Abstracts / Human Immunology 76 (2015) 38–167
COMPOUND HLA/KIR GENOTYPES INFLUENCE RISK OF NASOPHARYNGEAL CARCINOMA (NPC) IN A SOUTHERN CHINESE COHORT. Xiaojiang Gao a, Minzhong Tang b, Pat Martin a, Yi Zeng b, Mary Carrington a. a National Cancer Institute, Frederick, MD, United States; b Beijing University of Technology, Beijing, China. Aim: Southern China has recorded some of the highest incidents of NPC. The risk of NPC in China has long been associated with HLA polymorphism. In the present study, we extended the genetic analysis to HLA interactions with KIR. Methods: The study cohort included three groups from Guangxi, southern China: 1405 NPC cases, 1362 healthy individuals who are negative for EBV/IgA/VCA (a precursor for NPC onset), and 1288 healthy individuals who are positive for EBV/IgA/VCA. Four-digit HLA typing was performed using SBT. The KIR proﬁles (presence and absence of KIR genes) were examined using SSP. Results: HLA-A⁄11:01 was conﬁrmed to be the major projective allele (OR = 0.58, P < 0.0001). A⁄11:02, on the other hand, showed no effect on NPC risk despite the fact that the two A⁄11 subtypes differ by a single amino acid at position 19, which is outside the peptide binding groove and therefore unlikely to inﬂuence peptide presentation. However, this single replacement is known to inﬂuence HLA interaction with KIR2DS4, with A⁄11:02 serving as a better ligand than A⁄11:01. The analysis of compound HLA/KIR genotypes showed that the presence or absence of KIR2DS4 did not change the protective effect of A⁄11:01 but affected A⁄11:02 association with NPC risk and EBV/IgA/VCA conversion. In the absence of KIR2DS4, A⁄11:02 showed an increased risk for NPC onset as compared to EBV/IgA/VCA positive healthy individuals (OR = 5.3, P < 0.0001). On the other hand the same compound genotype showed a decreased odds ratio for EBV/IgA/VCA seroconversion (OR = 0.25, P = 0.002). In other words, in the absence of KIR2DS4, individuals having A⁄11:02 are less likely to convert to EBV/IgA/VCA positivity,but among EBV/IgA/VCA positive individuals the A⁄11:02+/ KIR2DS4- genotype confers an increased risk for NPC onset. Conclusion: HLA/KIR interaction may play an important role in NPC pathogenesis.
COMPARING FRAGMENT SIZE WITH READ-LENGTH AND THEIR EFFECT ON HLA GENOTYPING BY NEXT-GENERATION SEQUENCING (NGS). Tracie Profaizer, Eszter Lazar-Molnar, Julio C. Delgado, Attila Kumanovics. ARUP Laboratories, Salt Lake City, UT, United States. Aim: We have previously studied the effect of shorter DNA fragments (less than 600 bp) on HLA typing by NGS of HLA-A, B, C, DRB1 and DQB on the Illumina MiSeq and found that 100–300 bp fragments produce more ambiguous typing results. Here, we evaluated the same reagents but altered the conditions to produce longer DNA fragments in order to determine if the number of ambiguous typing results could be reduced. We have previously shown that Illumina 2 250 sequencing resulted in less ambiguity than 2 150 bp sequencing. Here, we asked if further increasing the sequencing read length (2 300 bp) leads to additional gains in accuracy. Methods: Ten IHWG samples had long-range PCR performed for HLA-A, B, C, DRB1 and DQB1 using a combination of in-house and commercial primers. After PCR clean-up and quantiﬁcation, each sample’s 5 loci were pooled. Using the NEBNext dsDNA Fragmentase kit and various incubation times, the long-range PCR products were digested to produce 600–1200 bp fragments size selected with magnetic beads (AMPure Beads, Beckman Coulter) or the Blue PippinPrep (Sage Science). The NEBNext Library Prep Kit was used to complete library preparation. Unique indices were ligated onto each sample. Each sample library was measured for size and quantiﬁed. Next, all sample libraries were equimolarly pooled and two Illumina Reagent kits were run, v2 (500 cycle) and v3 (600 cycle), each loaded at a 10 pM concentration. Fastq ﬁles were analyzed using Omixon’s Target v.1.8.1 software for HLA genotyping. Results: Ninety-six percent of the IHWG samples, each with 5 loci and an average fragment size of 684 73 bp, typed correctly without any ambiguity. Samples with an average fragment size of 973 132 bp performed the same, 96%. There was no signiﬁcant difference in results between the 2 250 and 2 300 read length kits using samples with shorter (684 bp) or longer (973 bp) fragment sizes. Conclusions: We have now compared fragment sizes from 100–300 bp, to 900–1200 bp, and found that generally, fragment sizes 600 bp or greater produce the most correct and unambiguous HLA typing assignments. We have previously determined that 2 250 read length kit performs better than the 2 150 kit, and now we show that the 2 250 read length kit is preferred over the 2 300 bp kit due to its shorter run time (40 h vs. 60 h) with equal performance.