's t-test between the two groups were extracted by volcano plot. , 13 molecules in the acute phase of SJS / TEN (Fig. 1A) and 41 molecules in the acute phase of DIHS (Fig. 1B) were identified (
(６)重症薬疹急性期患者におけるバイオマーカー候補miRNAsの発現変動の検証
探索用検体セットを用いた上述のmiRCURY LNA microRNA PCR測定結果が、測定手法に依存せず普遍的であることを示すため、探索用検体セットとは別群である検証用検体セットにおけるhsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p及び hsa-222-3pの発現量(ΔCq値)をTaqman Advanced miRNA Assayにより測定し、重症薬疹の急性期と回復期における発現量比について解析した。その結果、全ての重症薬疹バイオマーカー候補miRNAsについて、探索群で認められていた発現上昇プロファイルと同様の発現量変動が検証群においても確認された(表1)。このことから、重症薬疹と回復期の間におけるこれらmiRNAs分子の発現量変化は普遍的かつ再現性を有することが考えられた。
(6) Verification of expression fluctuations of biomarker candidate miRNAs in patients with severe drug eruption To show that the above-mentioned miRCURY LNA microRNA PCR measurement results using the search sample set are universal regardless of the measurement method. , Expression levels of hsa-miR-205-5p, hsa-miR-21-3p, hsa-miR-21-5p and hsa-222-3p in the verification sample set, which is a separate group from the search sample set (ΔCq) The value) was measured by the Taqman Advanced miRNA Assay, and the expression level ratio between the acute phase and the convalescent phase of severe drug eruption was analyzed. As a result, for all the severe drug eruption biomarker candidate miRNAs, the same expression level fluctuation as the expression increase profile observed in the search group was confirmed in the verification group (Table 1). From this, it was considered that the change in the expression level of these miRNAs molecules between the severe drug eruption and the convalescent period was universal and reproducible.
次に、重症薬疹病型ごとにおける各バイオマーカー候補miRNAの発現量と、軽症薬疹、回復期及び健常成人におけるそれらの発現量をドットプロットにより比較した(図2)。各群の中央値算出したところ、hsa-miR-205-5pはSJS/TEN急性期患者において最も高く発現していた(図2)。hsa-miR-21-3p, hsa-miR-21-5p及びhsa-miR-222-3pについてはDIHS急性期患者において最も高く発現することが明らかとなった(図2)。一方、一部のEM major急性期患者において、SJS/TENやDIHSと同様にこれらmiRNAsの過剰発現が認められるものの、その中央値は軽症薬疹と同程度であることが示された。 Next, the expression levels of each biomarker candidate miRNA in each severe drug eruption type and their expression levels in mild drug eruption, convalescent and healthy adults were compared by dot plot (Fig. 2). When the median value of each group was calculated, hsa-miR-205-5p was most highly expressed in SJS / TEN acute phase patients (Fig. 2). It was revealed that hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p are most highly expressed in patients in the acute phase of DIHS (Fig. 2). On the other hand, in some EM major acute phase patients, overexpression of these miRNAs was observed as in SJS / TEN and DIHS, but the median value was shown to be similar to that of mild drug eruption.
重症薬疹の重症化診断の観点から、SJS/TEN急性期と軽症薬疹の間で発現変動を示すmiRNAを解析したところ、統計学的に有意な発現量変化を示したmiRNAはhsa-miR-205-5pのみであった(図2)。一方、DIHSの急性期と軽症薬疹の間で統計学的に有意な発現量の変化を示したmiRNAsはhsa-miR-21-3p, hsa-miR-21-5p及びhsa-miR-222-3pであった(図2)。したがって、各重症薬疹病型において有意差が認められたmiRNAはその病型の重症化診断に利用可能であることが考えられた(図2)。 From the viewpoint of diagnosing the aggravation of severe drug eruption, we analyzed miRNAs showing expression fluctuations between the acute phase of SJS / TEN and mild drug eruptions. It was only -205-5p (Fig. 2). On the other hand, the miRNAs that showed a statistically significant change in the expression level between the acute phase of DIHS and mild drug eruption were hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-. It was 3p (Fig. 2). Therefore, it was considered that miRNAs that showed significant differences in each severe drug eruption type could be used for diagnosing the aggravation of that type (Fig. 2).
重症薬疹の病勢診断や治療効果の確認の観点から、各miRNA分子の重症薬疹と回復期並びに健常成人との間における発現量の差について解析した。その結果、hsa-miR-205-5pについては、全ての重症薬疹病型と回復期の間で有意な差異が認められた(図2)。一方、hsa-miR-21-3p, hsa-miR-21-5p及びhsa-miR-222-3pについては、他の重症薬疹病型と比べDIHS急性期患者において、回復期との間でより顕著な発現量の差異が認められた(図2)。一方、健常成人と重症薬疹の比較において、hsa-miR-205-5pはSJS/TEN及びDIHSの急性期患者と、hsa-miR-21-3p及びhsa-miR-222-3pはDIHSの急性期患者との間に発現量の有意差が認められた(図2)。これら結果は、本miRNAsが重症薬疹の病勢診断や重症薬疹に対する治療効果の確認等を目的とする検査に利用可能であることを示唆している。 From the viewpoint of diagnosing the condition of severe drug eruption and confirming the therapeutic effect, the difference in the expression level of each miRNA molecule between the severe drug eruption and the convalescent period and healthy adults was analyzed. As a result, for hsa-miR-205-5p, a significant difference was observed between all severe drug eruption types and the convalescent period (Fig. 2). On the other hand, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p were more effective in the acute phase of DIHS than in other severe drug eruption types during the recovery phase. A remarkable difference in the expression level was observed (Fig. 2). On the other hand, in comparison between healthy adults and severe drug eruption, hsa-miR-205-5p is an acute phase patient of SJS / TEN and DIHS, and hsa-miR-21-3p and hsa-miR-222-3p are acute DIHS. A significant difference in the expression level was observed with the stage patients (Fig. 2). These results suggest that these miRNAs can be used for tests aimed at diagnosing the pathology of severe drug eruption and confirming the therapeutic effect on severe drug eruption.
(７)重症薬疹バイオマーカー候補miRNAsのバイオマーカー性能
表2及び表3は、それぞれ探索用検体セットと検証用検体セットの測定値を用いた各病型の重症薬疹の急性期と、回復期、軽症薬疹及び健常成人(表3のみ)の間における、4種類のバイオマーカー候補miRNA、既報重症薬疹マーカー(Granulysin(表2のみ), TARC)、並びにそれらを組み合わせたAUROC値を示している。探索用検体セットを用いた病勢診断能について評価を行ったところ、hsa-miR-205-5pは、GranulysinやTARC等の既存マーカーでは鑑別不良である回復期とSJS/TEN急性期の間において高いAUROC値(0.93)を示した(表2)。また、hsa-miR-205-5p、hsa-miR-21-3p及びhsa-miR-222-3pはDIHSの病勢診断において既存マーカー(Granulysin: 0.84, TARC; 0.88)と同レベルの診断能(0.86-0.87)を示した(表2)。さらに、検証用データセットにおいても同様の結果が認められた(表3)。検証用データセットにおいて、hsa-miR-205-5pのSJS/TENの病勢診断におけるAUROC値は0.96と、著しく高値を示した(表3)。さらに、４種の重症薬疹バイオマーカー候補miRNAsのDIHSの病勢診断におけるAUROC値はすべて0.89以上であった(表3)。これら結果より、hsa-miR-205-5pはSJS/TENに関し、全4種のmiRNAsはDIHSに関し、病勢診断に有用なバイオマーカーとなることが示された。
(7) Biomarker performance of candidate miRNAs for severe drug eruption Tables 2 and 3 show the acute phase and recovery of severe drug eruption of each disease type using the measured values of the search sample set and the verification sample set, respectively. Shows four biomarker candidate miRNAs, previously reported severe drug eruption markers (Granulysin (Table 2 only), TARC), and combined AUROC values between the stage, mild drug eruption and healthy adults (Table 3 only). ing. When the disease diagnosis ability using the search sample set was evaluated, hsa-miR-205-5p was high between the recovery phase and the SJS / TEN acute phase, which are poorly differentiated by existing markers such as Granulysin and TARC. The AUROC value (0.93) is shown (Table 2). In addition, hsa-miR-205-5p, hsa-miR-21-3p and hsa-miR-222-3p have the same level of diagnostic ability (0.86) as existing markers (Granulysin: 0.84, TARC; 0.88) in the diagnosis of DIHS pathology. -0.87) is shown (Table 2). Furthermore, similar results were observed in the verification data set (Table 3). In the validation data set, the AUROC value of hsa-miR-205-5p in the disease diagnosis of SJS / TEN was 0.96, which was extremely high (Table 3). Furthermore, the AUROC values of the four severe drug eruption biomarker candidate miRNAs in the diagnosis of DIHS were all 0.89 or higher (Table 3). From these results, it was shown that hsa-miR-205-5p is related to SJS / TEN, and all four miRNAs are related to DIHS, which are useful biomarkers for disease diagnosis.
続いて、各miRNAの重症薬疹に対する重症化診断能を重症薬疹急性期と軽症薬疹の間のAUROC値を算出することにより評価した。探索群において、hsa-miR-205-5pがSJS/TENの重症化診断において、中程度ではあるものの有意な診断能(AUROC 0.75)を示した(表2)。DIHSの急性期予測では、hsa-miR-21-5p及びhsa-miR-222-3pは、AUROC値が0.70で有意であったが、GranulysinとTARCがAUROC 0.85以上の高い予測能を示したため、4種のmiRNAsでこれを超える予測能を示す分子は認められなかった(表2)。一方、上記miRNAsについて検証用群では、探索群と比べより有望な結果が認められた。解析の結果、SJS/TENの重症化診断におけるhsa-miR-205-5pのAUROC値は0.86と高値を示した(表3)。また、DIHSの重症化診断において、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-3pはAUROC値0.88以上を示し、その値は既存マーカーのTARCより高値であった(表3)。EM majorについて、AUROC値0.8以上を示す重症化予測miRNAバイオマーカーは探索群及び検証群の両方において認められなかった(表2、表3)。以上の結果から、hsa-miR-205-5pはSJS/TEN、hsa-miR-21-5p及びhsa-miR-222-3pはDIHSの重症化診断バイオマーカーとなりうることが示唆された。 Subsequently, the aggravation diagnostic ability of each miRNA for severe drug eruption was evaluated by calculating the AUROC value between the acute stage of severe drug eruption and the mild drug eruption. In the exploration group, hsa-miR-205-5p showed moderate but significant diagnostic ability (AUROC 0.75) in the diagnosis of SJS / TEN aggravation (Table 2). In the acute phase prediction of DIHS, hsa-miR-21-5p and hsa-miR-222-3p were significant at AUROC value of 0.70, but Granulysin and TARC showed high predictive ability of AUROC 0.85 or higher. No molecules with higher predictive potential were found in the four miRNAs (Table 2). On the other hand, more promising results were observed in the verification group than in the search group for the above miRNAs. As a result of the analysis, the AUROC value of hsa-miR-205-5p in the diagnosis of aggravation of SJS / TEN was as high as 0.86 (Table 3). In addition, in the diagnosis of aggravation of DIHS, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-3p showed an AUROC value of 0.88 or higher, which is higher than the existing marker TARC. There was (Table 3). For EM major, no aggravation-predicted miRNA biomarkers showing an AUROC value of 0.8 or higher were observed in both the exploration group and the validation group (Tables 2 and 3). From the above results, it was suggested that hsa-miR-205-5p could be SJS / TEN, and hsa-miR-21-5p and hsa-miR-222-3p could be biomarkers for diagnosing the severity of DIHS.
(８)miRNA組み合わせ重症薬疹診断モデルによる診断精度の向上
複数のバイオマーカー候補miRNAを組み合わせることで、診断精度の向上が期待される。そこで次に、探索用検体セットの重症薬疹全病型の急性期と軽症薬疹の間におけるhsa-miR-205-5p、hsa-miR-21-3p及びhsa-miR-21-5pのΔCq値を用いて多重ロジスティック回帰により重症薬疹診断モデル[診断スコア＝0.23486537×ΔCq(hsa-miR-205-5p)+0.34814324×ΔCq(hsa-miR-21-3p)-0.0088437×ΔCq(hsa-miR-21-5p) -3.1236303]を作製した。本モデルから得られる診断スコアをもとに、重症薬疹と軽症薬疹（重症化診断）並びに回復期（病勢診断）との比較をしたところ、軽症薬疹との鑑別においては僅かな診断能の向上が認められたのに対し、回復期とすべての重症薬疹の鑑別においてAUROC値0.89以上の診断能を示した(表2)。また、本診断モデルに既存マーカーであるTARCを追加することにより、AUROC値0.9以上ですべての重症薬疹病型の病勢診断が可能であることが示された(表2)。
続いて、検証用検体セットの測定値を用いて、上記と同様の手法によりmiRNAsの組み合わせによる重症薬疹診断モデル[診断スコア＝0.34042857×ΔCq(hsa-miR-205-5p)+ 2.65078716×ΔCq(hsa-miR-21-3p)-1.8524265×ΔCq(hsa-miR-21-5p)-18.211688]を構築した。本診断モデルから得られた診断スコア値をプロットしたところ、健常成人や回復期、さらに軽症薬疹と比較し、SJS/TEN及びDIHS等重症薬疹群において顕著な診断スコア値の低下が認められた(図3)。次に、重症薬疹と健常成人、軽症薬疹及び回復期との間のROC曲線解析を実施した。その結果、3種のmiRNAsを組み合わせることにより、全ての重症薬疹と健常成人をAUROC値0.9以上で鑑別可能であり、特にDIHSとEM majorについては各miRNAs単体で得られたAUROC値をよりも高値が得られた(表3)。さらに、本診断モデルは各miRNAマーカーの単独利用時よりも、DIHSの病勢診断及び重症化予測における診断能を向上させることが認められた(表3)。さらに、本診断モデルに既報マーカーTARCを組み込んだ場合、AUROC値0.98以上の精度で、DIHSの病勢診断及び重症化予測を行うことが可能であることが明らかとなった(表3)。以上より、本研究において同定された重症薬疹バイオマーカーmiRNAsの組み合わせや、それら組み合わせにTARCを追加した診断モデルの構築および利用が重症薬疹の病勢診断や重症化診断の精度の向上につながることが示された。一方で、miRNAs組み合わせモデルにおいて診断能の低下が確認された二群間の鑑別診断では、最も診断能が高いmiRNA単独での利用が良いと考えられた。
(8) Improvement of diagnostic accuracy by miRNA combination severe drug eruption diagnostic model It is expected that the diagnostic accuracy will be improved by combining multiple biomarker candidate miRNAs. Therefore, next, ΔCq of hsa-miR-205-5p, hsa-miR-21-3p and hsa-miR-21-5p between the acute phase and mild drug eruption of all types of severe drug eruption in the search sample set. Severe drug eruption diagnosis model by multiple logistic regression using values [Diagnosis score = 0.23486537 × ΔCq (hsa-miR-205-5p) +0.34814324 × ΔCq (hsa-miR-21-3p) -0.0088437 × ΔCq (hsa-miR) -21-5p) -3.1236303] was prepared. Based on the diagnostic score obtained from this model, a comparison between severe drug eruption and mild drug eruption (severe diagnosis) and convalescent period (disease diagnosis) revealed that there was a slight diagnostic ability in differentiating from mild drug eruption. However, it showed a diagnostic ability with an AUROC value of 0.89 or higher in the recovery period and the differentiation of all severe drug eruptions (Table 2). In addition, it was shown that by adding the existing marker TARC to this diagnostic model, it is possible to diagnose the pathology of all severe drug eruption types with an AUROC value of 0.9 or higher (Table 2).
Then, using the measured values of the sample set for verification, a severe drug eruption diagnosis model by combining miRNAs by the same method as above [diagnosis score = 0.34042857 × ΔCq (hsa-miR-205-5p) + 2.65078716 × ΔCq ( hsa-miR-21-3p) -1.8524265 × ΔCq (hsa-miR-21-5p) -18.211688] was constructed. When the diagnostic score values obtained from this diagnostic model were plotted, a marked decrease in the diagnostic score values was observed in the severe drug eruption group such as SJS / TEN and DIHS compared with healthy adults, convalescent period, and mild drug eruption. (Fig. 3). Next, ROC curve analysis between severe drug eruption and healthy adults, mild drug eruption and convalescent period was performed. As a result, by combining three types of miRNAs, it is possible to distinguish between all severe drug eruptions and healthy adults with an AUROC value of 0.9 or higher, and especially for DIHS and EM major, the AUROC values obtained with each miRNAs alone are higher than those obtained. High prices were obtained (Table 3). Furthermore, it was found that this diagnostic model improves the diagnostic ability in DIHS disease diagnosis and aggravation prediction compared to the case of using each miRNA marker alone (Table 3). Furthermore, it was clarified that when the previously reported marker TARC is incorporated into this diagnostic model, it is possible to diagnose the pathology of DIHS and predict its aggravation with an accuracy of AUROC value of 0.98 or higher (Table 3). Based on the above, the combination of the severe drug eruption biomarkers miRNAs identified in this study and the construction and utilization of a diagnostic model in which TARC is added to these combinations will lead to improvement in the accuracy of the diagnosis of severe drug eruption and the diagnosis of aggravation. It has been shown. On the other hand, in the differential diagnosis between the two groups in which a decrease in diagnostic ability was confirmed in the miRNAs combination model, it was considered that miRNA alone, which has the highest diagnostic ability, should be used alone.
(９)重症薬疹と対照皮膚疾患と間におけるバイオマーカー候補miRNAsの発現量の比較
重症薬疹及び対照皮膚疾患症例における、各バイオマーカー候補miRNAの血清中発現量(ΔCq値)をプロットし、各群間における発現量の中央値を統計学的に比較した(図4)。対照皮膚疾患と比べ、hsa-miR-205-5pはSJS/TENの急性期、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-5pはDIHSの急性期において高く発現する傾向があることが明らかとなった(図4)。SJS/TENに着目した統計学検定の結果、hsa-miR-205-5pについては、アトピー性皮膚炎・自己免疫性水疱症以外の全ての対照皮膚疾患患者と比べ、SJS/TENの急性期において有意な発現量上昇が認められた(図4)。一方、hsa-miR-21-3p、hsa-miR-21-5p及びhsa-miR-222-5pについて、SJS/TEN急性期と統計学上有意な発現量差が認められたのは、自己免疫性水疱症と水痘(hsa-miR-222-3pのみ)あった。次に、DIHSに着目したところ、hsa-miR-21-3pは全ての対照皮膚疾患との間において統計学上有意な発現量差が認められた(図4)。一方、hsa-miR-21-5p及びhsa-miR-222-5pは、アトピー性皮膚炎以外すべての皮膚疾患と有意な発現量差が認められた(図4)。EM majorの急性期と対照皮膚疾患の間において、明瞭な発現量差はほとんど認められなかった。
(9) Comparison of expression levels of biomarker candidate miRNAs between severe drug eruption and control skin disease The serum expression level (ΔCq value) of each biomarker candidate miRNA in severe drug eruption and control skin disease cases was plotted. The median expression levels among the groups were statistically compared (Fig. 4). Compared with control skin diseases, hsa-miR-205-5p in the acute phase of SJS / TEN, hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-5p in the acute phase of DIHS It was revealed that it tends to be highly expressed (Fig. 4). As a result of statistical tests focusing on SJS / TEN, hsa-miR-205-5p was found in the acute phase of SJS / TEN compared to all control skin disease patients except atopic dermatitis and autoimmune vesicular disease. A significant increase in the expression level was observed (Fig. 4). On the other hand, for hsa-miR-21-3p, hsa-miR-21-5p and hsa-miR-222-5p, a statistically significant difference in expression level was observed between the acute phase of SJS / TEN and autoimmunity. There was vesicular vesicular disease and chickenpox (hsa-miR-222-3p only). Next, focusing on DIHS, a statistically significant difference in the expression level of hsa-miR-21-3p was observed between all control skin diseases (Fig. 4). On the other hand, hsa-miR-21-5p and hsa-miR-222-5p showed a significant difference in expression level from all skin diseases except atopic dermatitis (Fig. 4). There was almost no clear difference in expression between the acute phase of EM major and control skin disease.
(１０)重症薬疹バイオマーカー候補miRNAsによる対照皮膚疾患との鑑別診断能
上記４種のバイオマーカー候補miRNAsについて、重症薬疹とそれ以外の皮膚疾患との鑑別における診断能の評価を行った。まずSJS/TENと対照皮膚疾患との鑑別において、最も高い診断能を示したmiRNAはhsa-miR-205-5pであった。本分子は、自己免疫性水疱症以外すべての対照皮膚疾患で、AUROC値0.85を超える高い診断能を示した(表4)。また、hsa-miR-205-5pでは中程度の診断能(AUROC 0.77)しか出せないSJS/TENと自己免疫性水疱症の鑑別において、hsa-miR-21-3p(AUROC 0.90)及びhsa-miR-21-5p(AUROC 0.89)が良好な診断能を示すことを見出した(表4)。
(10) Differential diagnosis ability of severe drug eruption biomarker candidate miRNAs from control skin diseases The above four types of biomarker candidate miRNAs were evaluated for their diagnostic ability in differentiation from severe drug eruption and other skin diseases. First, in the differentiation between SJS / TEN and control skin disease, the miRNA showing the highest diagnostic ability was hsa-miR-205-5p. This molecule showed high diagnostic ability with an AUROC value of over 0.85 in all control skin diseases except autoimmune vesicular disease (Table 4). In addition, hsa-miR-21-3p (AUROC 0.90) and hsa-miR are used to distinguish between SJS / TEN and autoimmune vesicular disease, which can only provide moderate diagnostic ability (AUROC 0.77) with hsa-miR-205-5p. It was found that -21-5p (AUROC 0.89) showed good diagnostic ability (Table 4).
DIHSと対照皮膚疾患との鑑別において最も有望な結果を示したmiRNAはhsa-miR-21-3pであり、アトピー性皮膚炎、乾癬、自己免疫性水疱症および水痘とのAUROC値は0.9以上であった(表4)。また、診断能としては中程度であるものの、DIHSと中毒疹の間におけるhsa-miR-21-3pのAUROC値は0.79であった。さらに本miRNA分子は、DIHSとアトピー性皮膚炎及び自己免疫性水疱症との鑑別において、既報マーカーであるTARCより高い診断能を示した(表4)。一方、hsa-miR-21-5p及びhsa-miR-222-5pは、hsa-miR-21-3pと同様の診断能のプロファイルを示したものの、それらAUROC値は同程度あるいはやや低値を示した(表4)。 The miRNA that showed the most promising results in differentiating DIHS from control skin diseases was hsa-miR-21-3p, with an AUROC value of 0.9 or higher for atopic dermatitis, psoriasis, autoimmune vesicular disease and varicella. There was (Table 4). The AUROC value of hsa-miR-21-3p between DIHS and toxic eruption was 0.79, although the diagnostic ability was moderate. Furthermore, this miRNA molecule showed higher diagnostic ability than the previously reported marker TARC in differentiating DIHS from atopic dermatitis and autoimmune vesicular disease (Table 4). On the other hand, hsa-miR-21-5p and hsa-miR-222-5p showed the same diagnostic ability profile as hsa-miR-21-3p, but their AUROC values showed similar or slightly lower values. (Table 4).
重症薬疹と対照皮膚疾患との鑑別診断能の向上に向けて、3種miRNAsの組み合わせによる診断モデル、あるいはそれに既存マーカーを加えた診断モデルの診断能の評価を行った。その結果、これら診断モデルは全重症薬疹病型のうち、DIHSと対照皮膚疾患との鑑別診断において最も有用であることが明らかとなった(表4)。本診断モデルはDIHSにおいて、全ての対照皮膚疾患との鑑別においてAUROC値0.84を超える高い診断能を示した。また、本診断モデルにTARCを加えることにより、鑑別診断能のさらなる向上が認められた(表4)。 In order to improve the differential diagnosis ability between severe drug eruption and control skin disease, we evaluated the diagnostic ability of a diagnostic model using a combination of three miRNAs or a diagnostic model with existing markers added. As a result, it was clarified that these diagnostic models are most useful in the differential diagnosis between DIHS and control skin disease among all severe drug eruption types (Table 4). This diagnostic model showed high diagnostic ability in DIHS with an AUROC value of over 0.84 in differentiation from all control skin diseases. In addition, the addition of TARC to this diagnostic model further improved the differential diagnosis ability (Table 4).
以上の結果から、本研究により同定したmiRNAsは、単独あるいは組み合わせることで、重症薬疹を特異的に検出することが可能で、かつ既報マーカー単体では識別困難である皮膚疾患との鑑別を補助できる新規重症薬疹バイオマーカーである。 From the above results, the miRNAs identified in this study can be used alone or in combination to specifically detect severe drug eruption, and can assist in differentiation from skin diseases that are difficult to identify with previously reported markers alone. It is a new severe drug eruption biomarker.
表１．探索群および検証群における各種重症薬疹バイオマーカー候補miRNAの発現量比
表２．探索群における重症薬疹バイオマーカー候補miRNAsの病勢診断能および重症化予測能
(i)重症薬疹急性期と軽症薬疹症例との間におけるhsa-miR-250-5p, hsa-miR-21-3及びhsa-miR-21-5pのΔCq値を用いて、多重ロジスティック回帰により構築した重症薬疹診断モデルから得られるスコア値を用いてROC解析を実施した。
(ii)重症薬疹急性期と軽症薬疹症例との間における(i)のモデルに用いた3種のmiRNAのΔCq値と、既報薬疹マーカーであるTARCの臨床検査値を用いて多重ロジスティック回帰により構築した重症薬疹診断モデルから得られるスコア値を用いてROC解析を実施した。臨床検査が入手不可であった検体（軽症薬疹1例）は解析から除外した。
(iii)定量結果が入手不可であった検体（回復期2例）はROC解析から除外した。
(iv)定量結果が入手不可であった検体（軽症薬疹1例）はROC解析から除外した。
Table 2. Disease diagnosis ability and aggravation prediction ability of miRNAs, which are candidates for severe drug eruption biomarkers in the exploration group.
(i) Multiple logistic regression using the ΔCq values of hsa-miR-250-5p, hsa-miR-21-3 and hsa-miR-21-5p between acute and mild drug eruption cases. ROC analysis was performed using the score values obtained from the severe drug eruption diagnosis model constructed by.
(ii) Multiple logistic using the ΔCq values of the three miRNAs used in the model (i) between the acute phase and mild drug eruption cases and the clinical laboratory test values of TARC, a previously reported drug eruption marker. ROC analysis was performed using the score values obtained from the severe drug eruption diagnosis model constructed by regression. Specimens for which clinical tests were not available (1 case of mild drug eruption) were excluded from the analysis.
(iii) Specimens for which quantitative results were not available (2 convalescent cases) were excluded from ROC analysis.
(iv) Specimens for which quantitative results were not available (1 case of mild drug eruption) were excluded from ROC analysis.
表３．検証群における重症薬疹バイオマーカー候補miRNAsの病勢診断能および重症化予測能
(i)重症薬疹急性期と軽症薬疹症例との間におけるhsa-miR-250-5p, hsa-miR-21-3 及びhsa-miR-21-5pのΔCq値を用いて、多重ロジスティック回帰により構築した重症薬疹診断モデルから得られるスコア値を用いてROC解析を実施した。
(ii)定量結果が入手不可であった検体（軽症薬疹4例、健常成人全例）はROC解析から除外した。
(iii)重症薬疹急性期と軽症薬疹症例との間における(i)のモデルに用いた3種のmiRNAsのΔCq値と、既存薬疹マーカーであるTARCの臨床検査値を用いて多重ロジスティック回帰分析により構築した重症薬疹診断モデルから得られるスコア値を用いてROC解析を実施した。臨床検査が入手不可であった（軽症薬疹4例、健常成人全例）は解析から除外した。
Table 3. Disease diagnosis ability and aggravation prediction ability of miRNAs, which are candidates for severe drug eruption biomarkers in the verification group.
(i) Multiple logistic regression using the ΔCq values of hsa-miR-250-5p, hsa-miR-21-3 and hsa-miR-21-5p between the acute phase and mild drug eruption cases. ROC analysis was performed using the score values obtained from the severe drug eruption diagnosis model constructed by.
(ii) Specimens for which quantitative results were not available (4 cases of mild drug eruption, all cases of healthy adults) were excluded from ROC analysis.
(iii) Multiple logistic using the ΔCq values of the three miRNAs used in the model (i) between the acute phase and mild drug eruption cases and the clinical laboratory test values of TARC, which is an existing drug eruption marker. ROC analysis was performed using the score values obtained from the severe drug eruption diagnosis model constructed by regression analysis. Laboratory tests were not available (4 mild drug eruptions, all healthy adults) were excluded from the analysis.
表４．対照皮膚疾患に対する重症薬疹バイオマーカー候補miRNAsの鑑別診断能
(i)TARCの臨床検査値が入手不可であった検体（アトピー性皮膚炎, 9例; 乾癬, 9例; 水痘, 9例; 中毒疹, 4例）はROC解析から除外した。
(ii)表3において構築した各重症薬疹診断モデルを用い、各重症薬疹病型と他の対照皮膚疾患の識別診断能の評価をROC曲線により解析した。
Table 4. Differential diagnosis ability of miRNAs, a candidate for severe drug eruption biomarkers for control skin diseases
(i) Specimens for which clinical laboratory test values for TARC were not available (atopic dermatitis, 9 cases; psoriasis, 9 cases; chickenpox, 9 cases; toxic eruption, 4 cases) were excluded from ROC analysis.
(ii) Using each severe drug eruption diagnostic model constructed in Table 3, the evaluation of the discriminative diagnostic ability of each severe drug eruption type and other control skin diseases was analyzed by ROC curve.
本発明は、体外診断薬、臨床検査などに利用できる。 INDUSTRIAL APPLICABILITY The present invention can be used for in vitro diagnostic agents, clinical tests, and the like.
＜配列番号１＞
hsa-miR-205-5pの塩基配列を示す。
＜配列番号２＞
hsa-miR-21-3pの塩基配列を示す。
＜配列番号３＞
hsa-miR-21-5pの塩基配列を示す。
＜配列番号４＞
hsa-miR-222-3pの塩基配列を示す。
＜配列番号５＞
hsa-let-7d-3pの塩基配列を示す。
＜配列番号６＞
hsa-miR-23b-3pの塩基配列を示す。
＜配列番号７＞
hsa-miR-24-3pの塩基配列を示す。
＜配列番号８＞
hsa-miR-30d-5pの塩基配列を示す。
＜配列番号９＞
hsa-miR-361-5pの塩基配列を示す。
＜配列番号１０＞
cel-miR-54-3pの塩基配列を示す。
<SEQ ID NO: 1>
The base sequence of hsa-miR-205-5p is shown.
<SEQ ID NO: 2>
The base sequence of hsa-miR-21-3p is shown.
<SEQ ID NO: 3>
The base sequence of hsa-miR-21-5p is shown.
<SEQ ID NO: 4>
The base sequence of hsa-miR-222-3p is shown.
<SEQ ID NO: 5>
The base sequence of hsa-let-7d-3p is shown.
<SEQ ID NO: 6>
The base sequence of hsa-miR-23b-3p is shown.
<SEQ ID NO: 7>
The base sequence of hsa-miR-24-3p is shown.
<SEQ ID NO: 8>
The base sequence of hsa-miR-30d-5p is shown.
<SEQ ID NO: 9>
The base sequence of hsa-miR-361-5p is shown.
<SEQ ID NO: 10>
The base sequence of cel-miR-54-3p is shown.
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