HLA-H*02:07 Is a Membrane-Bound Ligand of Denisovan Origin That Protects against Lysis by Activated Immune Effectors.

The biological relevance of genes initially categorized as "pseudogenes" is slowly emerging, notably in innate immunity. In the HLA region on chromosome 6, HLA-H is one such pseudogene; yet, it is transcribed, and its variation is associated with immune properties. Furthermore, two HLA-H alleles, H*02:07 and H*02:14, putatively encode a complete, membrane-bound HLA protein. Here we thus hypothesized that HLA-H contributes to immune homeostasis similarly to tolerogenic molecules HLA-G, -E, and -F. We tested if HLA-H*02:07 encodes a membrane-bound protein that can inhibit the cytotoxicity of effector cells. We used an HLA-null human erythroblast cell line transduced with HLA-H*02:07 cDNA to demonstrate that HLA-H*02:07 encodes a membrane-bound protein. Additionally, using a cytotoxicity assay, our results support that K562 HLA-H*02:07 inhibits human effector IL-2-activated PBMCs and human IL-2-independent NK92-MI cell line activity. Finally, through in silico genotyping of the Denisovan genome and haplotypic association with Denisovan-derived HLA-A*11, we also show that H*02:07 is of archaic origin. Hence, admixture with archaic humans brought a functional HLA-H allele into modern European and Asian populations.

Resolution of RHCE Haplotype Ambiguities in Transfusion Settings.

Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The RHCE mRNA length is compatible with full-length analysis and haplotype discrimination, but the RHCE mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an RHCE-specific RT-PCR followed by RHCE allele-specific sequencing enables analysis of cDNA RHCE haplotypes. All samples analyzed show full concordance between RHCE phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel RHCE allele (RHCE*03 c.340C>T).

HLA-F and LILRB1 Genetic Polymorphisms Associated with Alloimmunisation in Sickle Cell Disease.

Red blood cell (RBC) transfusion remains a critical component in caring for the acute and chronic complications of sickle cell disease (SCD). Patient alloimmunisation is the main limitation of transfusion, which can worsen anaemia and lead to delayed haemolytic transfusion reaction or transfusion deadlock. Although biological risk factors have been identified for immunisation, patient alloimmunisation remains difficult to predict. We aimed to characterise genetic alloimmunisation factors to optimise the management of blood products compatible with extended antigen matching to ensure the self-sufficiency of labile blood products. Considering alloimmunisation in other clinical settings, like pregnancy and transplantation, many studies have shown that HLA Ib molecules (HLA-G, -E, and -F) are involved in tolerance mechanism; these molecules are ligands of immune effector cell receptors (LILRB1, LILRB2, and KIR3DS1). Genetic polymorphisms of these ligands and receptors have been linked to their expression levels and their influence on inflammatory and immune response modulation. Our hypothesis was that polymorphisms of HLA Ib genes and of their receptors are associated with alloimmunisation susceptibility in SCD patients. The alloimmunisation profile of thirty-seven adult SCD patients was analysed according to these genetic polymorphisms and transfusion history. Our results suggest that the alloimmunisation of SCD patients is linked to both HLA-F and LILRB1 genetic polymorphisms located in their regulatory region and associated with their protein expression level.

Genetic and Functional Profiling of CD16-Dependent Natural Killer Activation Identifies Patients at Higher Risk of Cardiac Allograft Vasculopathy.

BACKGROUND: Cardiac transplantation is an effective therapy for end-stage heart failure. Because cardiac allograft vasculopathy (CAV) is the major cause of late mortality after heart transplant (HT), there is a need to identify markers that reflect inflammatory or cytotoxic immune mechanisms contributing to its onset. Noninvasive and early stratification of patients at risk remains a challenge for adapting individualized therapy. The CD16 (Fc-gamma receptor 3A [FCGR3A]) receptor was recently identified as a major determinant of antibody-mediated natural killer (NK) cell activation in HT biopsies; however, little is known about the role of CD16 in promoting allograft vasculopathy. This study aimed to investigate whether markers that reflect CD16-dependent circulating NK cell activation may identify patients at higher risk of developing CAV after HT. METHODS: Blood samples were collected from 103 patients undergoing routine coronarography angiography for CAV diagnosis (median 5 years since HT). Genomic and phenotypic analyses of FCGR3A/CD16 Fc-receptor profiles were compared in CAV-positive (n=52) and CAV-free patients (n=51). The levels of CD16 expression and rituximab-dependent cell cytotoxic activity of peripheral NK cells in HT recipients were evaluated using a noninvasive NK-cellular humoral activation test. RESULTS: Enhanced levels of CD16 expression and antibody-dependent NK cell cytotoxic function of HT recipients were associated with the FCGR3A-VV genotype. The frequency of the FCGR3A-VV genotype was significantly higher in the CAV+ group (odds ratio, 3.9; P=0.0317) than in the CAV- group. The FCGR3A-VV genotype was identified as an independent marker correlated with the presence of CAV at the time of coronary angiography by using multivariate logistic regression models. The FCGR3A-VV genotype was also identified as a baseline-independent predictor of CAV risk (odds ratio, 4.7; P=0.023). CONCLUSIONS: This study unravels a prominent role for the CD16-dependent NK cell activation pathway in the complex array of factors that favor the progression of transplant arteriosclerosis. It highlights the clinical potential of a noninvasive evaluation of FCGR3A/CD16 in the early stratification of CAV risk. The recognition of CD16 as a major checkpoint that controls immune surveillance may promote the design of individualized NK cell-targeted therapies to limit vascular damage in highly responsive sensitized patients. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01569334.

Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

Validation of KIR3DL1 alleles assigned by next generation sequencing.

We describe the novel KIR3DL1*182 allele and confirmed the 3DL1*15002 allele.

Posttranscriptional deregulation of MYC via PTEN constitutes a major alternative pathway of MYC activation in T-cell acute lymphoblastic leukemia.

Cumulative evidence indicates that MYC, one of the major downstream effectors of NOTCH1, is a critical component of T-cell acute lymphoblastic leukemia (T-ALL) oncogenesis and a potential candidate for targeted therapy. However, MYC is a complex oncogene, involving both fine protein dosage and cell-context dependency, and detailed understanding of MYC-mediated oncogenesis in T-ALL is still lacking. To better understand how MYC is interspersed in the complex T-ALL oncogenic networks, we performed a thorough molecular and biochemical analysis of MYC activation in a comprehensive collection of primary adult and pediatric patient samples. We find that MYC expression is highly variable, and that high MYC expression levels can be generated in a large number of cases in absence of NOTCH1/FBXW7 mutations, suggesting the occurrence of multiple activation pathways in addition to NOTCH1. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down-regulation of PTEN, and that NOTCH1(m) might play a dual transcriptional and posttranscriptional role in this process. Altogether, our data lend further support to the significance of therapeutic targeting of MYC and/or the PTEN/AKT pathways, both in GSI-resistant and identified NOTCH1-independent/MYC-mediated T-ALL patients.

Underestimation of glyphosate intake by the methods currently used by regulatory agencies.

The acceptable daily intake (ADI) is an estimate of the amount of a substance in food or beverages that can be consumed daily over a lifetime without presenting an appreciable risk to health. To assess the risk of ingesting glyphosate, regulatory agencies compare glyphosate daily intake to ADI. Based on published data on urine glyphosate levels measured according to known quantities of ingested glyphosate, our objectives were to test the robustness of the mathematical model currently used to calculate glyphosate daily intake, and to propose alternative models based on urinary excretion kinetics. Our results support that the quantity of ingested glyphosate is systematically underestimated by the model currently used by regulatory agencies, whereas the other models evaluated showed better estimations, with differences according to gender. Our results also show a great variability between individuals, leading to some uncertainties notably with regards to the ADI, and further support that glyphosate excretion varies significantly among individuals who follow a similar dosing regimen. In conclusion, our study highlights the lack of reliability of assessment processes carried out by regulatory agencies for glyphosate in particular, and pesticides in general, and questions the relevance of such processes supposed to safeguard human health and the environment.

HLA-F transcriptional and protein differential expression according to its genetic polymorphisms.

Many specificities single out HLA-F: its structure, expression regulation at cell membrane and function. HLA-F mRNA is detected in the most cell types and the protein is localized in the ER and Golgi apparatus. When expressed at cell surface, HLA-F may be associated to ß2-microglobulin and peptide or expressed as an open-conformer molecule. HLA-F reaches the membrane upon activation of different primary cell types and cell-lines. HLA-F has its highest affinity for the KIR3DS1-activating NK receptor, but also binds inhibitory immune receptors. Some studies reported that HLA-F expression is associated with its genotype. Higher HLA-F mRNA expression associated with F*01:01:02, and 3 noncoding SNPs, rs1362126, rs2523405, and rs2523393, located in HLA-F-AS1 or upstream the HLA-F sequence were associated with HLA-F mRNA expression. Given the implication of HLA-F in many clinical setting, and the undisclosed process of its expression regulation, we aim to confirm the effect of the aforementioned SNPs with HLA-F transcriptional and protein expression. We analyzed the distribution, frequency and linkage disequilibrium of these SNPs at worldwide scale in the 1000 Genomes Project samples. Influence on the genotype of each SNP on HLA-F expression was explored using RNAseq data from the 1000 Genomes Project, and using Q-PCR and intracellular cytometry in PBMC from healthy individuals. Our results show that the SNPs under studied displayed remarkably different allelic proportion according to geography and confirm that rs1362126, rs2523405, and rs2523393 displayed the most concordant results, with the highest effect size and a double-dose effect.

HSFY genes and the P4 palindrome in the AZFb interval of the human Y chromosome are not required for spermatocyte maturation.

BACKGROUND: Recurrent AZFb deletions on the human Y chromosome are associated with an absence of ejaculated spermatozoa consequent to a meiotic maturation arrest that prevents the progression of germ cells to haploid stages. The extreme rarity of partial deletions has hampered the identification of the AZFb genes required for normal meiotic stages. The critical interval, refined by two overlapping deletions associated with full spermatogenesis (AZFc and b1/b3), measures over 4 Mb and contains 13 coding genes: CDY2, XKRY, HSFY1, HSFY2, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2 and four copies of RBMY. METHODS AND RESULTS: We screened 1186 men from infertile couples for Y chromosome deletions, and identified three unrelated oligozoospermic men and one azoospermic man who carry an identical 768 kb deletion resulting in loss of the entire P4 palindrome, including both HSFY genes, the only coding genes within the deletion interval. This 768 kb deletion was not found in 1179 control men. The deletion breakpoints share only 4 bp of nucleotide identity, revealing that the deletions are not recurrent, but are descendants of a founding deletion. Confirming this, we find that all four men carry a Y chromosome of the same highly defined haplogroup (R1b1b1a1b) (incidence 30% in Southern France), although further haplotype analyses showed that they were not closely related. CONCLUSIONS: Although the HSFY deletion is restricted to our infertile group, it has been transmitted naturally over many generations, indicating that HSFY genes make only a slight contribution to male fertility. Importantly, our study formally excludes HSFY genes as the AZFb factor required for progression through meiosis.

Quantifiable urine glyphosate levels detected in 99% of the French population, with higher values in men, in younger people, and in farmers.

France is the first pesticide-consuming country in Europe. Glyphosate is the most used pesticide worldwide and glyphosate is detected in the general population of industrialized countries, with higher levels found in farmers and children. Little data was available concerning exposure in France. Our objective was to determine glyphosate levels in the French general population and to search for an association with seasons, biological features, lifestyle status, dietary habits, and occupational exposure. This study includes 6848 participants recruited between 2018 and 2020. Associated data include age, gender, location, employment status, and dietary information. Glyphosate was quantified by a single laboratory in first-void urine samples using ELISA. Our results support a general contamination of the French population, with glyphosate quantifiable in 99.8% of urine samples with a mean of 1.19 ng/ml + / - 0.84 after adjustment to body mass index (BMI). We confirm higher glyphosate levels in men and children. Our results support glyphosate contamination through food and water intake, as lower glyphosate levels are associated with dominant organic food intake and filtered water. Higher occupational exposure is confirmed in farmers and farmers working in wine-growing environment. Thus, our present results show a general contamination of the French population with glyphosate, and further contribute to the description of a widespread contamination in industrialized countries.

Mobilisation of HLA-F on the surface of bronchial epithelial cells and platelets in asthmatic patients.

Uncontrolled inflammation of the airways in chronic obstructive lung diseases leads to exacerbation, accelerated lung dysfunction and respiratory insufficiency. Among these diseases, asthma affects 358 million people worldwide. Human bronchial epithelium cells (HBEC) express both anti-inflammatory and activating molecules, and their deregulated expression contribute to immune cell recruitment and activation, especially platelets (PLT) particularly involved in lung tissue inflammation in asthma context. Previous results supported that HLA-G dysregulation in lung tissue is associated with immune cell activation. We investigated here HLA-F expression, reported to be mobilised on immune cell surface upon activation and displaying its highest affinity for the KIR3DS1-activating NK receptor. We explored HLA-F transcriptional expression in HBEC; HLA-F total expression in PBMC and HBEC collected from healthy individuals at rest and upon chemical activation and HLA-F membrane expression in PBMC, HBEC and PLT collected from healthy individuals at rest and upon chemical activation. We compared HLA-F transcriptional expression in HBEC from healthy individuals and asthmatic patients and its surface expression in HBEC and PLT from healthy individuals and asthmatic patients. Our results support that HLA-F is expressed by HBEC and PLT under healthy physiological conditions and is retained in cytoplasm, barely expressed on the surface, as previously reported in immune cells. In both cell types, HLA-F reaches the surface in the inflammatory asthma context whereas no effect is observed at the transcriptional level. Our study suggests that HLA-F surface expression is a ubiquitous post-transcriptional process in activated cells. It may be of therapeutic interest in controlling lung inflammation.

FCGR2A-HH Gene Variants Encoding the Fc Gamma Receptor for the C-Reactive Protein Are Associated with Enhanced Monocyte CD32 Expression and Cardiovascular Events' Recurrence after Primary Acute Coronary Syndrome.

Fcγ receptors (FcγRs) interact with the C-reactive protein (CRP) and mediate activation of inflammation-related pathogenic mechanisms affecting cardiovascular health. Our study evaluated whether FcγRIIA and FcγRIIIA profiles are associated with the recurrence of adverse cardiovascular events during the first year after a primary acute coronary syndrome (ACS). The primary endpoint was the recurrence of cardiovascular events (RCE), identified as a composite outcome comprising acute heart failure (AHF) and major adverse cardiovascular events (MACE). We obtained blood samples of 145 ACS patients to measure hsCRP circulating levels, to identify FcγRIIA-131RH rs1801274 and FcγRIIIA-158FV rs396991 polymorphisms, to analyze circulating monocytes and NK cell subsets expressing CD16 and CD32, and to detect serum-mediated FCGR2A-HH activation by luciferase reporter assays. The hsCRP, CD32-expression, and Fc-R mediated activation levels were similar in all patients regardless of their MACE risk. In contrast, the hsCRP levels and the proportion of CD14+ circulating monocytes expressing the CD32 receptor for CRP were significantly higher in the patients who developed AHF. The FCGR2A rs1801274 HH genotype was significantly more common in patients who developed RCE and MACE than in RCE-free patients and associated with an enhanced percentage of circulating CD32+CD14+ monocytes. The FCGR2A-HH genotype was identified as an independent predictor of subsequent RCE (OR, 2.7; p = 0.048; CI, 1.01-7.44) by multivariate analysis. These findings bring preliminary evidence that host FCGR2A genetic variants can influence monocyte CD32 receptor expression and may contribute to the fine-tuning of CD32-driven chronic activating signals that affect the risk of developing RCEs following primary ACS events.

The coming of the Greeks to Provence and Corsica: Y-chromosome models of archaic Greek colonization of the western Mediterranean.

BACKGROUND: The process of Greek colonization of the central and western Mediterranean during the Archaic and Classical Eras has been understudied from the perspective of population genetics. To investigate the Y chromosomal demography of Greek colonization in the western Mediterranean, Y-chromosome data consisting of 29 YSNPs and 37 YSTRs were compared from 51 subjects from Provence, 58 subjects from Smyrna and 31 subjects whose paternal ancestry derives from Asia Minor Phokaia, the ancestral embarkation port to the 6th century BCE Greek colonies of Massalia (Marseilles) and Alalie (Aleria, Corsica). RESULTS: 19% of the Phokaian and 12% of the Smyrnian representatives were derived for haplogroup E-V13, characteristic of the Greek and Balkan mainland, while 4% of the Provencal, 4.6% of East Corsican and 1.6% of West Corsican samples were derived for E-V13. An admixture analysis estimated that 17% of the Y-chromosomes of Provence may be attributed to Greek colonization. Using the following putative Neolithic Anatolian lineages: J2a-DYS445 = 6, G2a-M406 and J2a1b1-M92, the data predict a 0% Neolithic contribution to Provence from Anatolia. Estimates of colonial Greek vs. indigenous Celto-Ligurian demography predict a maximum of a 10% Greek contribution, suggesting a Greek male elite-dominant input into the Iron Age Provence population. CONCLUSIONS: Given the origin of viniculture in Provence is ascribed to Massalia, these results suggest that E-V13 may trace the demographic and socio-cultural impact of Greek colonization in Mediterranean Europe, a contribution that appears to be considerably larger than that of a Neolithic pioneer colonization.

Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: a transfusion support for sickle cell disease patients.

To lower the alloimmunization risk following transfusion in blacks, we developed two genotyping assays for large-scale screening of Comorian and Afro-Caribbean donors. One was a multiplex SNaPshot assay designed to identify ce(s) (340), ceMO/AR/EK/BI/SM, ce(s) , ce(s) (1006) and KEL*6/*7 alleles. The other was a multiplex fragment assay designed to detect RHD, RHDψ and RHCE*C and 455A>C transversion consistent with (C)ce(s) Type 1 and DIII Type5 ce(s) . Variant RHCE*ce alleles or RH haplotypes were detected in 58·69% of Comorians and 41·23% of Afro-Caribbeans. The ce(s) allele, (C)ce(s) Type 1, and DIII Type 5 ce(s) haplotypes were identified respectively in 39·13%, 14·67% and 4·88% of Comorians and 32·23%, 5·28% and 1·76% of Afro-Caribbeans. Genotypes consistent with partial D, C, c and/or e antigen expression were observed in 26·08% of Comorians and 14·69% of Afro-Caribbeans. No homozygous genotype corresponding to the RH:-18, -34, and -46 phenotypes were found. However, over 50% of genotypes produced low-prevalence antigens at risk for negative recipients, i.e., V, VS, JAL, and/or KEL6. One new variant RHCE*ce(s) (712) allele was identified. This is the first determination of variant RHCE and KEL allele frequencies. Results indicate the most suitable targets for molecular assay screening to optimize use of compatible blood units and lower immunization risk.

Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles.

BACKGROUND: Molecular RHD blood group typing is very efficient for managing donors and patients carrying any of the various molecular types of weak D and DEL. The purpose of the work was to develop a multiplex polymerase chain reaction (PCR) SNaPshot assay for simultaneous detection of weak D and DEL alleles that are prevalent in Europeans, Africans, and Asians. STUDY DESIGN AND METHODS: Preliminary profiling was carried out on single-nucleotide polymorphisms (SNPs) associated with 13 prevalent RHD alleles, that is, weak D Types 1, 2, 3, 4.0, 4.0.1, 4.1, 4.2, 5, 11, 15, and 17; RHD(IVS3+1g>a); and RHD(K409K). Multiplex PCR was used to amplify six RHD regions encompassing 14 SNPs. Identification was obtained by incorporation of the complementary dye single base at the 3'-end of each probe-primer. A prospective analysis was then carried out on 152 blood samples from patients (n = 53) and donors (n = 88) with equivocal RhD serology and pregnant women (n = 11). RESULTS: After validation, our SNaPshot assay allowed direct genotyping of 82.9% of samples overall and 100% of samples harboring weak D Types 1, 2, 3, and 4.1 alleles. In the remaining 17.1% of samples overall, sequence investigation allowed accurate genotyping. In addition, four novel RHD alleles were identified, that is, RHD(S256P), RHD(L390L), RHD(F410V), and RHD(IVS4-2a>g). CONCLUSION: The SNaPshot assay described herein is a helpful supplementary tool for resolving doubtful RhD serology. By allowing accurate identification of weak D and DEL alleles this assay should allow better management of the donors and the patients genotyped weak D Types 1, 2, 3, and 4.1 who can receive D+ blood units.

Validation of new HLA-J alleles assigned by next generation sequencing.

We describe nine novel HLA-J alleles validated by in silico and experimental data.

Identification of 11 novel HLA-A, -B, -C, -DRB1, and -DQB1 alleles in the 1000 Genomes Project panel.

We isolated 11 novel HLA alleles in the 1000 Genomes Project using PolyPheMe software.

Maternal HLA Ib Polymorphisms in Pregnancy Allo-Immunization.

During pregnancy the formation of alloreactive anti-human leukocyte antigen (HLA) antibodies are a major cause of acute rejection in organ transplantation and of adverse effects in blood transfusion. The purpose of the study was to identify maternal HLA class Ib genetic factors associated with anti-HLA allo-immunization in pregnancy and the degree of tolerance estimated by IgG4 expression. In total, 86 primiparous women with singleton pregnancies were included in the study. Maternal blood samples and umbilical cord samples were collected at delivery. Clinical data were obtained. Maternal blood serum was screened for HLA class I and II antibodies, identification of Donor Specific Antibody (DSA), activation of complement measured by C1q and IgG4 concentrations. Mothers were genotyped for HLA class Ib (HLA-E, -F and -G). Anti-HLA class I and II antibodies were identified in 24% of the women. The maternal HLA-E*01:06 allele was significantly associated with a higher fraction of anti-HLA I immunization (20.0% vs. 4.8%, p = 0.048). The maternal HLA-G 3'-untranslated region UTR4-HLA-G*01:01:01:05 haplotype and the HLA-F*01:03:01 allele were significantly associated with a low anti-HLA I C1q activation (16.7% vs. 57.1%, p = 0.028; 16.7% vs. 50.0%, p = 0.046; respectively). Both HLA­G and HLA-F*01:03:01 showed significantly higher levels of IgG4 compared with the other haplotypes. The results support an association of certain HLA class Ib alleles with allo-immunization during pregnancy. Further studies are needed to elucidate the roles of HLA-E*01:06, HLA-F*01:03 and HLA­G UTR4 in reducing the risk for allo-immunization.

Determination of the phylogenetic origins of the Árpád Dynasty based on Y chromosome sequencing of Béla the Third.

We set out to identify the origins of the Árpád Dynasty based on genome sequencing of DNA derived from the skeletal remains of Hungarian King Béla III (1172-1196) and eight additional individuals (six males, two females) originally interred at the Royal Basilica of Székesfehérvár. Y-chromosome analysis established that two individuals, Béla III and HU52 assign to haplogroups R-Z2125 whose distribution centres near South Central Asia with subsidiary expansions in the regions of modern Iran, the Volga Ural region and the Caucasus. Out of a cohort of 4340 individuals from these geographic areas, we acquired whole-genome data from 208 individuals derived for the R-Z2123 haplogroup. From these data we have established that the closest living kin of the Árpád Dynasty are R-SUR51 derived modern day Bashkirs predominantly from the Burzyansky and Abzelilovsky districts of Bashkortostan in the Russian Federation. Our analysis also reveals the existence of SNPs defining a novel Árpád Dynasty specific haplogroup R-ARP. Framed within the context of a high resolution R-Z2123 phylogeny, the ancestry of the first Hungarian royal dynasty traces to the region centering near Northern Afghanistan about 4500 years ago and identifies the Bashkirs as their closest kin, with a separation date between the two populations at the beginning of the first millennium CE.