Reanalysis of unclear CMV status results from buccal swab samples of potential stem cell donors is an efficient donor registry strategy.

Many stem cell donor registries determine the cytomegalovirus (CMV) IgG serostatus at donor recruitment as it is an important marker for donor selection in the context of hematopoietic stem cell transplantation. To make sample collection less uncomfortable for the donor, we have developed a method that allows CMV status determination from buccal swab samples, thus avoiding blood drawing. However, the determination fails in some cases which leads to new donors being listed for donor search without CMV status, thus hindering donor searches. In this work, we evaluated the success rate of repeating CMV status analysis from a new swab. Our results show that about 90% of the samples could be successfully determined. Due to the great importance of the CMV status in donor search, we consider the retesting approach to be highly recommended for stem cell donor registries.

Estimation of German KIR Allele Group Haplotype Frequencies.

The impact of the highly polymorphic Killer-cell immunoglobulin-like receptor (KIR) gene cluster on the outcome of hematopoietic stem cell transplantation (HCST) is subject of current research. To further understand the involvement of this gene family into Natural Killer (NK) cell-mediated graft-versus-leukemia reactions, knowledge of haplotype structures, and allelic linkage is of importance. In this analysis, we estimate population-specific KIR haplotype frequencies at allele group resolution in a cohort of n = 458 German families. We addressed the polymorphism of the KIR gene complex and phasing ambiguities by a combined approach. Haplotype inference within first-degree family relations allowed us to limit the number of possible diplotypes. Structural restriction to a pattern set of 92 previously described KIR copy number haplotypes further reduced ambiguities. KIR haplotype frequency estimation was finally accomplished by means of an expectation-maximization algorithm. Applying a resolution threshold of ½ n, we were able to identify a set of 551 KIR allele group haplotypes, representing 21 KIR copy number haplotypes. The haplotype frequencies allow studying linkage disequilibrium in two-locus as well as in multi-locus analyses. Our study reveals associations between KIR haplotype structures and allele group frequencies, thereby broadening our understanding of the KIR gene complex.

High-Throughput MICA/B Genotyping of Over Two Million Samples: Workflow and Allele Frequencies.

MICA and MICB are ligands of the NKG2D receptor and thereby influence NK and T cell activity. MICA/B gene polymorphisms, expression levels and the amount of soluble MICA/B in the serum have been linked to autoimmune diseases, infections, and cancer. In hematopoietic stem cell transplantation, MICA matching between donor and patient has been correlated with reduced acute and chronic graft-vs.-host disease and improved survival. Hence, we developed an extremely cost-efficient high-throughput workflow for genotyping MICA/B for newly registered potential stem cell donors. Since mid-2017, we have genotyped over two million samples using NGS amplicon sequencing for MICA/B exons 2-5. In donors of German origin, MICA*008 is the most common MICA allele with a frequency of 42.3%. It is followed by MICA*002 (11.7%) and MICA*009 (8.8%). The three most common MICB alleles are MICB*005 (43.9%), MICB*004 (21.7%), and MICB*002 (18.9%). In general, MICB is less diverse than MICA and only 6 alleles, instead of 15, account for a cumulative allele frequency of 99.5%. In 0.5% of the samples we observed at least one allele of MICA or MICB which has so far not been reported to the IPD/IMGT-HLA database. By providing MICA/B typed voluntary donors, clinicians become empowered to include MICA/B into their donor selection process to further improve unrelated hematopoietic stem cell transplantation.

Allele-Level KIR Genotyping of More Than a Million Samples: Workflow, Algorithm, and Observations.

The killer-cell immunoglobulin-like receptor (KIR) genes regulate natural killer cell activity, influencing predisposition to immune mediated disease, and affecting hematopoietic stem cell transplantation (HSCT) outcome. Owing to the complexity of the KIR locus, with extensive gene copy number variation (CNV) and allelic diversity, high-resolution characterization of KIR has so far been applied only to relatively small cohorts. Here, we present a comprehensive high-throughput KIR genotyping approach based on next generation sequencing. Through PCR amplification of specific exons, our approach delivers both copy numbers of the individual genes and allelic information for every KIR gene. Ten-fold replicate analysis of a set of 190 samples revealed a precision of 99.9%. Genotyping of an independent set of 360 samples resulted in an accuracy of more than 99% taking into account consistent copy number prediction. We applied the workflow to genotype 1.8 million stem cell donor registry samples. We report on the observed KIR allele diversity and relative abundance of alleles based on a subset of more than 300,000 samples. Furthermore, we identified more than 2,000 previously unreported KIR variants repeatedly in independent samples, underscoring the large diversity of the KIR region that awaits discovery. This cost-efficient high-resolution KIR genotyping approach is now applied to samples of volunteers registering as potential donors for HSCT. This will facilitate the utilization of KIR as additional selection criterion to improve unrelated donor stem cell transplantation outcome. In addition, the approach may serve studies requiring high-resolution KIR genotyping, like population genetics and disease association studies.

Quantum theory of fermion production after inflation.

We show that quantum effects dramatically enhance the production of fermions following preheating after inflation in the early Universe in the presence of high excitations of bosonic quanta. As a consequence, fermions rapidly approach a quasistationary distribution with a thermal occupancy in the infrared, while the inflaton enters a turbulent scaling regime. The failure of standard semiclassical descriptions based on the Dirac equation with a homogeneous background field is caused by nonperturbatively high boson occupation numbers. During preheating the inflaton occupation number increases, thus leading to a dynamical mechanism for the enhanced production of fermions from the rescattering of the inflaton quanta. We comment on related phenomena in heavy-ion collisions for the production of quark matter fields from highly occupied gauge bosons.