BackgroundCarbamazepine, an anticonvulsant also used as a mood stabilizer and for trigeminal neuralgia, is associated with serious, sometimes fatal cutaneous adverse drug reactions, including Stevens Johnson Syndrome and toxic epidermal necrolysis1. Current literature demonstrates a genetic predisposition linked to specific class I and II human leukocyte antigen (HLA) types in various ethnic populations2. HLA-A*31:01 is one such HLA type, and is routinely identified by the tag SNP rs1061235. However, rs1061235 has poor specificity for HLA*31:01 due to interference of HLA-A*33 types3. We investigated the false positive rate in our population and developed a novel real-time PCR assay that distinguishes HLA-A*31:01 from other HLA-A types including HLA-A*33.Methods120 unique samples were tested in triplicate during the validation of this assay and were sent to a reference lab for HLA next generation sequencing (NGS) typing, including 89 in-house samples and 31 Coriell samples with documented HLA typing results. The results from our real-time PCR assay were compared to the HLA typing results. HLA typing results were also compared to the tag SNP rs1061235 results to calculate the false positive rate.ResultsThere was 100% concordance between our real-time PCR results and expected results based on HLA typing. 89 sample results for tag SNP rs1061235 were compared to HLA typing results. 75/89 samples had a rs1061235 variant, but 31/75 (41%) samples did not have the HLA-A*31:01 type, thus defining the false positive rate of the tag SNP for our population. We theorized there would be a small subset of rare HLA-A types that would interfere with the assay and we tested the three types available to us. We confirmed that 3 of the HLA types (HLA-A*31:04, 31:12, and 31:16) result falsely positive due to sequence homology with 31:01. There is no known literature indicating whether these rare HLA-A*31 subtypes are associated with cutaneous adverse reactions. These 3 HLA types and the other suspected interfering HLA types have limited frequency data sets and are expected to occur rarely in our patient population; we expect these HLA types make up less than 0.003% of the our population. Our assay specificity for the validation is >99%.ConclusionsOur custom real-time PCR assay for detection of HLA-A*31:01 is significantly more specific than the commonly used tag SNP rs1061235. Clinicians considering carbamazepine therapy for their patients will have a better understanding of cutaneous adverse reaction risk and can make improved personalized treatment decisions. This quick, cost effective assay allows more patients in need of carbamazepine treatment to benefit from its use.FundingGenomind, Inc.

中文翻译：

---

一种新的实时 PCR 检测方法，用于检测考虑接受卡马西平治疗的个体中的 HLA-A*31:01

背景卡马西平是一种抗惊厥药，也用作情绪稳定剂和三叉神经痛，与严重的、有时是致命的皮肤不良药物反应有关，包括 Stevens Johnson 综合征和中毒性表皮坏死松解症1。目前的文献表明，与不同种族人群中特定的 I 类和 II 类人类白细胞抗原 (HLA) 类型相关的遗传易感性。HLA-A*31:01 就是这样一种 HLA 类型，通常由标签 SNP rs1061235 识别。然而，由于 HLA-A*33 类型的干扰，rs1061235 对 HLA*31:01 的特异性较差。我们调查了我们人群中的假阳性率，并开发了一种新的实时 PCR 检测方法，将 HLA-A*31:01 与其他 HLA-A 类型（包括 HLA-A*33）区分开来。方法 120 个独特的样本在该测定的验证过程中一式三份进行了测试，并被送到参考实验室进行 HLA 下一代测序 (NGS) 分型，其中包括 89 个内部样本和 31 个具有记录 HLA 分型结果的 Coriell 样本。我们将实时 PCR 检测的结果与 HLA 分型结果进行了比较。HLA分型结果也与标签SNP rs1061235结果进行比较，计算假阳性率。结果我们的实时PCR结果与基于HLA分型的预期结果有100%的一致性。将标签 SNP rs1061235 的 89 个样本结果与 HLA 分型结果进行了比较。75/89 个样本具有 rs1061235 变体，但 31/75 (41%) 个样本没有 HLA-A*31:01 类型，因此定义了我们人群中标签 SNP 的假阳性率。我们推测会有一小部分罕见的 HLA-A 类型会干扰检测，我们测试了我们可用的三种类型。我们确认了 3 种 HLA 类型（HLA-A*31:04、31:12 和 31:16）由于与 31:01 的序列同源性而导致假阳性。没有已知文献表明这些罕见的 HLA-A*31 亚型是否与皮肤不良反应有关。这 3 种 HLA 类型和其他疑似干扰 HLA 类型的频率数据集有限，预计在我们的患者群体中很少发生；我们预计这些 HLA 类型占我们人口的不到 0.003%。我们用于验证的检测特异性 > 99%。结论我们用于检测 HLA-A*31:01 的定制实时 PCR 检测比常用标签 SNP rs1061235 的特异性明显更高。考虑对其患者进行卡马西平治疗的临床医生将更好地了解皮肤不良反应风险，并可以做出改进的个性化治疗决策。这种快速、具有成本效益的检测方法使更多需要卡马西平治疗的患者受益于其使用。FundingGenomind, Inc.