TW202012634A - Methods for identifying and treating kawasaki disease patients with intravenous immunoglobulin resistance - Google Patents

Abstract

Methods for identifying Kawasaki Disease subjects resistant to intravenous immunoglobulin (IVIG) are provided, comprising the step of detecting at least one risk allele of the SNPs disclosed herein or calculating a weighted genetic risk score based on the presence of the risk alleles. Also provided are methods of treating Kawasaki Disease subjects with IVIG resistance, by administering a first dose of IVIG and an adjunctive therapy.

Description

用於識別及治療患有靜脈注射免疫球蛋白抗性的川崎氏病患者的方法Method for identifying and treating patients with Kawasaki disease with intravenous immunoglobulin resistance

本發明係關於一種用於識別及治療患有靜脈注射免疫球蛋白抗性的川崎氏病患者的方法。The present invention relates to a method for identifying and treating patients with Kawasaki's disease suffering from intravenous immunoglobulin resistance.

川崎氏病(KD)為一種急性系統性血管炎症候群。此疾病主要影響5歲以下的孩童。KD被認為是造成發展中國家孩童的後天心臟疾病的主要原因。目前公認2g/kg的高劑量靜脈注射免疫球蛋白(IVIG)是川崎氏病患者的標準治療方法。在疾病發作後10天內接受單一高劑量的IVIG可顯著降低冠狀動脈病變(CALs)的風險18.4%至49.0%。然而，在初始IVIG治療後，10%至20%的川崎氏病患者出現復發熱，可能的原因包括同時存在感染(intercurrent infection)或IVIG抗性。對IVIG抗性的患者具有較高的CAL風險以及成年期的後天心臟併發症。Kawasaki disease (KD) is an acute systemic vascular inflammation syndrome. This disease mainly affects children under 5 years of age. KD is considered to be the leading cause of acquired heart disease in children in developing countries. It is currently accepted that a high-dose intravenous immunoglobulin (IVIG) of 2 g/kg is the standard treatment for patients with Kawasaki disease. Receiving a single high-dose IVIG within 10 days after the onset of the disease can significantly reduce the risk of coronary artery disease (CALs) by 18.4% to 49.0%. However, after initial IVIG treatment, 10% to 20% of patients with Kawasaki disease have recurrent fever. Possible causes include concurrent infection (intercurrent infection) or IVIG resistance. Patients with resistance to IVIG have a higher risk of CAL and acquired cardiac complications in adulthood.

在診斷時，識別具有潛在性IVIG抗性或不應性(refractory)之川崎氏病患者以及藉由輔助性治療醫治IVIG抗性川崎氏病患者以降低冠狀動脈病變風險的需求仍未被滿足。本發明係解決此需求及其他需求。At the time of diagnosis, the need to identify patients with Kawasaki disease with potential IVIG resistance or refractory and to treat IVIG-resistant Kawasaki disease patients with adjuvant therapy to reduce the risk of coronary artery disease has not been met. The present invention addresses this and other needs.

在一實施例中，本發明揭露一種用於治療個體的川崎氏病的方法，其包含以下步驟：(a)檢測個體的樣本中選自rs9380548、rs742490、rs7634、rs1250301、rs2797915、rs4130857、rs4585205、rs4602399、rs4286440、rs16867137或rs1250302的至少一單核苷酸多型性(single nucleotide polymorphism, SNP)的風險對偶基因的存在；(b)將具有步驟(a)中至少一風險對偶基因的個體識別為對IVIG具有抗性；以及(c)對該個體施予第一劑(first dose)IVIG以及輔助性治療。In one embodiment, the present invention discloses a method for treating Kawasaki's disease of an individual, which includes the following steps: (a) a sample selected from an individual is selected from rs9380548, rs742490, rs7634, rs1250301, rs2797915, rs4130857, rs4585205, rs4602399, rs4286440, rs16867137, or rs1250302 of at least one single nucleotide polymorphism (SNP) risk dual genes; (b) identify individuals with at least one risk dual gene in step (a) as Resistant to IVIG; and (c) Administer a first dose of IVIG and adjuvant therapy to the individual.

在另一實施例中，本發明揭露一種治療個體的川崎氏病的方法，其包含以下步驟：(a)檢測個體的樣本中單核苷酸多型性(SNPs)的風險對偶基因的存在，該SNPs選自： (i) rs9380548； (ii) rs742490； (iii) rs7634； (iv) 選自rs1250301或rs2797915的至少一SNP； (v) 選自rs4130857、rs4585205或rs4602399的至少一SNP； (vi) rs4286440； (vii) rs16867137；以及 (viii) rs1250302； (b)基於下列公式計算加權遺傳風險評分(weighted genetic risk score, wGRS)；

(wGRS公式) 其中，n為8至11的整數，k為步驟(a)的SNP，w^k 為SNP的對應加權(ln(OR))以及X^k 為SNP的風險對偶基因的數目(0、1或2)；(c)若步驟(b)的wGRS等於或大於0.01，則將個體識別為IVIG抗性；以及(d) 對該個體施予第一劑IVIG以及輔助性治療。In another embodiment, the present invention discloses a method for treating Kawasaki's disease of an individual, which includes the following steps: (a) detecting the presence of risk dual genes for single nucleotide polymorphisms (SNPs) in the sample of the individual, The SNPs are selected from: (i) rs9380548; (ii) rs742490; (iii) rs7634; (iv) at least one SNP selected from rs1250301 or rs2797915; (v) at least one SNP selected from rs4130857, rs4585205 or rs4602399; (vi ) rs4286440; (vii) rs16867137; and (viii) rs1250302; (b) Calculate the weighted genetic risk score (wGRS) based on the following formula;

(wGRS formula) where n is an integer from 8 to 11, k is the SNP of step (a), w ^k is the corresponding weight of the SNP (ln(OR)) and X ^k is the number of risk dual genes of the SNP (0, 1 or 2); (c) if the wGRS of step (b) is equal to or greater than 0.01, the individual is identified as IVIG resistant; and (d) the first dose of IVIG and adjuvant therapy are administered to the individual.

本發明亦揭露一種套組用於識別對IVIG抗性的川崎氏病個體的用途：該套組包含用於檢測個體的樣本中選自rs9380548、rs742490、rs7634、rs1250301、rs2797915、rs4130857、rs4585205、rs4602399、rs4286440、rs16867137或rs1250302的至少一SNP的風險對偶基因的試劑。The present invention also discloses the use of a kit for identifying individuals with Kawasaki disease resistant to IVIG: the kit contains samples for testing individuals selected from rs9380548, rs742490, rs7634, rs1250301, rs2797915, rs4130857, rs4585205, rs4602399 , Rs4286440, rs16867137 or rs1250302 at least one SNP risk dual gene reagent.

本發明亦提供一種用以識別對IVIG抗性的川崎氏病個體的方法，其包含以下步驟：(a)檢測個體的樣本中選自rs9380548、rs742490、rs7634、rs1250301、rs2797915、rs4130857、rs4585205或rs4602399的至少一SNP的風險對偶基因的存在；以及(b)將具有步驟(a)中至少一風險對偶基因的個體識別為對IVIG具有抗性。The present invention also provides a method for identifying individuals with Kawasaki's disease resistant to IVIG, which includes the following steps: (a) a sample selected from the individuals selected from rs9380548, rs742490, rs7634, rs1250301, rs2797915, rs4130857, rs4585205 or rs4602399 The existence of a risk dual gene of at least one SNP; and (b) identifying individuals with at least one risk dual gene in step (a) as being resistant to IVIG.

更提供一種用以識別對IVIG抗性的川崎氏病個體的方法，其包含以下步驟：(a)檢測個體的樣本中單核苷酸多型性(SNPs)的風險對偶基因的存在，該SNPs選自： (i) rs9380548； (ii) rs742490； (iii) rs7634； (iv) 選自rs1250301或rs2797915的至少一SNP； (v) 選自rs4130857、rs4585205或rs4602399的至少一SNP； (vi) rs4286440； (vii) rs16867137；以及 (viii) rs1250302； (b) 基於本文所述的wGRS公式計算加權遺傳風險評分(wGRS)；以及(c)若步驟(b)的wGRS等於或大於0.01，則將個體識別為IVIG抗性。It also provides a method for identifying individuals with Kawasaki disease resistant to IVIG, which includes the following steps: (a) detecting the presence of a dual gene for risk of single nucleotide polymorphisms (SNPs) in the sample of the individual, the SNPs Selected from: (i) rs9380548; (ii) rs742490; (iii) rs7634; (iv) at least one SNP selected from rs1250301 or rs2797915; (v) at least one SNP selected from rs4130857, rs4585205 or rs4602399; (vi) rs4286440 ; (Vii) rs16867137; and (viii) rs1250302; (b) Calculate the weighted genetic risk score (wGRS) based on the wGRS formula described herein; and (c) If the wGRS of step (b) is equal to or greater than 0.01, the individual Recognized as IVIG resistance.

在此專利中所使用之用語「發明(invention)」、「該發明(the invention)」、「此發明(this invention)」及「本發明(the present invention」」旨在大致代表本專利之所有標的以及以下的發明申請專利範圍。包含此些用語之論述應理解為非限制本文所述標的或限制發明申請專利範圍之意思或範疇。由本專利所涵蓋之本發明實施例是由以下發明申請專利範圍所定義，而非此發明內容所定義。此發明內容是本發明各種態樣的上位概述且引介將進一步在下列實施方式說明之一些概念。此發明內容非旨在認定所主張標的之關鍵或必要特徵，亦非旨在用於獨立地判斷所主張標的之範疇。標的應參照整個說明書、任何或所有圖式及各請求項之適當部分而理解。The terms "invention", "the invention", "this invention" and "the present invention" used in this patent are intended to represent substantially all of this patent Subject and the following patent application scope of the invention. The discussion including these terms should be understood as not limiting the meaning or scope of the subject matter or limiting the patent application scope of the invention described herein. The embodiments of the invention covered by this patent are patent application by the following invention The scope is not defined by this summary. This summary is a general overview of various aspects of this invention and introduces some concepts that will be further explained in the following embodiments. This summary is not intended to identify the key or The essential features are not intended to be used to independently judge the scope of the claimed subject matter. The subject matter should be understood with reference to the entire specification, any or all drawings, and appropriate parts of each request.

本發明在一同閱覽附圖及下列詳細說明下將變得更加顯而易見。The present invention will become more apparent after reading the accompanying drawings and the following detailed description.

如本文所使用，冠詞「一(a)」以及「一(an)」指稱冠詞的語法對象的一個或一個以上(即，至少一個)。As used herein, the articles "a" and "an" refer to one or more (ie, at least one) of the grammatical objects of the article.

用語「個體(subject)」及「患者(patient)」可互換使用且係指稱為診斷患有川崎氏病或疑似患有川崎氏病的脊椎動物。個體包括靈長類，且較佳地是人類。The terms "subject" and "patient" are used interchangeably and refer to vertebrates that are diagnosed with or suspected of having Kawasaki disease. Individuals include primates, and preferably humans.

如本文可互換使用的「IVIG抗性(IVIG-resistance)」、「IVIG抵抗性(IVIG resistant)」或「對IVIG的不應性(refractory to IVIG) 本文定義為在施予首次IVIG後發燒(體溫38°C以上)48小時或48小時以上。As used interchangeably herein, "IVIG-resistance", "IVIG resistant" or "refractory to IVIG" is defined as fever after the first IVIG is administered ( (Body temperature above 38°C) 48 hours or more.

本文中的所有數字可理解為由「約(about)」修飾。如本文所使用的用語「約」表示涵蓋特定值±10的變異。All numbers in this article can be understood as modified by "about". The term "about" as used herein means to cover a variation of a specific value of ±10.

根據本發明的實施例之用於識別對IVIG不應性或抗性的川崎氏病患者的多重單核苷酸多型性(SNPs)包含SEQ ID NOS: 1至11中的至少一多核苷酸序列。Multiple single nucleotide polymorphisms (SNPs) for identifying Kawasaki disease patients who are refractory or resistant to IVIG according to embodiments of the present invention include at least one polynucleoside in SEQ ID NOS: 1 to 11 Acid sequence.

表1

Table 1

SNP的風險對偶基因描述於表2中。在染色體3上的rs4130857、rs4585205以及rs4602399與在染色體10上的rs1250301以及 rs2797915為高度連鎖不平衡(linkage disequilibrium)。The SNP risk dual genes are described in Table 2. Rs4130857, rs4585205 and rs4602399 on chromosome 3 and rs1250301 and rs2797915 on chromosome 10 are highly linkage disequilibrium.

國家生物技術資訊中心(National Center for Biotechnology Information, NCBI)資料庫中的SNP的基因庫登錄編號(GenBank accession No.)表示SNP的序列和位置。所屬領域中具有通常知識者使用基因庫登錄編號可容易地識別SNP的序列和位置。對應於在NCBI中註冊的SNP的rs No.的特定序列可能隨著時間改變。對於所屬領域中具有通常知識者顯而易見的是，即使對應的rs 數目改變，序列也在本發明的範圍內。SEQ ID NOS: 1 至11的核苷酸序列為多型性序列(polymorphic sequences)。多型性序列為包含代表SNP的多型性位點的多核苷酸序列。多核苷酸序列可為DNA或RNA。用於識別具有 IVIG 抗性的川崎氏病患者的方法 The GenBank accession number (GenBank accession No.) of the SNP in the National Center for Biotechnology Information (NCBI) database indicates the sequence and position of the SNP. Those with ordinary knowledge in the field can easily identify the sequence and position of the SNP using the gene bank accession number. The specific sequence of rs No. corresponding to the SNP registered in the NCBI may change over time. It is obvious to those with ordinary knowledge in the art that even if the corresponding number of rs changes, the sequence is within the scope of the present invention. The nucleotide sequences of SEQ ID NOS: 1 to 11 are polymorphic sequences. A polymorphic sequence is a polynucleotide sequence that contains polymorphic sites that represent SNPs. The polynucleotide sequence may be DNA or RNA. Method for identifying patients with Kawasaki disease with IVIG resistance

該識別方法包含自具有川崎氏病或疑似患有川崎氏病的患者的樣本分離出DNA、定義DNA的多型性位點的鹼基序列、以及當鹼基序列包含選自SEQ ID NO:1至SEQ ID NO:11的至少一SNP或列於表2中的至少一風險對偶基因時，判定該病患對IVIG具有抗性。The identification method includes separating DNA from a sample of a patient with Kawasaki disease or suspected of having Kawasaki disease, a base sequence defining a polymorphic site of DNA, and when the base sequence contains a sequence selected from SEQ ID NO: 1 When at least one SNP to SEQ ID NO: 11 or at least one risk dual gene listed in Table 2, the patient is determined to be resistant to IVIG.

在一實施例中，提供一種用於識別對IVIG抗性的川崎氏病患者的方法，該方法包含以下步驟：(a)檢測個體的樣本中選自rs9380548、rs742490、rs7634、rs1250301、rs2797915、rs4130857、rs4585205、rs4602399、rs4286440、rs16867137或rs1250302的至少一單核苷酸多型性(SNP)的風險對偶基因的存在；以及(b)將具有步驟(a)中至少一風險對偶基因的個體識別為對IVIG具有抗性。In one embodiment, a method for identifying a patient with Kawasaki disease resistant to IVIG is provided. The method includes the following steps: (a) a sample of an individual is selected from rs9380548, rs742490, rs7634, rs1250301, rs2797915, rs4130857 , Rs4585205, rs4602399, rs4286440, rs16867137, or rs1250302, the presence of a risk dual gene for at least one single nucleotide polymorphism (SNP); and (b) identifying individuals with at least one risk dual gene in step (a) as Resistant to IVIG.

在另一實施例中，提供一種用於識別對IVIG抗性的川崎氏病患者的方法，該方法包含以下步驟：(a)檢測個體的樣本中單核苷酸多型性(SNPs)的風險對偶基因的存在，該SNPs選自： (i) rs9380548； (ii) rs742490； (iii) rs7634； (iv) 選自rs1250301或rs2797915的至少一SNP； (v) 選自rs4130857、rs4585205或rs4602399的至少一SNP； (vi) rs4286440； (vii) rs16867137；以及 (viii) rs1250302； (b) 基於下列公式計算加權遺傳風險評分(wGRS)：

(wGRS公式) 其中，n為8至11的整數，k 為步驟(a)的SNP，w ^k 為SNP的對應加權(ln(OR))以及X ^k 為SNP的風險對偶基因的數目(0、1或2)；以及(c)若步驟(b)的wGRS等於或大於0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.3、1.4、1.5、1.6或1.65，則將個體識別為IVIG抗性。在例示性實施例中，SNPs的對應加權(ln(OR))列於表1中。In another embodiment, a method for identifying patients with Kawasaki disease resistant to IVIG is provided, the method comprising the following steps: (a) detecting the risk of single nucleotide polymorphisms (SNPs) in a sample of an individual For the presence of a dual gene, the SNPs are selected from: (i) rs9380548; (ii) rs742490; (iii) rs7634; (iv) at least one SNP selected from rs1250301 or rs2797915; (v) at least one selected from rs4130857, rs4585205 or rs4602399 One SNP; (vi) rs4286440; (vii) rs16867137; and (viii) rs1250302; (b) Calculate the weighted genetic risk score (wGRS) based on the following formula:

(wGRS formula) where n is an integer from 8 to 11, k is the SNP of step (a), w ^k is the corresponding weight of the SNP (ln(OR)) and X ^k is the number of risk dual genes of the SNP (0, 1 or 2); and (c) if the wGRS of step (b) is equal to or greater than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8 , 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 or 1.65, the individual is identified as IVIG resistant. In the exemplary embodiment, the corresponding weights (ln(OR)) of SNPs are listed in Table 1.

使用wGRS來集合本申請的SNPs的勝算比(odds ratio)而顯著改善IVIG抗性的預測準確性。Using wGRS to aggregate the odds ratio of the SNPs of the present application significantly improves the prediction accuracy of IVIG resistance.

本發明的部分實施例涉及基於測試樣本中的特定SNP的識別來評估川崎氏病個體是否具有IVIG抗性或具有IVIG抗性的風險的方法。樣本的非限制例包含自周邊白血球細胞、血清、細胞、組織或生物檢體的DNA。Some embodiments of the present invention relate to a method for assessing whether an individual with Kawasaki disease has IVIG resistance or the risk of having IVIG resistance based on the identification of a specific SNP in a test sample. Non-limiting examples of samples include DNA from peripheral white blood cells, serum, cells, tissues, or biological specimens.

多型性可藉由任意可獲得的方法進行檢測，其包含基因擴增、與探針雜合、微陣列分析、即時聚合酶鏈鎖反應(real-time PCR)、定序或其類似方式，參照B Sobrino et al, SNPs in forensic genetics: a review on SNP typing methodologies, Forensic Science International, Volume 154, Issues 2–3, 25 November 2005, Pages 181-194。在一特定實施例中，SNP偵測包含擴增多型性、連鎖基因座或與其相關的序列(例如，毗鄰序列(flanking sequences)、轉錄序列(transcribed sequences)等)並檢測所得的擴增子。例如，在一實施例中，擴增包含(a) 將擴增引子或擴增引子對與從生物體或生物樣本分離的核酸模板混合。引子或引子對可與接近或包含多型性或連鎖基因座的區域互補或部分互補，並且可餮由核酸模板上的聚合酶啟使核酸聚合。引子或引子對在包含聚合酶和模板核酸的DNA聚合反應中延伸以產生擴增子。在部分態樣中，擴增子可選地藉由包含將擴增子與陣列雜交、以限制酶消化擴增子或real-time PCR分析的過程進行檢測。可選地，擴增子可完全或部分例如藉由雜交進行定序。通常，擴增可包含使用自生物體或生物樣本分離的核酸作為PCR、RT-PCR或LCR的模板來執行聚合酶鏈反應(PCR)、反轉錄酶PCR(RT-PCR)或連接酶鏈反應(LCR)。其他技術可替代擴增，例如使用支鏈DNA(bDNA)探針。Polymorphism can be detected by any available method, including gene amplification, hybridization with probes, microarray analysis, real-time PCR, sequencing or the like, Refer to B Sobrino et al, SNPs in forensic genetics: a review on SNP typing methodologies, Forensic Science International, Volume 154, Issues 2–3, 25 November 2005, Pages 181-194. In a particular embodiment, SNP detection includes amplification of polymorphisms, linked loci or sequences related thereto (eg, flanking sequences, transcribed sequences, etc.) and detection of the resulting amplicons . For example, in one embodiment, the amplification comprises (a) mixing the amplification primer or the pair of amplification primers with the nucleic acid template isolated from the organism or biological sample. The primer or primer pair may be complementary or partially complementary to a region close to or containing a polymorphic or linked locus, and may be activated by a polymerase on the nucleic acid template to cause the nucleic acid to polymerize. The primer or primer pair is extended in a DNA polymerization reaction containing a polymerase and template nucleic acid to produce an amplicon. In some aspects, the amplicon is optionally detected by a process that includes hybridizing the amplicon to the array, digesting the amplicon with restriction enzymes, or real-time PCR analysis. Alternatively, the amplicons can be sequenced in whole or in part, for example by hybridization. In general, the amplification may include performing the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), or ligase chain reaction using nucleic acid isolated from the organism or biological sample as a template for PCR, RT-PCR, or LCR (LCR). Other techniques can replace amplification, such as the use of branched DNA (bDNA) probes.

這些SNP與下列基因連鎖，因此這些基因與川崎氏病中IVIG抗性的多型性相關：ADTRP (rs9380548)；KLF6 (rs7634)；EXOC2 (EXOC2 ；ZNF438-ZEB1 (s1250301及rs2797915)或MIR548A3-ZPLD1 (rs4130857、rs4585205及rs4602399)。用於檢測具有 IVIG 抗性的川崎氏病患者的方法 These SNPs are linked to the following genes, so these genes are associated with the polymorphism of IVIG resistance in Kawasaki disease: ADTRP (rs9380548); KLF6 (rs7634); EXOC2 ( EXOC2 ; ZNF438-ZEB1 (s1250301 and rs2797915) or MIR548A3-ZPLD1 (rs4130857, rs4585205, and rs4602399). Method for detecting patients with Kawasaki disease with IVIG resistance

在基於本文所揭露的方法識別具有IVIG抗性的川崎氏病患者後，輔助性治療可與第一劑IVIG施予，以減少川崎氏病的併發症，特別是冠狀動脈病變。After identifying patients with Kawasaki disease with IVIG resistance based on the methods disclosed herein, adjuvant therapy can be given with the first dose of IVIG to reduce the complications of Kawasaki disease, especially coronary artery disease.

輔助性治療的非限制例包含第二劑的IVIG、抗TNF-α試劑、皮質類固醇、環孢靈(cyclosporin)、IL-1抑制劑(阿那白滯素(anakinra)及卡那單抗(canakinumab))、環磷酸醯胺(cyclophosphamide)、利妥昔單抗(Rituximab)、托珠單抗(Tocilizumab)、配妥西菲林(Pentoxifylline)以及血漿除去術(plasmapheresis)或其組合。Non-limiting examples of adjuvant therapy include a second dose of IVIG, anti-TNF-α agents, corticosteroids, cyclosporin, cyclosporin, IL-1 inhibitors (anakinra) and canastuzumab ( canakinumab)), cyclophosphamide, Rituximab, Tocilizumab, Pentoxifylline, and plasmapheresis or combinations thereof.

所屬領域中具有通常知識者可根據年齡、體重、待治療個體的症狀、任何現有的醫學症狀以及醫學專業人員的判斷來確定輔助性治療的劑量。輔助性治療可在施予第一劑IVIG之前、同時或之後進行施予。用於識別具有 IVIG 抗性的川崎氏病患者的套組 Those with ordinary knowledge in the art can determine the dosage of adjuvant therapy based on age, weight, symptoms of the individual to be treated, any existing medical symptoms, and the judgment of a medical professional. Adjuvant therapy can be administered before, at the same time, or after the first dose of IVIG. Kit for identifying patients with Kawasaki disease with IVIG resistance

本發明亦提供一種套組用於識別具有IVIG抗性的川崎氏病患者的用途。該套組包含用於檢測患者的樣本中選自rs9380548、rs742490、rs7634、rs1250301、rs2797915、rs4130857、rs4585205、rs4602399、rs4286440、rs16867137或rs1250302的至少一SNP的風險對偶基因之試劑。The invention also provides the use of a kit for identifying patients with Kawasaki disease having IVIG resistance. The kit contains reagents for detecting at least one SNP risk dual gene selected from rs9380548, rs742490, rs7634, rs1250301, rs2797915, rs4130857, rs4585205, rs4602399, rs4286440, rs16867137, or rs1250302 in the sample of the patient.

試劑的非限制例包含用於分離和擴增DNA的引子組或可檢測至少一SNP的雜交探針。用語「引子(primer)」係指用於聚合酶鏈反應(PCR)的寡核苷酸。適當的引子組可容易由所屬領域中具有通常知識者參照根據本發明的實施例的序列來設計。Non-limiting examples of reagents include primer sets for separating and amplifying DNA or hybridization probes that can detect at least one SNP. The term "primer" refers to an oligonucleotide used in polymerase chain reaction (PCR). A suitable primer set can be easily designed by a person having ordinary knowledge in the art with reference to the sequence according to the embodiment of the present invention.

在一實施例中，該套組包含微陣列。微陣列為是核酸、蛋白質、小分子、細胞或其他物質的微觀有序陣列，可以對複雜的生化樣本進行平行分析。微陣列可使用各種技術製造，其包含用玻璃載玻片上的細尖針印刷、使用預製遮罩的光蝕刻法、使用動態微鏡裝置的光蝕刻法、噴墨印刷或微電極陣列上的電化學。In one embodiment, the kit includes a microarray. Microarrays are microscopic ordered arrays of nucleic acids, proteins, small molecules, cells, or other substances that can be used for parallel analysis of complex biochemical samples. Microarrays can be manufactured using a variety of techniques, including printing with fine-tip needles on glass slides, photoetching using prefabricated masks, photoetching using dynamic micromirror devices, inkjet printing, or electrochemistry on microelectrode arrays learn.

根據本發明的部分實施例的微陣列包含對偶基因特異寡核苷酸(allele-specific oligonucleotide, ASO)探針(亦即，多核苷酸與SEQ ID NOS:1-11的多核苷酸雜交)。ASO探針可固定(immobilized)在塗佈有選自胺矽烷(amino-silane)、聚-L-離胺酸以及醛的活性基團的基板上。此外，基板可由矽晶片、玻璃、石英、金屬或塑料所構成。將多核苷酸固定在基板的方法可為使用壓電的微量移液或使用針形點樣器(pin-shaped spotter)的方法。A microarray according to some embodiments of the present invention includes an allele-specific oligonucleotide (ASO) probe (ie, the polynucleotide hybridizes with the polynucleotides of SEQ ID NOS: 1-11). The ASO probe can be immobilized on a substrate coated with a reactive group selected from amino-silane, poly-L-lysine, and aldehyde. In addition, the substrate may be composed of silicon wafer, glass, quartz, metal or plastic. The method of fixing the polynucleotide to the substrate may be a method of using a micropipette using piezoelectric or a method of using a pin-shaped spotter.

本發明的實施例將由下列的實例所描述，但不以任何方式解釋成強加對其範圍的限制性。相反地，應當清楚地認知到本申請可具有各種其他實施例、其修飾、以及其等效物，在閱讀本文的描述之後，對於所屬技術領域中具有通常知識者而言可建議該些實施例、修飾及等效物而不背離本發明的精神。在下列實例描述的研究期間，除非另有說明，否則將遵照常規程序。以下僅為了說明性的目的而描述一些程序。實例 1 ： 方法 The embodiments of the present invention will be described by the following examples, but are not to be construed in any way to impose limitations on its scope. On the contrary, it should be clearly recognized that the present application may have various other embodiments, modifications thereof, and equivalents thereof, and after reading the description herein, those with ordinary knowledge in the technical field may suggest these embodiments , Modifications and equivalents without departing from the spirit of the invention. During the studies described in the following examples, unless otherwise stated, conventional procedures will be followed. The following describes some procedures for illustrative purposes only. Example 1 : Method

在高雄長庚紀念醫院醫療中心，於2001年至2007年收集診斷為川崎氏病且接受IVIG治療的孩童的DNA。川崎氏病的診斷可由臨床醫師使用美國心臟協會提出的川崎氏病標準進行：發燒持續＞ 5天且具有以下四項標準：具有草莓舌和唇裂的瀰漫性黏膜發炎(diffuse mucosal inflammation)、雙側非膿性結膜炎(bilateral nonpurulent conjunctivitis)、手足誘發性血管性水腫(indurative angioedema over the hands and feet)、變形性皮疹(dysmorphic skin rashes)以及單側頸淋巴結腫大(unilateral cervical lymphadenopathy)。排除來自急性發燒＜5天的孩童樣本。In the medical center of Kaohsiung Chang Gung Memorial Hospital, DNA of children diagnosed with Kawasaki's disease and treated with IVIG was collected from 2001 to 2007. Diagnosis of Kawasaki disease can be performed by clinicians using the Kawasaki disease standard proposed by the American Heart Association: fever lasts> 5 days and has the following four criteria: diffuse mucosal inflammation with strawberry tongue and cleft lip, bilateral Non-purulent conjunctivitis, inducible angioedema over the hands and feet, dysmorphic skin rashes, and unilateral cervical lymphadenopathy. Samples from children with acute fever <5 days were excluded.

診斷為川崎氏病的每個患者在12小時內以單劑IVIG輸注(2 g/kg)治療。給予阿斯匹靈(每日每公斤3至5 mg)直到所有發炎徵象解除或藉由顯影主動脈的胸骨旁短軸視圖(parasternal short-axis view)上的冠狀動脈(包含左側和右側)直徑的二維超聲心動圖顯象測試(2-dimensional echocardiography test)下CAL的消退。根據日本衛生部的規範，我們將CAL定義為內徑3 mm（≤5歲）或4 mm(＞ 5歲)的增加(increment)或比相鄰段的內徑大1.5倍。我們將IVIG反應性(IVIG responsiveness)定義成在首次IVIG施予後48小時內的退熱。將持續發燒超過48小時的川崎氏病患者定義成IVIG抗性。Each patient diagnosed with Kawasaki's disease was treated with a single dose of IVIG infusion (2 g/kg) within 12 hours. Give aspirin (3 to 5 mg per kg daily) until all signs of inflammation are resolved or by visualizing the diameter of the coronary arteries (including the left and right sides) on the parasternal short-axis view of the aorta CAL subsided under the 2-dimensional echocardiography test. According to the regulations of the Japanese Ministry of Health, we define CAL as an increment of 3 mm (≤5 years old) or 4 mm (>5 years old) or 1.5 times larger than the inner diameter of the adjacent segment. We defined IVIG responsiveness as the reduction of fever within 48 hours after the first IVIG administration. Patients with Kawasaki's disease who continued to have a fever for more than 48 hours were defined as IVIG resistant.

川崎氏病患者的血球細胞首先以0.5%十二烷基硫酸鈉溶解緩衝液(sodium dodecylsulfate lysis buffer)處理，接著以蛋白酶K(1 mg/mL)在60°C下4小時。使用Gentra萃取套組(Qiagen, USA)後，以70%乙醇沉澱及Gentra Puregene Blood套組來萃取DNA。單核苷酸多型性(SNPs)使用Affymetrix全基因組人類SNP陣列6.0平台(Affymetrix Genome-Wide Human SNP Array 6.0 platform) (Affymetrix, Inc, USA)進行檢測。The blood cells of patients with Kawasaki disease were first treated with 0.5% sodium dodecylsulfate lysis buffer, followed by proteinase K (1 mg/mL) at 60°C for 4 hours. After using Gentra extraction kit (Qiagen, USA), DNA was extracted with 70% ethanol precipitation and Gentra Puregene Blood kit. Single nucleotide polymorphisms (SNPs) were detected using the Affymetrix Genome-Wide Human SNP Array 6.0 platform (Affymetrix, Inc, USA).

PLINK31(S. Purcellet al ., Am J Hum Genet. 2007;81:559-575)用於全基因組掃描數據的質量控制。我們排除缺失判讀率(call rates)超過1.0%、Hardy–Weinberg平衡(Hardy–Weinberg equilibrium)的P 值＜1×10^{− 05} 且少數對偶基因頻率(minor allele frequency) ＜5.0%的SNPs。參照Affymetrix全基因組人類SNP陣列6.0平台的SNPs為NCBI36 (hg18)。CrossMap(版本0.1.5)用於將數據提升至NCBI37(hg19)。SHAPEIT(O. Delaneau et al.,Nat Methods . 2011;9:179–181.)及IMPUTE2 (BN Howie et al.,PLoS Genet . 2009;5:e1000529.)用以單倍型定相(haplotype phasing)及基因型填補(genotype imputation)。HapMap 3基因型數據(The International HapMap Consortium.Nature . 2003;426:789–796)與台灣數據合併以執行主要組成分析(principal component analysis, PCA)。PCA藉由使用全基因組複雜性狀分析 35(Genome-wide Complex Trait Analysis,35)執行，其藉由在EIGENSTRAT中實施的相同演算法進行PCA並輸出對應的特徵值(eigenvalues)和特徵向量(eigenvectors)，以識別體染色體基因型數據上的樣本次結構。PLINK31 (S. Purcell et al ., Am J Hum Genet. 2007; 81:559-575) is used for the quality control of whole genome scan data. Our interpretation of negative deletion (call rates) exceeds 1.0%, Hardy-Weinberg equilibrium (Hardy-Weinberg equilibrium) P-value <1 × 10 ^{- 05} and the minority allele frequency (minor allele frequency) <5.0% of SNPs. The SNPs referring to the Affymetrix genome-wide human SNP array 6.0 platform are NCBI36 (hg18). CrossMap (version 0.1.5) is used to promote the data to NCBI37 (hg19). SHAPEIT (O. Delaneau et al., Nat Methods . 2011; 9:179–181.) and IMPUTE2 (BN Howie et al., PLoS Genet . 2009; 5:e1000529.) are used for haplotype phasing ) And genotype imputation. HapMap 3 genotype data (The International HapMap Consortium. Nature . 2003;426:789–796) was merged with Taiwan data to perform principal component analysis (PCA). PCA is performed by using Genome-wide Complex Trait Analysis 35 (Genome-wide Complex Trait Analysis, 35), which performs PCA by the same algorithm implemented in EIGENSTRAT and outputs corresponding eigenvalues and eigenvectors To identify the sample substructure on somatic chromosome genotype data.

為了使603698 SNPs滿足篩選標準，使用在全基因組複雜性狀分析中實施的混合線性模型演算法執行關聯分析，其解釋了關聯測試期間所有SNP的多基因效應。所有SNP的固定效應藉由排除候選標記(排除候選標記的混合線性模型)來計算，由於候選標記的雙重擬合(double fitting)而防止功率損失。曼哈頓圖(Manhattan plot)藉由Haploview軟體繪製(JC Barrett et al.,Bioinformatics . 2005;21:263–265)。殘留人口分層(residual population stratification)藉由計算基因組膨脹λ值(genomic inflation λ value)並使用R中的分位數-分位數圖可視化對應的測試統計來評估(http://www.r-project.org/)。To meet the screening criteria for 603698 SNPs, a hybrid linear model algorithm implemented in genome-wide complex trait analysis was used to perform association analysis, which explained the polygenic effects of all SNPs during the association test. The fixed effect of all SNPs is calculated by excluding candidate markers (mixed linear model excluding candidate markers), preventing power loss due to double fitting of candidate markers. The Manhattan plot was drawn by Haploview software (JC Barrett et al., Bioinformatics . 2005; 21:263–265). Residual population stratification is evaluated by calculating the genomic inflation λ value and using the quantile-quantile graph in R to visualize the corresponding test statistics (http://www.r -project.org/).

wGRS系統(PL.De Jager et al.,Lancet Neurol . 2009;8:1111–1119)用於計算候選SNPs的累積效應。對偶基因勝算比是自然對數轉換成每個SNP的加權。wGRS是藉由將每個SNP的加權((ln(OR))乘以風險對偶基因數目(0、1或2)並將至少8個SNP的總和相乘來計算，如下列公式所示：

其中，n為8至11的整數，k 為SNP，w ^k 為SNP的對應加權(ln(OR))以及X ^k 為風險對偶基因的數目(0、1或2)。IVIG反應者和IVIG無反應者的wGRS藉由威爾卡森等級和檢定(Wilcoxon rank-sum test)與連續性校正進行比較。接著將川崎氏病患者由對應的wGRS分類成4個群組：群組1(wGRS＜|平均−SD|)、群組2(|平均−SD| ≤wGRS＜中位數)、群組3(中位數≤wGRS＜|平均+SD|)以及群組4(wGRS≥|平均+SD|)。亦比較群組之間的wGRS，並相關統計參數使用群組1作為參考來計算。進一步執行亞組分析以確認IVIG反應者和IVIG無反應者之間的wGRS的組內差異。The wGRS system (PL. De Jager et al., Lancet Neurol . 2009; 8:1111–1119) is used to calculate the cumulative effect of candidate SNPs. The odds ratio of the dual genes is the natural logarithm converted to the weight of each SNP. wGRS is calculated by multiplying the weight of each SNP ((ln(OR)) by the number of risk dual genes (0, 1, or 2) and multiplying the sum of at least 8 SNPs, as shown in the following formula:

Where n is an integer from 8 to 11, k is SNP, w ^k is the corresponding weight of SNP (ln(OR)) and X ^k is the number of risk dual genes (0, 1 or 2). The wGRS of IVIG responders and IVIG non-responders were compared with continuity correction by Wilcoxon rank-sum test. Then, the patients with Kawasaki's disease were classified into 4 groups according to the corresponding wGRS: group 1 (wGRS<|average−SD|), group 2 (|average−SD| ≤wGRS<median), group 3 (Median≤wGRS<|average+SD|) and group 4 (wGRS≥|average+SD|). The wGRS between groups is also compared, and related statistical parameters are calculated using group 1 as a reference. A subgroup analysis was further performed to confirm the intragroup differences in wGRS between IVIG responders and IVIG non-responders.

結果result

GWAS 的樣本次結構評估： 150位川崎氏病患者 (n=150)包含在此研究中。總體而言，在24位IVIG無反應者(男性=58.33%，年齡=2.24±2.82歲)及126位IVIG反應者(男性=60.32%，年齡=2.16±2.41歲)對867 877 SNP進行基因分型。在標記水平質量控制之後，篩選出264、179的867、877(30.44%)標記者。SNP被定義為通過具有下列標準的質量控制：少數對偶基因頻率＞ 5.0％、基因分型判讀率＞ 99％以及在對照中通過Hardy-Weinberg平衡測試(P ＜1×10^{− 05} )。603、698的867、877的SNP(69.56%)仍有待進一步分析。在樣本水平質量控制步驟中，所有個體通過樣本判讀率的標準＞99%且仍有待進一步分析。執行PCA以評估樣本結構且沒有發現明顯的人口分層。來自對偶基因關聯測試的P 值顯示沒有膨脹並且顯示樣本次結構的低可能性(基因組膨脹因子λ值λGC= 1.015)。 GWAS sample substructure evaluation: 150 patients with Kawasaki disease (n=150) were included in this study. Overall, 867 877 SNPs were genotyped in 24 IVIG non-responders (male=58.33%, age=2.24±2.82 years) and 126 IVIG responders (male=60.32%, age=2.16±2.41 years). type. After quality control of the labeling level, 867 and 877 (30.44%) markers of 264 and 179 were selected. SNP is defined as passing quality control with the following criteria: frequency of a few dual genes> 5.0%, genotyping interpretation rate> 99%, and passing Hardy-Weinberg balance test in the control ( P <1×10 ^{− 05} ). The SNPs of 603, 698, 867 and 877 (69.56%) still need further analysis. In the sample level quality control step, all individuals pass the sample interpretation rate standard >99% and still need further analysis. PCA was performed to evaluate the sample structure and no obvious population stratification was found. The P value from the dual gene association test showed no swelling and showed a low probability of sample substructure (genomic swelling factor λ value λGC = 1.015).

影響IVIG反應性的風險基因座：Risk loci affecting IVIG reactivity:

為了識別影響IVIG反應性的風險基因座，執行混合線性模型相關測試。識別出11個SNP具有P ＜1×10^{− 05} (表2)。In order to identify risk loci that affect the responsiveness of IVIG, a mixed linear model related test was performed. 11 SNPs were identified as having P <1×10 ^{− 05} (Table 2).

表2，自與IVIG抗性相關的川崎氏病及非川崎氏病組識別出的SNP

Chr., 染色體位置；^a 風險對偶基因，^b 非風險對偶基因，^c 病例中的風險對偶基因頻率以及對照組^{. d} PMLMA 藉由混合線性模型相關測試計算。^e 風險對偶基因的勝算比^f 加權是使用勝算比的自然對數變換計算Table 2. SNPs identified from the Kawasaki disease and non-Kawasaki disease groups related to IVIG resistance

Chr., chromosome position; ^a risk dual gene, ^b non-risk dual gene, ^c risk dual gene frequency in case and control group ^{. d} PMLMA is calculated by mixed linear model correlation test. The odds ratio of ^e risk dual genes ^f weighting is calculated using the natural log transformation of the odds ratio

基於由De Jager et al提出的wGRS的評分系統基於表1中的11個SNP計算。wGRS在IVIG反應組與IVIG無反應組之間顯示有顯著差異(威爾卡森等級和檢定，P =1.071×10^{− 10} )。將患者分類成4個層級：群組1(wGRS＜0.042)、群組2(0.042≤wGRS＜0.636)、群組3 (0.636≤wGRS＜1.681)及群組4 (wGRS≥1.681)。如表3所示，群組1的所有患者(42位中的42位)顯示對IVIG治療具有良好反應性，而群組4的21位中的14位為IVIG無反應者。該結果顯示與群組1相比，被分類成群組4的患者將估計有164.33倍較高風險的IVIG抗性(P =8.224×10^{− 10} ; 95% CI, 8.83~3059.50)。與第1組相比，第4組分類的患者IVIG耐藥風險估計高164.33倍。相對於群組1的群組4的靈敏度及特異性分別為100%及85.7%。The scoring system based on wGRS proposed by De Jager et al is calculated based on the 11 SNPs in Table 1. The wGRS showed a significant difference between the IVIG response group and the IVIG non-response group (Wilcarson grade and test, P = 1.071×10 ^{− 10} ). The patients were classified into 4 levels: group 1 (wGRS<0.042), group 2 (0.042≤wGRS<0.636), group 3 (0.636≤wGRS<1.681) and group 4 (wGRS≥1.681). As shown in Table 3, all patients in Group 1 (42 of 42) showed good response to IVIG treatment, while 14 of 21 in Group 4 were IVIG non-responders. This result shows that patients classified as group 4 will be estimated to have a higher risk of IVIG resistance of 164.33 times compared with group 1 ( P = 8.224×10 ^{− 10} ; 95% CI, 8.83~3059.50). Compared with group 1, the risk of IVIG resistance in patients classified in group 4 is estimated to be 164.33 times higher. The sensitivity and specificity of group 4 relative to group 1 are 100% and 85.7%, respectively.

表3，wGRS分析

P值及勝算比(OR)是在Haldane校正後計算。CI表示信賴區間；以及wGRS，加權遺傳風險評分。 *加權遺傳風險評分 †雙尾P值由費雪正確性測試計算Table 3, wGRS analysis

P value and odds ratio (OR) are calculated after Haldane correction. CI stands for confidence interval; and wGRS, weighted genetic risk score. *Weighted genetic risk score† Two-tailed P value calculated by Fisher's correctness test

群組4與群組1之間的進一步分析顯示0.667的陽性預測值、1.000的陰性預測值、6.993的陽性似然比(適度證據以排入疾病(moderate evidence to rule in disease))以及0的陰性似然比(強力證據以排除為疾病(strong evidence to rule out disease))。Further analysis between Cohort 4 and Cohort 1 showed a positive predictive value of 0.667, a negative predictive value of 1.000, a positive likelihood ratio of 6.993 (moderate evidence to rule in disease), and 0 Negative likelihood ratio (strong evidence to rule out disease).

建構ROC曲線以評估wGRS在江川崎氏病患者分類成IVIG反應族群或IVIG無反應族群的表現。該結果顯示wGRS的最佳預測值為1.3，且對應的特異性及靈敏度分別為88.9%及79.2%。在調整性別影響後，wGRS的表現保持良好，具有2.8的最佳值。對應的特異性及靈敏度分別為81.7%及79.2%。2個ROC之間的比較顯示，在AUC的真實差異的虛無假設(null hypothesis)下0.10502的P 值(Z統計量為1.621)等於0。A ROC curve was constructed to evaluate the performance of wGRS in the classification of IVIG-responsive or IVIG-unresponsive groups in patients with Jiangchuanqi disease. The results show that the best predictive value of wGRS is 1.3, and the corresponding specificity and sensitivity are 88.9% and 79.2%, respectively. After adjusting for gender effects, wGRS's performance remained good, with an optimal value of 2.8. The corresponding specificity and sensitivity were 81.7% and 79.2%, respectively. A comparison between the 2 ROCs shows that under the null hypothesis of the true difference of AUC, the P value of 0.10502 (Z statistic is 1.621) is equal to zero.

由於染色體3上的rs4130857、rs4585205及rs4602399與染色體10上的rs1250301及rs2797915為高度連鎖不平衡，因此(總共8個SNP中)選擇rs4130857及rs1250301用於wGRS分析。表4的結果顯示基於8個SNP的wGRS與表3中基於11個SNP的結果相似。Because rs4130857, rs4585205, and rs4602399 on chromosome 3 and rs1250301 and rs2797915 on chromosome 10 are highly linkage disequilibrium, rs4130857 and rs1250301 (in total 8 SNPs) were selected for wGRS analysis. The results in Table 4 show that the wGRS based on 8 SNPs is similar to the results based on 11 SNPs in Table 3.

表4，使用8個SNP的加權遺傳風險評分(wGRS)分析

a加權遺傳風險評分。b雙尾P值由費雪正確性測試計算。P值及勝算比是在Haldane校正後計算。Table 4. Weighted genetic risk score (wGRS) analysis using 8 SNPs

a Weighted genetic risk score. b Two-tailed P value is calculated by Fisher's correctness test. P value and odds ratio are calculated after Haldane correction.

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＜110＞ 長庚醫療財團法人高雄長庚紀念醫院 ＜120＞ 用於識別及治療患有靜脈注射免疫球蛋白抗性的川崎氏病患者的方法 ＜130＞ CGMFK-18003-TWI ＜160＞ 11 ＜170＞ PatentIn version 3.5 ＜210＞ 1 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人(Homo sapiens) ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs9380548; n 是 a 或 t ＜400＞ 1 cttctttgga tttgcagtgc ctacangact ctcacagcgc tactaggagc t 51 ＜210＞ 2 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs742490; n 是 g 或 c ＜400＞ 2 tggccgatgc tgtaagctcc agtttnaatg ccagccggct cgagacctaa g 51 ＜210＞ 3 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs7634; n 是 a 或 t ＜400＞ 3 aaaagtagtt tgaatgtttt gtgtgnaagg agtataccat gagatgagat g 51 ＜210＞ 4 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs1250301; n 是 g 或 a ＜400＞ 4 gcctgggcga gagagtggat ccttgnctct aaaaaataaa tttataaaat c 51 ＜210＞ 5 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs2797915;n 是 t 或 c ＜400＞ 5 cttgttcagg ctgtgaggca actaanattg taaccatttg taagtgtgca g 51 ＜210＞ 6 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs4130857; n 是 a 或 g ＜400＞ 6 gaaggaaaaa gaaagaaagg acactnagtt tcaaatctgt ccctgagtca t 51 ＜210＞ 7 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs4585205; n 是 t 或 c ＜400＞ 7 tgcgatacct agaataatgt tactgnccag aagaaaagtc caatacacaa a 51 ＜210＞ 8 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs4602399; n 是 g 或 t ＜400＞ 8 tgtgtaccaa tttactatac tagtcncttc tcacactgct ataaaaaact g 51 ＜210＞ 9 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs4286440; n 是 g 或 t ＜400＞ 9 agaacattgg aaaaatacta ggatancttg cctctaatct tagctttatg t 51 ＜210＞ 10 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs16867137; n 是 c 或 t ＜400＞ 10 gaactctgtc tagctccacc catttntaag ttaggctgaa agacaggtta a 51 ＜210＞ 11 ＜211＞ 51 ＜212＞ DNA ＜213＞ 智人 ＜220＞ ＜221＞ misc_feature ＜222＞ (26)..(26) ＜223＞ SNPrs1250302; n 是 g 或 a ＜400＞ 11 catacaggga attcagtatt ggaaangggt tgaatgaggt gacttcgagg g 51＜110＞ Kaohsiung Chang Gung Memorial Hospital, a subsidiary of Chang Gung Medical Foundation <120> A method for identifying and treating patients with Kawasaki's disease who are resistant to intravenous immunoglobulin <130> CGMFK-18003-TWI <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26)..(26) <223> SNPrs9380548; n is a Or t <400> 1 cttctttgga tttgcagtgc ctacangact ctcacagcgc tactaggagc t 51 <210> 2 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26)..(26) <223 > SNPrs742490; n is g or c <400> 2 tggccgatgc tgtaagctcc agtttnaatg ccagccggct cgagacctaa g 51 <210> 3 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26). .(26) <223> SNPrs7634; n is a or t <400> 3 aaaagtagtt tgaatgtttt gtgtgnaagg agtataccat gagatgagat g 51 <210> 4 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature ＜ 222> (26)..(26) <223> SNPrs1250301; n is g or a <400> 4 gcctgggcga gagagtggat ccttgnctct aaaaaataaa tttataaaat c 51 <210> 5 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26).. (26) <223> SNPrs2797915; n is t or c <400> 5 cttgttcagg ctgtgaggca actaanattg taaccatttg taagtgtgca g 51 <210> 6 < 211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26)..(26) <223> SNPrs4130857; n is a or g <400> 6 gaaggaaaaa gaaagaaagg acactnagtt tcaaatctgt ccctgagtca t 51 <210> 7 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26)..(26) <223> SNPrs4585205; n is t or c <400> 7 tgcgatacct agaataatgt tactgnccag aagaaaagtc caatacacaa a 51 <210> 8 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26)..(26) <223> SNPrs4602399; n is g Or t <400> 8 tgtgtaccaa tttactatac tagtcncttc tcacactgct ataaaaaact g 51 <210> 9 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26)..(26) <223 ＞ SNPrs4286440; n is g or t ＜400＞ 9 agaacattgg aaaaatacta ggatancttg cctctaatct tagctttatg t 51 <210> 10 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26).. (26) <223> SNPrs16867137; n is c or t <400> 10 gaactctgtc tagctccacc catttntaag ttaggctgaa agacaggtta a 51 <210> 11 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (26).. (26) <223> SNPrs1250302; n is g Or a ＜400＞ 11 catacaggga attcagtatt ggaaangggt tgaatgaggt gacttcgagg g 51

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Claims (11)

一種用於治療個體的川崎氏病的方法，其包含以下步驟： (a)檢測一個體的樣本中選自rs9380548、rs742490、rs7634、rs1250301、rs2797915、rs4130857、rs4585205、rs4602399、rs4286440、rs16867137或rs1250302的至少一單核苷酸多型性(SNP)的風險對偶基因的存在； (b)將具有步驟(a)中至少一風險對偶基因的該個體識別為對IVIG具有抗性；以及 (c)對該個體施予第一劑的IVIG以及一輔助性治療。A method for treating Kawasaki's disease of an individual, comprising the following steps: (a) detecting a sample selected from rs9380548, rs742490, rs7634, rs1250301, rs2797915, rs4130857, rs4585205, rs4602399, rs4286440, rs16867137 or rs1250302 The presence of at least one risk dual gene for single nucleotide polymorphism (SNP); (b) identifying the individual with at least one risk dual gene in step (a) as being resistant to IVIG; and (c) The individual is given the first dose of IVIG and an adjunct therapy. 如申請專利範圍第1項所述之方法，其中該輔助性治療係為第二劑的IVIG、抗TNF-α試劑、皮質類固醇、環孢靈(cyclosporin)、IL-1抑制劑(阿那白滯素(anakinra)及卡那單抗(canakinumab))、環磷酸醯胺(cyclophosphamide)、利妥昔單抗(Rituximab)、托珠單抗(Tocilizumab)、配妥西菲林(Pentoxifylline)、血漿除去術(plasmapheresis)或其組合。The method as described in item 1 of the patent application scope, wherein the adjuvant therapy is a second dose of IVIG, anti-TNF-α agent, corticosteroids, cyclosporin, IL-1 inhibitor (Anabe (Anakinra and canakinumab), cyclophosphamide, rituximab, tocilizumab, pentoxifylline, plasma removal Technique (plasmapheresis) or a combination thereof. 如申請專利範圍第2項所述之方法，其中該輔助性治療係為第二劑的IVIG。The method as described in item 2 of the patent application scope, wherein the adjuvant therapy is a second dose of IVIG. 一種治療個體的川崎氏病的方法，其包含以下步驟： (a)檢測一個體的樣本中單核苷酸多型性(SNP)的風險對偶基因的存在，該單核苷酸多型性係選自： (i) rs9380548； (ii) rs742490； (iii) rs7634； (iv) 選自rs1250301或rs2797915的至少一SNP； (v) 選自rs4130857、rs4585205或rs4602399的至少一SNP； (vi) rs4286440； (vii) rs16867137；以及 (viii) rs1250302； (b)基於下列公式計算加權遺傳風險評分(wGRS)；

其中，n為8至11的整數，k為該步驟(a)的SNP，w^k 為SNP的對應加權(ln(OR))以及X^k 為SNP的風險對偶基因的數目(0、1或2)； (c)若該步驟(b)的wGRS等於或大於0.01，則將該個體識別為IVIG抗性；以及 (d) 對該個體施予第一劑IVIG以及一輔助性治療。A method for treating Kawasaki's disease of an individual, comprising the following steps: (a) Detecting the presence of a dual gene for the risk of single nucleotide polymorphism (SNP) in a sample of an individual Selected from: (i) rs9380548; (ii) rs742490; (iii) rs7634; (iv) at least one SNP selected from rs1250301 or rs2797915; (v) at least one SNP selected from rs4130857, rs4585205 or rs4602399; (vi) rs4286440 ; (Vii) rs16867137; and (viii) rs1250302; (b) Calculate the weighted genetic risk score (wGRS) based on the following formula;

Where n is an integer from 8 to 11, ^k is the SNP of this step (a), w ^k is the corresponding weight of the SNP (ln(OR)) and X ^k is the number of SNP risk dual genes (0, 1 or 2 ); (c) If the wGRS of step (b) is equal to or greater than 0.01, the individual is identified as IVIG resistant; and (d) the individual is given a first dose of IVIG and an adjuvant treatment. 如申請專利範圍第4項所述之方法，其中該輔助性治療係為第二劑的IVIG、抗TNF-α試劑、皮質類固醇、環孢靈或其組合。The method according to item 4 of the patent application scope, wherein the adjuvant therapy is a second dose of IVIG, anti-TNF-α agent, corticosteroid, cyclosporine, or a combination thereof. 如申請專利範圍第4項所述之方法，其中該wGRS等於或大於0.04。The method as described in item 4 of the patent application scope, wherein the wGRS is equal to or greater than 0.04. 一種用以識別對IVIG抗性的川崎氏病個體的方法，其包含以下步驟： (a)檢測一個體的樣本中單核苷酸多型性(SNP)的風險對偶基因的存在，該SNP選自： (i) rs9380548； (ii) rs742490； (iii) rs7634； (iv) 選自rs1250301或rs2797915的至少一SNP； (v) 選自rs4130857、rs4585205或rs4602399的至少一SNP； (vi) rs4286440； (vii) rs16867137；以及 (viii) rs1250302； (b) 基於下列公式計算加權遺傳風險評分(wGRS)：

其中，n為8至11的整數，k為該步驟(a)的SNP，w^k 為SNP的對應加權(ln(OR))以及X^k 為SNP的風險對偶基因的數目(0、1或2)；以及 (c)若該步驟(b)的wGRS等於或大於0.01，則將該個體識別為IVIG抗性。A method for identifying individuals with Kawasaki's disease resistant to IVIG, which includes the following steps: (a) To detect the presence of a dual gene of single nucleotide polymorphism (SNP) risk in a sample of an individual, the SNP is selected From: (i) rs9380548; (ii) rs742490; (iii) rs7634; (iv) at least one SNP selected from rs1250301 or rs2797915; (v) at least one SNP selected from rs4130857, rs4585205 or rs4602399; (vi) rs4286440; (vii) rs16867137; and (viii) rs1250302; (b) Calculate the weighted genetic risk score (wGRS) based on the following formula:

Where n is an integer from 8 to 11, ^k is the SNP of this step (a), w ^k is the corresponding weight of the SNP (ln(OR)) and X ^k is the number of SNP risk dual genes (0, 1 or 2 ); and (c) If the wGRS of step (b) is equal to or greater than 0.01, the individual is identified as IVIG resistant. 如申請專利範圍第7項所述之方法，其中該wGRS等於或大於0.04。The method as described in item 7 of the patent application scope, wherein the wGRS is equal to or greater than 0.04. 一種用以識別對IVIG抗性的川崎氏病個體的方法，其包含以下步驟： (a)檢測一個體的樣本中選自rs9380548、rs742490、rs7634、rs1250301、rs2797915、rs4130857、rs4585205、rs4602399、rs4286440、rs16867137或rs1250302的至少一單核苷酸多型性(SNP)的風險對偶基因的存在；以及 (b)將具有該步驟(a)中至少一風險對偶基因的該個體識別為對IVIG具有抗性。A method for identifying individuals with Kawasaki's disease resistant to IVIG, which includes the following steps: (a) detecting a sample selected from rs9380548, rs742490, rs7634, rs1250301, rs2797915, rs4130857, rs4585205, rs4602399, rs4286440, the presence of a risk dual gene for at least one single nucleotide polymorphism (SNP) of rs16867137 or rs1250302; and (b) identifying the individual with at least one risk dual gene in step (a) as being resistant to IVIG . 一種套組用於識別對IVIG抗性的川崎氏病個體的用途，該套組包含用於檢測一個體的樣本中選自rs9380548、rs742490、rs7634、rs1250301、rs2797915、rs4130857、rs4585205或rs4602399的至少一單核苷酸多型性(SNP)的風險對偶基因的試劑。A kit for identifying individuals with Kawasaki disease resistant to IVIG, the kit contains at least one selected from rs9380548, rs742490, rs7634, rs1250301, rs2797915, rs4130857, rs4585205, or rs4602399 in a sample for detecting an individual Single nucleotide polymorphism (SNP) risk dual gene reagent. 如申請專利範圍第10項所述之用途，其中該套組係為微陣列。The use as described in item 10 of the patent application scope, wherein the set is a microarray.