Acquired aplastic anemia (aAA) is a non-malignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20–25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin in 22 patients. We found somatic mutations in sixteen patients (72.7%) with a median disease duration of 1 year; twelve (66.7%) were patients with pediatriconset aAA. Fifty-eight mutations in 51 unique genes were primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with 7 mutations. Only two mutations were in genes recurrently-mutated in MDS. Two patients had oligoclonal loss of HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B(p.N642H), CAMK2G(p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging― immune escape and increased proliferation. Our findings expand conceptual understanding of this non-neoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes, and for designing personalized treatment strategies.

The high degree of polymorphism at HLA class I and class II loci makes high resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results due to incomplete genomic coverage and inability to set phase for HLA haplotype determination. The 454 Life Sciences GS FLX next generation sequencing system coupled with Conexio ATF software can provide very high resolution HLA genotyping. High throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, and DRB3, DRB4 and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs (bp) and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and Conexio ATF software allows reliable identification of HLA genotypes at high resolution.

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Summary Human leukocyte antigens (HLA) genes play an important role in the success of organ transplantation and are associated with autoimmune and infectious diseases. Current DNA based genotyping methods, including Sanger sequence-based typing (SSBT), have identified a high degree of polymorphism. This level of polymorphism makes high-resolution HLA genotyping challenging, resulting in ambiguous typing results due to an inability to resolve phase and/or defining polymorphisms lying outside the region amplified. Next generation sequencing (NGS) may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods. The NGS HLA sequencing project of the 16IHIW aimed to discuss the different approaches to; (i) template preparation including short and long range PCR amplicons, exome capture and whole genome; (ii) sequencing platforms, including GS 454 FLX, Ion Torrent PGM, Illumina MiSeq/HiSeq and Pacific Biosciences SMRT; (iii) data analysis, specifically allele calling software. The pilot studies presented at the workshop demonstrated that although individual sequencers have very different performance characteristics, all produced sequence data suitable for the resolution of HLA genotyping ambiguities. The developments presented at this workshop clearly highlight the potential benefits of NGS in the HLA laboratory.