Abstract
INTRODUCTION: Labcorp’s DNA Identification division offers HLA typing at various levels of resolution using commercially available kits as well as lab developed assays. A patient sample was received by the lab requiring HLA typing for a disease association screen, and was found to have anomalous HLA-A results. OBJECTIVES: To investigate a patient typing with results initially showing multiple variants to any known reference sequence. METHODS: This sample was sequenced using a lab developed whole gene assay that utilizes Pacific Biosciences’s® (PacBio) SMRT® NGS sequencing technology on a Sequel® I with LAA2 contig generation. Due to ambiguous results, the sample was repeated for confirmation using the same test, but a more sensitive data pipeline was used. Subreads coming off of the sequencer were converted to high fidelity or Closed Circular Consensus (CCS) reads using the PBAA tool from PacBio®. The sample was also processed with the GenDx NGSgo®-MX11-3 HLA typing kit with Illumina® sequencing, and the results were sent to GenDx for analysis. RESULTS: Initial testing yielded a high resolution fourth field result for HLA Class I, though the result remained ambiguous. Repeat sequencing resulted three HLA-A alleles with a similar number of CCS reads. The sample was typed as A*01:01:01:01, A*02:01:01:01, and A*02:New which contained a large exonic deletion compared to the A*02:01:01:01 reference. GenDX output confirmed previous findings, indicating a clear first allele of A*01:01:01:01, and reads for the second allele split between A*02:01:01:01 and A*02:New. DISCUSSION: Using Pacbio® sequencing technology with CCS analysis, and confirmation with GenDx technology, it was determined that this individual possesses 3 HLA-A alleles. When aligned with 02:01:01:01, both Pacbio® and GenDx assays determined the A*02:New allele to have a 34bp deletion in exon 2 (position 196-229). As a result of the frame shift caused by this deletion, a premature stop codon was found 43bp downstream from the anomaly, potentially rendering this allele null. Possible explanations for the presence of three HLA-A alleles in one patient include partial allele loss during an autoimmune event, a mutation within a chromosomal duplication of the region, or the patient has undergone a hematopoietic stem cell transplant.