HLA alleles and haplotypes observed in 263 US families.

The 17th International HLA and Immunogenetics Workshop (IHIW) conducted a project entitled "The Study of Haplotypes in Families by NGS HLA". We investigated the HLA haplotypes of 1017 subjects in 263 nuclear families sourced from five US clinical immunogenetics laboratories, primarily as part of the evaluation of related donor candidates for hematopoietic stem cell and solid organ transplantation. The parents in these families belonged to five broad groups - African (72 parents), Asian (115), European (210), Hispanic (118) and "Other" (11). High-resolution HLA genotypes were generated for each subject using next-generation sequencing (NGS) HLA typing systems. We identified the HLA haplotypes in each family using HaplObserve, software that builds haplotypes in families by reviewing HLA allele segregation from parents to children. We calculated haplotype frequencies within each broad group, by treating the parents in each family as unrelated individuals. We also calculated standard measures of global linkage disequilibrium (LD) and conditional asymmetric LD for each ethnic group, and used untruncated and two-field allele names to investigate LD patterns. Finally we demonstrated the utility of consensus DNA sequences in identifying novel variants, confirming them using HLA allele segregation at the DNA sequence level.

Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop.

The 17th International HLA and Immunogenetics Workshop (IHIW) organizers conducted a Pilot Study (PS) in which 13 laboratories (15 groups) participated to assess the performance of the various sequencing library preparation protocols, NGS platforms and software in use prior to the workshop. The organizers sent 50 cell lines to each of the 15 groups, scored the 15 independently generated sets of NGS HLA genotyping data, and generated "consensus" HLA genotypes for each of the 50 cell lines. Proficiency Testing (PT) was subsequently organized using four sets of 24 cell lines, selected from 48 of 50 PS cell lines, to validate the quality of NGS HLA typing data from the 34 participating IHIW laboratories. Completion of the PT program with a minimum score of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci satisfied the requirements to submit NGS HLA typing data for the 17th IHIW projects. Together, these PS and PT efforts constituted the 17th IHIW Quality Control project. Overall PT concordance rates for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 were 98.1%, 97.0% and 98.1%, 99.0%, 98.6%, 98.8%, 97.6%, 96.0%, 99.1%, 90.0% and 91.7%, respectively. Across all loci, the majority of the discordance was due to allele dropout. The high cost of NGS HLA genotyping per experiment likely prevented the retyping of initially failed HLA loci. Despite the high HLA genotype concordance rates of the software, there remains room for improvement in the assembly of more accurate consensus DNA sequences by NGS HLA genotyping software.

Expression of activating KIR2DS2 and KIR2DS4 genes after hematopoietic cell transplantation: relevance to cytomegalovirus infection.

The important role of activating killer immunoglobulin-like receptors (KIRs) in protecting against cytomegalovirus (CMV) reactivation has been described previously in patients undergoing hematopoietic cell transplantation (HCT). More specifically, the presence of multiple activating KIRs and the presence of at least KIR2DS2 and KIR2DS4 in the donor genotype identified a group of HCT patients at low risk for CMV reactivation. However, CMV infection still occurs in patients with the KIR protective genotype, and the question has been raised as to whether this is related to the lack of KIR expression. In this report, expression of the KIR2DS2 and KIR2DS4 genes, as measured by mRNA-based quantitative polymerase chain reaction in both the donor cells and the HCT recipient cells, was studied relative to CMV reactivation. In the control samples from healthy donors, the median range for KIR2DS2 and KIR2DS4 expression was low, with 35% of donors considered null-expressers. Interestingly, KIR2DS2 and KIR2DS4 expression was elevated after HCT compared with donor expression before HCT, and was significantly elevated in CMV viremic compared with CMV nonviremic HCT recipients. The CMV seropositivity of donors was not associated with activating KIR expression, and donor null expression in those with the KIR2DS2 or KIR2DS4 genotype was not predictive for CMV reactivation in the recipient. After controlling for other transplant factors, including donor type (sibling or unrelated), transplant source (bone marrow or peripheral blood stem cells), and acute GVHD grade, regression analysis of elevated KIR gene expression found an association for both KIR2DS2 and KIR2DS4, with a 7-fold increase in risk for CMV reactivation. We speculate that the elevated activating KIR expression in CMV-viremic HCT recipients is either coincidental with factors that activate CMV or is initiated by CMV or cellular processes responsive to such CMV infection reactivation.

The effect of single and combined activating killer immunoglobulin-like receptor genotypes on cytomegalovirus infection and immunity after hematopoietic cell transplantation.

It has been shown that activating killer Ig-like receptor (aKIR) genes are important for control of cytomegalovirus (CMV) reactivation after hematopoietic cell transplantation (HCT). To date, using the broad classification of KIR haplotypes A and B, the precise role of individual KIR genes in the control of infection cannot be discerned. To address this, a consecutive case series of 211 non-T cell-depleted HCT patients all at risk for CMV were monitored biweekly for CMV DNA in plasma by quantitative polymerase chain reaction (Q-PCR) and at intervals for CMV-specific T cell immunity. Comparing patients with CMV reactivation (n = 152) to those with no reactivation (n = 59), the presence of specific aKIR haplotypes in the donor, but not in the recipient, were associated with protection from CMV reactivation and control of peak plasma CMV DNA (P < .001). A donor aKIR profile, predictive for low risk of CMV reactivation, contained either aKIR2DS2 and aKIR2DS4 or had >/=5 aKIR genes. Neither donor nor recipient inhibitory KIR (iKIR) played a role in a protective effect. CD4(+)- and CD8(+)-specific CMV immunity did not explain reduced CMV infection. The initial control of CMV infection after HCT is managed by aKIR functions, and donor aKIR haplotypes deserve further evaluation in donor selection for optimized HCT outcome.

A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling.

In RNA site-directed spin labeling (SDSL) studies, structural and dynamic information at the individual RNA nucleotide level is derived from the observed electron paramagnetic resonance spectrum of a covalently attached nitroxide. A systematic approach for RNA SDSL is to establish a library that categorizes observed spectral lineshapes based on known RNA structures, thus enabling lineshape-based structure identification at any RNA site. To establish the first RNA SDSL library, selective secondary structure elements have been systematically engineered into a model RNA. Nitroxide lineshapes reporting features specific to each element were obtained utilizing a new avidin-tethering scheme for suppressing spectral effects due to uniform RNA tumbling. The data demonstrated two key features required for a SDSL library with a predicting power: (i) spectral divergence--distinctive lineshape for different elements; and (ii) spectral convergence--similar lineshape for the same element in different contexts. This sets the foundation for further RNA SDSL library development.