中國人群別嘌呤醇致嚴重皮膚不良反應的遺傳標志物研究
發(fā)布時間：2018-08-17 08:46
【摘要】：背景：別嘌呤醇在臨床上廣泛用于痛風、高尿酸血癥及其并發(fā)癥等的治療。2004年首次有文獻報道HLA-B*5801等位基因攜帶與中國臺灣人群別嘌呤醇所致致死性嚴重皮膚不良反應(severe cutaneous adverse reaction,SCAR)強烈相關,該發(fā)現先后在歐洲、泰國、韓國、日本、葡萄牙、中國香港以及大陸人群中均得到驗證。薈萃分析結果表明,中國人群中HLA-B*5801等位基因攜帶率尤其高,攜帶率約為8.9%。HLA分型由于技術、成本及基因本身的復雜性等原因而無法在實驗室普及。近年來基于日本人群的GWAS研究發(fā)現與HLA-B相鄰的PSORS1C1基因內的單核苷酸多態(tài)性(single nucleotide polymorphism, SNP) rs9263726位點A等位基因與HLA-B*5801呈完全連鎖不平衡,并在小樣本人群建立了對該SNP位點進行分型的聚合酶鏈反應-限制性片段長度多態(tài)性(PCR-RFLP)法,提示rs9263726在日本人群可能作為別嘌呤醇導致SJS/TEN的替代分子標志物。中國人群中rs9263726是否可替代HLA-B*5801作為預測別嘌呤醇致嚴重過敏反應的預測因子目前沒有相關的研究報道。焦磷酸測序(Pyrosequencing)是一種基于聚合原理的DNA測序方法,是新一代DNA序列分析技術,且分型方法準確、快速、簡便、經濟,易于在實驗室普及用作SNP分型。 目的：在中國大陸人群中驗證HLA-B*5801等位攜帶與別嘌呤醇致嚴重皮膚不良不良反應發(fā)生間的相關性；探索在中國人群中PSORS1C1rs9263726(GA) A等位基因替代HLA-B*5801作為預測別嘌呤醇致嚴重皮膚過敏反應發(fā)生的分子標記物的可行性；建立可用于對rs9263726位點進行分型的焦磷酸測序法。 方法：通過多中心回顧性病例-對照研究,收集了來自全國各地17家醫(yī)院別嘌呤醇致嚴重皮膚不良反應患者血液標本92例,同時在廣州、廣西、四川、湖南和山西等地收集連續(xù)或累計服用別嘌呤醇3個月以上未出現皮膚不良反應(別嘌呤醇耐受組病例)的患者血液標本81例,收集長沙地區(qū)健康志愿者外周血標本99例。提取DNA后通過直接測序分型法(sequencing based typing, SBT)對所有標本進行進行HLA-B基因分型。Sanger測序法分型對三組人群的PSORS1C1rs9263726(GA)位點進行分型,計算HLA-B*5801和rs9263726(GA)位點間的連鎖不平衡強度參數D,和r2；通過設計PCR擴增引物和生物素標記的測序引物,建立可用于對rs9263726位點進行分型的焦磷酸測序分型方法。 結果：本研究收集到92例出現別嘌呤醇所致SCAR的患者,SCAR的具體類型包括伴嗜酸性粒細胞增多性全身癥狀的藥物反應(Drug reaction with eosinophilia and systemic symptoms, DRESS)、史蒂文斯-約翰遜綜合征(Stevens-Johnson syndrome, SJS)和中毒性表皮壞死松解癥(Toxic epidermal necrolysis, TEN)。在92例DRESS/SJS/TEN患者中,有87例患者(87/92,94.57%)攜帶HLA-B*5801等位基因,別嘌呤醇耐受組患者中該等位基因的攜帶率為11.11%(9/81),HLA-B*5801攜帶增加別嘌呤醇至嚴重皮膚不良反應發(fā)生風險的比值比(odds ratio,OR=139.2,95％CI:44.7-433.9,p=3.02×10-28)。84例(91.30%)出現別嘌呤醇所致DRESS/SJS/TEN的患者攜帶rs9263726A等位基因,而別嘌呤醇耐受組人群中rs9263726A等位基因的攜帶率為11.11%,以rs9263726多態(tài)性位點預測別嘌呤醇致嚴重皮膚不良反應的比值比(OR)為84.0(95%CI：30.8-229.0,p=4.75×10-26)。99名健康志愿者中rs9263726A等位基因攜帶者的頻率為14.14%。HLA-B*5801和PSORS1C1rs9263726(GA)多態(tài)位點間呈強連鎖不平衡(D'=0.966,r2=0.902)。HLA-B*5801用于預測該人群別嘌呤醇SCAR發(fā)生的靈敏度和特異度分別為94.57%和88.89%；陽性預測值(PPV)和陰性預測值(NPV)分別為90.61%和93.51%；rs9263726(GA)多態(tài)位點A等位基因用于預測該人群別嘌呤醇致SCAR發(fā)生的靈敏度和特異度分別為91.30%和88.89%；PPV和NPV分別為90.32%和90.00%。將攜帶HLA-B*5801和rs9263726A等位基因作為診斷指標分析ROC曲線下面積AUC分別為0.911和0.895。本研究所建立的rs9263726(GA)位點焦磷酸測序分型方法與Sanger測序法所得基因型的一致率達100%。 結論：1)證實在中國大陸人群中HLA-B*5801等位基因攜帶與別嘌呤醇所致嚴重不良反應(SJS/TEN/DRESS)的發(fā)生風險強相關；2)中國人群中HLA-B*5801與PSORS1C1rs9263726A等位基因呈高度連鎖不平衡,rs9263726A等位基因可作為在中國人群中預測別嘌呤醇所致SCAR風險的替代分子遺傳標志物；3)建立了可用于對rs9263726多態(tài)性位點用于分型的焦磷酸測序方法,可用于在應用別嘌呤醇前對嚴重皮膚過敏反應的發(fā)生進行預測,指導個體化用藥。圖4幅,表9個,參考文獻72篇。
[Abstract]:BACKGROUND: Allopurinol is widely used in the treatment of gout, hyperuricemia and its complications. In 2004, it was first reported that HLA-B*5801 allele carriers were strongly associated with severe cutaneous adverse reactions (SCAR) caused by allopurinol in Taiwanese population in China. This finding was found in Europe successively. The results of meta-analysis showed that the carrying rate of HLA-B*5801 allele was particularly high in the Chinese population, with a carrying rate of about 8.9%. HLA typing could not be popularized in laboratories because of the complexity of technology, cost and gene itself. The GWAS study found that the single nucleotide polymorphism (SNP) allele A at rs9263726 in the PSORS1C1 gene adjacent to HLA-B was completely linked to HLA-B*5801, and a polymerase chain reaction-restriction fragment length polymorphism (PC) was established in a small sample population. R-RFLP method suggested that rs9263726 might be used as an alternative molecular marker for allopurinol-induced SJS/TEN in Japanese population. Whether rs9263726 could replace HLA-B*5801 as a predictor of severe allergic reaction induced by allopurinol in Chinese population has not been reported. Pyrosequencing is a polymerization-based method. Principle of DNA sequencing method is a new generation of DNA sequence analysis technology, and typing method is accurate, fast, simple, economical, easy to popularize in the laboratory as a SNP typing.
Objective: To verify the association between HLA-B*5801 allele carrier and severe skin adverse reactions induced by allopurinol in mainland China, and to explore the feasibility of substituting allele PSORS1C1rs9263726 (GA) A for HLA-B*5801 as a molecular marker for predicting severe skin allergic reactions induced by allopurinol in Chinese population. A pyrosequencing method was developed for typing rs9263726 sites.
Methods: A multicenter retrospective case-control study was conducted to collect 92 blood samples of patients with severe skin adverse reactions caused by allopurinol from 17 hospitals across the country. At the same time, no skin adverse reactions (allopurinol) were observed in Guangzhou, Guangxi, Sichuan, Hunan and Shanxi for more than 3 months. The peripheral blood samples of 81 patients in tolerance group were collected from 99 healthy volunteers in Changsha. After DNA extraction, HLA-B genotyping was performed on all samples by sequencing based typing (SBT). Linkage disequilibrium strength parameters D, and R2 between - B * 5801 and rs9263726 (GA) loci were designed, and a pyrophosphatic acid sequencing typing method for rs9263726 loci was established by designing PCR amplification primers and biotin-labeled sequencing primers.
RESULTS: 92 patients with allopurinol-induced SCAR were enrolled. The specific types of SCAR included Drug reaction with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS) and toxic epidermis. Toxic epidermal necrolysis (TEN). Among 92 DRESS/SJS/TEN patients, 87 (87/92, 94.57%) carried the HLA-B*5801 allele, 11.11% (9/81) in the allopurinol-tolerant group, and odds ratio (odds r) in the HLA-B*5801 group which increased the risk of severe skin adverse reactions to allopurinol. 84 (91.30%) patients with allopurinol-induced DRESS/SJS/TEN carried the rs9263726A allele, while the rs9263726A allele carrying rate was 11.11% in the allopurinol tolerance group. The rs9263726A polymorphism was used to predict the ratio of severe skin adverse reactions induced by allopurinol (O R) was 84.0 (95% CI: 30.8-229.0, P = 4.75 *10-26). The frequency of rs9263726A allele was 14.14% in 99 healthy volunteers. There was a strong linkage imbalance between HLA-B * 5801 and PSORS1C1rs9263726 (GA) polymorphisms (D'= 0.966, R2 = 0.902). HLA-B * 5801 was used to predict the occurrence of allopurinol SCAR in this population with a sensitivity and specificity of 94.57% respectively. The positive predictive value (PPV) and negative predictive value (NPV) were 90.61% and 93.51% respectively; the sensitivity and specificity of allele A of rs9263726 (GA) for predicting SCAR induced by allopurinol were 91.30% and 88.89% respectively; the PPV and NPV were 90.32% and 90.00% respectively. HLA-B*5801 and rs9263726A alleles will be carried for screening. The area of AUC under ROC curve was 0.911 and 0.895, respectively. The similarity between the pyrophosphatic acid sequencing method of rs9263726 (GA) and Sanger sequencing was 100%.
Conclusion: 1) HLA-B*5801 allele was strongly associated with the risk of severe adverse reactions caused by allopurinol (SJS/TEN/DRESS) in mainland China; 2) HLA-B*5801 was highly linked to PSORS1C1rs9263726A allele in Chinese population, and rs9263726A allele could be used as a predictor of allopurine in Chinese population. 3) A pyrophosphate sequencing method was developed for the identification of rs9263726 polymorphism loci, which could be used to predict the occurrence of severe skin allergies before allopurinol was used to guide individualized medication. 4 figs, 9 tables, 72 references.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96
本文編號：2187088
[Abstract]:BACKGROUND: Allopurinol is widely used in the treatment of gout, hyperuricemia and its complications. In 2004, it was first reported that HLA-B*5801 allele carriers were strongly associated with severe cutaneous adverse reactions (SCAR) caused by allopurinol in Taiwanese population in China. This finding was found in Europe successively. The results of meta-analysis showed that the carrying rate of HLA-B*5801 allele was particularly high in the Chinese population, with a carrying rate of about 8.9%. HLA typing could not be popularized in laboratories because of the complexity of technology, cost and gene itself. The GWAS study found that the single nucleotide polymorphism (SNP) allele A at rs9263726 in the PSORS1C1 gene adjacent to HLA-B was completely linked to HLA-B*5801, and a polymerase chain reaction-restriction fragment length polymorphism (PC) was established in a small sample population. R-RFLP method suggested that rs9263726 might be used as an alternative molecular marker for allopurinol-induced SJS/TEN in Japanese population. Whether rs9263726 could replace HLA-B*5801 as a predictor of severe allergic reaction induced by allopurinol in Chinese population has not been reported. Pyrosequencing is a polymerization-based method. Principle of DNA sequencing method is a new generation of DNA sequence analysis technology, and typing method is accurate, fast, simple, economical, easy to popularize in the laboratory as a SNP typing.
Objective: To verify the association between HLA-B*5801 allele carrier and severe skin adverse reactions induced by allopurinol in mainland China, and to explore the feasibility of substituting allele PSORS1C1rs9263726 (GA) A for HLA-B*5801 as a molecular marker for predicting severe skin allergic reactions induced by allopurinol in Chinese population. A pyrosequencing method was developed for typing rs9263726 sites.
Methods: A multicenter retrospective case-control study was conducted to collect 92 blood samples of patients with severe skin adverse reactions caused by allopurinol from 17 hospitals across the country. At the same time, no skin adverse reactions (allopurinol) were observed in Guangzhou, Guangxi, Sichuan, Hunan and Shanxi for more than 3 months. The peripheral blood samples of 81 patients in tolerance group were collected from 99 healthy volunteers in Changsha. After DNA extraction, HLA-B genotyping was performed on all samples by sequencing based typing (SBT). Linkage disequilibrium strength parameters D, and R2 between - B * 5801 and rs9263726 (GA) loci were designed, and a pyrophosphatic acid sequencing typing method for rs9263726 loci was established by designing PCR amplification primers and biotin-labeled sequencing primers.
RESULTS: 92 patients with allopurinol-induced SCAR were enrolled. The specific types of SCAR included Drug reaction with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS) and toxic epidermis. Toxic epidermal necrolysis (TEN). Among 92 DRESS/SJS/TEN patients, 87 (87/92, 94.57%) carried the HLA-B*5801 allele, 11.11% (9/81) in the allopurinol-tolerant group, and odds ratio (odds r) in the HLA-B*5801 group which increased the risk of severe skin adverse reactions to allopurinol. 84 (91.30%) patients with allopurinol-induced DRESS/SJS/TEN carried the rs9263726A allele, while the rs9263726A allele carrying rate was 11.11% in the allopurinol tolerance group. The rs9263726A polymorphism was used to predict the ratio of severe skin adverse reactions induced by allopurinol (O R) was 84.0 (95% CI: 30.8-229.0, P = 4.75 *10-26). The frequency of rs9263726A allele was 14.14% in 99 healthy volunteers. There was a strong linkage imbalance between HLA-B * 5801 and PSORS1C1rs9263726 (GA) polymorphisms (D'= 0.966, R2 = 0.902). HLA-B * 5801 was used to predict the occurrence of allopurinol SCAR in this population with a sensitivity and specificity of 94.57% respectively. The positive predictive value (PPV) and negative predictive value (NPV) were 90.61% and 93.51% respectively; the sensitivity and specificity of allele A of rs9263726 (GA) for predicting SCAR induced by allopurinol were 91.30% and 88.89% respectively; the PPV and NPV were 90.32% and 90.00% respectively. HLA-B*5801 and rs9263726A alleles will be carried for screening. The area of AUC under ROC curve was 0.911 and 0.895, respectively. The similarity between the pyrophosphatic acid sequencing method of rs9263726 (GA) and Sanger sequencing was 100%.
Conclusion: 1) HLA-B*5801 allele was strongly associated with the risk of severe adverse reactions caused by allopurinol (SJS/TEN/DRESS) in mainland China; 2) HLA-B*5801 was highly linked to PSORS1C1rs9263726A allele in Chinese population, and rs9263726A allele could be used as a predictor of allopurine in Chinese population. 3) A pyrophosphate sequencing method was developed for the identification of rs9263726 polymorphism loci, which could be used to predict the occurrence of severe skin allergies before allopurinol was used to guide individualized medication. 4 figs, 9 tables, 72 references.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96
【共引文獻】
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