中国人群别嘌呤醇致严重皮肤不良反应的遗传标志物研究

发布时间：2018-08-17 08:46

【摘要】：背景：别嘌呤醇在临床上广泛用于痛风、高尿酸血症及其并发症等的治疗。2004年首次有文献报道HLA-B*5801等位基因携带与中国台湾人群别嘌呤醇所致致死性严重皮肤不良反应(severe cutaneous adverse reaction,SCAR)强烈相关,该发现先后在欧洲、泰国、韩国、日本、葡萄牙、中国香港以及大陆人群中均得到验证。荟萃分析结果表明,中国人群中HLA-B*5801等位基因携带率尤其高,携带率约为8.9%。HLA分型由于技术、成本及基因本身的复杂性等原因而无法在实验室普及。近年来基于日本人群的GWAS研究发现与HLA-B相邻的PSORS1C1基因内的单核苷酸多态性(single nucleotide polymorphism, SNP) rs9263726位点A等位基因与HLA-B*5801呈完全连锁不平衡,并在小样本人群建立了对该SNP位点进行分型的聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法,提示rs9263726在日本人群可能作为别嘌呤醇导致SJS/TEN的替代分子标志物。中国人群中rs9263726是否可替代HLA-B*5801作为预测别嘌呤醇致严重过敏反应的预测因子目前没有相关的研究报道。焦磷酸测序(Pyrosequencing)是一种基于聚合原理的DNA测序方法,是新一代DNA序列分析技术,且分型方法准确、快速、简便、经济,易于在实验室普及用作SNP分型。目的：在中国大陆人群中验证HLA-B*5801等位携带与别嘌呤醇致严重皮肤不良不良反应发生间的相关性；探索在中国人群中PSORS1C1rs9263726(GA) A等位基因替代HLA-B*5801作为预测别嘌呤醇致严重皮肤过敏反应发生的分子标记物的可行性；建立可用于对rs9263726位点进行分型的焦磷酸测序法。方法：通过多中心回顾性病例-对照研究,收集了来自全国各地17家医院别嘌呤醇致严重皮肤不良反应患者血液标本92例,同时在广州、广西、四川、湖南和山西等地收集连续或累计服用别嘌呤醇3个月以上未出现皮肤不良反应(别嘌呤醇耐受组病例)的患者血液标本81例,收集长沙地区健康志愿者外周血标本99例。提取DNA后通过直接测序分型法(sequencing based typing, SBT)对所有标本进行进行HLA-B基因分型。Sanger测序法分型对三组人群的PSORS1C1rs9263726(GA)位点进行分型,计算HLA-B*5801和rs9263726(GA)位点间的连锁不平衡强度参数D,和r2；通过设计PCR扩增引物和生物素标记的测序引物,建立可用于对rs9263726位点进行分型的焦磷酸测序分型方法。结果：本研究收集到92例出现别嘌呤醇所致SCAR的患者,SCAR的具体类型包括伴嗜酸性粒细胞增多性全身症状的药物反应(Drug reaction with eosinophilia and systemic symptoms, DRESS)、史蒂文斯-约翰逊综合征(Stevens-Johnson syndrome, SJS)和中毒性表皮坏死松解症(Toxic epidermal necrolysis, TEN)。在92例DRESS/SJS/TEN患者中,有87例患者(87/92,94.57%)携带HLA-B*5801等位基因,别嘌呤醇耐受组患者中该等位基因的携带率为11.11%(9/81),HLA-B*5801携带增加别嘌呤醇至严重皮肤不良反应发生风险的比值比(odds ratio,OR=139.2,95％CI:44.7-433.9,p=3.02×10-28)。84例(91.30%)出现别嘌呤醇所致DRESS/SJS/TEN的患者携带rs9263726A等位基因,而别嘌呤醇耐受组人群中rs9263726A等位基因的携带率为11.11%,以rs9263726多态性位点预测别嘌呤醇致严重皮肤不良反应的比值比(OR)为84.0(95%CI：30.8-229.0,p=4.75×10-26)。99名健康志愿者中rs9263726A等位基因携带者的频率为14.14%。HLA-B*5801和PSORS1C1rs9263726(GA)多态位点间呈强连锁不平衡(D'=0.966,r2=0.902)。HLA-B*5801用于预测该人群别嘌呤醇SCAR发生的灵敏度和特异度分别为94.57%和88.89%；阳性预测值(PPV)和阴性预测值(NPV)分别为90.61%和93.51%；rs9263726(GA)多态位点A等位基因用于预测该人群别嘌呤醇致SCAR发生的灵敏度和特异度分别为91.30%和88.89%；PPV和NPV分别为90.32%和90.00%。将携带HLA-B*5801和rs9263726A等位基因作为诊断指标分析ROC曲线下面积AUC分别为0.911和0.895。本研究所建立的rs9263726(GA)位点焦磷酸测序分型方法与Sanger测序法所得基因型的一致率达100%。结论：1)证实在中国大陆人群中HLA-B*5801等位基因携带与别嘌呤醇所致严重不良反应(SJS/TEN/DRESS)的发生风险强相关；2)中国人群中HLA-B*5801与PSORS1C1rs9263726A等位基因呈高度连锁不平衡,rs9263726A等位基因可作为在中国人群中预测别嘌呤醇所致SCAR风险的替代分子遗传标志物；3)建立了可用于对rs9263726多态性位点用于分型的焦磷酸测序方法,可用于在应用别嘌呤醇前对严重皮肤过敏反应的发生进行预测,指导个体化用药。图4幅,表9个,参考文献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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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