Abstract 22 Anomalous HLA-A Typing Found in Patient Using PacBio Sequencing Technology

Abstract

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.