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. 2022 Dec;100(6):610-616.
doi: 10.1111/tan.14767. Epub 2022 Aug 26.

A low-cost, sensitive and specific PCR-based tool for rapid clinical detection of HLA-B*35 alleles associated with delayed drug hypersensitivity reactions

Yueran Li  1 , Pooja Deshpande  1 , Abha Chopra  1   2 , Linda Choo  1 , Andrew Gibson  1 , Elizabeth J Phillips  1   2
Affiliations

A low-cost, sensitive and specific PCR-based tool for rapid clinical detection of HLA-B*35 alleles associated with delayed drug hypersensitivity reactions

Yueran Li et al. HLA. 2022 Dec.

Abstract

HLA (HLA) alleles are risk factors for CD8+ T-cell-mediated drug hypersensitivity reactions. However, as most HLA associations are incompletely predictive and/or involve risk alleles at low frequency, costly sequence-based typing can elude an economically productive cost: benefit ratio for clinical validation studies and diagnostic and/or preventative screening. Hence rapid and low-cost detection assays are now required, both for single alleles but also across risk loci associated with broader multi-disease risk; exemplified by associations with diverse alleles in HLA-B*35, including HLA-B*35:01 and green tea- or co-trimoxazole-induced liver injury. Here, we developed a cost-effective (<$10USD) qPCR assay for rapid (<2.5 h) clinical detection of HLA-B*35 alleles. The assay was validated using 430 DNA samples with previous American society for histocompatibility and immunogenetics-accredited sequence-based high-resolution HLA typing, positively detecting all HLA-B*35 allelic variants in our cohort, and as expected by primer design, the six samples that expressed low-frequency B*78:01. The assay did not result in positive detection for any negative control allele. With expected detection of B*35 and B*78, our assay sensitivity (95% CI, 95.07%-100.00%) and specificity (95% CI, 98.97%-100.00%) of 100% using as low as 10 ng of DNA provides a reliable HLA-B*35 screening tool for clinical validation and HLA-risk-based prevention and diagnostics.

Keywords: HLA (HLA); Immunogenetics; drug hypersensitivity reactions; real-time quantitative polymerase chain reaction.

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Conflict of interest statement

Elizabeth J. Phillips receives royalties from UpToDate and consulting fees from Biocryst, Janssen and Vertex. She is co‐director of IIID Pty Ltd. that holds a patent for HLA‐B*57:01 testing for abacavir hypersensitivity, and she holds a patent with AC for detection of HLA‐A*32:01 in connection with Diagnosing Drug Reaction with Eosinophilia and Systemic Symptoms without financial remuneration. All the other authors have no conflict of interest to disclose.

Figures

FIGURE 1
FIGURE 1
Targeted sequence design for discrimination of HLA‐B*35 from other HLA‐B alleles at four‐digit resolution. Binding sites of HLA‐B*35 primers/probe aligned with HLA‐B*35:01 allele. The HLA‐B sequences from IMGT/HLA database were aligned with HLA‐B*35:01 using Bioedit 3.0. Primer and probe positions are indicated by the boxes, with the arrows indicating direction. The forward primer spans Exon 2 at positions 117–133 and is locked at position 133. The reverse primer is complementary to Exon 2 at positions 229–244 and is locked complementary to position 229. The HLA‐B*35 probe is complementary to Exon 2 at positions 178–204, with the targeted region between the dye (FAM) and quencher (ZEN), which allows optimization
FIGURE 2
FIGURE 2
HLA‐B*35 assay real time PCR result. (A). HLA‐B*35 positive and negative samples were distinguished by the HLA‐B*35 assay. Fluorescence of HLA‐B*35 (and B*78) positive samples (100 ng) plateaued at higher levels (>18 × 103 RFU) after 40 cycles of PCR. HLA‐B*18 alleles that are distinguished by one base pair within the probe showed elevated but lower‐level fluorescence (10 × 103 RFUs). (B). Amplification of housekeeping gene RNAseP showed HEX fluorescence (plateaued at 10 × 103 RFUs) after 30 cycles of PCR
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References

    1. Mallal S, Phillips E, Carosi G, et al. HLA‐B* 5701 screening for hypersensitivity to abacavir. N Engl J Med. 2008;358(6):568‐579. - PubMed
    1. Ferrell PB Jr, McLeod HL. Carbamazepine, HLA‐B*1502 and risk of Stevens‐Johnson syndrome and toxic epidermal necrolysis: US FDA recommendations. Pharmacogenomics. 2008;9(10):1543‐1546. doi:10.2217/14622416.9.10.1543 - DOI - PMC - PubMed
    1. Chiu MLS, Hu M, Ng MHL, et al. Association between HLA‐B58:01 allele and severe cutaneous adverse reactions with allopurinol in Han Chinese in Hong Kong. Br J Dermatol. 2012;167(1):44‐49. doi:10.1111/j.1365-2133.2012.10894.x - DOI - PubMed
    1. Faulkner L, Meng X, Park BK, Naisbitt DJ. The importance of hapten–protein complex formation in the development of drug allergy. Curr Opin Allergy Clin Immunol. 2014;14(4):293‐300. - PubMed
    1. Pichler WJ. Immune pathomechanism and classification of drug hypersensitivity. Allergy. 2019;74(8):1457‐1471. - PubMed

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