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Aim Swabs created from BLCLs transformed from peripheral blood mononuclear cells (PBMCs) or expanded from existing BLCLs are used in the Be The Match Registry (BTMR) HLA QC program. The aim of this project was to document mutation as a cause of HLA typing discrepancies observed in 2 BLCLs. The first discrepancy arose in expanded BLCLs, sequenced in 2002 on a donor-derived BLCL. BLCL swabs were routinely typed multiple times using SSO and SBT methodologies as B∗49:01; however, in 2009, an SBT lab reported a potential new allele, B∗49:15, which was described in 2010 and confirmed in 2011 on the expanded BLCL by two labs. B∗49:15 differs from B∗49:01:01 by a single non-synonymous nucleotide polymorphism at NT 176 in exon 2, G/C. In 2013, a lab reported that the B49-specific amplicon showed a double peak, suggesting two alleles were present. Typing of other loci ruled out contamination. The second discrepancy arose in a BLCL transformed from patient-derived PBMC, originally typed as C∗02:09 using DNA extracted from PBMC in 2004. BLCL swabs repeatedly typed as C∗02:02 by SSO, SBT, and NGS. C∗02:09 differs from C∗02:02:01 by a synonymous nucleotide polymorphism at NT 315 in exon 2, A/G, and a non-synonymous polymorphism at NT 613 in exon 3, C/T. Methods PBMCs, fresh buccal swabs and expanded BLCL swabs were obtained for SBT and NGS of B∗49:15. Archived extracted DNA, PBMCs, failed cell culture, and transformed BLCL swabs were obtained for SBT of C∗02:09. Results Three labs confirmed B∗49:01:01 by SBT and NGS on archived PBMCs and newly obtained buccal swabs. NGS revealed the BLCL DNA contained both B∗49:15 and B∗49:01:01 in a 64/36 ratio and a 75/25 ratio in 2 samples. C∗02:09 was confirmed in archived extracted DNA used for the original allele description and in archived PBMC. SBT confirmed C∗02:02 on a failed cell culture from the PBMC to BLCL expansion and on additional transformed BLCL swabs. Conclusion It is likely these discrepancies arose as the result of mutations that occurred during the transformation of PBMC to BLCL and/or during the expansion of existing BLCLs. As a result of this work, B∗49:15 was deleted from the IMGT/HLA database in 6/2014. C∗02:09 was typed by 2 labs using DNA from the same cell, but should now be confirmed by SBT on a second subject. Purified DNA extracted from whole blood has begun to replace BLCL swabs in the BTMR QC program. |
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