P161 The road less traveled: Calling a novel HLA-DPB1 allele against the ASHI proficiency testing consensus

We describe a novel HLA-DPB1 allele identified in an ASHI proficiency testing (PT) sample. We tested the sample by rSSO (One Lambda), SSP (One Lambda), Real Time-PCR (Linkage Biosciences), SBT (Conexio) and NGS (Immucor). Initial results by rSSO and SSP resulted in no assignment because probes and primers detected shared recognition sites between DPB1 ∗ 01:01 & 40:01 in exon 2 and a group of alleles including DPB1 ∗ 01:01 but not 40:01 in exon 3. Typing by Real Time-PCR & SBT resulted in DPB1 ∗ 01:01:01, 40:01 based on exon 2 sequences (Fig. 1-A). Mia Fora software auto-assigned DPB1 ∗ 01:01:01, 40:01 based on exon 2 as IMGT currently does not have reference sequences for DPB1 ∗ 40:01 beyond exon 2. However Mia Fora provides sequence data for exon 3 and most of exon 4 for DPB1 ∗ 40:01 (Fig. 1-B). The non-DPB1 ∗ 01:01:01 allele has 0 mismatches in exon 2 but 7 mismatches in exon 3 with DPB1 ∗ 40:01. We identified DPB1 ∗ 39:01 to have 0 mismatches in exon 3 and 1 mismatch in exon 2. This variant has a novel non-synonymous G  >  T substitution at the 2nd nucleotide of codon 84 (Gly  >  Val) in DPB1 ∗ 39:01 or a recombination between exon 2 of DPB1 ∗ 40:01 and exon 3 of DPB1 ∗ 39:01. DPB1 ∗ 39:01v also has a synonymous C  >  T substitution at the 3rd position of codon 3 in exon 1 (Data not shown). Exclusion of exon 3 probes in rSSO assigns DPB1 ∗ 01:01:01, 40:01 (Fig. 1-C). We designed hemizygous primers to sequence DPB1 ∗ 39:01v by Sanger’s SBT and results are concordant with NGS for both substitutions (Fig. 1-D). The sequence was submitted to Gene Bank (KX121039) and IMGT (HWS10026456). The haplotype carrying DPB1 ∗ 39:01v is predicted as A ∗ 30:01, C ∗ 04:01, B ∗ 53:01, DRB1 ∗ 03:02, DRB3 ∗ 02:02, DQA1 ∗ 04:01, DQB1 ∗ 04:02, DPA1 ∗ 02:01. With exception of our lab 56/57 labs participating in this PT who reported results for this allele made the assignment of DPB1 ∗ 01:01, 40:01 (98% consensus). Table 1 shows unsupervised assignments by different methods in our, number of adjustments needed to make different assignments & number of labs using various methods who participated in this HLA Class II PT. Download high-res image (470KB) Download full-size image