Validation of a Rapid, Robust, Inexpensive Screening Method for Detecting the HLA-B^*58:01 Allele in the Prevention of Allopurinol-Induced Severe Cutaneous Adverse Reactions

Allopurinol, a xanthine oxidase inhibitor, is more commonly used as a first-line therapy for the gout,1 asymptomatic hyperuricemia-related cell-lysing therapy in malignancy diseases, and nephrolithiasis-associated hyperuricsuria.2 However, allopurinol has been reported as a cause of cutaneous adverse reaction in 2%-3% of allopurinol users3 and as the most common culprit medication of severe cutaneous adverse drug reactions (SCARs), including Steven-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and drug reactions with eosinophilia and systemic symptoms (DRESS) in Europe4 and Asia.5

Since 2005, the HLA B^*58:01 allele has been worldwide reported as a pharmacogenetic susceptibility to allopurinol-induced SJS/TEN/DRESS in diffirent populations.6, 7 The strongest association has been observed in a Taiwanese population (OR 580.3 [95% CI: 34.4-9,780.9]).8 Although there are other risk factors for allopurinol hypersensitivity, such as higher dose, concurrent renal disease, and the use of diuretic medications2 the HLA-B^*58:01 allele contributes to the susceptibility and pathogenesis in a significant proportion of cases of allopurinol-induced SCARs. Subsequently, in 2012, the American College of Rheumatology recommended that HLA-B^*58:01 be screened prior to the initiation of allopurinol treatment.1

To evaluate the benefit of screening tests, a number of studies were conducted and the results showed that screening for the HLA-B^*5801 allele could potentially prevent SCARs, allopurinol - related fatalities, and be more cost-effective in comparison to the initiation of treatment without HLA screening.9, 10, 11

To apply HLA-B screening routinely in clinical practice, the key property of screening tests is simple and cost-effective,12 while current various methodologies for HLA-B typing, such as polymerase chance reaction (PCR) using specific sequencing primers (SSP), sequencing specific oligonucleotide probe (SSO), and sequencing based-typing (SBT), are usually expensive and time-consuming13 with the turnaround time up to 3 weeks.14 In order to reduce time and cost of test, real time PCR might be a candidate for the screening method. Recently, we have just proposed a newly developed method for the detection of both HLA-B^*58:01 and HLA-B^*57:01 by using TaqMan® probe realtime PCR.15 Herein, we present a rapid, robust, inexpensive approach to screen HLA-B^*58:01 using SBYR® real time PCR.

Following the initial optimization of the reaction, testing on positive controls with the HLA-B^*58:01 allele and negative control to identify an annealing temperature was performed for each set of primers for target and reference genes detection. Results demonstrated that 65℃ was the ideal annealing temperature. To optimize the multiplex PCR, different ratios of the HLA-B^*58:01 allele and ACTB primers concentrations were tested (data not shown). In comparing dissociation curves, the best ratio observed was 2:1 (400 nM of each B^*58:01 specific primers and 200 nM of each ACTB primers) and PCR efficiencies for HLA-B^*58:01 was 107% (the lowest Ct: 28.53; highest Ct: 34.48; slope: -3.15; r²=0.99), ACTB was 107% (the lowest Ct: 27.8; highest Ct: 39.2; slope: -3.15; r²=0.95) (Fig. 2). The dissociation curve was used to determine the HLA-B^*58:01 allele and ACTB with the theoretical TM of 91.7℃ and 81.4℃, respectively. The threshold for melting curves was set at 0.45 to avoid a false negative result. The assay was able to detect the target allele in a DNA sample with a concentration as low as 0.8 ng/µL, making it possible to use this assay for samples, such as buccal swabs.

Fig. 2
PCR efficiencies of the HLA-B^*58:01 allele and ACTB.

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The PCR product was confirmed by direct sequencing using BigDye terminators v3.1. The sequence of product had 100% matching with the sequence of the HLA-B^*58:01 allele (HM 543722.1-EMBL-Bank Sequence Database).

From the total 119 samples tested, we detected 10 positive samples by using the present method. Two peaks were observed at 91.26℃±0.01℃ (mean±standard error of mean; range 91.25℃-91.40℃) (HLA-B^*58:01 allele) and 80.97±0.04 (ACTB). The 109 negative samples showed only 1 peak for ACTB (81.42℃±0.16℃) (Fig. 3). The comparative analysis between this assay using SYBR® Green real time PCR and Luminex SSO/SBT/SSP was performed by using MedCalc Software. Our screening assay had a sensitivity of 100% (95% CI: 69.15%–100%), a specificity of 100% (95% CI: 96.67%-100%), a positive predictive value of 100% (95% CI: 69.15%-100.0%), and a negative predictive value of 100% (95% CI: 96.67%-100%) (Table). HLA-B^*58:01 genotyping results showed 100% agreement with those obtained from Luminex SSO/SBT/SSP.

Fig. 3
Detection of the HLA-B^*58:01 allele using dissociation curves of our screening method. The positive samples have 2 peaks of melting temperatures (ACTB and B^*58:01); the negative samples have only 1 peak of ACTB.

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Table
Comparison of the present method with Luminex SSO/SBT/SSP

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In this study, we have reported a novel screening method for detecting the HLA-B^*58:01 allele to prevent allopurinol-induced SCARs. Using SYBR® Green real time PCR, the target allele was amplified successfully and cost-effectively enabling it to be utilized in routine clinical practice.

In 2010, Virakul et al.17 presented a method for detecting the HLA-B^*58:01 allele using a touch down PCR-SSP. Although this method detected successfully the target allele from 200 samples, limitations of PCR-SSP are more time-consuming because post-PCR analysis is required. More importantly, we modified the reverse primer by removing 2 bases at the end of 3' and 2 bases at the end of 5' (Fig. 1). This advancement allowed our set of primers to amplify specifically the HLA-B^*58:01 allele and minimized the risk of non-specific detection of the HLA-B^*57:01 allele because a mismatch at the middle of the primer binding site was not robust enough for the latter one to eliminate the amplification of the HLA-B^*57:01 allele (Fig. 1).

Recently, a similar approach was independently published for the detection of the HLA-B^*58:01 allele using real time TaqMan® probe and specific primers.3, 13 Although real time PCR was also used, TaqMan® probe is less sensitive than SYBR® Green because of the probability of having false negative results due to mismatches at the probe binding site. To prevent SCARs, the crucial properties of screening method are sensitivity and negative predictive values. A false negative result potentially could result in a life-threatening SCAR if a patient has been prescribed the medication. In our study, there were no false negative results. In addition, the studies using the TaqMan® probe were compared to the commercial SSP kit to determine validation of results. PCR–SSP is not a standard method of genotyping. In contrast, our study used high-resolution HLA-B typing using Luminex SSO/SBT/SSP and was performed independently and blindly at the New South Wales Transplantation and Immunogenetics Service (Australian Red Cross Blood Service). There was 100% agreement between our method and the Luminex SSO/SBT/SSP.

As compared to the commercially available testing kit (PG5801 Detection Kit; Pharmigene, Inc., Taipei, Taiwan), our method uses multiplexing reducing both time and costs. Multiplexing is also an optimal way to reduce the chances of obtaining a false negative result due to low quality of DNA or the presence of any potential PCR inhibitors. Further, it is more accurate and robust to detect the target PCR product using the melting curves rather than calculating delta Ct or loading PCR products on an agarose gel. Additionally, using our method, genomic DNA can be used to detect the target gene rather than to adjust concentration. Our assays are a robust and very sensitive technique that is able to detect the target allele at a very low amount of DNA (as low as 2 ng DNA/reaction), while the commercial kit requires 25-100 ng/reaction. Therefore, this method can be used for DNA that is extracted from specimens obtained by non-invasive methods, such as buccal swabs, which can have a lower yield of DNA. Finally, we calculated the unit price of our test, including DNA extraction kit, reagents for PCR that is only approximately 3.8 USD, being much cheaper than commercial kit (15 USD/kit), SBT (32 USD), SSO based typing (38 USD), and Loop-mediated isothermal amplification (6.4 USD) for the detection of the HLA-B^*58:01 allele.18

A limitation of our assays is that since we tested and validated only 1 commercially available master mix (SensiMix™ SYBR No-ROX Kit), re-validation in an alternative mix will be required if this master mix is discontinued. Re-validation of the assay will also be required prior to implementation on other real time PCR cyclers although this could be of lesser concern because our method does not require the analysis of results using delta Ct and is only a qualitative one. Another limitation common to any other existing molecular test for HLA-alleles is related to the continuous need for sequence updates owing to the highly polymorphic nature of these genes. New polymorphisms that could be present in the primer-binding sites of the assay can always results in both false negative and false positive results.