New Discovery Of Risk Factor Of Dapsone Hypersensitivity Syndrome

BackgroundDapsone(4,4’-diaminodiphenylsulfone)was one kind of sulfone compounds.It was only applicated in German dye industry in the early twentieth century.From 1937,scientists started medical researches on sulfones.Using dapsone for the treatment of leprosy was firstly reported in 1949.Later,as one of the components,dapsone was used in the multi-drug therapy of leprosy world-widely.Because its antibacterial and anti-inflammatory effects,dapsone is also widely used to the treatment of malaria,pneumocystiscarinii pneumonia(PCP)in AIDS patients,and many chronic inflammatory diseases such as dermatitis herpetiformis,linear IgA bullous dermatosis,pemphigoid,pemphigus and chronic bullous dermatosis of childhood,nodulocystic acne,bullous eruption of systemic lupus erythematosus,erythema elevatum diutinum,leukocytoclastic and other kinds of vasculitis and some diseases out of dermatology,such as rheumatoid arthritis,eosinophilic fasciitis,immune thrombocytopenia,stroke,epilepsy and so on.The side effects of dapsone can affect hematologic system,skin,central nervous system,gastrointestinal,liver,eyes and other organs.Dapsone hypersensitivity syndrome(DHS)is the most serious side effect of dapsone,and it is a rare yet potentially fatal adverse drug reaction caused by dapsone.It usually manifested 3 to 6 weeks after the initiation of treatment,characterized by sudden onset of papule or exfoliative dermatitis,with fever,lymphadenopathy and systemic involvement(including liver,kidney,hematological system,etc.).Total DHS prevalence was estimated to be about 1.4%.In Chinese leprosy patients,the prevalence of DHS is estimated as about 1.5%with a mortality of 9.6%.At present,DHS was diagnosed according to the criteria proposed by Richardus and Smith:presence of at least two of the following signs or symptoms:fever,skin eruption,lymphadenopathy,and liver abnormalities(hepatomegaly,hepatitis,jaundice,and/or deranged liver function tests)after taking dapsone.Pharmacogenomics is a field of rapid development in recent years.It has been applied in clinical practice and will become more and more important in the coming years.In recent years,lots of candidate genes studies and genome wide association studies have shown human leukocyte antigen(HLA)genes are associated with susceptibility to specific drug hypersensitivity syndromes.HLA molecules are highly polymorphic and are,therefore,involved in variable interactions with drug antigens.Immunological responses to certain drug antigens may be triggered in patients carrying specific HLA alleles,leading to T-cell activation and clonal expansion.HLA-B*57:01 was a strong risk factor of abacavir-induced hypersensitivity.HLA-B*15:02(Asian populations)and HLA-A*31:01 were significantly associated with carbamazepine-induced Stevens-Johnson syndrome.Some treatment guidelines operated by international authoritative organizations recommended to have HLA-B*58:01 and HLA-B*15:02 allele test before taking allopurinol or carbamazepine,to reduce the incidence of the adverse reaction induced by these drugs.Many non-MHC genes are also likely to be involved in the pathogenesis of severe drug adverse reactions.ST6GAL1 is an additional susceptibility factor in HLA-B*5701-positive flucloxacillin-induced liver injury cases.CFLAR is involved in the mechanism of pathogenesis of allopurinol-induced SCAR.TNF and HSP70 genes are associated with carbamazepine-induced hypersensitivity reactions,and a strong association with SLC01B1 is for simvastatin-related myotoxicity.In 2013,using genome-wide association and subsequent next generation sequencing based HLA-typing method,our group identified HLA-B*13:01 was strongly associated with DHS in Chinese population(odds ratio(OR)= 20.53,P =6.84 × 10-25).Using this allele to predict DHS,the sensitivity and specificity of the presence of HLA-B*13:01 were 85.5%and 85.7%,respectively.The negative predictive value of genotyping the biomarker was calculated as 99.8%,however,the positive predictive value was observed as only 7.8%.It means only 7.8%carriers of HLA-B*13:01 would develop DHS after taking DDS.Low positive predictive value of genotyping HLA-B*13:01 would restrict the clinical utility of pre-screen test of HLA-B*13:01 before taking DDS to prevent the development of dapsone hypersensitivity syndrome.In addition,HLA-B*13:01 is ethnically heterogeneous,which is a specific allele in Asians,but is absent in European and African populations.Thus,it is conceivable that additional genetic variants located within HLA loci or elsewhere in the genome may be associated with the onset of dapsone hypersensitivity syndrome.In addition,according to HLA allele database,for HLA-B*13:01,there are 12 subtypes at six-digit resolution with different mutations located on exons 2,3 and 4 of HLA-B gene.Thus,we also hypothesized DHS patients may carry special HLA-B*13:01 subtype at six-digit resolution.This study devided to two parts.In Part 1,we discovered genetic variants located within HLA loci associated with DHS in additon to HLA-B*13:01 using HLA genotyping based on next-generation sequencing technology;and discovered whether DHS patients carry special HLA-B*13:01 subtype at six-digit resolution.In Part 2,we performed a GWAS to investigate the association between non-MHC genomic polymorphisms and dapsone hypersensitivity syndrome.Part 1 Association Study between HLA Genomic Polymorphismsand Dapsone Hypersensitivity SyndromeObjectiveThis study was to discover contributing genetic variants within HLA loci in addition to HLA-B*13:01 associated with dapsone hypersensitivity syndrome and to discover whether DHS patients carry special HLA-B*13:01 subtype at six-digit resolution.MethodsThis was a case-control study.All subjects enrolled in this study were leprosy patients with HLA-B*13:01 positive and were of Chinese population.They were obtained from multiple hospitals and regional centers responsible for leprosy control in China from 2009 to 2017.DHS was diagnosed according to the criteria proposed by Richardus and Smith with a little modification:(1)showing at least two of the following symptoms or signs:1)fever,2)eruptions,3)lymphadenopathy,4)hepatic function abnormalities(hepatomegaly,jaundice and/or deranged liver function tests);(2)manifestations linked with the administration of DDS,and clinical improvement along with the discontinuation of the drug;(3)symptoms not attributed to prescribed drugs other than DDS,or leprosy reactions;(4)excluding other diseases causing similar clinical appearance.Controls included 857 leprosy patients who did not experience any adverse reactions after treatment of multidrug including DDS for at least 6 months.Genomic DNA was extracted,the presence of the HLA-B*13:01 allele was detected using Taqman-probe-based real-time PCR assay.High resolution genotyping of HLA-A,-B.-C,HLA-DRB1 and-DQB1 was performed using next-generation sequencing technology.After power calculation,only markers the common variants with MAF ≥15%were included for analysis.Fisher’s exact test was applied to compare the proportions of DHS patients and those of DDS-tolerant controls carrying certain variations.Bonferroni correction was performed according to the number of analyzed variants.The statistical analyses were carried out using R and SPSS software.ResultsClinical characteristics of 103 dapsone hypersensitivity syndrome patients were summarized.A total of 409 amino acid variants and 167 classical 4-digit HLA alleles were genotyped.After calculation,we found only with the current sample size our study only has statistical power(>50%)to analyze common variants(MAF>15%).138 HLA variants including 130 amino acid variants and 8 HLA alleles that have MAF≥ 15%were evaluated for associations with DHS.After Bonferroni correction,five amino acid variants in HLA-DRB1(positions:133,142,-17,11,13)were found to be significantly associated with DHS.These five associated variants were in strong linkage disequilibrium.All these significantly associated amino acid variants tagged the classical alleles HLA-DRB1*16:02 and DRB1*15:01 The carrier frequencies of HLA-DRB1*16:02 and DRB1*15:01 were higher in DHS cases than in DDS-tolerant controls.HLA-DRB1*16:02,-DRB1*15:01 and-A*24:02 were nominally significant in association with the susceptibility of DHS.However,none of them reached the significance threshold after Bonferroni correction.According to the clinical features of dapsone hypersensitivity syndrome,DHS patients with presence of all 4 cardinal symptoms of diagnostic criteria(fever,eruptions,lymphadenopathy and hepatic function abnormalities)belonged to complete DHS type,and other patients that the 4 cardinal symptoms could not appear simultaneously were incomplete DHS type.According to the latency of DHS,DHS patients were devided into two groups:long-latency group(with latency>28 days)and short-latency group(with latency≤ 28 days).We compared the carrier frequencies of the 138 HLA variants between complete and incomplete groups,long-latency group and short-latency group.None of the variants was found to be significant in association with the complete/incomplete type of DHS and latency of DHS.For HLA-B*13:01,there are 12 subtypes at six-digit resolution with different mutations located on exons 2,3 and 4 of HLA-B gene.Our analysis showed that all the cases and controls carried the HLA-B*13:01:01,and none of other 11 subtypes of HLA-B*13:01 at 6-digit resolution were detected in our sample.ConclusionsOur study indicated that the amino acid variants in strong linkage disequilibrium of HLA-DRB1 are risk factors for dapsone hypersensitivity syndrome in addition to HLA-B*13:01.6-digit resolution of HLA-B*13:01 showed no influence on the association pattern with DHS in Chinese population.Additional studies with a larger number of samples in different ethnic groups should be undertaken to confirm the current finding and discover additional non-HLA-related genetic risk factors.Part 2 Association study between non-MHC genomic polymorphisms and Dapsone hypersensitivity syndromeObjectiveThis study was to investigate the association between non-MHC genomic polymorphisms and dapsone hypersensitivity syndrome.MethodsWe conducted a two-stage GWAS of DHS in Chinese population including discovery stage and replication stage.All subjects in this study were leprosy patients in Chinese population.The diagnosis of cases met the diagnosis criteria proposed by Richardus and Smith with a little modification.Some leprosy patients who did not experience any adverse reactions after treatment of multidrug including DDS for at least 6 months were recruited as controls.The genome-wide discovery cohort included 39 DHS cases and 88 controls from previously published GWAS dataset 5 and a new added discovery analysis data with 12 cases and 130 controls.Additional 62 DHS cases and 319 controls were recruited as a replication cohort.Illumina Omni Zhonghua chips were used for genotyping in the discovery stage.After sample and SNPs quality control,phasing was performed using SHAPEIT software;and SNP imputation was carried out with IMPUTE software.The SNPs were genotyped in an additional replication cohort with Sequenom MassARRAY platform.The statistical analyses were carried out using SNPTEST and PLINK software.ResultsWe first analyzed the genotyping data for 51 cases and 218 controls genotyped with Illumina Omni Zhonghua chips.After QC filtering,a total of 258,961 overlapped SNPs were analyzed for the genome wide association analysis.After imputation and quality control,3,574,594 SNPs outside the MHC region were performed a log-additive test.Among them,41 loci outside the MHC locus showed suggestive association with DHS(P<1.0 × 10-4),which were selected for the validation study.62 DHS cases and 319 controls were recruited for the subsequent validation analysis.A total of 19 candidate SNPs with P<1.0 × 10-4 were successfully genotyped in the validation study.None of these 19 non-MHC SNPs showed significant association with DHS in the replication analysis,although 18 SNPs were observed association with nominal significance(P<0.05)in combined analysis.ConclusionsOur study did not identify any non-MHC SNPs associated with DHS in Chinese population at a genome-wide significant level.