Validation of Single Nucleotide Variant Assays for Human Leukocyte Antigen Haplotypes HLA-B*15:02 and HLA-A*31:01 Across Diverse Ancestral Backgrounds

Abstract

The human leukocyte antigen haplotypes HLA-B*15:02 and HLA-A*31:01 have been linked to life-threatening adverse drug reactions to the anticonvulsants carbamazepine and oxcarbazepine. Identification of these haplotypes via pharmacogenetic techniques facilitates implementation of precision medicine to prevent such reactions. Using reference samples from diverse ancestral origins, we investigated the test analytical validity (i.e., ability to detect whether or not the haplotypes were present or absent) of TaqMan assays for single nucleotide variants previously identified as potentially being able to “tag” these haplotypes. A TaqMan custom assay for rs10484555 and an inventoried assay for rs17179220 and were able to identify with 100% sensitivity and 100% specificity HLA-B*15:02 and HLA-A*31:01 respectively. A custom assay for rs144012689 that takes into account a neighboring single nucleotide variant with manual calling was also able to identify HLA-B*15:02 with 100% sensitivity and 100% specificity. A custom assay for rs106235 identified HLA-A*31:01 with 100% sensitivity and 95% specificity. The slight reduction in specificity for the latter was owing to another haplotype (HLA-A*33:03) also being detected. While any positive call using the rs106235 assay could therefore be further investigated, as the presence of the HLA-A*31:01 haplotype confers adverse drug reaction risk, the absence of false negatives (indexed by sensitivity) is more important than false positives. In summary, we present validated TaqMan assay methodology for efficient detection of HLA haplotypes HLA-B*15:02 and HLA-A*31:01. Our data are relevant for other genotyping technologies that identify, or have the potential to identify, these haplotypes using single nucleotide variants.

Single Nucleotide Variants as Proxies for HLA-A*31:01 in Native American Populations

“…In 1 KG_AMR, specificity was nearly complete (0.96) for rs1061235, and somewhat lower for rs17179220 (0.86), whereas PPV ranged between 0.42 (rs17179220) and 0.70 (rs1061235). All false positives for rs1061235 were due to the presence of this SNV in the HLA-A*33:01 haplotype, as previously reported for other populations ( Amstutz et al, 2013 ; He et al, 2015 ; Buchner et al, 2021 ). False positives for rs17179220, however, were not linked to HLA-A*33:01.…”

“…Two SNVs, namely rs1061235 (GRCh38.p13 chr 6, NC_000006.12:g.29945521A>T) and rs17179220 (GRCh38.p13 chr 6, NC_000006.12:g.29853458G>A) have been proposed for tagging the HLA-A*31:01 haplotype: de Bakker et al (2006) first reported complete linkage disequilibrium (LD) between rs1061235 and HLA-A*31:01 in the HapMap European (CEU) sample. Subsequent work revealed that rs1061235 is also linked to HLA-A*33 haplotypes, which impacts the predictive performance of rs1061235 to identify carriers of HLA-A*31:01 ( Amstutz et al, 2013 ; Thorstensen et al, 2014 ; Buchner et al, 2021 ). High LD of rs17179220 with HLA-A*31:01 was initially detected in Han Chinese ( Zhou et al, 2016 ), and subsequently verified in a large set of ancestrally diverse populations ( Erlichster et al, 2019 ).…”