Analysis of Genomic DNA from Medieval Plague Victims Suggests Long-Term Effect of Yersinia pestis on Human Immunity Genes.

Pathogens and associated outbreaks of infectious disease exert selective pressure on human populations, and any changes in allele frequencies that result may be especially evident for genes involved in immunity. In this regard, the 1346-1353 Yersinia pestis-caused Black Death pandemic, with continued plague outbreaks spanning several hundred years, is one of the most devastating recorded in human history. To investigate the potential impact of Y. pestis on human immunity genes, we extracted DNA from 36 plague victims buried in a mass grave in Ellwangen, Germany in the 16th century. We targeted 488 immune-related genes, including HLA, using a novel in-solution hybridization capture approach. In comparison with 50 modern native inhabitants of Ellwangen, we find differences in allele frequencies for variants of the innate immunity proteins Ficolin-2 and NLRP14 at sites involved in determining specificity. We also observed that HLA-DRB1*13 is more than twice as frequent in the modern population, whereas HLA-B alleles encoding an isoleucine at position 80 (I-80+), HLA C*06:02 and HLA-DPB1 alleles encoding histidine at position 9 are half as frequent in the modern population. Simulations show that natural selection has likely driven these allele frequency changes. Thus, our data suggest that allele frequencies of HLA genes involved in innate and adaptive immunity responsible for extracellular and intracellular responses to pathogenic bacteria, such as Y. pestis, could have been affected by the historical epidemics that occurred in Europe.

Immunogenetics in stem cell donor registry work: The DKMS example (Part 1).

Currently, stem cell donor registries include more than 35 million potential donors worldwide to provide HLA-matched stem cell products for patients in need of an unrelated donor transplant. DKMS is a leading stem cell donor registry with more than 9 million donors from Germany, Poland, the United States, the United Kingdom, India and Chile. DKMS donors have donated hematopoietic stem cells more than 80,000 times. Many aspects of donor registry work are closely related to topics from immunogenetics or population genetics. In this two-part review article, we describe, analyse and discuss these areas of donor registry work by using the example of DKMS. Part 1 of the review gives a general overview on DKMS and includes typical donor registry activities with special focus on the HLA system: high-throughput HLA typing of potential stem cell donors, HLA haplotype frequencies and resulting matching probabilities, and donor file optimization with regard to HLA diversity.

Immunogenetics in stem cell donor registry work: The DKMS example (Part 2).

DKMS is a leading stem cell donor registry with more than 9 million donors. Donor registry activities share many touch points with topics from immunogenetics or population genetics. In this two-part review article, we deal with these aspects of donor registry work by using the example of DKMS. In the second part of the review, we focus on donor typing of non-HLA genes, the impact of donor age, gender and CMV serostatus on donation probabilities, the identification of novel HLA, KIR and MIC alleles by high-throughput donor typing, the activities of the Collaborative Biobank and pharmacogenetics in the donor registry context.

HLA allele and haplotype frequencies of registered stem cell donors in Chile.

Patients in need of hematopoietic stem cell transplantation often rely on unrelated stem cell donors matched in certain human leukocyte antigen (HLA) genes. Donor search is complicated by the extensive allelic variability of the HLA system. Therefore, large registries of potential donors are maintained in many countries worldwide. Population-specific HLA characteristics determine the registry benefits for patients and also the need for further regional donor recruitment. In this work, we analyzed HLA allele and haplotype frequencies of donors of DKMS Chile, the first Chilean donor registry, with self-assessed "non-Indigenous" (n=92,788) and "Mapuche" (n=1,993) ancestry. We identified HLA alleles that were distinctly more abundant in the Chilean subpopulations than in worldwide reference populations, four of them particularly characteristic for the Mapuche subpopulation, namely B*39:09g, B*35:09, DRB1*04:07g, and DRB1*16:02g. Both population subsamples carried haplotypes of both Native American and European origin at high frequencies, reflecting Chile's complex history of admixture and immigration. Matching probability analysis revealed limited benefits for Chilean patients (both non-Indigenous and Mapuche) from donor registries of non-Chilean donors, thus indicating a need for ongoing significant donor recruitment efforts in Chile.

5 years DKMS Chile: approach, results and impact of the first unrelated stem cell donor center in Chile.

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed worldwide to treat blood cancer and other life-threatening blood disorders. As successful transplantation requires an HLA-compatible donor, unrelated donor centers and registries have been established worldwide to identify donors for patients without a family match. Ethnic minorities are underrepresented in large donor registries. Matching probabilities are higher when donors and patients share the same ethnic background, making it desirable to increase the diversity of the global donor pool by recruiting donors in new regions. Here, we report the establishment and the first 5 years of operation of the first unrelated stem cell donor center in Chile, a high-income country in South America with a population of over 19 million. Methods: We used online and in-person donor recruitment practices through patient appeals and donor drives in companies, universities, the armed forces, and public services. After confirmatory typing donors were subjected to medical work-up and cleared for donation. Results: We recruited almost 170,000 donors in 5 years. There were 1,488 requests received for confirmatory typing and donor availability checks, of which 333 resulted in medical work-up, leading to 194 stem cell collections. Products were shipped to Chile (48.5%) and abroad. Even when the COVID-19 pandemic challenged our activities, the number of donors recruited and shipped stem cell products remained steady. In Chile there was an almost 8-fold increase in unrelated donor transplantation activity from 16 procedures in 2016-2018 to 124 procedures in 2019-2021, mainly for pediatric patients following the center's establishment. We estimate that 49.6% of Chilean patients would find at least one matched unrelated donor in the global DKMS donor pool. Discussion: Establishing a DKMS donor center in Chile has significantly increased donor availability for Chilean patients and contributed to an increase of unrelated donor stem cell transplant activity.

Large case-control study indicates no association of KIR genotype and risk of developing acute myeloid leukemia.

Immunogenetic association studies may give rise to new hypotheses on the immune surveillance of cancer. We hypothesized that certain combinations of killer immunoglobulin-like receptor (KIR) and HLA genotypes may enhance natural killer (NK) cell immunity against nascent acute myeloid leukemia (AML) and, thereby, lead to a skewed genotype distribution among patients. For this purpose, we analyzed KIR and HLA genotypes of 1767 German patients with AML and compared the results with that of the data of 51 890 German volunteers who had registered with German bone marrow donor file (DKMS). Patient samples were retrieved from the Collaborative Biobank and the biorepository of the Study Alliance Leukemia. All samples were genotyped with high-resolution amplicon-based next-generation sequencing. Because of the large number of controls, this study was very sensitive to detect the impact of KIR genotype. Knowledge on KIRs and their cognate HLA ligands allowed for testing of several hypotheses of NK cell-mediated endogenous leukemia surveillance. We did not find significant differences between the 2 cohorts in regard to the presence or absence of single KIR genes. When grouped based on telomeric or centromeric gene content, the major haplotypes A/A, A/B, and B/B were equally distributed among patients and control subjects. Using information on KIRs and their HLA ligands, we further tested receptor-ligand models and summation models without revealing markedly significant differences between patients and controls, albeit we observed a trend pointing at a minor protective effect of a low number of inhibitory KIR/KIR-ligand pairs. The results suggest that the KIR/KIR-ligand genotype has no effect on the susceptibility for the development of de novo AML.

Graphical user interface for the haplotype frequency estimation software Hapl-o-Mat.

Population-specific human leukocyte antigen (HLA) haplotype frequencies are an essential basis of advanced algorithms for donor selection in unrelated hematopoietic stem cell transplantation. In 2016, we introduced Hapl-o-Mat, a versatile tool for haplotype frequency estimation based on an expectation-maximization algorithm (https://github.com/DKMS/hapl-o-Mat). Hapl-o-Mat is specifically tailored to the analysis of HLA genes and able to cope with the heterogeneous genotyping data usually found in donor registries. To make Hapl-o-Mat accessible to a wider range of users, we designed a graphical user interface module that considerably facilitates the interaction with the application (https://github.com/DKMS/hapl-o-Mat_GUI). We further provide a precompiled version of Hapl-o-Mat that can be used on Windows personal computers without dependency on additional software libraries (https://github.com/DKMS/hapl-o-Mat_WinBin). We are confident that these new, user-oriented features will encourage more researchers to apply Hapl-o-Mat to their data, thereby increasing knowledge and public availability of population-specific HLA haplotype frequencies.

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.

Frequencies of gene variant CCR5-Δ32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national DKMS donor centers.

Homozygous carriers of CCR5-Δ32, a gene variant of CC-type chemokine receptor 5 (CCR5), are highly resistant to infections with human immunodeficiency virus type 1 (HIV-1) and therefore preferred stem cell donors for HIV-infected patients. We analyzed CCR5 typing data of 1,333,035 potential hematopoietic stem cell donors enlisted with three national DKMS donor centers. Allele and genotype frequencies were determined for 87 countries of origin as self-assessed by the donors. CCR5-Δ32 allele frequencies ranged from 16.4% in the Norwegian sample to 0 in donors from Ethiopia. The highest CCR5-Δ32/Δ32 genotype frequency was found in the sample from the Faroe Islands (2.3%), whereas in 27 samples, predominantly of donors from Africa, Asia and South America, none of the individuals carried this genotype. The characteristic CCR5-Δ32 allele frequency decline from Northern to Southeastern Eurasia supports findings of earlier studies. With available HLA haplotype frequency information for the patient's ethnicity, our data allows upfront estimation of the probability that an HLA-matched donor with CCR5-Δ32/Δ32 genotype can be found for a patient in need of hematopoietic stem cell transplantation.

Simulation shows that HLA-matched stem cell donors can remain unidentified in donor searches.

The heterogeneous nature of HLA information in real-life stem cell donor registries may hamper unrelated donor searches. It is even possible that fully HLA-matched donors with incomplete HLA information are not identified. In our simulation study, we estimated the probability of these unnecessarily failed donor searches. For that purpose, we carried out donor searches in several virtual donor registries. The registries differed by size, composition with respect to HLA typing levels, and genetic diversity. When up to three virtual HLA typing requests were allowed within donor searches, the share of unnecessarily failed donor searches ranged from 1.19% to 4.13%, thus indicating that non-identification of completely HLA-matched stem cell donors is a problem of practical relevance. The following donor registry characteristics were positively correlated with the share of unnecessarily failed donor searches: large registry size, high genetic diversity, and, most strongly correlated, large fraction of registered donors with incomplete HLA typing. Increasing the number of virtual HLA typing requests within donor searches up to ten had a smaller effect. It follows that the problem of donor non-identification can be substantially reduced by complete high-resolution HLA typing of potential donors.

High-resolution HLA haplotype frequencies of stem cell donors in Germany with foreign parentage: how can they be used to improve unrelated donor searches?

In hematopoietic stem cell transplantation, human leukocyte antigens (HLA), usually HLA loci A, B, C, DRB1 and DQB1, are required to check histocompatibility between a potential donor and the recipient suffering from a malignant or non-malignant blood disease. As databases of potential unrelated donors are very heterogeneous with respect to typing resolution and number of typed loci, donor registries make use of haplotype frequency-based algorithms to provide matching probabilities for each potentially matching recipient/donor pair. However, it is well known that HLA allele and haplotype frequencies differ significantly between populations. We estimated high-resolution HLA-A, -B, -C, -DRB1 haplotype and allele frequencies of donors within DKMS German Bone Marrow Donor Center with parentage from 17 different countries: Turkey, Poland, Italy, Russian Federation, Croatia, Greece, Austria, Kazakhstan, France, The Netherlands, Republic of China, Romania, Portugal, USA, Spain, United Kingdom and Bosnia and Herzegovina. 5-locus haplotypes including HLA-DQB1 are presented for Turkey, Poland, Italy and Russian Federation. We calculated linkage disequilibria for each sample. Genetic distances between included countries could be shown to reflect geography. We further demonstrate how genetic differences between populations are reflected in matching probabilities of recipient/donor pairs and how they influence the search for unrelated donors as well as strategic donor center typings.

Regional HLA differences in Poland and their effect on stem cell donor registry planning.

Regional HLA frequency differences are of potential relevance for the optimization of stem cell donor recruitment. We analyzed a very large sample (n = 123,749) of registered Polish stem cell donors. Donor figures by 1-digit postal code regions ranged from n = 5,243 (region 9) to n = 19,661 (region 8). Simulations based on region-specific haplotype frequencies showed that donor recruitment in regions 0, 2, 3 and 4 (mainly located in the south-eastern part of Poland) resulted in an above-average increase of matching probabilities for Polish patients. Regions 1, 7, 8, 9 (mainly located in the northern part of Poland) showed an opposite behavior. However, HLA frequency differences between regions were generally small. A strong indication for regionally focused donor recruitment efforts can, therefore, not be derived from our analyses. Results of haplotype frequency estimations showed sample size effects even for sizes between n≈5,000 and n≈20,000. This observation deserves further attention as most published haplotype frequency estimations are based on much smaller samples.

Support of unrelated stem cell donor searches by donor center-initiated HLA typing of potentially matching donors.

Large registries of potential unrelated stem cell donors have been established in order to enable stem cell transplantation for patients without HLA-identical related donors. Donor search is complicated by the fact that the stored HLA information of many registered donors is incomplete. We carried out a project that was aimed to improve chances of patients with ongoing donor searches to find an HLA-matched unrelated donor. For that purpose, we carried out additional donor center-initiated HLA-DRB1 typing of donors who were only typed for the HLA loci A and B so far and were potential matches for patients in need of a stem cell transplant. In total, 8,861 donors were contacted for donor center-initiated HLA-DRB1 typing within 1,089 donor searches. 12 of these donors have donated stem cells so far, 8 thereof for their respective target patients. We conclude that chances of patients with ongoing donor searches to find an HLA-matched unrelated donor can indeed be improved by donor-center initiated typing that is carried out in addition to the standard donor search process. Our results also raise questions regarding the appropriate use of incompletely typed donors within unrelated donor searches.

High-resolution human leukocyte antigen allele and haplotype frequencies of the Polish population based on 20,653 stem cell donors.

We present high-resolution allele and haplotype frequency (HF) estimations of the Polish population based on more than 20,000 registered stem cell donors. Sequencing-based donor human leukocyte antigen (HLA) typing led to unambiguous typing results in most cases (between 94.3% for HLA-DRB1 and 96.9% for HLA-B). HF estimations were carried out with a new, validated implementation of the expectation-maximization algorithm that allowed processing of data with ambiguities. Our results confirm several earlier results, for example, the relative commonness of the haplotype A*25:01 g, B*18:01 g, C*12:03, DRB1*04:01 in the Polish population. Because of the large sample size, we were able to obtain results of unprecedented accuracy. The estimated population-specific HFs were then used to analyze questions of strategic donor registry planning. Simulated matching probabilities by donor file size suggest that there is a need for intense donor recruitment efforts in Poland despite the large German donor registry and the genetic relatedness of both populations. Based on the current German registry size of approximately 4 million donors, the recruitment of 100,000 Polish donors would produce a stronger increase in matching probabilities for Polish patients than the recruitment of 3.3 million additional German donors.

Estimation of high-resolution HLA-A, -B, -C, -DRB1 allele and haplotype frequencies based on 8862 German stem cell donors and implications for strategic donor registry planning.

Human leukocyte antigen (HLA) haplotype frequency distributions in specific populations can be applied to optimize both individual stem cell donor searches and donor registry planning. We present allele and haplotype frequencies derived from a data set of 8862 German stem cell donors who were typed at high resolution for the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes upon registration. Calculated haplotype frequencies were used to estimate the probability p to find matching donors subject to donor registry size n. The impact of various matching standards on p(n) was analyzed. When high-resolution matching for HLA-A, HLA-B, HLA-C, and HLA-DRB1 is required, p(1,000,000) is 0.678. The corresponding value for n = 7,000,000 is 0.859. In a scenario with low-resolution matching and no consideration of HLA-C, p(1,000,000) is 0.863 and thus larger than p(7,000,000) in the scenario with stricter matching requirements. As recent findings support the importance of high-resolution matching of HLA-A, HLA-B, HLA-C, and HLA-DRB1 for outcomes of hematopoietic stem cell transplantation, our results are highly relevant for strategic planning and resource allocation of donor centers and registries.