TWI755027B - Method for predicting the response to glucagon-like peptide-1 analogue therapy - Google Patents
Method for predicting the response to glucagon-like peptide-1 analogue therapy Download PDFInfo
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本發明關於一種用於評估療程之反應的方法，更具體而言係用於評估糖尿病患者對類升糖素胜肽1(GLP-1)類似物療法之反應的方法。 The present invention relates to a method for assessing the response to a course of treatment, and more particularly to a method for assessing the response of a diabetic patient to Glucagon-Like Peptide 1 (GLP-1) analog therapy.
類升糖素胜肽1(GLP-1)係由腸黏膜的腸內分泌L細胞分泌，並對進食做出反應而經由激素原轉化酶將升糖素原(proglucagon)在其分泌細胞中的轉錄後加工釋放到門脈循環。GLP-1以葡萄糖依賴性方式增強胰島素分泌並抑制升糖素釋放，促進以GLP-1為基礎的療法用於糖尿病之治療的發展。以GLP-1為基礎的療法經由多種機制影響血糖控制，包含減緩胃排空、餐後升糖素的控制、食物攝取的減少以及葡萄糖依賴性胰島素分泌的強化而無低血糖之風險。 Glucagon-like peptide 1 (GLP-1) is secreted by enteroendocrine L cells of the intestinal mucosa, and in response to feeding, transcribes proglucagon in its secretory cells via prohormone convertase Postprocessing is released into the portal circulation. GLP-1 enhances insulin secretion and inhibits glucagon release in a glucose-dependent manner, facilitating the development of GLP-1-based therapies for the treatment of diabetes. GLP-1-based therapy affects glycemic control through multiple mechanisms, including slowing gastric emptying, control of postprandial glucagon, reduction in food intake, and potentiation of glucose-dependent insulin secretion without the risk of hypoglycemia.
然而，在患有第二型糖尿病的患者當中對於GLP-1類似物的臨床反應性不盡相同，意味著遺傳因素可能對這些患者的藥理反應至關重要。由於臨床規範以及這些新藥物的高價格，釐清第二型糖尿病患者的這個關鍵問題是非常重要。
However, clinical responsiveness to GLP-1 analogs varies among patients with
因此，仍有需要評估對GLP-1類似物療法之反應並提供個人化的治療方法。本發明意在解決這些需求與其他需求。 Therefore, there remains a need to assess response to GLP-1 analog therapy and provide a personalized treatment approach. The present invention is intended to address these needs and others.
在一實施例中，本發明揭露一種用於評估受試者對GLP-1類似物療法之反應的方法，其包含：(a)測定至少一個選自由cg06536614(SEQ ID NO：1)、cg26328633(SEQ ID NO：2)以及cg25340688(SEQ ID NO：3)所組成之群組的CpG二核苷酸之胞嘧啶甲基化狀態，或測定來自受試者之樣品的單核苷酸多型性(SNP)rs2346018(風險型等位基因為等位基因A)；以及(b)當在步驟(a)的SNP或在步驟(a)的至少一個CpG二核苷酸之胞嘧啶甲基化相較於對GLP-1類似物療法有反應之受試者低時，鑑定受試者為對GLP-1類似物療法具有低度反應。 In one embodiment, the present invention discloses a method for assessing a subject's response to GLP-1 analog therapy, comprising: (a) determining at least one selected from the group consisting of cg06536614 (SEQ ID NO: 1), cg26328633 ( Cytosine methylation status of CpG dinucleotides of the group consisting of SEQ ID NO: 2) and cg25340688 (SEQ ID NO: 3), or determination of single nucleotide polytypes in samples from subjects (SNP) rs2346018 (the allele at risk is allele A); and (b) when the SNP in step (a) or the cytosine methylation phase of at least one CpG dinucleotide in step (a) A subject is identified as having a low response to GLP-1 analog therapy when compared to subjects who are less responsive to GLP-1 analog therapy.
本發明將在閱讀下列所附圖式及下文的詳細說明而變得更顯而易見。 The present invention will become more apparent upon reading the accompanying drawings and the detailed description below.
第1圖為經由(A)火山圖以及(B)熱圖顯示本發明之訓練群(training group)在Infinium® Human MethylationEPIC BeadChip分析中的性別差異。每個編號代表登錄的患者、編號-1代表GLP-1類似物療法之前萃取的基因組DNA，而編號-2代表GLP-1類似物療法之後萃取的基因組DNA。 Figure 1 shows gender differences in the Infinium ® Human MethylationEPIC BeadChip analysis of the training group of the present invention via (A) a volcano plot and (B) a heat map. Each number represents an enrolled patient, number -1 represents genomic DNA extracted before GLP-1 analog therapy, and number -2 represents genomic DNA extracted after GLP-1 analog therapy.
第2圖為比較反應組與無反應組在GLP-1類似物治療之前及之後甲基化差異的火山圖。x軸：在GLP-1類似物治療之前及之後反應組與無反應組之β值的差異，y軸：(A)-log10(P)值以及(B)-log10(Q)值。P值為以t test比較在GLP-1 類似物治療之前及之後的β值之間的差異。Q值為錯誤發現率調整的P值。在多重測試調整Q值下檢測到的基因甲基化無差異。 Figure 2 is a volcano plot comparing the differences in methylation before and after GLP-1 analog treatment between responder and non-responders. x-axis: difference in beta values between responders and non-responders before and after GLP-1 analog treatment, y-axis: (A)-log 10 (P) values and (B)-log 10 (Q) values. P values were compared by t test for the difference between beta values before and after GLP-1 analog treatment. The Q value is the false discovery rate adjusted P value. No differences in gene methylation detected under multiple-test adjusted Q-values.
第3圖顯示DNA甲基化程度的不同能夠區分以GLP-1類似物治療之患有第二型糖尿病的反應組與無反應組患者。(A)非監督式(Unsupervised)階層分群36個甲基化探針。圖譜使用Cluster 3.0以及採用平均連結分群演算法(average-linkage clustering algorithms)的Tree View programs進行可視化。將所有參與者與最重要的差異甲基化區域(DMRs)的熱圖以及基因列表。基於t test選擇最差異表達的差異甲基化點(DMPs)。基於t test(P<0.0001)以及四倍表達變化選擇在甲基化中最差異表達的β值的火山圖(紅色代表高β值；綠色代表低β值)。每個編號代表登錄的患者、編號-1及編號-2分別代表在GLP-1類似物治療之前及之後萃取的基因組DNA。(B)經由甲基化特異性PCR反應區分VTRNA2-1的甲基化狀態(NR：無反應；R：有反應；M：甲基化；U：未甲基化)。(C)在VTRNA2-1啟動子區域中各CpG二核苷酸之亞硫酸鹽定序分析。每一列表示在每個CpG二核苷酸上甲基化狀態的百分比(+1代表轉錄起始點；NR：無反應；R：有反應)。實體位置係基於GRCh37/hg19染色體。
Figure 3 shows that differences in the degree of DNA methylation can differentiate between responders and non-responders with
第4圖顯示VTRNA2-1啟動子的GLP-1藥物反應與甲基化百分比之間的關聯性。(A)衍生自患者(泳道1-7)的亞硫酸鹽基因組DNA經由經設計的焦磷酸定序PCR引子放大，甲基化控制組(100%甲基化，泳道8)、未甲基化控制組(0%甲基化，泳道9)以及未修飾基因組DNA(泳道10)也使用相同引子組放大。本發明的負控制組為供應有PCR試劑及引子的ddH2O(泳道11)。M：100bp marker。隨後PCR產物將進行焦磷酸定序反應(pyrosequencing reaction)。(B)至(D)為使用Q96 platform(Qiagen)的PCR產物定序結果之熱解圖(pyrogram)。在各CpG位點的甲基 化百分比以灰框表示。由左至右為各樣品中cg06536614、chr5：135416388、cg26328633、cg 25340688、cg 26896946的甲基化百分比。箭頭代表在焦磷酸定序反應中應為無訊號的非特異性控制。衍生自100%甲基化、0%甲基化以及未修飾基因組DNA的PCR焦磷酸定序反應結果分別示於(B)、(C)以及(D)。(E)在VTRNA2-1之啟動子四個位點：cg06536614、chr5：135416388、cg 26328633、cg25340688、cg26896946中平均甲基化百分比(Y軸：四個位點的甲基化%之平均)以及對GLP-1治療之藥物反應(無反應、有反應；X軸)之間的關聯性，*P<0.05。(F)經由年齡、性別及BMI調整之VTRNA2-1啟動子區域中，甲基化程度的預測模型之AUC。箭頭代表根據Youden’s法之1.542的最佳切點(optimal cutoff point)。 Figure 4 shows the correlation between GLP-1 drug response and percent methylation of the VTRNA2-1 promoter. (A) Sulfite genomic DNA derived from patients (lanes 1-7) amplified via designed pyrosequencing PCR primers, methylated control (100% methylated, lane 8), unmethylated Control (0% methylation, lane 9) and unmodified genomic DNA (lane 10) were also amplified using the same primer set. The negative control group of the present invention was ddH2O supplied with PCR reagents and primers (lane 11 ). M: 100bp marker. The PCR product will then undergo a pyrosequencing reaction. (B) to (D) are pyrograms of PCR product sequencing results using the Q96 platform (Qiagen). The percentage of methylation at each CpG site is represented by a grey box. From left to right are the percent methylation of cg06536614, chr5: 135416388, cg26328633, cg 25340688, cg 26896946 in each sample. Arrows represent non-specific controls that should be signalless in the pyrosequencing reaction. The results of PCR pyrosequencing reactions derived from 100% methylated, 0% methylated and unmodified genomic DNA are shown in (B), (C) and (D), respectively. and Association between drug responses to GLP-1 treatment (non-responding, responding; X-axis), *P<0.05. (F) AUC of the prediction model for methylation degree in the promoter region of VTRNA2-1 adjusted for age, sex and BMI. The arrow represents the optimal cutoff point of 1.542 according to Youden's method.
第5圖顯示經由年齡、性別及BMI調整之VTRNA2-1啟動子區域中，CpG位點甲基化狀態的預測模型之ROC曲線。(A)進一步針對rs2346018將vtRNA2-1中4個CpG位點的平均甲基化百分比進行調整(1：CC；2：CA或AA)。(B)在vtRNA2-1啟動子中的cg06536614。(C)在vtRNA2-1啟動子中的chr5：135416388。(D)cg26328633。(E)cg25340688。(F)在vtRNA2-1中的4個CpG位點的平均甲基化百分比。 Figure 5 shows the ROC curve of the prediction model for the methylation status of CpG sites in the VTRNA2-1 promoter region adjusted for age, sex and BMI. (A) The mean percent methylation of the 4 CpG sites in vtRNA2-1 was further adjusted for rs2346018 (1: CC; 2: CA or AA). (B) cg06536614 in vtRNA2-1 promoter. (C) chr5:135416388 in vtRNA2-1 promoter. (D) cg26328633. (E) cg25340688. (F) Mean percent methylation of 4 CpG sites in vtRNA2-1.
第6圖顯示根據皮爾森相關係數(Pearson's correlation coefficient)vtRNA2-1的表達與VTRNA2-1啟動子的甲基化百分比相關。在本發明中用5S RNA的Ct值對vtRNA2-1的表達進行標準化。 Figure 6 shows that the expression of vtRNA2-1 correlates with the percent methylation of the VTRNA2-1 promoter according to Pearson's correlation coefficient. The expression of vtRNA2-1 was normalized with the Ct value of 5S RNA in the present invention.
第7圖顯示作為對GLP-1類似物治療的血糖反應之預測子的TGF-β1之血清含量。(A)VTRNA2-1啟動子甲基化與TGF-β1之血清含量的關聯性。(B)TGF-β1之血清含量可以作為針對GLP-1類似物治療之血糖反應的預測子。 Figure 7 shows serum levels of TGF-beta1 as a predictor of glycemic response to GLP-1 analog treatment. (A) Association of VTRNA2-1 promoter methylation with serum levels of TGF-β1. (B) Serum levels of TGF-β1 can be used as a predictor of glycemic response to GLP-1 analog therapy.
用語「受試者(subject)」可指的是具有糖尿病的脊椎動物。受試者包含如哺乳類的溫血動物，如靈長類，且更佳為人類。用語受試者包含如貓、狗等的馴養動物、家畜(例如，牛、馬、豬、綿羊、山羊等)及實驗動物(例如，小鼠、兔、大鼠、沙鼠、天竺鼠等)。 The term "subject" may refer to a vertebrate having diabetes. Subjects include warm-blooded animals such as mammals, such as primates, and more preferably humans. The term subject includes domestic animals such as cats, dogs, etc., domestic animals (eg, cows, horses, pigs, sheep, goats, etc.) and experimental animals (eg, mice, rabbits, rats, gerbils, guinea pigs, etc.).
用語「樣品(sample)」指的是含有能夠被偵測的材料之組合物，並包含例如「生物樣品(biological samples)」指的是任何取自生命源的材料，例如人類或其他可能患有糖尿病的哺乳類之動物。生物樣品可以存在任何形式，包含如組織、細胞、細胞沉澱物(cell pellet)、細胞萃取物、手術樣品、活體組織切片(biopsy)或細針抽吸物(fine needle aspirate)的固體材料，或其可以是如尿液、全血、血漿或血清的生物流體之形式，或如唾液(saliva)之由受試者產生的其他任何流體樣品。 The term "sample" refers to a composition containing material that can be detected, and includes, for example, "biological samples" refers to any material taken from a source of life, such as a human or other Diabetic mammals. A biological sample may exist in any form, comprising solid material such as tissue, cells, cell pellet, cell extract, surgical sample, biopsy or fine needle aspirate, or It may be in the form of a biological fluid such as urine, whole blood, plasma or serum, or any other fluid sample produced by a subject such as saliva.
當將用語「甲基化程度(methylation level)」應用於基因時指的是CpG環境中存在的一個或多個胞嘧啶殘基是否具有甲基化基團。甲基化程度也可以指的是樣品中的細胞萃段之胞嘧啶C5或N4位置上有或沒有甲基化基團。CpG二核苷酸的甲基化可以駐留在非編碼轉錄控制序列中(例如啟動子、增強子等)或在包含相關基因的內含子和外顯子的編碼序列中。 The term "methylation level" when applied to a gene refers to whether one or more cytosine residues present in the CpG environment have a methylation group. The degree of methylation can also refer to the presence or absence of methylated groups at the C5 or N4 positions of cytosines in the cell extracts in the sample. Methylation of CpG dinucleotides can reside in non-coding transcriptional control sequences (eg, promoters, enhancers, etc.) or in coding sequences comprising introns and exons of related genes.
用語「低甲基化(hypomethylation)」指的是相對於正常對照DNA樣本中相應之CpG二核苷酸處發現的5-mCyt數量的平均甲基化狀態，該狀態對應於在測試DNA樣本的DNA序列中一個或多個CpG二核苷酸處5-mCyt的存在少。 The term "hypomethylation" refers to the average methylation status relative to the amount of 5-mCyt found at the corresponding CpG dinucleotide in a normal control DNA sample that corresponds to the amount of methylation in a test DNA sample. The presence of 5-mCyt at one or more CpG dinucleotides in the DNA sequence is low.
用語CpG基因座也稱為CpG二核苷酸或CpG位點。 The term CpG locus is also referred to as a CpG dinucleotide or CpG site.
本發明提供一種評估受試者對GLP-1類似物療法之反應的方法，其包含：a)測定選自主要由SEQ ID NO：1、SEQ ID NO：2以及SEQ ID NO：3所組成之群組的CpG二核苷酸之胞嘧啶甲基化狀態，或測定來自受試者之樣品的單核苷酸多型性(SNP)rs2346018；以及(b)當在步驟(a)的SNP或在步驟(a)的至少一個CpG二核苷酸之胞嘧啶甲基化相較於對GLP-1類似物療法有反應之受試者低時，鑑定受試者為對GLP-1類似物療法具有低度反應。 The present invention provides a method of assessing a subject's response to GLP-1 analog therapy, comprising: a) determining a method selected from the group consisting essentially of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 the cytosine methylation status of the CpG dinucleotides of the cohort, or the determination of the single nucleotide polytype (SNP) rs2346018 in a sample from the subject; and (b) when the SNP in step (a) or A subject is identified as responsive to GLP-1 analog therapy when the cytosine methylation of at least one CpG dinucleotide of step (a) is low compared to a subject responsive to GLP-1 analog therapy Has a low degree of reactivity.
根據本發明之實施例用於評估對GLP-1類似物療法的單核苷酸多型性(SNP)包含rs2346018(此為國家生物技術信息中心數據庫中的GenBank登錄號)之SEQ ID NO：4的多核苷酸序列。所屬技術領域具有通常知識者可以輕易使用GenBank登錄號辨識SNP的序列及位置。在NCBI中註冊的SNP之rs編號對應的特定序列可能隨時間改變。對所屬技術領域具有通常知識者顯而易見的是即使對應的rs編號改變，這些序列也在本發明的範圍內。SEQ ID NO：4的核苷酸序列為多型性序列(polymorphic sequences)(包含代表SNP的多型性位點)。 The single nucleotide polytype (SNP) used to assess GLP-1 analog therapy according to embodiments of the present invention comprises SEQ ID NO: 4 of rs2346018 (this is the GenBank accession number in the National Center for Biotechnology Information Database) the polynucleotide sequence. One of ordinary skill in the art can readily identify the sequence and position of SNPs using GenBank accession numbers. The specific sequences corresponding to the rs numbers of SNPs registered in NCBI may change over time. It will be apparent to those of ordinary skill in the art that these sequences are within the scope of the present invention even if the corresponding rs numbers are changed. The nucleotide sequence of SEQ ID NO: 4 is a polymorphic sequence (comprising a polymorphic site representing a SNP).
低甲基化程度或低甲基化為相對的用語，且可以藉由將糖尿病患者之測試樣品的SEQ ID NO：1、SEQ ID NO：2以及SEQ ID NO：3之CpG基因座的甲基化程度與參考之控制程度(對GLP-1類似物有反應的糖尿病患者)的比較而決定。在一些實施例中，測試樣品中SEQ ID NO：1至3之CpG基因座的甲基化程度為低於對GLP-1類似物有反應的糖尿病患者的1%、5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或100%。表1顯示染色體位置、SEQ ID編號以及DNA序列。 The degree of hypomethylation or hypomethylation is a relative term and can be determined by comparing the methyl groups of the CpG loci of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 of a test sample from a diabetic patient It was determined by comparison of the degree of lipolysis with a reference degree of control (diabetic patients responsive to GLP-1 analogs). In some embodiments, the degree of methylation of the CpG loci of SEQ ID NOs: 1 to 3 in the test sample is 1%, 5%, 10%, 15% lower than that of diabetic patients responsive to GLP-1 analogs %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. Table 1 shows the chromosomal location, SEQ ID number and DNA sequence.
任何用於評估DNA甲基化程度的方法皆可用於本發明的方法中。評估甲基化程度的方法之非限制性實例包含甲基化敏感性限制酶分析、藉由使用包含亞硫酸鹽、焦亞硫酸鹽、亞硫酸氫鹽或其組合的試劑處理的化學分析或焦磷酸定序。 Any method for assessing the degree of DNA methylation can be used in the methods of the present invention. Non-limiting examples of methods of assessing the degree of methylation include methylation-sensitive restriction enzyme assays, chemical assays by treatment with reagents comprising sulfite, metabisulfite, bisulfite, or a combination thereof, or pyrolysis. Phosphate sequencing.
當亞硫酸氫鈉(sodium bisulfite)接觸DNA時，未甲基化的胞嘧啶轉變成尿嘧啶，同時甲基化的胞嘧啶不會被修飾。如此附加的實施例包含如Illumina® Human Methylation450 BeadChip以及多重PCR分析之基於陣列的分析之使用。 When sodium bisulfite contacts DNA, unmethylated cytosines are converted to uracils, while methylated cytosines are not modified. Such additional examples include the use of array-based assays such as the Illumina® Human Methylation450 BeadChip and multiplex PCR assays.
定量擴增法(Quantitative amplification)可用於定量限制性內切酶消化後側接擴增引物的基因座中完整DNA的數量。定量擴增的方法揭露於例如美國專利號6,180,349；6,033,854以及5,972,602中。 Quantitative amplification can be used to quantify the amount of intact DNA in the loci flanked by amplification primers following restriction endonuclease digestion. Methods of quantitative amplification are disclosed, for example, in US Pat. Nos. 6,180,349; 6,033,854; and 5,972,602.
其他甲基化程度的測定方法包含但不限於核酸擴增、聚合酶鏈鎖反應(PCR)、甲基化特異性PCR(MCP)、甲基化CpG島恢復分析(methylated-CpG island recovery assay；MIRA)、結合亞硫酸氫鹽限制分析(combined bisulfite-restriction analysis；COBRA)、單股結構多形性(single-strand conformation polymorphism；SSCP)分析、限制性分析及微陣列分析(restriction analysis and microarray analysis)(Lian ZQ et al，使用基於微陣列的甲基化CpG島回收法篩選乳腺癌中明顯高甲基化的基因並鑑定其表達程度，國際腫瘤學雜誌，41：629-638,2012)。 Other methods for measuring the degree of methylation include but are not limited to nucleic acid amplification, polymerase chain reaction (PCR), methylation-specific PCR (MCP), and methylated- CpG island recovery assay; MIRA), combined bisulfite-restriction analysis (COBRA), single-strand conformation polymorphism (SSCP) analysis, restriction analysis and microarray analysis ) (Lian ZQ et al, Screening and identification of significantly hypermethylated genes in breast cancer using microarray-based recovery of methylated CpG islands, International Journal of Oncology, 41:629-638, 2012).
GLP-1類似物的非限制性實例包含利拉鲁肽(liraglutide)、艾塞那肽(exenatide)或度拉魯肽(dulaglutide)。 Non-limiting examples of GLP-1 analogs include liraglutide, exenatide, or dulaglutide.
在一些實施例中，用於評估受試者對GLP-1類似物療法之反應的方法包含測定基本上選自SEQ ID NO：1、SEQ ID NO：2以及SEQ ID NO：3的CpG二核苷酸的胞嘧啶甲基化狀態，以及測定來自受試者之樣品的rs2346018。 In some embodiments, the method for assessing a subject's response to GLP-1 analog therapy comprises assaying a CpG binuclear substantially selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 Cytosine methylation status of nucleotides, and determination of rs2346018 in samples from subjects.
測定受試者中選自SEQ ID NO：1、SEQ ID NO：2以及SEQ ID NO：3中的至少一個CpG基因座的差異甲基化程度及/或rs2346018的存在能使對GLP-1類似物有反應的受試者迅速使用這種類似物治療。 Determining the degree of differential methylation of at least one CpG locus selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the subject and/or the presence of rs2346018 enables analogs to GLP-1 Responding subjects were rapidly treated with this analog.
本發明之實施例將藉由下列實例進行說明，但不應以任何方式解釋為對其範圍加以限制。除非另有指明，在描述下列實例的研究中，將遵循習知程序。一些程序將在以下描述以用於說明目的。 Embodiments of the present invention will be illustrated by the following examples, which should not be construed in any way as limiting the scope thereof. Unless otherwise indicated, in the studies describing the following examples, conventional procedures will be followed. Some procedures are described below for illustrative purposes.
實例 example
研究參與者：在本研究中登錄138位患有第2型糖尿病且接受GLP-1類似物療法的患者。GLP-1類似物選自利拉鲁肽或度拉魯肽。
Study participants: 138 patients with
納入條件為：(a)年齡>20歲；(b)經診斷患有糖尿病>2年；以及(c)8%至12%的HbA1c量。排除條件為：(a)近期有藥物或酒精成癮之病史；(b)對類似物產品敏感；(c)嚴重心血管疾病；(d)參與其他臨床研究；(e)持續流感、自體免疫疾病或其他代謝病症；以及(f)懷孕或哺乳期婦女。此研究規程由台灣長庚紀念醫院的人體試驗倫理委員會(Institutional Review Board)核准。已取得每個受試者的知情同意書(IRB No.104-9629B)。 Inclusion criteria were: (a) age >20 years; (b) diagnosed with diabetes >2 years; and (c) HbA1c amount of 8% to 12%. Exclusion criteria were: (a) recent history of drug or alcohol addiction; (b) sensitivity to analog products; (c) severe cardiovascular disease; (d) participation in other clinical studies; (e) persistent influenza, autologous Immune diseases or other metabolic disorders; and (f) pregnant or breastfeeding women. This study protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan. Informed consent was obtained from each subject (IRB No. 104-9629B).
在訓練階段，藉由Infinium® HumanMethylation450及MethylationEPIC BeadChip platform對10位對治療產生GLP-1反應的參與者進行DNA甲基化分析。分析了在GLP-1類似物治療之前及之後，來自10位受試者的基因組DNA的全甲基化圖譜。平均年齡、平均BMI、糖尿病持續期間以及基線A1C量在GLP-反應組及無反應組之間並沒有顯著差異。在數據分析之前，根據性別檢查在X及Y染色體基因中的表現差異。結果顯示於第1圖中。可以清楚分辨X及Y染色體特定基因甲基化程度在男性與女性受試者之間的分布。 During the training phase, 10 participants who responded to treatment with GLP-1 were subjected to DNA methylation analysis using the Infinium® HumanMethylation450 and MethylationEPIC BeadChip platforms. Pan-methylation profiles of genomic DNA from 10 subjects before and after GLP-1 analog treatment were analyzed. Mean age, mean BMI, duration of diabetes, and baseline A1C levels were not significantly different between GLP-responsive and non-responsive groups. Before data analysis, differences in expression in X and Y chromosome genes were examined by sex. The results are shown in Figure 1. The distribution of X- and Y-chromosome-specific gene methylation levels between male and female subjects can be clearly distinguished.
首先我們分析治療前及治療後的組之間有關甲基化表現的甲基化差異。第2圖中所示的結果顯示在火山圖及熱圖分析中，反應組及無反應組在GLP-1類似物治療之前及之後的甲基化並沒有顯著變化。 First, we analyzed the methylation differences in relation to methylation performance between the groups before and after treatment. The results shown in Figure 2 show that in the volcano plot and heat map analysis, there were no significant changes in methylation between responder and non-responders before and after GLP-1 analog treatment.
參照第3圖的(A)部分，VTRNA2-1的遺傳性甲基化狀態在GLP-1反應組及無反應組之間最具顯著差異(P<1x10-10)。VTRNA2-1的遺傳性甲基化狀態在GLP-1類似物治療前後無明顯變化。參照下列表2，除了VTRNA2-1其他遺傳性DMR區域也顯示了在無反應組及反應組之間不同的甲基化狀態。在候選的 DMRs當中，VTRNA2-1在反應組及無反應組之間顯示出最明顯的差異(P<1x10-10)。 Referring to part (A) of Figure 3, the genetic methylation status of VTRNA2-1 was most significantly different between the GLP-1 reactive and non-responsive groups (P< 1x10-10 ). The genetic methylation status of VTRNA2-1 did not change significantly before and after GLP-1 analog treatment. Referring to Table 2 below, in addition to VTRNA2-1 , other inherited DMR regions also showed different methylation status between the non-responding group and the responding group. Among the candidate DMRs, VTRNA2-1 showed the most significant difference (P< 1x10-10 ) between responder and non-responder groups.
為了確認VTRNA2-1啟動子的甲基化，使用甲基化特異性PCR以及隨後進行DNA定序反應的亞硫酸鹽生物複製(bisulfite cloning following DNA sequencing reactions)分析在VTRNA2-1啟動子(從-7至-202)中16個CpGs中的每一個之DNA甲基化狀態。在第3圖的(B)部分中，VTRNA2-1啟動子的差異性甲基化由MS-PCR分析進行確認。在反應組中只有來自對甲基化狀態具有特異性的引子對的PCR產物是明顯的(甲基化狀態)，而在無反應組中則相反(未甲基化狀態)。如第3圖的(C)部分所示，在無反應組中幾乎16個CpGs為未甲基化，而在反應組中16個CpGs中的大部分為過甲基化。總之，在訓練組中VTRNA2-1啟動子的甲基化與對GLP-1類似物治療的血糖反相關，且VTRNA2-1啟動子的甲基化狀態經由甲基化特異性PCR以及亞硫酸鹽生物複製進行確認。 To confirm the methylation of the VTRNA2-1 promoter, methylation-specific PCR and bisulfite cloning following DNA sequencing reactions were used to analyze the methylation at the VTRNA2-1 promoter (from- 7 to -202) DNA methylation status of each of the 16 CpGs. In part (B) of Figure 3, differential methylation of the VTRNA2-1 promoter was confirmed by MS-PCR analysis. Only PCR products from primer pairs specific for methylation state were evident in the reactive group (methylated state), whereas the opposite was true in the non-reactive group (unmethylated state). As shown in part (C) of Figure 3, almost 16 CpGs were unmethylated in the non-reactive group, while most of the 16 CpGs in the reactive group were hypermethylated. In conclusion, methylation of the VTRNA2-1 promoter was inversely correlated with blood glucose to GLP-1 analog treatment in the training group, and the methylation status of the VTRNA2-1 promoter was determined by methylation-specific PCR as well as sulfite Biological replicates were confirmed.
在驗證階段，登錄接受結合GLP-1類似物與其他抗糖尿病藥物的128位患者。表3顯示從基線至3個月之A1C的變化。A1C的變化<0.5%定義為對GLP-1類似物無反應。 During the validation phase, 128 patients who received GLP-1 analogs combined with other antidiabetic drugs were enrolled. Table 3 shows the change in A1C from baseline to 3 months. A change in A1C of <0.5% was defined as non-response to GLP-1 analogs.
如上表3所示，驗證組中登錄了總共為128位患者進行焦磷酸定序分析。在128位患者中，52%每日一次以利拉鲁肽治療，32%每日兩次以艾塞那肽治療，以及16%每周一次以度拉魯肽治療。其中年齡、性別、BMI、糖尿病持續期間以及基線A1C量在反應組及無反應組之間沒有顯著差異。在GLP-1治療之後A1C量在反應組及無反應組之間具有顯著差異(P<0.0001)。 As shown in Table 3 above, a total of 128 patients were enrolled in the validation group for pyrosequencing analysis. Of the 128 patients, 52% were treated with liraglutide once daily, 32% with exenatide twice daily, and 16% with duraglutide once weekly. There were no significant differences in age, gender, BMI, duration of diabetes, and baseline A 1 C amount between responder and non-responder groups. There was a significant difference (P<0.0001) in the amount of A1C between the responder and non-responder groups after GLP- 1 treatment.
DNA萃取 DNA extraction
在GLP-1類似物治療之前及之後，使用基因組DNA萃取套組根據製造商(RBC Bioscience,Taiwan)的建議從138位患者的周邊血液的白血球中萃取基因組DNA。 Genomic DNA was extracted from leukocytes of peripheral blood of 138 patients before and after GLP-1 analog treatment using a genomic DNA extraction kit according to the manufacturer's recommendations (RBC Bioscience, Taiwan).
全基因組甲基化分析 Genome-wide methylation analysis
在進行分析之前，使用EZ DNA甲基化套組根據製造商(Zymo Research,Orange,A,USA)的指示將1μg的基因組DNA經過亞硫酸鹽修飾。修飾之後，經亞硫酸鹽轉化的DNA樣品使用Infinium® Human MethylationEPIC BeadChip根據製造商(Illumina Inc,CA,USA)的建議進行評估。由Illumina GenomeStudio software V2011.1萃取每個探針的原始訊號強度(不含背景校正或標準化)。所得的原始數據經過標準化(控制標準化)並藉由製造商軟體進行背景校正以產生甲基化β值。 Prior to analysis, 1 μg of genomic DNA was sulfite modified using the EZ DNA Methylation Kit according to the manufacturer's instructions (Zymo Research, Orange, A, USA). After modification, sulfite-converted DNA samples were evaluated using the Infinium® Human MethylationEPIC BeadChip according to the manufacturer's recommendations (Illumina Inc, CA, USA). The raw signal intensity (without background correction or normalization) of each probe was extracted by Illumina GenomeStudio software V2011.1. The resulting raw data were normalized (control normalized) and background corrected by the manufacturer's software to generate methylation beta values.
為分析數據，進行火山圖並以MATLAB(版本2015a)進行統計分析(t-test)。使用Cluster 3.0以及具有平均鏈接聚類算法的Tree View軟體將差異甲基化位點(DMPs)可視化。在本發明中，表現最具差異的選擇被用來選擇探針。 To analyze the data, volcano plots were performed and statistical analysis (t-test) was performed with MATLAB (version 2015a). Differentially methylated sites (DMPs) were visualized using Cluster 3.0 and Tree View software with an average link clustering algorithm. In the present invention, selections that exhibit the most differential are used to select probes.
甲基化特異性PCR(MS-PCR) Methylation-specific PCR (MS-PCR)
使用MethPrimer software設計針對VTRNA2-1的MS-PCR引子及用於MS-PCR的溫度條件。所述引子列於下表4中。 The MS-PCR primers for VTRNA2-1 and the temperature conditions for MS-PCR were designed using MethPrimer software. The primers are listed in Table 4 below.
亞硫酸鹽PCR、生物複製以及DNA定序 Sulfite PCR, biological replication, and DNA sequencing
使用特定引子藉由PCR將經亞硫酸鹽處理的基因組DNA進行放大(Titanium Taq DNA Polymerase,Takara Bio Inc.USA)。經放大的PCR產物經過純化並選殖到pCRII-TOPO載體中(Invitrogen,CarIsbad,CA)以選擇殖株(clones)。DNA定序在至少10個獨立的殖株上使用Automatic Sequencer(Applied Biosystems,Foster City,CA,US)進行。引子列於下表4中。 Sulfite-treated genomic DNA was amplified by PCR using specific primers (Titanium Taq DNA Polymerase, Takara Bio Inc. USA). The amplified PCR product was purified and cloned into pCRII-TOPO vector (Invitrogen, CarIsbad, CA) for selection of clones. DNA sequencing was performed on at least 10 independent clones using the Automatic Sequencer (Applied Biosystems, Foster City, CA, US). The primers are listed in Table 4 below.
亞硫酸鹽焦磷酸定序反應 Sulfite Pyrophosphate Sequencing Reaction
在VTRNA2-1之CpG位點特異性DNA甲基化的定量分析使用亞硫酸鹽焦磷酸定序(Qiagen PyroMark Q96)進行。焦磷酸定序分析使用由100%甲基 化、0%未甲基化以及未修飾的人類基因組DNA(EpiTect Control DNA以及Control DNA Set,Qiagen)組成的標準驗證。使用EpiTect Fast BISULFITE Conversino Kit(Qiagen)根據製造商的建議對1ug的基因組DNA進行修飾。大約10至20ng的亞硫酸鹽DNA被用於與焦磷酸測序vtRNA2-1正向和反向引子進行PCR反應，引子係使用PyroMark Assay desingn software(Qiagen)設計並列於下表4中。PCR使用經過濾的(0.22um)無核酸酶水(Invitrogem)在PCR Workstation(AirClean®Systems,Inc)上進行。在進行MS-PCR和焦磷酸測序之前應將胜肽校正及清除。每個生物素化的PCR產物(20ul)與1ul的Streptavidin SepharoseTM(GE Healthcare)以及40ul的結合緩衝液(Qiagen)混合，然後搖動10分鐘以進行固定化。使用vacuum workstation(Qiagen)根據製造商的建議將固定化的PCR產物純化程單股DNA、在黏接緩衝液中以0.3uM的濃度與定序引子混合、加熱至80℃ 5分鐘，然後冷卻至室溫以使定序引子黏接(annealing)。用於焦磷酸定序反應的模板、酵素、基質及核苷酸在PyroMark Q96(Qiagen)上設定。 Quantitative analysis of CpG site-specific DNA methylation at VTRNA2-1 was performed using sulfite pyrosequencing (Qiagen PyroMark Q96). Pyrosequencing analysis was performed using standard validation consisting of 100% methylated, 0% unmethylated and unmodified human genomic DNA (EpiTect Control DNA and Control DNA Set, Qiagen). 1 ug of genomic DNA was modified using the EpiTect Fast BISULFITE Conversino Kit (Qiagen) according to the manufacturer's recommendations. Approximately 10 to 20 ng of sulfite DNA was used for PCR reactions with pyrosequencing vtRNA2-1 forward and reverse primers designed using PyroMark Assay desingn software (Qiagen) and listed in Table 4 below. PCR was performed on a PCR Workstation (AirClean® Systems, Inc) using filtered (0.22um) nuclease-free water (Invitrogem). Peptides should be corrected and cleared prior to MS-PCR and pyrosequencing. Each biotinylated PCR product (20 ul) was mixed with 1 ul of Streptavidin Sepharose ™ (GE Healthcare) and 40 ul of binding buffer (Qiagen), followed by shaking for 10 minutes for immobilization. The immobilized PCR product was purified from single-stranded DNA using a vacuum workstation (Qiagen) according to the manufacturer's recommendations, mixed with sequencing primers at a concentration of 0.3 uM in adhesion buffer, heated to 80°C for 5 min, and cooled to Room temperature to allow sequencing primers to annealing. Templates, enzymes, substrates and nucleotides for pyrosequencing reactions were set up on a PyroMark Q96 (Qiagen).
定量反轉錄聚合酶連鎖反應(qRT-PCR) Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
RNA係使用Tempus Blood RNA Tubes(Thermo Fisher Scientific Inc.,US)以及QIAamp RNA Blood Mini Kit(Qiagen,Hilden,Germany)從患者的周邊血液樣品萃取。藉由5ug的總RNA及具隨機六具體的反轉錄酶(Promega,Madison,WI,USA)合成cDNA，接著使用含vtRNA2-1及5S RNA引子的SYBR Green Master Mix(Applied Biosystems,USA)進行定量PCR。選擇5S RNA作為內參標準化基因。 RNA was extracted from peripheral blood samples of patients using Tempus Blood RNA Tubes (Thermo Fisher Scientific Inc., US) and the QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized by 5ug of total RNA and reverse transcriptase with random six specificity (Promega, Madison, WI, USA), followed by quantification using SYBR Green Master Mix (Applied Biosystems, USA) containing vtRNA2-1 and 5S RNA primers PCR. 5S RNA was selected as the internal reference normalization gene.
表4：用於甲基化特異性PCR(MS-PCR)、亞硫酸鹽定序引子(BSP)以及焦磷酸定序反應的引子
在第4圖的(A)部分中，設計PCR與定序引子以放大表4中chr5：135416381(cg06536614)(SEQ ID NO：1)、chr5：135416388、chr5：135416394(cg26328633)(SEQ ID NO：2)以及chr5：135416398(cg25340688)(SEQ ID NO：3)的區域，用以偵測來自患者之基因組DNA中VTRNA2-1啟動子的甲基化狀態。使用我們具有甲基化/未甲基化對照基因組(EpiTech control DNA and Control DNA set,Qiagen)的引子或亞硫酸鹽對照基因組DNA的引子放大VTRNA2-1啟動子，並修飾癌自患者的基因組DNA，但在未修飾的基因組DNA中無法放大(第4圖的(A))。這些PCR產物進一步使用焦磷酸定序進行分析，且那些衍生自控制組DNA的甲基化(100%)及未甲基化控制(0%)顯示出相同的甲基化狀態。此外，參照第4圖的(B)至(D)部分，從未經修飾的基因組DNA衍生的PCR反應中仍然沒有明顯的訊號，表示所設計之引子具有特異性。 In part (A) of Figure 4, PCR and sequencing primers were designed to amplify chr5: 135416381 (cg06536614) (SEQ ID NO: 1), chr5: 135416388, chr5: 135416394 (cg26328633) (SEQ ID NO: Table 4) : 2) and the region of chr5: 135416398 (cg25340688) (SEQ ID NO: 3) to detect the methylation status of VTRNA2-1 promoter in genomic DNA from patients. Amplify the VTRNA2-1 promoter using our primers with methylated/unmethylated control genomic DNA (EpiTech control DNA and Control DNA set, Qiagen) or primers for sulfite control genomic DNA and modify the genomic DNA from patients with cancer , but could not be amplified in unmodified genomic DNA (Fig. 4(A)). These PCR products were further analyzed using pyrosequencing, and those derived from control group DNA that were methylated (100%) and unmethylated controls (0%) showed identical methylation status. In addition, referring to parts (B) to (D) of Figure 4, there is still no obvious signal in the PCR reaction derived from unmodified genomic DNA, indicating that the designed primers are specific.
藉由焦磷酸定性在反應與非反應性基團之間的VTRNA2-1之啟動子區域上的平均甲基化百分比示於第4圖的(E)部分中。在反應組中的平均甲基化程度高於無反應性組(40.07±2.78 vs 37.48±4.52%,P=0.025)。我們將平均甲基化在40%的甲基化處進行二分化，定義高度甲基化(
參照表6，在多元邏輯回歸中針對年齡、性別及BMI調整之後，勝算比增加至5.264(95% CI 1.743-15.899,P=0.003)。進一步地，vtRNA2-1的甲基化狀態藉由CTCF結合位點所接鄰(flanked)。中心粒CTCF結合位點含有A/C多型性(rs2346018)，且已經報導了A等位基因的存在可以增加VTRNA2-1中的DNA甲基化百分比。當VTRNA2-1啟動子區域之甲基化程度對GLP-1類似物反應的影響使用rs2346018的隱性模型(即，主要等位基因C與具有一個或多個次要等位基因A的個體)分析時，與主要等位基因C的純合子相比，次要等位基因A的雜合子或純合子根據啟動子區域的甲基化程度表現出更可預測的GLP-1類似物反應。因此，如表5所示，在具有A等位基因的患者中的勝算比增加至4.048(95% CI.438-11.389,P=0.007)。總之，在VTRNA2-1啟動子低度甲基化(<40%)及rs2346018的A等位基因時傾向為對GLP-1治療無反應。 Referring to Table 6, after adjusting for age, sex, and BMI in multiple logistic regression, the odds ratio increased to 5.264 (95% CI 1.743-15.899, P=0.003). Further, the methylation status of vtRNA2-1 is flanked by the CTCF binding site. The centriole CTCF binding site contains an A/C polymorphism (rs2346018), and the presence of the A allele has been reported to increase the percentage of DNA methylation in VTRNA2-1. A recessive model of rs2346018 was used when the degree of methylation in the promoter region of VTRNA2-1 influences the response to GLP-1 analogs (ie, major allele C versus individuals with one or more minor alleles A) When analyzed, heterozygotes or homozygotes for the minor allele A exhibited a more predictable GLP-1 analog response based on the degree of methylation in the promoter region compared to homozygotes for the major allele C. Thus, as shown in Table 5, the odds ratio in patients with the A allele increased to 4.048 (95% CI.438-11.389, P=0.007). In conclusion, the VTRNA2-1 promoter was hypomethylated (<40%) and the A allele of rs2346018 tended to be unresponsive to GLP-1 treatment.
在VTRNA2-1啟動子的甲基化程度對GLP-1類似物治療的反應性的預測分數 Predictive fraction of responsiveness to GLP-1 analog treatment by methylation levels at the VTRNA2-1 promoter
在針對表6中的年齡、性別及BMI調整之後，VTRNA2-1啟動子中的高度遺傳性甲基化程度與第2型糖尿病患者對GLP-1類似物治療有更大反應機會相關。我們進一步使用操作特性(ROC)曲線(AUC)評估了VTRNA2-1的甲基化狀態作為GLP-1治療的預測指標的表現。預測分數的公式計算如下：[0.022×年齡(歲)-0.042×BMI-0.608×性別(男性=1，女性=0)+1.661×甲基化程度(高甲基化=1，低甲基化=0)+1.095]。如第4圖的(E)部分所示，VTRNA2-1啟動子的甲基化狀態
用於評估GLP-1反應性具有良好的表現(具有95% CI 0.628-0.863之AUC,0.745)。使用Youden’s法之1.542的最佳切點敏感性及特異性分別為53%及86%。藉由VTRNA2-1啟動子區域中每個CpG位點的甲基化狀態的預測模型的其他ROC曲線也藉由年齡、性別及BMI或或rs2346018的已登記多態性調整，且第5圖所示的結果具有類似的預測能力。總之，控制性差的第2型糖尿病患者對GLP-1類似物治療的反應性可能會使用羅吉斯模型(logistic model)及VTRNA2-1的可遺傳甲基化狀態進行預測。
After adjustment for age, sex, and BMI in Table 6, a high degree of hereditary methylation in the VTRNA2-1 promoter was associated with a greater chance of responding to GLP-1 analog therapy in patients with
TGF-β1 ELISA分析 TGF-β1 ELISA analysis
取得血液樣品並在離心之後立刻收及血清。直到分析之前將血清樣品儲存於-20℃。使用TGF-β1 ELISA kit(R&D System Inc.,Minneapolis,MN)測定血清TGF-β1。 Blood samples were taken and serum was collected immediately after centrifugation. Serum samples were stored at -20°C until analysis. Serum TGF-β1 was measured using a TGF-β1 ELISA kit (R&D System Inc., Minneapolis, MN).
統計分析 Statistical Analysis
使用配對的Student’s t-test對連續變量的組間差異進行檢驗。適當地使用卡方檢驗(chi-square test)或費雪精確檢驗(Fisher’s exact test)評估比例差異。結果以平均值±標準差或百分比表示。統計學上顯著設定為P值為0.05。使用R package“ChAMP”進行MethylationEPIC BeadChip標準化(使用參數“BMIQ”將樣品標準化)、calling DMPs以及calling DMRs(使用參數“Bumphunter”)。操作特性(ROC)曲線分析及其他統計分析使用SAS(v9.3,SAS Institute,Cary,NC,USA)進行。 Group differences in continuous variables were tested using paired Student’s t-test. Differences in proportions were assessed using the chi-square test or Fisher's exact test, as appropriate. Results are expressed as mean ± standard deviation or percentage. Statistical significance was set at a P value of 0.05. MethylationEPIC BeadChip normalization using the R package "ChAMP" (sample normalization using parameter "BMIQ"), calling DMPs and calling DMRs (using parameter "Bumphunter"). Operating characteristic (ROC) curve analysis and other statistical analyses were performed using SAS (v9.3, SAS Institute, Cary, NC, USA).
VTRNA2-1啟動子的甲基化程度基因表現之的相關性 Correlation between methylation degree of VTRNA2-1 promoter and gene expression
如第6圖所示，甲基化水平與VTRNA2-1的基因表達適度相關(皮爾森值=0.315,P=0.014)，代表VTRNA2-1啟動子中較高的甲基化百分比抑制了其
基因表達。此外，VTRNA2-1啟動子位於人類第5染色體並由乙型轉化生長因子(transforming growth factor beta induced,TGFBI)以及SMAD Family Member 5(SMAD5)接鄰，兩者都與TGF-β訊號路徑有關。如第7圖的(A)部分所示，VTRNA2-1啟動子的甲基化百分比與血清TGF-β1水平呈正相關，但相關係數為中等(皮爾森值=0.458,P=0.005)。如第7圖的(B)部分所示，使用血清TGF-β1的含量預測對GLP-1類似物組的血糖反應，表示多種因素可調節人體TGF-β1的分泌量。
As shown in Figure 6, methylation levels were moderately correlated with the gene expression of VTRNA2-1 (Pearson value = 0.315, P = 0.014), representing that a higher percentage of methylation in the VTRNA2-1 promoter repressed its gene Express. In addition, the VTRNA2-1 promoter is located on
在表觀遺傳甲基化經晶片分析中，VTRNA2-1啟動子區域的低度甲基化狀態(<40%)接近於GLP-1類似物的無反應性。VTRNA2-1為RNA聚合酶III轉錄的非編碼RNA，其大小為101bp，又稱為nc886。在某些治療藥物耐藥性研究中已顯示出VTRNA2-1受其啟動子甲基化調節，但他可以與被TGF-β1誘導而能夠與Dicer結合以抑制miRNA的成熟並隨後抑制miRNA路徑。在我們近期研究中，我們也發現以腸促素(incretin)為基礎的方案可能涉及某些抗糖尿病治療的不同結果。VTRNA2-1啟動子區域的甲基化狀態在GLP-1類似物反應者與無反應者之間具有明顯不同的模式。因此，在時下精密醫學的時代對患者對GLP-1類似物的反應進行詳細研究至關重要。 In epigenetic methylation microarray analysis, the hypomethylation status (<40%) of the VTRNA2-1 promoter region was close to the anergy of GLP-1 analogs. VTRNA2-1 is a non-coding RNA transcribed by RNA polymerase III, its size is 101bp, also known as nc886 . VTRNA2-1 has been shown to be regulated by its promoter methylation in some therapeutic drug resistance studies, but it can be induced by TGF-β1 to bind to Dicer to inhibit miRNA maturation and subsequent inhibition of the miRNA pathway. In our recent study, we also found that incretin-based regimens may be involved in different outcomes of certain antidiabetic treatments. The methylation status of the VTRNA2-1 promoter region had a distinct pattern between GLP-1 analog responders and non-responders. Therefore, a detailed study of patient responses to GLP-1 analogs is critical in the current era of precision medicine.
本發明中VTRNA2-1啟動子區域上的低甲基化狀態與GLP-1類似物無反應性密切相關。經報導隨後向上調節的VTRNA2-1與其靶基因TGF-β1相關，可能對β細胞存活產生影響。已知GLP-1用於刺激胰島β細胞產生葡萄糖依賴性胰島素的產生和分泌。初步證據代表GLP-1也可以影響胰腺祖細胞(progenitor)的內分泌分化。此外，TGF-β訊號也可以藉由抑制內分泌祖細胞增殖及增強其分化成成熟的β細胞控制內分泌分化。一些研究代表同功型TGF-β在早期胰腺發育 中於朝向內分泌分化和遠離腺泡分化的GLP-1訊號路徑中具有特定的協同作用。TGF-β/SMAD3訊號路徑在調節葡萄糖與能量平衡(energy homeostasis)方面相當重要。肥胖中TGF-β訊號的流失會導致發炎；然而，近期研究中血清TGF-β1含量不是GLP-1反應的預測因子。這可能是由於TGF-β訊號的調控網路複雜。VTRNA2-1與TGF-β1之間對GLP-1反應之相互作用的評估可能不僅僅是藉由患者的TGF-β1量來評估。 The hypomethylation state on the VTRNA2-1 promoter region in the present invention is closely related to GLP-1 analog anergy. The subsequent up-regulation of VTRNA2-1 has been reported to be associated with its target gene TGF-β1 and may have an effect on β-cell survival. GLP-1 is known to stimulate islet beta cells to produce glucose-dependent insulin production and secretion. Preliminary evidence indicates that GLP-1 can also affect the endocrine differentiation of pancreatic progenitors. In addition, TGF-β signaling can also control endocrine differentiation by inhibiting the proliferation of endocrine progenitor cells and enhancing their differentiation into mature β cells. Several studies suggest that the isoform of TGF-β has a specific synergistic role in the GLP-1 signaling pathway towards endocrine differentiation and away from acinar differentiation in early pancreatic development. The TGF-β/SMAD3 signaling pathway is important in regulating glucose and energy homeostasis. Loss of TGF-β signaling in obesity leads to inflammation; however, serum TGF-β1 levels were not a predictor of GLP-1 response in recent studies. This may be due to the complex regulatory network of TGF-β signaling. Assessment of the interaction between VTRNA2-1 and TGF-β1 in response to GLP-1 may not be solely based on the amount of TGF-β1 in patients.
DMRs是與其他樣品比較具有不同DNA甲基化模式的DNA片段與生物體基因組。這些樣品可以為相同個體中的不同細胞或組織、不同時間點的相同細胞或來自不同個體的細胞。在多個個體當中已經報導了具有不同甲基化圖譜的個體間DMRs(inter-DMRs)。這種遺傳表觀遺傳標記可以被用於評估疾病及藥物反應。有趣的是，VTRNA2-1為介穩的表觀等位基因(metastable epiallele)，其對如孕婦營養之類的臨孕期環境敏感，可以指的是在臨孕期階段母體的飲食可以對小孩包含癌症風險、代謝狀態或甚至是藥物反應的終身健康產生影響。 DMRs are DNA fragments with different DNA methylation patterns compared to other samples and the genome of an organism. These samples can be different cells or tissues in the same individual, the same cells at different time points, or cells from different individuals. Inter-individual DMRs (inter-DMRs) with different methylation profiles have been reported in multiple individuals. Such genetic epigenetic markers can be used to assess disease and drug response. Interestingly, VTRNA2-1 is a metastable epiallele, which is sensitive to pregnancy-related conditions such as maternal nutrition, which may mean that the maternal diet during pregnancy can be associated with cancer in the child. Risk, metabolic status, or even lifelong health effects of drug response.
綜上所述，GLP-1類似物的血糖反應與VTRNA2-1啟動子的可遺傳甲基化及rs2346018的多態性有關。篩選鄰近VTRNA2-1之DMRs的臨床應用可以在開始治療之前識別患者可能對GLP-1類似物無反應。這可以助於在現代以腸促素為基礎的第2型糖尿病治療方案中有效避免此昂貴的藥物被無效地使用。總之，在VTRNA2-1附近的DMR中，對GLP-1類似物療法的可變反應與可遺傳的甲基化狀態相關。可以藉由精確篩選該表觀遺傳標記來製定個人化治療方案。
In conclusion, the glycemic response of GLP-1 analogs is associated with heritable methylation of the VTRNA2-1 promoter and the polymorphism of rs2346018. The clinical utility of screening for DMRs adjacent to VTRNA2-1 can identify patients who may not be responsive to GLP-1 analogs before initiating therapy. This could help avoid the ineffective use of this expensive drug in modern incretin-based treatment regimens for
<110> 長庚紀念醫院，林口長庚大學 <110> Chang Gung Memorial Hospital, Chang Gung University, Linkou
<120> 評估GLP-1類似物療法之反應的方法 <120> Methods of Assessing Response to GLP-1 Analog Therapy
<130> CGMH-LK-20003-TWI <130> CGMH-LK-20003-TWI
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| Vaiserman, Alexander, and Oleh Lushchak. "Developmental origins of type 2 diabetes: focus on epigenetics." Ageing research reviews 55 (2019): 100957, p. 1-17. * |
| Van Dijk, S. J., et al. "DNA methylation in blood from neonatal screening cards and the association with BMI and insulin sensitivity in early childhood." International Journal of Obesity 42.1 (2018): 28-35.; * |
| 林嘉鴻，從昇糖素類似胜肽(GLP-1)治療到診斷探討糖尿病的基因標記，長庚大學臨床醫學研究所博士論文(2015)，上架日2017/08/17 * |
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