A Detailed View of KIR Haplotype Structures and Gene Families as Provided by a New Motif-based Multiple Sequence Alignment

Human chromosome 19q13.4 contains genes encoding killer-cell immunoglobulin-like receptors (KIR). Reported haplotype lengths range from 67 to 269 kilobases and contain 4 to 18 genes. The region has certain properties such as single nucleotide variation, structural variation, homology, and repetitive elements that make it hard to align accurately beyond single gene alleles. To the best of our knowledge, a multiple sequence alignment of KIR haplotypes has never been published or presented. Such an alignment would be useful to precisely define KIR haplotypes and loci, provide context for assigning alleles (especially fusion alleles) to genes, infer evolutionary history, impute alleles, interpret and predict co-expression, and generate markers. In order to extend the framework of KIR haplotype sequences in the human genome reference, 27 new sequences were generated including 24 haplotypes from 12 individuals of African American ancestry that were selected for genotypic diversity and novelty to the reference, to bring the total to 68 full length genomic KIR haplotype sequences. We leveraged these data and tools from our long-read KIR haplotype assembly algorithm to define and align KIR haplotypes at <5 kb resolution on average. We then used a standard alignment algorithm to refine that alignment down to single base resolution. This processing demonstrated that the high-level alignment recapitulates human-curated annotation of the human haplotypes as well as a chimpanzee haplotype. Further, assignments and alignments of gene alleles were consistent with their human curation in haplotype and allele databases. These results define KIR haplotypes as 14 loci containing 9 genes. The multiple sequence alignments have been applied in two software packages as probes to capture and annotate KIR haplotypes and as markers to genotype KIR from WGS.

Efficient Sequencing, Assembly, and Annotation of Human KIR Haplotypes

The homology, recombination, variation, and repetitive elements in the natural killer-cell immunoglobulin-like receptor (KIR) region has made full haplotype DNA interpretation impossible without physical separation of chromosomes. Here, we present a new approach using long-read sequencing to efficiently capture, sequence, and assemble diploid human KIR haplotypes. Sequences for capture probe design were derived from public full-length gene and haplotype sequences. IDT xGen® Lockdown probes were used to capture 2-8 kb of sheared DNA fragments followed by sequencing on a PacBio Sequel. The sequences were error corrected, binned, and then assembled using the Canu assembler. The assembly was evaluated on 16 individuals (8 African American and 8 Europeans) from whom ground truth was known via long-range sequencing on fosmid-isolated chromosomes. Using only 18 capture probes, the results show that the assemblies cover 97% of the GenBank reference, are 99.97% concordant, and it takes only 1.8 contigs to cover 75% of the reference. We also report the first assembly of diploid KIR haplotypes from long-read WGS, including the first sequencing of cB05∼tB01, which pairs a KIR2DS2/KIR2DS3 fusion with the tB01 region. Our targeted hybridization probe capture and sequencing approach is the first of its kind to fully sequence and phase all diploid human KIR haplotypes, and it is efficient enough for population-scale studies and clinical use.

Interactions between Donor Activating Killer Immunoglobulin-like Receptors (KIRs) and Somatic Mutations and Their Association with Outcomes after Allogeneic Hematopoietic Cell Transplant for Acute Myeloid Leukemia (AML)

Background: Graft-versus-leukemia (GVL) responses after allogeneic hematopoietic cell transplantation (alloHCT) for AML are mediated by alloreactive donor-derived immune effector cells including T lymphocytes and natural killer (NK) cells. The function of NK cells is regulated by inhibitory and activating signals mediated through cell-surface receptors, including KIRs. Various models of NK cell alloreactivity have been associated with post-transplant outcomes, including leukemia relapse. However, these results have varied widely between different investigators employing similar models of NK cell alloreactivity. Assessment of somatic mutations in AML on post-transplant outcomes has not been investigated in the context of KIR profiles. Methods: In this single-institution retrospective cohort study, we investigated KIR haplotypes (haplotype AA vs. Bx [associated with multiple activating KIRs]; Cooley S., et al. Blood. 113:726-732. 2009) in the context of somatic mutations. We included 34 adult patients with AML who underwent alloHCT from a matched related donor from 2006 to 2013. A targeted multi-amplicon deep NGS panel of 79 commonly mutated genes in myeloid neoplasia was performed. Post-HCT outcomes were assessed based on mutational status and KIR haplotype with Kaplan-Meier method and log-rank test. Results: Median age at transplant was 54 (range 31-73). Cytogenetic risk groups were 9% favorable, 56% intermediate, and 35% poor based on 2017 ELN classification. HCT-CI scores included 26% low, 32% intermediate, and 41% high. Disease risk group defined by ASTCT included 71% low, 26% intermediate, and 3% high. Disease status at HCT included 74% CR1 and 26% CR2. Frequencies of somatic mutations prior to HCT were: 21% DNMT3A, 18% IDH2, 9% each for STAG2 and NRAS, 6% each for ASXL1, JAK2, PHF6, RUNX1, TET2, and 3% each for CBL, FLT3, NPM1, and U2AF1. Overall, 53% of patients had at least 1 mutation: 24%, 18%, 9%, and 3% of patients had 1, 2, 3, and 4 mutations, respectively. 41% were carriers of KIR haplotype AA, and 59% were haplotype Bx. Relapse (p=0.40), relapse-free (p=0.33), and overall survival (p=0.30) between haplotypes AA and Bx were not statistically different. However, when considering somatic mutations in the context of KIR haplotypes, those with any somatic mutation (n= 18) present had inferior relapse-free (p=0.002) and overall survival (p=0.002; figures A-B) as compared to those with none. Further assessment of outcomes was then considered for those who had the following poor prognostic mutations (n=12): ASXL1, DNMT3A, FLT3, NRAS, RUNX1, and TET2. KIR haplotype AA with one or more of these mutations was associated with inferior relapse-free (p=0.05) and overall survival (p=0.008). At median follow-up of 83 (range 66-137) months, 38% were alive. Non-relapse mortality rates were 21% (9-36) at 1 year and 29% (15-39) at 3 years. The most common causes of death for all patients were relapse (48%) followed by infection (33%). Conclusion: In presence of somatic mutations, carrying KIR haplotypes Bx was associated with better survival in AML post-alloHCT. The presence of multiple activating KIRs may also help mitigate the worse prognosis associated with some of the more deleterious somatic mutations in AML. These observations may have implications for improving patient risk stratification prior to transplant and optimizing donor selection. Future investigation with larger cohorts interrogating KIR haplotypes in the context of pre-transplant AML somatic mutations on post-transplant outcomes may further elucidate which patients may benefit most from transplant. Disclosures Nazha: Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee. Mukherjee:Bristol-Myers Squibb: Speakers Bureau; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Advani:Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Abbvie: Research Funding. Gerds:Sierra Oncology: Research Funding; Incyte: Consultancy, Research Funding; CTI Biopharma: Consultancy, Research Funding; Imago Biosciences: Research Funding; Celgene Corporation: Consultancy, Research Funding; Roche: Research Funding; Pfizer: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Majhail:Mallinckrodt: Honoraria; Atara Bio: Consultancy; Anthem, Inc.: Consultancy; Nkarta: Consultancy; Incyte: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.