80-P: DETECTION OF A NOVEL HLA-C∗04 ALLELE CONTAINING SEQUENCES FROM THE C∗07:02 ALLELE GROUP IN EXON 3

Abstract

HLA typing of two siblings of Asian descent, using sequence-specific oligonucleotide probes (SSOP) and sequence-based typing (SBT), resulted in the detection of a new allele. Detailed analysis of the probe and sequence data showed the new allele to be a recombination of sequences from HLA-C∗04 and HLA-C∗07 alleles. To confirm these results, the DNA samples were tested using sequence-specific primers (SSP) and an alternate sequencing-based method. DNA was extracted from whole blood using the QIAGEN EZ1 DNA Blood 350 μ l kit and BioRobot® EZ1 instrument. Low resolution HLA typing was obtained by performing DNA amplification and probe hybridization using the One Lambda LABType SSO HLA-C Locus kit. High resolution HLA typing was achieved by the sequencing of amplified DNA using the Celera AlleleSEQR® HLA-C PLUS SBT reagents. Confirmation testing of the novel C∗04 allele was performed by amplifying the DNA samples using the Olerup SSP HLA-C∗04, C∗07 kits and Invitrogen AllSet+™ Gold SSP Cw Locus High Res kits. Finally, HLA-C∗04 group specific DNA amplification (GSA) was performed and sequenced using the Texas BioGene HLAssure™ SBT HLA-C Typing Kit. Results from all SSP, SSOP and SBT testing confirmed the loss of heterozygosity somewhere between positions 1084 and 1282 in exon 3. The sequence in this region matches exactly to a group of C∗07:02-related alleles. Positions in exon 3 specific for C∗04:03 (4 snps) and C∗04:06 (5 snps) were completely homozygous. Outside this region, there were heterozygous positions detected in exon 3. C∗07:02 is the most common HLA-C allele amongst Asian/Pacific Islanders. C∗04:03 and C∗04:06 are also common allele types in this population. In the two DNA samples we tested, exon 2 contains the sequences for C∗04:03/06. The recombination event between HLA-C∗04 and HLA-C∗07 likely occurred somewhere between positions 1084 and 1282 in exon 3. This case illustrates the need for use of differing technologies for the identification of new alleles.