Igation On Genetic Biomarker Associated With Allopurinol-induced Severe Cutaneous Adverse Reactions In Chinese Population

Background:Allopurinol is widely used as a primary therapy for the treatment of chronic gout, hyperuricaemia, and renal stones accompanied by hyperuricosuria, etc, Originaliy, Chen and colleagues in Taiwan reported a strong association between HLA-B*5801and allopurinol-induced serious life-threating skin allergy reactions. The find is replicated by following studies in Europe, Thailand, South Korea, Japan, Portugal, China, Hong Kong and Mainland China. The results of meta-analysis showed that the allele frequence of HLA-B*5801is very high in the Chinese population (allele frequency was8.9%indicated by the dbMHC database).HLA genotyping is far away to popularize in laboratories because of the complexity and cost-effectiveness technology as well as the particularity of HLA genes. A previous genome wide association study (GWAS) in Japan carried out more recently indicated that the single nucleotide polymorphism (SNP) rs9263726in the nearby PSORS1C1gene was in complete linkage disequilibrium(LD) with HLA-B*5801. Rs9263726genotyping based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) confirmed that rs9263726may serve as a surrogate marker of allopurinol-induced SJS/TEN in Japanese. It remains unknown whether the rs9263726can serve as a surrogate marker for HLA-B*5801to predict allopurinol-induced SCAR in Chinese population. Pyrosequencing is one of the next generation DNA sequencing technologies based on principles of aggregation, and is a simple, convenient, precise, and cheap SNP genotyping technology that can be popularized easily.Purpose:To validate the association of HLA-B*5801and allopurinol-induced SCAR in Chinese Han population, and to explore the possibility of genotyping PSORS1C1rs9263726(G>A) polymorphism as a surrogate to predict allopurinol-induced SCAR in this population. Then other aim was to establish a pyrosequencing method for rs9263726genotyping.Method:A multicenter retrospective case-control study was performed with92allopurinol-induced SCAR patients from17hospitals all over the country,81allopurinol tolerant patients, and99healthy individuals. Peripheral venous blood samples were drawn and DNA samples were extracted. Allopurinol tolerance were those patients t who hade underwent allopurinol therapy for at least three3consecutive months and showed no SCAR. HLA-B genotyping of the samples were carried out a sequencing based typing (SBT). PSORS1Cl-rs9263726(G>A) polymorphism was genotyped with Sanger sequencing. Degree of linkage disequilibrium (LD) between HLA-B*5801allele and rs9263726was analyzed by D’and r2. Pyrosequencing method for rs9263726genotyping was also developed by modification of the PCR and sequencing primers.Result:HLA-B*5801allele was observed in94.57%(87/92) of allopurinol-induced DRESS/SJS/TEN patients, whereas the allele frequency was11.11%(9/81) in allopurinol tolerance patients [Odds ratio (OR)=139.2,95%confidence interval (CI)=44.7-433.9,p=3.02×10-28). Eighty Two allopurinol-induced DRESS/SJS/TEN patients (84/92) carried the PSORS1C1rs9263726A allele, while the frequency of rs9263726A allele carriers was only11.11%(9/81)in the allopurinol tolerance patients (OR=84.0,30.8-229.0,p=4.75×10-26), and14.14%(14/99) in the healthy controls. In addition, an almost complete linkage disequilibrium between HLA-B*5801and PSORS1C1-rs9263726(G>A) polymorphism was observed (D=0.966, r2=0.902). Sensitivity and specialicity for HLA-B*5801to predict allopurinol-induced DRESS/SJS/TEN events in the population were94.57%and88.89%, respectively. Similarly, the sensitivity and specialicity of the PSORS1C1rs9263726A allele to predict allopurinol-induced DRESS/SJS/TEN events were91.30%and88.89%, respectively. When HLA-B*5801and s9263726A allele were use as diagnostic markers for allopurinol-induced hypersensitivity, and the area under curve (AUC) of Receiver operating characteristic (ROC) curve was0.911and0.895, respectively. A simple and convient pyrosequencing method was developed for s9263726genotyping, and the genotyping result was in accordance with Sanger sequencing completely.Conclusion:1) Both HLA-B*58:01allele and PSORS1C1rs9263726A allele are associated with allopurinol-induced SCARs in Chinese population.2) HLA-B*5801allele and PSORS1C1rs9263726are in extensive LD in Chinese population, and rs9263726A allele can be used as a surrogate for allopurinol-induced SCAR in this population.3) A pyrosequencing method for rs9263726was established, which could be used to predict allopurinol-induced SCARs and to guide individualized use of allopurinol.