OR46 Detecting low level clonal somatic mutation in HLA genes using next-generation sequencing in the presence of aplastic anemia

Abstract

Aim Next-generation sequencing (NGS) has assisted in the full characterization of HLA alleles, allowing in-depth studies of how these genes can play a direct role in the pathophysiology of many diseases outside of the context of transplantation. Here, we sought to determine the degree to which NGS can be used to characterize clonal somatic mutations acquired in HLA genes during the progression of aplastic anemia. Methods Omixon’s Holotype Kit was used to sequence HLA-A, B, C and DRB1 on the Illumina MiSeq targeting an ultra-high depth of coverage of 10,000 across the length of each gene. Omixon’s Twin, GenDx’s NGSengine and a custom pipeline analyzed the HLA allele data. The custom pipeline applies GATK’s best practices and tunes the ploidy parameter to lower the detection threshold of somatic mutations in each HLA allele to 10% of the depth. Results The pathogenesis of aplastic anemia, an autoimmune disease where cytotoxic T lymphocytes (CTL) destroy hematopoietic stem cells (HSC), most likely includes the recognition of a self-antigen in the context of a particular HLA allele by CTLs. Loss of heterozygosity of HLA alleles has been shown to allow HSCs to escape recognition of the CTLs, gaining a selective advantage in the disease state. In one mechanism of immune escape, HSCs acquire loss of function somatic mutations in the HLA allele(s) targeted by CTLs. By sequencing the HLA alleles of aplastic anemia patients, persistent somatic mutations were able to be detected above the background noise level. The frequency of mutations found in this dataset varied, but the lowest frequency mutation found was at 9% depth of coverage compared to the germline HLA allele. Mutations observed in HLA genes include the loss of a single or multiple HLA alleles in an individual by either frameshift mutations with insertions/deletions in exons or point mutations that introduce premature stop codons or mutate the translation start site. Interestingly, we can detect instances of different clones of somatic mutations causing loss of function in the same HLA allele of a single patient. Conclusion The combination of NGS with the Holotype HLA kit and multiple analysis tools, enables detection of clinically significant loss of function somatic mutations present in at least 10% of the depth for the targeted HLA allele in confirmed aplastic anemia cases. D.S. Monos: Scientific/Medical Advisor; Company/Organization; Omixon. Other (Identify); Company/Organization; Royalty from Omixon .