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Patients with sickle cell disease (SCD) with high fetal haemoglobin (HbF) tend to have a lower incidence of complications and longer survival due to inhibition of deoxyhaemoglobin S (HbS) polymerisation by HbF. HbF-containing cells, namely F cells, are strongly influenced by genetic factors. We measured the percentage of F cells (Fcells%) in 222 patients with SCD to evaluate the association of (i) Fcells% with genetic HbF-modifier variants and (ii) Fcells% with haematological parameters. There was a different distribution of Fcells% in females compared to males. The association of the B-cell lymphoma/leukaemia 11A (BCL11A) locus with Fcells% (β = 8·238; P < 0·001) and with HbF% (β = 2·490; P < 0·001) was significant. All red cell parameters except for Hb and mean corpuscular Hb concentration levels in males and females were significantly different. The Fcells% was positively associated with mean cell Hb, mean cell volume and reticulocytes. To explain the significant gender difference in Fcells%, we tested for associations with single nucleotide polymorphisms on the X chromosomal region Xp22.2, where a genetic determinant of HbF had been previously hypothesised. We found in males a significant association with a SNP in FERM and PDZ domain-containing protein 4 (FRMPD4) and adjacent to male-specific lethal complex subunit 3 (MSL3). Thus, we have identified an X-linked locus that could account for a significant fraction of the Fcells% variation in patients with SCD.

Background: Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common variants which differ across populations and hence do not fully account for HbF variation. Methods: We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A, MYB, HOXA9, HBB, HBG1, HBG2, CHD4, KLF1, MBD3, ZBTB7A and PGLYRP1.Results: Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified deletions with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis. Conclusions: This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD using multiple genomic variants associated with HbF in SCD.

The nucleotides ATP and NAD+ are released from stressed cells as endogenous danger signals. Ecto-enzymes in the tumor microenvironment hydrolyze these inflammatory nucleotides to immunosuppressive adenosine, thereby, hampering anti-tumor immune responses. The NAD+ hydrolase CD38 is expressed at high levels on the cell surface of multiple myeloma (MM) cells. Daratumumab, a CD38-specific monoclonal antibody promotes cytotoxicity against MM cells. With long CDR3 loops, nanobodies and nanobody-based heavy chain antibodies (hcAbs) might bind to cavities on CD38 and thereby inhibit its enzyme activity more potently than conventional antibodies. The goal of our study was to establish assays for monitoring the enzymatic activities of CD38 on the cell surface of tumor cells and to assess the effects of CD38-specific antibodies on these activities. We monitored the enzymatic activity of CD38-expressing MM and other tumor cell lines, using fluorometric and HPLC assays. Our results showed that daratumumab and hcAb MU1067 inhibit the ADPR cyclase but not the NAD+ hydrolase activity of CD38-expressing MM cells. We conclude that neither clinically approved daratumumab nor recently developed nanobody-derived hcAbs provide a second mode of action against MM cells. Thus, there remains a quest for &ldquo, double action&rdquo, CD38-inhibitory antibodies.

Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.

BACKGROUND Antibodies against phospholipase A2 receptor 1 (PLA2R1) are found in 80% of patients with membranous nephropathy, and previous studies described three autoantibody-targeted PLA2R1 epitope regions. Although anti-PLA2R1 antibody levels are closely associated with treatment response and disease prognosis, the clinical role of epitope regions targeted by autoantibodies is unclear. METHODS In a prospective cohort of 150 patients with newly diagnosed PLA2R1-associated membranous nephropathy, we investigated the clinical role of epitope-recognition patterns and domain-specific PLA2R1 antibody levels by western blot and ELISA. RESULTS We identified a fourth epitope region in the CTLD8 domain of PLA2R1, which was recognized by anti-PLA2R1 antibodies in 24 (16.0%) patients. In all study patients, anti-PLA2R1 antibodies bound both the N-terminal (CysR-FnII-CTLD1) region and the C-terminal (CTLD7-CTLD8) region of PLA2R1 at study enrollment. The total anti-PLA2R1 antibody levels of patients determined detection of domain-specific PLA2R1 antibodies, and thereby epitope-recognition patterns. A remission of proteinuria occurred in 133 (89%) patients and was not dependent on the domain-recognition profiles. A newly developed ELISA showed that domain-specific PLA2R1 antibody levels targeting CysR, CTLD1, and CTLD7 strongly correlate with the total anti-PLA2R1 antibody level (Spearman's rho, 0.95, 0.64, and 0.40; P

With the arrival of the Internet of Things (IoT) and the challenges arising from Big Data, neuromorphic chip concepts are seen as key solutions for coping with the massive amount of unstructured data streams by moving the computation closer to the sensors, the so-called “edge computing.” Augmenting these chips with emerging memory technologies enables these edge devices with non-volatile and adaptive properties which are desirable for low power and online learning operations. However, an energy- and area-efficient realization of these systems requires disruptive hardware changes. Memristor-based solutions for these concepts are in the focus of research and industry due to their low-power and high-density online learning potential. Specifically, the filamentary-type valence change mechanism (VCM memories) have shown to be a promising candidate In consequence, physical models capturing a broad spectrum of experimentally observed features such as the pronounced cycle-to-cycle (c2c) and device-to-device (d2d) variability are required for accurate evaluation of the proposed concepts. In this study, we present an in-depth experimental analysis of d2d and c2c variability of filamentary-type bipolar switching HfO2/TiOx nano-sized crossbar devices and match the experimentally observed variabilities to our physically motivated JART VCM compact model. Based on this approach, we evaluate the concept of parallel operation of devices as a synapse both experimentally and theoretically. These parallel synapses form a synaptic array which is at the core of neuromorphic chips. We exploit the c2c variability of these devices for stochastic online learning which has shown to increase the effective bit precision of the devices. Finally, we demonstrate that stochastic switching features for a pattern classification task that can be employed in an online learning neural network., Frontiers in Neuroscience, 15, ISSN:1662-453X, ISSN:1662-4548

CD38 is the major NAD+-hydrolyzing ecto-enzyme in most mammals. As a type II transmembrane protein, CD38 is also a promising target for the immunotherapy of multiple myeloma (MM). Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Using phage display technology, we isolated 13 mouse CD38-specific nanobodies from immunized llamas and produced these as recombinant chimeric mouse IgG2a heavy chain antibodies (hcAbs). Sequence analysis assigned these hcAbs to five distinct families that bind to three non-overlapping epitopes of CD38. Members of families 4 and 5 inhibit the GDPR-cyclase activity of CD38. Members of families 2, 4 and 5 effectively induce complement-dependent cytotoxicity against CD38-expressing tumor cell lines, while all families effectively induce antibody dependent cellular cytotoxicity. Our hcAbs present unique tools to assess cytotoxicity mechanisms of CD38-specific hcAbs in vivo against tumor cells and potential off-target effects on normal cells expressing CD38 in syngeneic mouse tumor models, i.e. in a fully immunocompetent background.

Plants as a platform for recombinant protein expression are now economically comparable to well-established systems, such as microbes and mammalian cells, thanks to advantages such as scalability and product safety. However, downstream processing accounts for the majority of the final product costs because plant extracts contain large quantities of host cell proteins (HCPs) that must be removed using elaborate purification strategies. Heat precipitation in planta (blanching) can remove ∼80% of HCPs and thus simplify further purification steps, but this is only possible if the target protein is thermostable. Here we describe a combination of blanching and chromatography to purify the thermostable transmission-blocking malaria vaccine candidate FQS, which was transiently expressed in Nicotiana benthamiana leaves. If the blanching temperature exceeded a critical threshold of ∼75 °C, FQS was no longer recognized by the malaria transmission-blocking monoclonal antibody 4B7. A design-of-experiments approach revealed that reducing the blanching temperature from 80 °C to 70 °C restored antibody binding while still precipitating most HCPs. We also found that blanching inhibited the degradation of FQS in plant extracts, probably due to the thermal inactivation of proteases. We screened hydrophobic interaction chromatography materials using miniature columns and a liquid-handling station. Octyl Sepharose achieved the highest FQS purity during the primary capture step and led to a final purity of ∼72% with 60% recovery via step elution. We found that 30–75% FQS was lost during ultrafiltration/diafiltration, giving a final yield of 9 mg kg−1 plant material after purification based on an initial yield of ∼49 mg kg−1 biomass after blanching.

Fetal haemoglobin (HbF) levels have a clinically beneficial effect on sickle cell disease (SCD). Patients with SCD demonstrate extreme variability in HbF levels (1-30%), a large part of which is likely genetically determined. The main genetic modifier loci for HbF persistence, HBS1L-MYB, BCL11A and the β-globin gene cluster in adults also act in SCD patients. Their effects are, however, modified significantly by a disease pathology that includes a drastically shortened erythrocyte lifespan with an enhanced survival of those red blood cells that carry HbF (F cells). We propose a model of how HbF modifier genes and disease pathology interact to shape HbF levels measured in patients. We review current knowledge on the action of these loci in SCD, their genetic architecture, and their putative functional components. At each locus, one strong candidate for a causative, functional DNA change has been proposed: Xmn1-HBG2 at the β-globin cluster, rs1427407 at BCL11A and the 3 bp deletion rs66650371 at HBS1L-MYB. These, however, explain only part of the impact of these loci and additional variants are yet to be identified. Further progress in understanding the genetic control of HbF levels requires that confounding factors inherent in SCD, such as ethnic complexity, the role of F cells and the influence of drugs, are suitably addressed. This will depend on international collaboration and on large, well-characterised patient cohorts with genome-wide single-nucleotide polymorphism or sequence data.

Background: Acute pain, the most prominent complication of sickle cell disease (SCD), results from vasoocclusion triggered by sickling of deoxygenated red blood cells (RBCs). A key factor influencing HbS oxygenation is the intracellular concentration of 2,3- diphosphoglycerate (2,3-DPG). 2,3-DPG, an intermediate substrate in the glycolytic pathway, decreases oxygen binding and stabilizes the deoxygenated hemoglobin. Pyruvate kinase (gene PKLR, protein PKR) is a rate-limiting enzyme in glycolysis; variants in PKLR may affect PKR activity, 2,3-DPG levels in RBCs, subsequent frequency of sickling and acute pain episodes (APE). There is thus a strong biological basis for exploring PKLR as a candidate gene affecting acute pain in SCD. Methods: The study population for genetic association consists of 2 cohorts: 1) 242 adults with HbSS from King's College Hospital (KCH), London, UK, with complete hospitalisation records over 10 years (2004-2013 inclusive) as the "discovery" cohort; 2) 977 children with HbSS or HbSb 0 thalassemia from the Silent Infarct Transfusion (SIT) trial, with a 3-year history of severe vasoocclusive pain based on hospitalization, as the "validation" cohort. Both studies were approved by the local Institutional Review Boards at KCH and Vanderbilt University Medical Center, respectively. An independent cohort comprises 52 adults with SCD enrolled under 3 protocols - NCT00011648, NCT00081523, and NCT03685721 - approved by the NHLBI Review Board (NIH), for evaluation of imbalance in allele expression. Genome scan for the KCH cohort was performed using llumina's Infinium "MEGA" chip (1.7m markers). The SIT DNA samples were genotyped using Illumina HumanHap650Y array 5 (661K markers) or Illumina Infinium HumanOmni1-Quad array (1.1m markers). The results were quality controlled followed by genotype imputation based on the 1000 Genomes Project phase 3 data. An annualised "hospitalisation rate" as a measure of pain incidence rate, was calculated by dividing the number of hospital admissions for severe acute pain by the number of years of observation for KCH and SIT cohorts (Fig A). We performed association analysis with common SNPs at PKLR locus using data from our genome-wide SNP set and a linear mixed modelling approach incorporating a genetic relatedness matrix to take account of relatedness, plus sex and age as fixed covariates. We corrected for multiple testing after quantifying the linkage disequilibrium (LD) within PKLR and used this to calculate appropriate significance levels. For the PKLR region and hospitalisation rate, the modified significance level was p Results: 7 of 47 variants evaluated in PKLR were associated with hospitalization rate (LnLnHospRate) in the discovery cohort: intron 4 - rs071053, and intron 2 - rs8177970, rs116244351, rs114455416, rs12741350, rs3020781, and rs8177964). All 7 were validated in Fisher's meta-analyses of the KCH and the SIT cohorts using p Conclusion: Intronic variants of PKLR are associated with hospitalization rate for acute pain episodes in adults and children with SCD. We show that the intronic variants are likely to influence acute pain by affecting expression of the PKLR gene using allele-specific expression analyses, although the causal variant is unclear. These results support PKLR as a genetic modifier of SCD. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Fetal hemoglobin (HbF) and peripheral hemoglobin oxygen saturation (SpO2) both predict clinical severity in sickle cell disease (SCD), while reticulocytosis is associated with vasculopathy, but there are few data on mechanisms. HbF, SpO2 and routine clinical and laboratory measures were available in a Tanzanian cohort of 1175 SCD individuals aged ≥ 5 years and the association with SpO2 (as response variable transformed to a Poisson distribution) was assessed by negative binomial model with age and sex as covariates. Increase in HbF was associated with increased SpO2 (rate ratio, RR = 1.19; 95% confidence intervals [CI] 1.04, 1.37 per natural log unit of HbF; p = 0.0004). In univariable analysis, SpO2 was inversely associated with age, reticulocyte count, and log (total bilirubin) and directly with pulse, SBP, hemoglobin, and log(HbF). In multivariable regression log(HbF) (RR 1.191; 95%CI 1.04, 1.37; p = 0.013), pulse (RR 1.01; 95%CI 1.00, 1.01; p = 0.026), SBP (RR 1.008; 95%CI 1.00, 1.02; p = 0.014), and hemoglobin (1.120; 95%CI 1.05, 1.19; p = 0.001) were positively and independently associated with SpO2 while reticulocyte count (RR 0.985; 95%CI 0.97, 0.99; p = 0.019) was independently inversely associated with SpO2. In SCD, improving SpO2, in part through cardiovascular compensation and associated with reduced reticulocytosis, may be a mechanism by which HbF reduces disease severity., Highlights • Fetal hemoglobin may moderate sickle cell disease through increased oxygen saturation. • Low oxygen saturation is associated with reticulocytosis which might moderate cerebral vasculopathy and stroke risk. • Higher pulse rate and systolic blood pressure in those with higher SpO2 suggests cardiovascular compensation for low SpO2. Fetal hemoglobin (HbF) is normally synthesized during intrauterine life and it starts to decline before birth being replaced by adult hemoglobin (HbA). However some individuals continue to synthesize HbF to adulthood and are relatively protected from severe sickle cell disease. The mechanism of HbF protection in SCD has not been entirely established. This study reports a positive association between HbF and oxygen saturation (SpO2). Higher SpO2 is associated with decreased reticulocytes but increased pulse rate and systolic blood pressure, suggesting SpO2 is maintained in part through cardiovascular compensation. Increasing HbF may reduce disease severity partly through increasing SpO2.

We investigated changes in the plasma proteome of children with sickle cell anaemia (SCA) associated with hydroxycarbamide (HC) use, to further characterize the actions of HC. Fifty-one children with SCA consented to take part in this study. Eighteen were taking HC at a median dose of 22 mg/kg, and 33 were not on HC. Plasma was analysed using an unbiased proteomic approach and a panel of 92 neurological biomarkers. HC was associated with increased haemoglobin (Hb) (89·8 vs. 81·4 g/l, P = 0·007) and HbF (6·7 vs. 15·3%, P 1·3 increased concentration. HC use was associated with reduced haemolysis (lower α, β, δ globin chains, haptoglobin-related protein, complement C9; higher haemopexin), reduced inflammation (lower α-1-acid glycoprotein, CD5 antigen-like protein, ceruloplasmin, factor XII, immunoglobulins, cysteine-rich secretory protein 3, vitamin D-binding protein) and decreased activation of coagulation (lower factor XII, carboxypeptidase B2, platelet basic protein). There was a significant correlation between the increase in HbF% on HC and haemopexin levels (r = 0·603, P = 0·023). This study demonstrated three ways in which HC may be beneficial in SCA, and identified novel proteins that may be useful to monitor therapeutic response.

The P2X4 channel is involved in different physiological and pathological conditions and functions in the nervous system. Despite the existence of several mouse models for which the expression of the gene was manipulated, there is still little information on the expression of the protein at the cellular level. In particular, supposedly specific available antibodies have often proved to recognize unrelated proteins in P2X4-deficient mice. Here, we used an in vivo DNA vaccine approach to generate a series of monoclonal antibodies and nanobodies specific for human, mouse, and rat P2X4 channels. We further characterized these antibodies and show that they solely recognize the native form of the proteins both in biochemical and cytometric applications. Some of these antibodies prove to specifically recognize P2X4 channels by immunostaining in brain or sensory ganglia slices, as well as at the cellular and subcellular levels. Due to their clonality, these different antibodies should represent versatile tools for further characterizing the cellular functions of P2X4 in the nervous system as well as at the periphery.

Rationale: CD38 is a target for the therapy of multiple myeloma (MM) with monoclonal antibodies such as daratumumab and isatuximab. Since MM patients exhibit a high rate of relapse, the development of new biologics targeting alternative CD38 epitopes is desirable. The discovery of single-domain antibodies (nanobodies) has opened the way for a new generation of antitumor therapeutics. We report the generation of nanobody-based humanized IgG1 heavy chain antibodies (hcAbs) with a high specificity and affinity that recognize three different and non-overlapping epitopes of CD38 and compare their cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. Methods: We generated three humanized hcAbs (WF211-hcAb, MU1067-hcAb, JK36-hcAb) that recognize three different non-overlapping epitopes (E1, E2, E3) of CD38 by fusion of llama-derived nanobodies to the hinge- and Fc-domains of human IgG1. WF211-hcAb shares the binding epitope E1 with daratumumab. We compared the capacity of these CD38-specific hcAbs and daratumumab to induce CDC and ADCC in CD38-expressing tumor cell lines in vitro and in patient MM cells ex vivo as well as effects on xenograft tumor growth and survival in vivo. Results: CD38-specific heavy chain antibodies (WF211-hcAb, MU1067-hcAb, JK36-hcAb) potently induced antibody-dependent cellular cytotoxicity (ADCC) in CD38-expressing tumor cell lines and in primary patient MM cells, but only little if any complement-dependent cytotoxicity (CDC). In vivo, CD38-specific heavy chain antibodies significantly reduced the growth of systemic lymphomas and prolonged survival of tumor bearing SCID mice. Conclusions: CD38-specific nanobody-based humanized IgG1 heavy chain antibodies mediate cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. These promising results of our study indicate that CD38-specific hcAbs warrant further clinical development as therapeutics for multiple myeloma and other hematological malignancies.

Targeting the P2X7 ion channel, a danger sensor for extracellular nucleotides, improves outcomes in models of inflammation, cancer, and brain-diseases. Antibodies and nanobodies have been developed that antagonize or potentiate gating of P2X7. Their potential advantages over small-molecule drugs include high specificity, lower off-target effects, and tunable in vivo half-life. Genetic fusion of P2X7-specific biologics to binding modules may enable targeting of specific cell subsets. Besides directly modulating P2X7 function, antibodies can also initiate specific depletion of P2X7-expressing cells. Adeno-associated viral vectors (AAV) can be used to express P2X7-specific antibodies in vivo to achieve long-lasting biological effects. Furthermore, if successfully targeted to P2X7-expressing cells, AAVs may enable modulation of the function of P2X7-expressing immune cells via encoded transgenic RNA or proteins.

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

Genetic variants at three quantitative trait loci (QTL) for fetal haemoglobin (HbF), BCL11A, HBS1L-MYB and the β-globin gene cluster, have attracted interest as potential targets of therapeutic strategies for HbF reactivation in sickle cell anaemia (SCA). We carried out the first systematic evaluation of critical single nucleotide polymorphisms at these disease modifier loci in Nigerian patients with SCA. Common variants for BCL11A and HBS1L-MYB were strongly associated with HbF levels. At both loci, secondary association signals were detected, illustrating the mapping resolution attainable in this population. For BCL11A, the two independent sites of association were represented by rs1427407 (primary site, p = 7.0 x 10−10) and rs6545816 (secondary site, conditioned on rs1427407: p = 0.02) and for HBS1L-MYB by rs9402686 (HMIP-2B, p = 1.23 x 10−4) and rs66650371 (HMIP-2A, p = 0.002). Hap-lotype analysis revealed similarities in the genetic architecture of BCL11A and HBS1L-MYB in Nigerian patients. Variants at both loci also alleviated anaemia. The variant allele for the γ globin gene promoter polymorphism XmnI-HBG2 was too infrequent in our patients to be evaluated in this relatively small study. Studying the large and diverse SCA patient populations in African countries such as Nigeria will be key for a clearer understanding of how these loci work and for the discovery of new disease modifier genes.

We report genome-wide association study results for the levels of A1, A2 and fetal hemoglobins, analyzed for the first time concurrently. Integrating high-density array genotyping and whole-genome sequencing in a large general population cohort from Sardinia, we detected 23 associations at 10 loci. Five signals are due to variants at previously undetected loci: MPHOSPH9, PLTP-PCIF1, ZFPM1 (FOG1), NFIX and CCND3. Among the signals at known loci, ten are new lead variants and four are new independent signals. Half of all variants also showed pleiotropic associations with different hemoglobins, which further corroborated some of the detected associations and identified features of coordinated hemoglobin species production.

Introduction: Fetal hemoglobin is an important factor in modulating the severity of sickle cell anemia. Its level in peripheral blood underlies strong genetic determination. Associated loci with increased levels of fetal hemoglobin display population-specific allele frequencies. Objective: We investigated the presence and effect of known common genetic variants promoting fetal hemoglobin persistence (rs11886868, rs9399137, rs4895441, and rs7482144) in 60 Colombian patients with sickle cell anemia. Materials and methods: Four single nucleotide polymorphisms (SNP) were genotyped by restriction fragment length polymorphisms (RFLP) and the use of the TaqMan procedure. Fetal hemoglobin (HbF) from these patients was quantified using the oxyhemoglobin alkaline denaturation technique. Genotype frequencies were compared with frequencies reported in global reference populations. Results: We detected genetic variants in the four SNPs, reported to be associated with higher HbF levels for all four SNPs in the Colombian patients. Genetic association between SNPs and HbF levels did not reach statistical significance. The frequency of these variants reflected the specific ethnic make-up of our patient population: A high prevalence of rs7482144-'A' reflects the West-African origin of the sickle cell mutation, while high frequencies of rs4895441-'G' and rs11886868-'C' point to a significant influence of an Amerindian ethnic background in the Colombian sickle cell disease population. Conclusion: These results showed that in the sickle cell disease population in Colombia there is not a unique genetic background, but two (African and Amerindian). This unique genetic situation will provide opportunities for a further study of these loci, such as fine-mapping and molecular-biological investigation. Colombian patients are expected to yield a distinctive insight into the effect of modifier loci in sickle cell disease. Introducción. La hemoglobina fetal es un importante factor modulador de la gravedad de la anemia falciforme, cuya expresión está muy condicionada por el factor genético. Los loci asociados con el incremento de la hemoglobina fetal pueden presentar frecuencias alélicas específicas para cada población. Objetivo. Investigar la presencia y el efecto de las variantes genéticas rs11886868, rs9399137, rs4895441 y rs7482144 asociadas con la persistencia de hemoglobina fetal, en 60 pacientes colombianos con anemia falciforme. Materiales y métodos. Se hizo la genotipificación de los polimorfismos de nucleótido simple ( Single Nucleotide Polymorphisms, SNP) mediante la técnica de polimorfismos de longitud de fragmentos de restricción ( Restriction Fragment Length Polymorphisms, RFLP) y el procedimiento TaqMan. La hemoglobina fetal (HbF) se cuantificó utilizando la técnica de desnaturalización alcalina de la oxihemoglobina. Las frecuencias genotípicas se compararon con las reportadas en poblaciones de referencia global. Resultados. Se observaron variantes genéticas ya reportadas para aumento de HbF en los cuatro SNP. La asociación genética entre los SNP y el incremento de la HbF no alcanzó significancia estadística. La frecuencia de estos alelos reflejó la siguiente composición específica en esta muestra de pacientes colombianos: una gran prevalencia de rs7482144-'A', lo que indica que el origen de la mutación para la anemia falciforme es África occidental, y una gran frecuencia de rs4895441-'G' y rs11886868-'C', lo que denota la influencia significativa del origen genético amerindio. Conclusión. Los resultados evidenciaron que la población con anemia falciforme de Colombia no tiene un único origen genético, sino que existen dos (africano y amerindio). Esta situación genética única ofrece la oportunidad de llevar a cabo un estudio más amplio de estos loci a nivel molecular. Se espera que el estudio de pacientes colombianos permita una visión diferente del efecto de los loci modificadores en esta enfermedad.

ARTC2.2 is a toxin-related, GPI-anchored ADP-ribosyltransferase expressed by murine T cells. In response to NAD+ released from damaged cells during inflammation, ARTC2.2 ADP-ribosylates and thereby gates the P2X7 ion channel. This induces ectodomain shedding of metalloprotease-sensitive cell surface proteins. In this study, we show that ARTC2.2 itself is a target for P2X7-triggered ectodomain shedding. We identify the metalloprotease cleavage site 3 aa upstream of the predicted GPI anchor attachment site of ARTC2.2. Intravenous injection of NAD+ increased the level of enzymatically active ARTC2.2 in serum, indicating that this mechanism is operative also under inflammatory conditions in vivo. Radio–ADP-ribosylation assays reveal that shedding refocuses the target specificity of ARTC2.2 from membrane proteins to secretory proteins. Our results uncover nucleotide-induced membrane-proximal proteolysis as a regulatory mechanism to control the substrate specificity of ARTC2.2.

Nanobodies are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. Nanobodies hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic nanobodies because they are accessible to systemically injected biologics. In order to be effective, therapeutic nanobodies must recognize their target membrane protein in native conformation. However, raising nanobodies against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and therefore are difficult to purify in native conformation. Here we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other co-stimulatory proteins. The plasmid is coated onto 1 µm gold particles which are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy chain antibodies. The VHH/nanobody coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific nanobodies are selected by phage display or by screening of nanobody-based heavy chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic and antagonistic nanobodies against several cell surface ecto-enzymes and ligand-gated ion channels.

Mammalian ecto-ADP-ribosyltransferases (ecto-ARTs or also ARTCs) catalyze the ADP-ribosylation of cell surface proteins using extracellular nicotinamide adenine dinucleotide (NAD+) as substrate. By this post-translational protein modification, ecto-ARTs modulate the function of various target proteins. A functional role of ARTC2 has been demonstrated for peripheral immune cells such as T cells and macrophages. Yet, little is known about the role of ecto-ARTs in the central nervous system and on microglia. Here, we identified ARTC2.1 as the major ecto-ART expressed on murine microglia. ARTC2.1 expression was strongly upregulated on microglia upon co-stimulation with LPS and an ERK1/2 inhibitor or upon IFNβ stimulation. We identified several target proteins modified by ARTC2.1 on microglia with a recently developed mass spectrometry approach, including two receptors for immunoglobulin G (IgG), FcγR1 and FcγR2B. Both proteins were verified as targets of ARTC2.1 in vitro using a radiolabeling assay with 32P-NAD+ as substrate. Moreover, ADP-ribosylation of both targets strongly inhibited their capacity to bind IgG. In concordance, ARTC2.1 induction in WT microglia and subsequent cell surface ADP-ribosylation significantly reduced the phagocytosis of IgG-coated latex beads, which was unimpaired in NAD+/DTT treated microglia from ARTC2.1−/− mice. Hence, induction of ARTC2.1 expression under inflammatory conditions, and subsequent ADP-ribosylation of cell surface target proteins could represent a hitherto unnoticed mechanism to regulate the immune response of murine microglia.

HMIP-2 is a human quantitative trait locus affecting peripheral numbers, size and hemoglobin composition of red blood cells, with a marked effect on the persistence of the fetal form of hemoglobin, HbF, in adults. The locus consists of multiple common variants in an enhancer region for MYB (chr 6q23.3), which encodes the hematopoietic transcription factor cMYB. Studying a European population cohort and four African-descended groups of patients with sickle cell anemia, we found that all share a set of two spatially separate HbF-promoting alleles at HMIP-2, termed "A" and "B." These typically occurred together ("A-B") on European chromosomes, but existed on separate homologous chromosomes in Africans. Using haplotype signatures for "A" and "B," we interrogated public population datasets. Haplotypes carrying only "A" or "B" were typical for populations in Sub-Saharan Africa. The "A-B" combination was frequent in European, Asian, and Amerindian populations. Both alleles were infrequent in tropical regions, possibly undergoing negative selection by geographical factors, as has been reported for malaria with other hematological traits. We propose that the ascertainment of worldwide distribution patterns for common, HbF-promoting alleles can aid their further genetic characterization, including the investigation of gene-environment interaction during human migration and adaptation.

Three quantitative trait loci (QTLs) modifying fetal hemoglobin (HbF) levels have been identified, and these have been shown to have a predictive value of disease severity in β thalassemia and sickle cell disease in diverse ethnic groups. One of the HbF QTLs which consists of a set of common intergenic single nucleotide polymorphisms (SNPs) in the HBS1L-MYB intergenic region on chromosome 6q23, has also been consistently identified as highly associated with clinically important human erythroid traits. Despite extensive genetic evidence, a clear mechanistic basis for the association between the intergenic SNPs and erythroid biology has remained elusive, although the two flanking genes (HBS1L and MYB) are candidate target genes. Here, we set out to characterize the regulatory potential of the human HBS1L-MYB intergenic region in detail and to investigate its functional impact on the erythroid phenotype-associated variants. We profiled chromatin occupancy of the key erythroid LDB1 transcription factor (TF) complex in primary human erythroid progenitors (HEPs) using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq) and quantitative PCR (ChIP-qPCR). We detected an intergenic cluster containing 7 binding sites for the LDB1–complex, characterized by strong binding and co-occupancy of core complex proteins LDB1, GATA1, TAL1 and ETO2. One of these sites was co-occupied by the erythroid-specific TF KLF1, a protein which was also found to bind the murine intergenic region. Depletion of LDB1, TAL1 and KLF1 in K562 cells using RNA interference resulted in a specific downregulation of MYB expression while leaving HBS1L levels unaffected, demonstrating that the erythroid TFs occupying the intergenic enhancers are required for MYB expression. Using chromosome conformation capture (3C) coupled to high-throughput sequencing (3C-Seq), we profiled higher order chromatin structure within the locus, and detected several strong chromatin co-associations between the MYB promoter and intergenic sequences, almost all of which correlated with TF binding. The binding activities were distributed over a conserved core region identical to a 24-kb interval containing genetic variants in strong genetic association with erythroid traits in human populations. This block of SNPs is referred to as HBS1L-MYB intergenic polymorphisms block-2 (HMIP-2). The SNPs within HMIP-2 clustered in 2 regions positioned directly under the 2 LDB1-complex ChIP-Seq peaks, at -84 and -71 kb from the MYB transcription start site. Using allele-specific ChIP in K562 cells, we observed diminished (25-50%) binding of LDB1, GATA1, TAL1 and KLF1 to the minor rs66650371 allele, showing that rs66650371 affects local TF binding. An allele-specific 3C analysis also showed reduced interactions between the minor rs66650371 allele at -84 and MYB. We validated and expanded observations made in K562 cells using primary human cells: 1. HEPs from high HbF individuals homozygous for all minor alleles of the phenotype-associated -84kb and -71kb intergenic variants in the conserved core (‘SNP/SNP’) showed consistently lower MYB levels throughout phase II of the culture as compared to wildtype control cells (‘WT/WT’); 37% lower MYB on average; 2. ChIP experiments on SNP/SNP and WT/WT HEPs showed reduced binding of GATA1 and KLF1 to the -84 and -71 regulatory elements (containing the associated variants), the results were further confirmed by allele-specific ChIP assays in SNP/WT HEPs; 3. 3C-qPCR assays on cultured SNP/SNP and WT/WT cells demonstrated diminished looping between the -84 element and the MYB promoter in SNP/SNP individuals; 4. SNP/WT HEPs also showed allelic imbalance of MYB but not HBS1L transcripts when compared to controls (WT/WT and SNP/SNP HEPs). In conclusion, we show that the HBS1L-MYB gene-free interval contains distal enhancer elements that interact with MYB, a critical regulator of erythroid development and HbF levels. Key variants in the intergenic interval affect MYB expression by reducing TF binding to its regulatory elements and disrupting long-range enhancer-gene interaction. Our study identifies the first causal link between the 6q23 HbF QTL and MYB regulation, provides novel insights into the molecular control of clinically important haematological traits and adds another layer of complexity to the regulation of MYB, suggesting potential targets for therapeutic intervention. Disclosures: Thein: Sangart: Consultancy; Shire: Research Funding; Novartis: Speakers Bureau.

The cell surface ecto-enzyme CD38 is a promising target antigen for the treatment of hematological malignancies, as illustrated by the recent approval of daratumumab for the treatment of multiple myeloma. Our aim was to evaluate the potential of CD38-specific nanobodies as novel diagnostics for hematological malignancies. We successfully identified 22 CD38-specific nanobody families using phage display technology from immunized llamas. Crossblockade analyses and in-tandem epitope binning revealed that the nanobodies recognize three different non-overlapping epitopes, with four nanobody families binding complementary to daratumumab. Three nanobody families inhibit the enzymatic activity of CD38 in vitro, while two others were found to act as enhancers. In vivo, fluorochrome-conjugated CD38 nanobodies efficiently reach CD38 expressing tumors in a rodent model within 2 hours after intravenous injection, thereby allowing for convenient same day in vivo tumor imaging. These nanobodies represent highly specific tools for modulating the enzymatic activity of CD38 and for diagnostic monitoring CD38-expressing tumors. Fil: Fumey, William. University Medical Center Hamburg-Eppendorf; Alemania Fil: Koenigsdorf, Julia. University Medical Center Hamburg-Eppendorf; Alemania Fil: Kunick, Valentin. University Medical Center Hamburg-Eppendorf; Alemania Fil: Menzel, Stephan. University Medical Center Hamburg-Eppendorf; Alemania Fil: Schütze, Kerstin. University Medical Center Hamburg-Eppendorf; Alemania Fil: Unger, Mandy. University Medical Center Hamburg-Eppendorf; Alemania Fil: Schriewer, Levin. University Medical Center Hamburg-Eppendorf; Alemania Fil: Haag, Friedrich. University Medical Center Hamburg-Eppendorf; Alemania Fil: Adam, Gerhard. University Medical Center Hamburg-Eppendorf; Alemania Fil: Oberle, Anna. University Medical Center Hamburg-Eppendorf; Alemania Fil: Binder, Mascha. University Medical Center Hamburg-Eppendorf; Alemania Fil: Fliegert, Ralf. University Medical Center Hamburg-Eppendorf; Alemania Fil: Guse, Andreas. University Medical Center Hamburg-Eppendorf; Alemania Fil: Zhao, Yong Juan. Peking University; China Fil: Lee, Hon Cheung. Peking University; China Fil: Malavasi, Fabio. Università di Torino; Italia Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Instituto Leloir; Argentina Fil: Van Hegelsom, Rob. Ablynx nv; Bélgica Fil: Stortelers, Catelijne. Ablynx nv; Bélgica Fil: Bannas, Peter. University Medical Center Hamburg-Eppendorf; Alemania Fil: Koch Nolte, Friedrich. University Medical Center Hamburg-Eppendorf; Alemania

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to these disorders, and then implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories 1. A total of 1,941 unique genetic variants in 37 genes, encoding globins (HBA2, HBA1, HBG2, HBG1, HBD, HBB) and other erythroid proteins (ALOX5AP, AQP9, ARG2, ASS1, ATRX, BCL11A, CNTNAP2, CSNK2A1, EPAS1, ERCC2, FLT1, GATA1, GPM6B, HAO2, HBS1L, KDR, KL, KLF1, MAP2K1, MAP3K5, MAP3K7, MYB, NOS1, NOS2, NOS3, NOX3, NUP133, PDE7B, SMAD3, SMAD6, and TOX) are currently documented in these databases with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants and now provides a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The large repository of previously reported data, together with more recent data, acquired by microattribution, demonstrates how the comprehensive documentation of human variation will provide key insights into normal biological processes and how these are perturbed in human genetic disease. Using the microattribution process set out here, datasets which took decades to accumulate for the globin genes could be assembled rapidly for other genes and disease systems. The principles established here for the globin gene system will serve as a model for other systems and the analysis of other common and/or complex human genetic diseases.

F cells measure the presence of fetal hemoglobin, a heritable quantitative trait in adults that accounts for substantial phenotypic diversity of sickle cell disease and beta thalassemia. We applied a genome-wide association mapping strategy to individuals with contrasting extreme trait values and mapped a new F cell quantitative trait locus to BCL11A, which encodes a zinc-finger protein, on chromosome 2p15. The 2p15 BCL11A quantitative trait locus accounts for 15.1% of the trait variance.

Purpose of review Much effort has been spent on understanding the regulation of fetal hemoglobin (HbF, α 2 γ 2 ) production and its reactivation in adults, as elevated HbF levels are correlated with reduced morbidity and mortality in sickle cell anemia and β thalassemia, diseases that represent a major public health problem. Interindividual HbF variation is largely genetically controlled, but the inheritance patterns are not clear. Recent findings HbF persistence, measured either as percentage HbF or as percentage F cells, is increasingly understood as a quantitative genetic trait; multiple genes together with an environmental component determine the measured value in any given individual. Recent genome-wide studies have shown that common genetic polymorphisms account for a large proportion of the common variation in HbF levels, not only in healthy adults but also in patients with these β hemoglobinopathies. Genetic variation at only three major loci (Xmn1 ―HBG2, HBS1L ―MYB intergenic region on chromosome 6q23 and BCL 11A on chromosome 2p16) account for a relatively large proportion (20―50%) of the phenotypic variation in HbF levels. Two of the major quantitative trait loci include oncogenes emphasizing the importance of cell proliferation and differentiation as an important contribution to the HbF phenotype. Summary The review traces our progress in the understanding of HbF persistence in adults as a quantitative trait and the changing genetic methodology that has helped in the dissection of the genetic architecture underlying HbF variability. We also speculate on the plausible mechanisms underlying the increased HbF production.

Individual variation in fetal hemoglobin (HbF, α 2 γ 2 ) response underlies the remarkable diversity in phenotypic severity of sickle cell disease and β thalassemia. HbF levels and HbF-associated quantitative traits (e.g., F cell levels) are highly heritable. We have previously mapped a major quantitative trait locus (QTL) controlling F cell levels in an extended Asian-Indian kindred with β thalassemia to a 1.5-Mb interval on chromosome 6q23, but the causative gene(s) are not known. The QTL encompasses several genes including HBS1L , a member of the GTP-binding protein family that is expressed in erythroid progenitor cells. In this high-resolution association study, we have identified multiple genetic variants within and 5′ to HBS1L at 6q23 that are strongly associated with F cell levels in families of Northern European ancestry ( P = 10 −75 ). The region accounts for 17.6% of the F cell variance in northern Europeans. Although mRNA levels of HBS1L and MYB in erythroid precursors grown in vitro are positively correlated, only HBS1L expression correlates with high F cell alleles. The results support a key role for the HBS1L -related genetic variants in HbF control and illustrate the biological complexity of the mechanism of 6q QTL as a modifier of fetal hemoglobin levels in the β hemoglobinopathies.

Serum bilirubin levels and predisposition to gallstones in sickle cell disease (SCD) are influenced by genetic variation in the hepatic uridine diphosphate (UDP)-glucuronosyltransferase (UGT1A1) gene, but the association is not consistent. This study investigated whether variation in the gene encoding haem oxygenase (HMOX1), a rate-limiting enzyme upstream of UGT1A in the haem catabolic pathway, and alpha-thalassaemia could explain some of the inconsistent effects. The UGT1A1 [TA](n) and HMOX1 [GT](n) promoter polymorphisms and alpha globin genotypes were determined in 263 SCD patients (199 HbSS, 5 HbS/beta(0), 59 HbSC). Detection of gallstones was based on ultrasound of the liver/biliary tree. Regression analysis showed that serum bilirubin levels and the incidence of gallstones were strongly associated with the number of UGT1A1 [TA] repeats in all subjects (P < 0.0001 and P < 0.01, respectively). While HMOX1 genotype had no effect, co-inheritance of alpha-thalassaemia reduced serum bilirubin levels in all SCD patients independently of the number of UGT1A1 [TA] repeats. Each additional [TA] repeat is associated with an increase in mean serum bilirubin levels of 21% and cholelithiasis risk of 87% in SCD.

Control of immunologic tolerance and homeostasis rely on Foxp3+CD4+CD25+ regulatory T cells (Tregs) that constitutively express the high affinity receptor for Interleukin-2, CD25. Tregs proliferate in response to injections of IL-2/anti-IL-2 antibody complexes or low doses of IL-2. However, little is known about endogenous mechanisms that regulate the sensitivity of CD25 to signaling by IL-2. Here we demonstrate that CD25 is ADP-ribosylated at Arg35 in the IL-2 binding site by ecto-ADP-ribosyltransferase ARTC2.2, a toxin-related GPI-anchored ecto-enzyme. ADP-ribosylation inhibits binding of IL-2 by CD25, IL-2- induced phosphorylation of STAT5 and IL-2-dependent cell proliferation. Our study elucidates an as-yet-unrecognized mechanism to tune IL-2 signaling. This newly found mechanism might thwart Tregs at sites of inflammation and thereby permit a more potent response of activated effector T cells.

BACKGROUND: Common genetic variants residing near upstream regulatory elements for MYB, the gene encoding transcription factor cMYB, promote the persistence of fetal hemoglobin (HbF) into adulthood. While they have no consequences in healthy individuals, high HbF levels have major clinical benefits in patients with sickle cell disease (SCD) or β thalassemia. Here, we present our detailed investigation of HBS1L-MYB intergenic polymorphism block 2 (HMIP-2), the central component of the complex quantitative-trait locus upstream of MYB, in 1,022 individuals with SCD in Tanzania. METHODS: We have looked at 1022 individuals with HbSS or HbS/β(0) in Tanzania. In order to achieve a detailed analysis of HMIP-2, we performed targeted genotyping for a total of 10 SNPs and extracted additional 528 SNPs information from a genome wide scan involving the same population. Using MACH, we utilized the existing YRI data from 1000 genomes to impute 54 SNPs situated within HIMP-2. RESULTS: Seven HbF-increasing, low-frequency variants (β > 0.3, p

A genome-wide linkage analysis of platelet count was carried out in a large Asian Indian kindred. Linkage analysis showed one marker (D3S1309) on chromosome 3q with a lod score of 3.26 and another (D3S1282) approximately 30 cM centromeric, with a lod score of 2.52. Multipoint analysis of chromosome 3q identified two peaks with maximum multipoint lod scores of 3.52 and 4.11 under markers D3S1309 and D3S1282, respectively. Two strong candidate genes for platelet variation were identified in the linked region; thrombopoietin (THPO) and glycoprotein IX (GPIX). Resequencing of four individuals revealed five single-nucleotide polymorphisms (SNPs) in THPO and one mutation in the transmembrane region of GPIX. Analysis of variance showed that the GPIX mutation and one THPO SNP accounted for 6 and 4% of the variation in platelet count, respectively. The THPO SNP lies in the 3' untranslated region of the gene and has not been previously reported. The G to A transition at nucleotide 653 resulted in an Ala 156 (GCC) to Thr (ACC) replacement in the GPIX protein. The GPIX mutation was recently identified in a Chinese patient with Bernard-Soulier syndrome (BSS), a rare recessive bleeding disorder characterized by thrombocytopenia and giant platelets. One copy of the GPIX mutation was found in 300 European individuals with platelet counts within the normal range. The results suggest that two QTLs on chromosome 3q influence platelet count variation in the Asian Indian kindred, with the GPIX transmembrane mutation and the 3' UTR SNP in THPO being strong candidates.

Maturity-onset diabetes of the young (MODY) is a heterogeneous single gene disorder characterized by non–insulin-dependent diabetes, an early onset and autosomal dominant inheritance. Mutations in six genes have been shown to cause MODY. Approximately 15–20% of families fitting MODY criteria do not have mutations in any of the known genes. These families provide a rich resource for the identification of new MODY genes. This will potentially enable further dissection of clinical heterogeneity and bring new insights into mechanisms of β-cell dysfunction. To facilitate the identification of novel MODY loci, we combined the results from three genome-wide scans on a total of 23 families fitting MODY criteria. We used both a strict parametric model of inheritance with heterogeneity and a model-free analysis. We did not identify any single novel locus but provided putative evidence for linkage to chromosomes 6 (nonparametric linkage [NPL]score 2.12 at 71 cM) and 10 (NPL score 1.88 at 169–175 cM), and to chromosomes 3 (heterogeneity LOD [HLOD] score 1.27 at 124 cM) and 5 (HLOD score 1.22 at 175 cM) in 14 more strictly defined families. Our results provide evidence for further heterogeneity in MODY.

The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease. Fil: Unger, Mandy. University Medical Center Hamburg Eppendorf; Alemania Fil: Eichhoff, Anna Marei. University Medical Center Hamburg Eppendorf; Alemania Fil: Schumacher, Lucas. University Medical Center Hamburg Eppendorf; Alemania Fil: Strysio, Moritz. University Medical Center Hamburg Eppendorf; Alemania Fil: Menzel, Stephan. University Medical Center Hamburg Eppendorf; Alemania Fil: Schwan, Carsten. University of Freiburg; Alemania Fil: Alzogaray, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Instituto Leloir; Argentina Fil: Zylberman, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Inmunova S.A; Argentina Fil: Seman, Michel. Normandy University; Francia Fil: Brandner, Johanna. University Medical Center Hamburg Eppendorf; Alemania Fil: Rohde, Holger. University Medical Center Hamburg Eppendorf; Alemania Fil: Zhu, Kai. Schrodinger Inc.; Estados Unidos Fil: Haag, Friedrich. University Medical Center Hamburg Eppendorf; Alemania Fil: Mittrücker, Hans Willi. University Medical Center Hamburg Eppendorf; Alemania Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Instituto Leloir; Argentina Fil: Aktories, Klaus. University of Freiburg; Alemania Fil: Koch Nolte, Friedrich. University Medical Center Hamburg Eppendorf; Alemania

Protein mono-ADP-ribosylation is a reversible post-translational modification of cellular proteins. This scheme of amino-acid modification is used not only by bacterial toxins to attack host cells, but also by endogenous ADP-ribosyltransferases (ARTs) in mammalian cells. These latter ARTs include members of three different families of proteins: the well characterised arginine-specific ecto-enzymes (ARTCs), two sirtuins, and some members of the poly(ADP-ribose) polymerase (PARP/ARTD) family. In the present study, we demonstrate that human ARTC1 is localised to the endoplasmic reticulum (ER), in contrast to the previously characterised ARTC proteins, which are typical GPI-anchored ecto-enzymes. Moreover, using the “macro domain” cognitive binding module to identify ADP-ribosylated proteins, we show here that the ER luminal chaperone GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) is a cellular target of human ARTC1 and hamster ARTC2. We further developed a procedure to visualise ADP-ribosylated proteins using immunofluorescence. With this approach, in cells overexpressing ARTC1, we detected staining of the ER that co-localises with GRP78/BiP, thus confirming that this modification occurs in living cells. In line with the key role of GRP78/BiP in the ER stress response system, we provide evidence here that ARTC1 is activated during the ER stress response, which results in acute ADP-ribosylation of GRP78/BiP paralleling translational inhibition. Thus, this identification of ARTC1 as a regulator of GRP78/BiP defines a novel, previously unsuspected, player in GRP78-mediated ER stress responses.

BACKGROUND: Fetal hemoglobin (HbF) is an important modulator of sickle cell disease (SCD). HbF has previously been shown to be affected by variants at three loci on chromosomes 2, 6 and 11, but it is likely that additional loci remain to be discovered. METHODS AND FINDINGS: We conducted a genome-wide association study (GWAS) in 1,213 SCA (HbSS/HbSβ0) patients in Tanzania. Genotyping was done with Illumina Omni2.5 array and imputation using 1000 Genomes Phase I release data. Association with HbF was analysed using a linear mixed model to control for complex population structure within our study. We successfully replicated known associations for HbF near BCL11A and the HBS1L-MYB intergenic polymorphisms (HMIP), including multiple independent effects near BCL11A, consistent with previous reports. We observed eight additional associations with P

We have recently shown that mutations in the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1alpha are the cause of one form of maturity-onset diabetes of the young (MODY3). Here, we report the exon-intron organization and partial sequence of the human HNF-1alpha gene. In addition, we have screened the ten exons and flanking introns of this gene for mutations in a group of 25 unrelated white subjects from Germany who presented with NIDDM before 35 years of age and had a first-degree relative with NIDDM. Mutations were identified in nine of these individuals, suggesting that mutations in the HNF-1alpha gene are a common cause of diabetes in German subjects with early-onset NIDDM and a family history of diabetes. Thus, screening for mutations in this gene may be indicated in subjects with early-onset NIDDM. Interestingly, three of the nine mutations occurred at the same site in exon 4 with insertion of a C in a polyC tract, centered around codon 290 (designated Pro291fsinsC), thereby resulting in a frameshift during translation and premature termination. Analyses of linked DNA polymorphisms in the HNF-1alpha gene indicated that the Pro291fsinsC mutation was present on a different haplotype in each subject, implying that the polyC tract represents a mutational hot spot. We have also identified the mutation in the HNF-1alpha gene in the Jutland pedigree, one of the original MODY pedigrees reported in the literature, as being a T--G substitution in codon 241, resulting in the replacement of a conserved Cys by Gly (C241G). The information on the sequence of the HNF-1alpha gene and its promoter region will facilitate the search for mutations in other subjects and studies of the role of the gene in determining normal beta-cell functions.

In 1901, the first Nobel Prize in Physiology or Medicine was awarded to Emil von Behring for his ground-breaking discovery of serum therapy: serum from horses vaccinated with toxin-containing culture medium of Corynebacterium diphtheriae contained life-saving 'antitoxins'. The molecular nature of the ADP-ribosylating toxin and the neutralizing antibodies were unraveled only 50 years later. Today, von Behring's antibody therapy is being refined with a new generation of recombinant antibodies and antibody fragments. Nanobodies, which are single-domain antibodies derived from the peculiar heavy-chain antibodies of llamas and other camelids, are emerging as a promising new class of highly specific enzyme inhibitors. In this review, we illustrate the potential of nanobodies as tools to block extracellular and intracellular ADP-ribosyltransferases (ARTs), using the toxin-related membrane-bound mammalian ecto-enzyme ARTC2 and the actin-ADP-ribosylating Salmonella virulence plasmid factor B toxin of Salmonella enterica as examples. Fil: Menzel, Stephan. University Medical Center Hamburg-Eppendorf; Alemania Fil: Rissiek, Björn. University Medical Center Hamburg-Eppendorf; Alemania Fil: Haag, Friedrich. University Medical Center Hamburg-Eppendorf; Alemania Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Koch Nolte, Friedrich. University Medical Center Hamburg-Eppendorf; Alemania

Despite the identical genotypes, the clinical course of sickle cell disease (SCD) is extremely variable, prompting the search for genetic and biological predictors of disease severity.1,2 Raised white blood counts (WBC) have been known to be a marker of disease severity in SCD since the 1990s.1–3 “Benign ethnic neutropenia” has long been observed in hematology clinics in peoples of African descent. Recent work has shown that this relates to a single nucleotide polymorphism (SNP) rs2814778 T/C position -33 in the FY gene promoter resulting in lack of Duffy expression on red blood cells, and also a lower WBC.4–7 Afenyi-Annan et al. also found that the association with FY status held with and without hydroxycarbamide therapy.5 Relationships between the Duffy phenotype and markers of disease severity or the postulated disease phenotypes in SCD have been investigated but no clear consensus has been reached.5,8–10 However, using a multi-organ chronic disease score, Afenyi-Annan et al. reported an overall increase in the number of organs damaged and also an association with macroalbuminuria as shown by 1+ urine dip positivity in the Duffy negative group.5 For this study, DNA was extracted from buffy coats obtained via the sickle cell gene bank based at King’s College Hospital, London, UK (REC 07/H0606/165). All patients were of African origin; they were genotyped for SNP rs2814778 in the DARC (Duffy Antigen Receptor for Chemokines) promoter region using a TaqMan allelic discrimination assay and proprietary PCR primers from ABI biosystems. Biological data including hemoglobin (Hb), lactate dehydrogenase (LDH), WBC, neutrophil count, HbF, reticulocyte count, ferritin, creatinine, urine albumin creatinine ratio (ACR), cystatin C and erythropoietin levels were collected from routine blood results in patients attending steady state clinic during a 2-year period from January 1st 2009 to December 31st 2010. Estimated glomerular filtration rates (eGFRs) were calculated using the 4-point Modification of Diet in Renal Disease (MDRD) formula. Clinical data, including the presence of specific complications (stroke, priapism, leg ulcers, acute chest syndrome, avascular necrosis, retinopathy, tricuspid regurgitant jet velocity ≥2.5 m/s and gallstones) were collected from the electronic patient record system and sickle cell database. Definitions of clinical complications were made using local diagnostic guidelines which are based on published literature as reviewed by Ballas et al.11 Diagnosis of chronic sickle lung disease was made on chest CT scans as reviewed by a consultant chest radiologist. Admission data were also collected for the 2-year study period, including length of stay, time to readmission and number of admissions. Only admissions lasting over 24 h requiring inpatient stay were included; visits to the Accident and Emergency Department were excluded. Patient age at the end of the study period was recorded. Variables were log transformed where appropriate to obtain a normal distribution. A total of 272 patients were genotyped for SNP rs2814778 (T/C) in the DARC promoter. The Duffy phenotype was predicted to be negative based on the absence of the DARC -33T allele. The genotype was DARC 33C/C in 243 (89%) of patients. The Duffy phenotype was predicted to be positive based on the presence of the DARC 33T allele, with DARC -33T/C and -33T/T in 26 (10%) and 3 (1%) patients, respectively (Table 1). We observed the percentage of Duffy negative subjects to be much higher than the 63% and 73% reported in African Americans by Nalls et al. and Afenyi-Annan et al., respectively. The difference is likely to be related to the different degrees of European admixture. The UK cohort is predominantly West African with a smaller number of Afro-Caribbeans (Table 1). Table 1. Summary of demographic data and Duffy genotypes/phenotypes for study group and admission cohort. Laboratory data were available on the whole cohort. The clinical characteristics and Duffy genotypes (and phenotypes) of the study group (and subgroups) are outlined in Tables 1 and ​and2.2. There was no significant difference between the frequencies of rs2814778 T or C alleles between the different sickle genotypes. The predicted Duffy phenotypes were used to look for associations with markers of disease severity. Table 2. Summary of demographic data and Duffy genotypes/phenotypes for sickle cell anemia patients with clinical and end-organ damage data available. Laboratory data were available for the entire study group. Multiple regression analysis using a random effects model was used to analyze the data, enabling the pooling of multiple observations for the same individual. These data included both acute and steady-state results. Analysis showed significantly lower WBC and neutrophil counts for Duffy negative patients (P=0.001). Interestingly, raised Cystatin C and lower estimated glomerular filtration measurement using the Hoek formula12 were significantly associated with the DARC positive genotype (P=0.002 and 0.001, respectively), although no association was found with macroalbuminuria as defined by a urine albumin:creatinine ratio over 30, in line with National Institute for Health and Clinical Excellence (NICE) guidelines.13 The WBC and neutrophil rise in the acute phase was proportional to the starting value. Duffy status had no additional effect on hematologic parameters in the acute phase. These results are summarized in Table 2. Table 3. Laboratory values in Duffy negative and Duffy positive patients. Difference between the groups is expressed as a percentage or absolute difference except for Hb where absolute difference was used as the parameter. Table 4. Clinical complications and end-organ damage in Duffy positive and Duffy negative patients. Admission data were available for the whole cohort. Among the 272 patients, 112 (41%) had at least one admission during the 2-year study period. There was no difference in the distribution of Duffy phenotypes between the admitted and non-admitted groups; 89% of the total cohort were Duffy negative compared to 87% of the admitted group. The effect of Duffy status was analyzed using logistical regression. There were a total of 313 admissions ranging from 1 to 19 per patient (mean 3). Length of stay ranged from 1 to 74 days (mean 7). Sixty-nine of the 112 patients (61%) were readmitted within the study period (time to readmission 3–627 days, mean 158). There were no significant effects of Duffy status on length of stay or time to readmission. Analysis of clinical complications was limited to the 180 patients with Hb SS and Hb Sβ0 as patients with Hb SC and Hb Sβ+ tend to have a milder disease with fewer complications (Table 2). The groups were compared using Fisher’s exact test. Notably, the incidence of leg ulcers was significantly higher in Duffy positive compared to Duffy negative patients (P=0.04). Age, gender and alpha globin genotype had no effect on the frequency of leg ulcers. We noted a difference in leg ulcers between the Fy −ve and Fy +ve patients in the study by Afenyi-Annan et al. when compared to our present study: the population studied by Afenyi-Annan et al. had 25% Fy +ve patients indicating a greater degree of European admixture. The number of sickle-related complications also appeared to be significantly associated with Duffy status, but this effect disappeared when corrected for age. All other associations were non-significant. Our results confirm the findings of seven reports that Duffy negative patients have lower WBCs and neutrophil counts both in healthy subjects and in individuals with SCD. Both Duffy negative and Duffy positive patients appear to be able to mount an increase in WBC during acute events with no difference in the amplitude of the WBC or neutrophil count from steady-state to acute state. Although the association between macroalbuminuria and Duffy status did not reach significance, the trend was towards an increased prevalence in the Duffy positive group that is in contrast to Afenyi-Annan et al.5 Cystatin C (another marker of renal function) was also significantly associated with Duffy positive status. This needs to be explored further, correcting for other known influences on renal function such as the role of hemolysis and other genetic polymorphisms. Several factors, genetic and environmental, contribute to the development and persistence of leg ulcers14,15 that we found to be significantly higher in the Duffy positive group. We speculate that the relatively higher white cell and neutrophil counts potentiate inflammation, predisposing to skin infarction and infection in the Duffy positive patients. Our study has some limitations. Our sample numbers are small for some complications (including leg ulcers) and our study period for the admission cohort was limited to two years. It does not include data on Accident and Emergency attendances or the number of painful episodes patients have at home. It is also well known that admission to hospital and duration of stay are influenced by a range of cultural and social issues as well as intermittent illness, such as infections. It is important to note the potential effect of ethnic stratification on interpretation of the data. The Duffy null genotype is exclusive to Africans; hence, presence of the Duffy antigen infers European admixture, and potentially, the inheritance of other polymorphisms that could be responsible for the association. Nonetheless, associations between the Duffy phenotype and markers of disease severity could benefit from further investigation. Further work is required to validate the hypothesis that the raised WBC associated with Duffy positive phenotype could potentiate increased rates of vaso-occlusion and potentially be a risk factor for the development of leg ulceration and renal dysfunction.

The disease non-insulin-dependent (type 2) diabetes mellitus (NIDDM) is characterized by abnormally high blood glucose resulting from a relative deficiency of insulin. It affects about 2% of the world's population and treatment of diabetes and its complications are an increasing health-care burden. Genetic factors are important in the aetiology of NIDDM, and linkage studies are starting to localize some of the genes that influence the development of this disorder. Maturity-onset diabetes of the young (MODY), a single-gene disorder responsible for 2-5% of NIDDM, is characterized by autosomal dominant inheritance and an age of onset of 25 years or younger. MODY genes have been localized to chromosomes 7, 12 and 20 (refs 5, 7, 8) and clinical studies indicate that mutations in these genes are associated with abnormal patterns of glucose-stimulated insulin secretion. The gene on chromosome 7 (MODY2) encodes the glycolytic enzyme glucokinases which plays a key role in generating the metabolic signal for insulin secretion and in integrating hepatic glucose uptake. Here we show that subjects with the MODY3-form of NIDDM have mutations in the gene encoding hepatocyte nuclear factor-1alpha (HNF-1alpha, which is encoded by the gene TCF1). HNF-1alpha is a transcription factor that helps in the tissue-specific regulation of the expression of several liver genes and also functions as a weak transactivator of the rat insulin-I gene.

The expansion of trinucleotide repeats has been associated with late-onset neurodegenerative disorders. Although the genes harbouring the triplet expansions may be widely expressed, the pathological expression of these diseases is restricted to specific tissues. Non-insulin-dependent diabetes mellitus (NIDDM) shares several features with diseases resulting from such dynamic mutations including late-onset and specific but limited sites of tissue pathology-muscle, fat, liver and insulin-secreting pancreatic beta cells. In order to examine the contribution of genes containing polymorphic CAG/CTG repeats to the development of NIDDM, we screened an adult human skeletal muscle cDNA library for expressed sequences containing tandem repeats of CAG and/ or CTG. Ten different loci with polymorphic CAG/ CTG repeats were identified, of which seven had a heterozygosity greater than 0.20. There was no evidence for linkage between these seven loci and NIDDM in a group of affected Mexican-American sib pairs. Nor was there a significant difference in the distribution of alleles between Caucasian patients with NIDDM and normal healthy control subjects or evidence for repeat expansion in diabetic subjects. Thus, muscle genes with polymorphic CAG/CTG repeats do not appear to play a significant role in the development of NIDDM.

Genetic factors contribute to the development of NIDDM, and genes involved in regulating pancreatic β-cell function and insulin's effects on glucose metabolism are good candidates for being NIDDM susceptibility loci. However, testing candidate genes for linkage to NIDDM depends on the identification of highly informative DNA polymorphisms in or near the candidate locus. Here we describe an approach for identifying highly polymorphic markers near candidate genes that utilizes the emerging physical map of the human genome. A sequence-tagged site from the candidate gene is used to screen the Centre d'Etude du Polymorphisme Humain megabase-insert yeast artificial chromosome library, which contains information on the physical localization of >3,000 genetically mapped simple sequence repeat DNA polymorphisms. Thus, identification of a yeast artificial chromosome containing the candidate locus will in many instances also identify a physically linked simple sequence repeat DNA polymorphism that can be used as a marker for the candidate gene in linkage studies. We have used this approach to identify a marker for the islet amyloid polypeptide gene on chromosome 12. The physical mapping of this gene to a yeast artificial chromosome showed that it was in the same yeast artificial chromosome as the gene encoding liver glycogen synthase, another possible NIDDM susceptibility gene. Affected sib pair studies using a simple sequence repeat DNA polymorphism physically linked to the islet amyloid polypeptide and liver glycogen synthase genes showed no evidence for linkage with NIDDM, indicating that they are not major genes contributing to NIDDM susceptibility.

Fetal hemoglobin (HbF) is strongly associated with clinical severity in the β-hemoglobinopathies, including sickle cell disease (SCD). In recent years, the three major HbF genetic loci (at BCL11A on chromosome 2p, HMIP-2 on chromosome 6q and Xmn1-HBG on chromosome 11p) have been more clearly characterized and mechanisms of the likely causal variants better defined. In this study, we have combined this new biological understanding with statistical methods to create a genetic "predictor" for HbF in SCD. We chose 7 variants to represent the 3 HbF quantitative trait loci (QTL) to investigate their utility in predicting HbF levels, and, in turn, clinical severity of SCD. For BCL11A, we used 2 markers: rs1427407 (62kb downstream of BCL11A) localizes to an erythroid-specific enhancer (Bauer et al. Science 2013) and rs6545816 tags a second signal 58kb downstream of BCL11A. The genetic architecture of HMIP-2 as a QTL comprises two elements, A and B (Menzel et al. Ann Hum Genet 2014). We have represented HMIP-2A withthe 3bp deletion rs66650371, shown as a causal variant (Stadhouders et al. JCI 2014) plus the ethnicity marker rs9376090. HMIP-2B is less well-characterized, we selected: rs9494142 (near the MYB enhancer) and rs9494145. For the β-globin locus, we used the long-established Xmn1 marker (rs7482144) in the proximal promoter 158kb upstream of HBG2. This is likely not the variant itself, but in tight linkage disequilibrium with the causal element. Of 892 initial patients (516 females, 376 males), we excluded 17 children aged under 5 because of the non-linear relationship between age and HbF at a young age (we confirmed this finding in our cohort). This left: 658 with HbSS, 206 with HbSC, 8 with HbSβ0 thalassemia, and 20 with HbSβ+ thalassemia. We then genotyped 666 patients with HbSS/HbSβ0 thalassemia for the 7 genetic variants. For each patient, we selected 'validated' HbF levels i.e. HbF not influenced by transfusion, drugs (especially hydroxyurea) or pregnancy. HbF levels were log-transformed (Ln). We then used multiple linear regression models to identify variants which were independently associated with Ln-HbF levels. Using only age and sex as covariates revealed predictive power r2~10% which was orthogonal to (i.e. additive) the predictive power of the variants, and so we did not include them in subsequent analysis. Also, by adding α-globin status to the model where known (N=272), the r2 remained unchanged and is not significant for α-globin status. We then normalized the 7 variants to take account of the mean allele count (a strongly predictive but rare variant may not explain much of the total population variance). We performed multiple linear regression to rationalize the 7 variants, and found 4 markers (rs6545816, rs1427407, rs66650371 and rs7482144) independently contributing HbF-boosting alleles (see table). Combining these 4 variants into a genetic risk model, as per the table, allows us to predict 21.8% of variability (r2) of HbF in our HbSS / HbSβ0 thalassemia patients. We validated the 4-variant risk score first with a 5-fold cross-validation within the cohort which demonstrated a mean r2=22% for the 5 folds. We then replicated the findings in the cohort of HbSC patients (N=206) and found the 4-variant model to predict HbF with variability r2=27.5% (i.e. towards r2=44% seen in non-anemic individuals). Thus, our 4-variant model provides a robust approach to genetic prediction of HbF in SCD. The predictive power appears to be larger for HbSC compared to HbSS (r2=27.5% vs 21.8%) which may be related to stress erythropoiesis in HbSS patients releasing immature erythrocytes as a non-genetic factor modifying HbF levels. This process is a first step towards creating a global genetic predictive score in SCD: stratifying patients with SCD early in life would enable us to offer curative therapy (i.e. hematopoietic stem cell transplant) to those identified as genetically severe. Disclosures No relevant conflicts of interest to declare.

Using a genome-wide association study, we found that common inter-individual differences in haemoglobin A(2) (HbA(2) , α(2) δ(2) ) levels are largely governed by genetic factors (42% of variability). The influence of age (1%) and sex (4%) was small. HbA(2) levels were influenced by two loci: the HBS1L-MYB locus on chromosome 6q, which has been shown to have pleiotropic effects on other haematological traits; and a second locus surrounding HBB, the gene encoding β-globin. Our results suggest that HbA(2) levels in adults are influenced by two different biological processes: one via kinetics of erythropoiesis, and the other, via competition between HBB and HBD activity.

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count-3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.

Cytotoxic precipitation of free α-globin monomers and its production of reactive oxygen species cause red cell membrane damage that leads to anemia and eventually ineffective erythropoiesis in β-thalassemia. Alpha hemoglobin stabilizing protein (AHSP) was found to bind only to free α-globin monomers creating a stable and inert complex which remains soluble in the cytoplasm thus preventing harmful precipitations. Alpha hemoglobin stabilizing protein was shown to bind nascent α-globin monomers with transient strength before transferring α-globin to β-globin to form hemoglobin tetramer. A classical twin study would be beneficial to investigate the role of genetics and environment in the variation of alpha hemoglobin stabilizing protein expression as this knowledge will enable us to determine further investigations with regards to therapeutic interventions if alpha hemoglobin stabilizing protein is to be a therapeutic agent for β-thalassemia. This study investigates the heritability influence of alpha hemoglobin stabilizing protein expression and factors that may contribute to this. Results indicated that a major proportion of alpha hemoglobin stabilizing protein expression was influenced by genetic heritability (46%) withcis-acting factors accounting for 19% andtrans-acting factors at 27%.

Fetal hemoglobin (HbF, α2γ2) is a major contributor to the remarkable phenotypic heterogeneity of sickle cell anemia (SCA). Genetic variation at 3 principal loci (HBB cluster on chromosome 11p, HBS1L-MYB region on chromosome 6q, and BCL11A on chromosome 2p) have been shown to influence HbF levels and disease severity in β-thalassemia and SCA. Previous studies in SCA, however, have been restricted to populations from the African diaspora, which include multiple genealogies. We have investigated the influence of these 3 loci on HbF levels in sickle cell patients from Tanzania and in a small group of African British sickle patients. All 3 loci have a significant impact on the trait in both patient groups. The results suggest the presence of HBS1L-MYB variants affecting HbF in patients who are not tracked well by European-derived markers, such as rs9399137. Additional loci may be identified through independent genome-wide association studies in African populations.