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Abstract: The G haplotype is a group of co-inherited single nucleotide variants in the F5 gene that reduce venous thromboembolism (VTE) risk. Although 7% of the population is homozygous for the G haplotype (F5-G/G), the underlying mechanism of VTE protection is poorly understood. Using RNA sequencing data from 4651 blood donors in the INTERVAL study, we detected a rare excision event at the factor V (FV)-short splice sites in 5% of F5-G/Gs carriers as compared with 2.16% of homozygotes for the F5 reference sequence (F5-ref; P = .003). Highly elevated (∼10-fold) FV-short, a FV isoform that lacks most of the B-domain, has been linked with increased tissue factor inhibitor α (TFPIα) levels in rare hemorrhagic diathesis, including East Texas bleeding disorder. To ascertain whether the enhanced FV-short splicing seen in F5-G/G INTERVAL participants translated to increased plasma FV-short levels, we analyzed plasma samples from 7 F5-G/G and 13 F5-ref individuals in a recall-by-genotype study. A ∼2.2-fold higher amount of FV-short was found in a plasma pool from F5-G/G participants when compared with the pool of F5-refs (P = .029), but there was no difference in the total FV levels. Although no significant difference in TFPI levels were found, F5-G/Gs showed a ∼1.4-fold TFPI-dependent increase in lag time to thrombin generation than F5-refs (P = .0085). Finally, in an analysis of 117 699 UK Biobank participants, we discovered that, although being protective against VTE, the G haplotype also confers an increase in bleeding episodes (P = .011). Our study provides evidence that the effect of the common G haplotype is mediated by the FV-short/TFPI pathway., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Obesity rates have nearly tripled in the past 50 years, and by 2030 more than 1 billion individuals worldwide are projected to be obese. This creates a significant economic strain due to the associated non-communicable diseases. The root cause is an energy expenditure imbalance, owing to an interplay of lifestyle, environmental, and genetic factors. Obesity has a polygenic genetic architecture; however, single genetic variants with large effect size are etiological in a minority of cases. These variants allowed the discovery of novel genes and biology relevant to weight regulation and ultimately led to the development of novel specific treatments., Methods: We used a case-control approach to determine metabolic differences between individuals homozygous for a loss-of-function genetic variant in the small integral membrane protein 1 (SMIM1) and the general population, leveraging data from five cohorts. Metabolic characterization of SMIM1 -/- individuals was performed using plasma biochemistry, calorimetric chamber, and DXA scan., Findings: We found that individuals homozygous for a loss-of-function genetic variant in SMIM1 gene, underlying the blood group Vel, display excess body weight, dyslipidemia, altered leptin to adiponectin ratio, increased liver enzymes, and lower thyroid hormone levels. This was accompanied by a reduction in resting energy expenditure., Conclusion: This research identified a novel genetic predisposition to being overweight or obese. It highlights the need to investigate the genetic causes of obesity to select the most appropriate treatment given the large cost disparity between them., Funding: This work was funded by the National Institute of Health Research, British Heart Foundation, and NHS Blood and Transplant., Competing Interests: Declaration of interests J.S. is the deputy CEO and 50% owner of BLUsang AB. He holds patents on Vel genotyping (inventors: Jill Storry, Magnus Jöud, Björn Nilsson, and Martin L. Olsson). J.S. has received speaker fees, royalties, and honoraria from the following companies: Grifols Diagnostic Solutions, QuidelOrtho Inc., and Biorad Laboratories. J.S. receives an honorarium for Section Editor work, Vox Sanguinis from John Wiley & Sons Ltd. J.S. is Vice President of the International Society of Blood Transfusion and married to Professor M.L.O. M.L.O. is CEO and 50% owner of BLUsang AB. M.L.O. holds patents on Vel genotyping (inventors: Jill Storry, Magnus Jöud, Björn Nilsson, and Martin L. Olsson). M.L.O. received speaker fees, royalties, and honoraria from the following companies: Grifols Diagnostic Solutions, QuidelOrtho Inc., and Biorad Laboratories. M.L.O. is married to Adjunct Professor J.S. W.N.E. is chair of the International Council for Standardization in Haematology. W.N.E. works as advisor for Scorpio Labs and is on the editorial board of the Journal of Clinical Pathology. W.H.O. is chair of the Blood Transfusion Genomics Consortium. W.H.O. is in receipt of an educational/research grant from Thermo Fisher Scientific. N.G. offers scientific consulting services to Thermo Fisher Scientific., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Restless legs syndrome (RLS) affects up to 10% of older adults. Their healthcare is impeded by delayed diagnosis and insufficient treatment. To advance disease prediction and find new entry points for therapy, we performed meta-analyses of genome-wide association studies in 116,647 individuals with RLS (cases) and 1,546,466 controls of European ancestry. The pooled analysis increased the number of risk loci eightfold to 164, including three on chromosome X. Sex-specific meta-analyses revealed largely overlapping genetic predispositions of the sexes (r g  = 0.96). Locus annotation prioritized druggable genes such as glutamate receptors 1 and 4, and Mendelian randomization indicated RLS as a causal risk factor for diabetes. Machine learning approaches combining genetic and nongenetic information performed best in risk prediction (area under the curve (AUC) = 0.82-0.91). In summary, we identified targets for drug development and repurposing, prioritized potential causal relationships between RLS and relevant comorbidities and risk factors for follow-up and provided evidence that nonlinear interactions are likely relevant to RLS risk prediction., (© 2024. The Author(s).)

Rare genetic diseases affect millions, and identifying causal DNA variants is essential for patient care. Therefore, it is imperative to estimate the effect of each independent variant and improve their pathogenicity classification. Our study of 140 214 unrelated UK Biobank (UKB) participants found that each of them carries a median of 7 variants previously reported as pathogenic or likely pathogenic. We focused on 967 diagnostic-grade gene (DGG) variants for rare bleeding, thrombotic, and platelet disorders (BTPDs) observed in 12 367 UKB participants. By association analysis, for a subset of these variants, we estimated effect sizes for platelet count and volume, and odds ratios for bleeding and thrombosis. Variants causal of some autosomal recessive platelet disorders revealed phenotypic consequences in carriers. Loss-of-function variants in MPL, which cause chronic amegakaryocytic thrombocytopenia if biallelic, were unexpectedly associated with increased platelet counts in carriers. We also demonstrated that common variants identified by genome-wide association studies (GWAS) for platelet count or thrombosis risk may influence the penetrance of rare variants in BTPD DGGs on their associated hemostasis disorders. Network-propagation analysis applied to an interactome of 18 410 nodes and 571 917 edges showed that GWAS variants with large effect sizes are enriched in DGGs and their first-order interactors. Finally, we illustrate the modifying effect of polygenic scores for platelet count and thrombosis risk on disease severity in participants carrying rare variants in TUBB1 or PROC and PROS1, respectively. Our findings demonstrate the power of association analyses using large population datasets in improving pathogenicity classifications of rare variants., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Genetic studies of platelet reactivity (PR) phenotypes may identify novel antiplatelet drug targets. However, such studies have been limited by small sample sizes (n < 5000) because of the complexity of measuring PR. We trained a model to predict PR from complete blood count (CBC) scattergrams. A genome-wide association study of this phenotype in 29 806 blood donors identified 21 distinct associations implicating 20 genes, of which 6 have been identified previously. The effect size estimates were significantly correlated with estimates from a study of flow cytometry-measured PR and a study of a phenotype of in vitro thrombus formation. A genetic score of PR built from the 21 variants was associated with the incidence rates of myocardial infarction and pulmonary embolism. Mendelian randomization analyses showed that PR was causally associated with the risks of coronary artery disease, stroke, and venous thromboembolism. Our approach provides a blueprint for using phenotype imputation to study the determinants of hard-to-measure but biologically important hematological traits., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Repetitive sequences represent about 45% of the human genome. Some are transposable elements (TEs) with the ability to change their position in the genome, creating genetic variability both as insertions or deletions, with potential pathogenic consequences. We used long-read nanopore sequencing to identify TE variants in the genomes of 24 patients with antithrombin deficiency. We identified 7 344 TE insertions and 3 056 TE deletions, 2 926 were not previously described in publicly available databases. The insertions affected 3 955 genes, with 6 insertions located in exons, 3 929 in introns, and 147 in promoters. Potential functional impact was evaluated with gene annotation and enrichment analysis, which suggested a strong relationship with neuron-related functions and autism. We conclude that this study encourages the generation of a complete map of TEs in the human genome, which will be useful for identifying new TEs involved in genetic disorders., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Blood cells contain functionally important intracellular structures, such as granules, critical to immunity and thrombosis. Quantitative variation in these structures has not been subjected previously to large-scale genetic analysis. We perform genome-wide association studies of 63 flow-cytometry derived cellular phenotypes-including cell-type specific measures of granularity, nucleic acid content and reactivity-in 41,515 participants in the INTERVAL study. We identify 2172 distinct variant-trait associations, including associations near genes coding for proteins in organelles implicated in inflammatory and thrombotic diseases. By integrating with epigenetic data we show that many intracellular structures are likely to be determined in immature precursor cells. By integrating with proteomic data we identify the transcription factor FOG2 as an early regulator of platelet formation and α-granularity. Finally, we show that colocalisation of our associations with disease risk signals can suggest aetiological cell-types-variants in IL2RA and ITGA4 respectively mirror the known effects of daclizumab in multiple sclerosis and vedolizumab in inflammatory bowel disease., (© 2023. Springer Nature Limited.)

Stroke is the second leading cause of death with substantial unmet therapeutic needs. To identify potential stroke therapeutic targets, we estimate the causal effects of 308 plasma proteins on stroke outcomes in a two-sample Mendelian randomization framework and assess mediation effects by stroke risk factors. We find associations between genetically predicted plasma levels of six proteins and stroke (P ≤ 1.62 × 10 -4 ). The genetic associations with stroke colocalize (Posterior Probability >0.7) with the genetic associations of four proteins (TFPI, TMPRSS5, CD6, CD40). Mendelian randomization supports atrial fibrillation, body mass index, smoking, blood pressure, white matter hyperintensities and type 2 diabetes as stroke risk factors (P ≤ 0.0071). Body mass index, white matter hyperintensity and atrial fibrillation appear to mediate the TFPI, IL6RA, TMPRSS5 associations with stroke. Furthermore, thirty-six proteins are associated with one or more of these risk factors using Mendelian randomization. Our results highlight causal pathways and potential therapeutic targets for stroke., (© 2022. The Author(s).)

The identification of inherited antithrombin deficiency (ATD) is critical to prevent potentially life-threatening thrombotic events. Causal variants in SERPINC1 are identified for up to 70% of cases, the majority being single-nucleotide variants and indels. The detection and characterization of structural variants (SVs) in ATD remain challenging due to the high number of repetitive elements in SERPINC1 . Here, we performed long-read whole-genome sequencing on 10 familial and 9 singleton cases with type I ATD proven by functional and antigen assays, who were selected from a cohort of 340 patients with this rare disorder because genetic analyses were either negative, ambiguous, or not fully characterized. We developed an analysis workflow to identify disease-associated SVs. This approach resolved, independently of its size or type, all eight SVs detected by multiple ligation-dependent probe amplification, and identified for the first time a complex rearrangement previously misclassified as a deletion. Remarkably, we identified the mechanism explaining ATD in 2 out of 11 cases with previous unknown defect: the insertion of a novel 2.4 kb SINE-VNTR-Alu retroelement, which was characterized by de novo assembly and verified by specific polymerase chain reaction amplification and sequencing in the probands and affected relatives. The nucleotide-level resolution achieved for all SVs allowed breakpoint analysis, which revealed repetitive elements and microhomologies supporting a common replication-based mechanism for all the SVs. Our study underscores the utility of long-read sequencing technology as a complementary method to identify, characterize, and unveil the molecular mechanism of disease-causing SVs involved in ATD, and enlarges the catalogue of genetic disorders caused by retrotransposon insertions., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Metabolite levels measured in the human population are endophenotypes for biological processes. We combined sequencing data for 3,924 (whole-exome sequencing, WES, discovery) and 2,805 (whole-genome sequencing, WGS, replication) donors from a prospective cohort of blood donors in England. We used multiple approaches to select and aggregate rare genetic variants (minor allele frequency [MAF] < 0.1%) in protein-coding regions and tested their associations with 995 metabolites measured in plasma by using ultra-high-performance liquid chromatography-tandem mass spectrometry. We identified 40 novel associations implicating rare coding variants (27 genes and 38 metabolites), of which 28 (15 genes and 28 metabolites) were replicated. We developed algorithms to prioritize putative driver variants at each locus and used mediation and Mendelian randomization analyses to test directionality at associations of metabolite and protein levels at the ACY1 locus. Overall, 66% of reported associations implicate gene targets of approved drugs or bioactive drug-like compounds, contributing to drug targets' validating efforts., Competing Interests: Declaration of interests John Danesh reports grants, personal fees, and non-financial support from Merck Sharp & Dohme (MSD); grants, personal fees, and non-financial support from Novartis; grants from Pfizer; and grants from AstraZeneca outside the submitted work. John Danesh sits on the International Cardiovascular and Metabolic Advisory Board for Novartis (since 2010); the Steering Committee of UK Biobank (since 2011); the MRC International Advisory Group (ING) member, London (since 2013); the MRC High Throughput Science ‘Omics Panel Member, London (since 2013); the Scientific Advisory Committee for Sanofi (since 2013); the International Cardiovascular and Metabolism Research and Development Portfolio Committee for Novartis; and the AstraZeneca Genomics Advisory Board (2018). Adam Butterworth reports institutional grants from AstraZeneca, Bayer, Biogen, BioMarin, Bioverativ, Merk and Sanofi. During the course of the project Praveen Surendran became an employee of GSK, Lorenzo Bomba became an employee of BioMarin, Mohd Karim became an employee of Variant Bio and Qi Guo became an employee of BenevolentAI., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The interindividual variation in the functional response of platelets to activation by agonists is heritable. Genome-wide association studies (GWASs) of quantitative measures of platelet function have identified fewer than 20 distinctly associated variants, some with unknown mechanisms. Here, we report GWASs of pathway-specific functional responses to agonism by adenosine 5'-diphosphate, a glycoprotein VI-specific collagen mimetic, and thrombin receptor-agonist peptides, each specific to 1 of the G protein-coupled receptors PAR-1 and PAR-4, in subsets of 1562 individuals. We identified an association (P = 2.75 × 10-40) between a common intronic variant, rs10886430, in the G protein-coupled receptor kinase 5 gene (GRK5) and the sensitivity of platelets to activate through PAR-1. The variant resides in a megakaryocyte-specific enhancer that is bound by the transcription factors GATA1 and MEIS1. The minor allele (G) is associated with fewer GRK5 transcripts in platelets and the greater sensitivity of platelets to activate through PAR-1. We show that thrombin-mediated activation of human platelets causes binding of GRK5 to PAR-1 and that deletion of the mouse homolog Grk5 enhances thrombin-induced platelet activation sensitivity and increases platelet accumulation at the site of vascular injury. This corroborates evidence that the human G allele of rs10886430 is associated with a greater risk for cardiovascular disease. In summary, by combining the results of pathway-specific GWASs and expression quantitative trait locus studies in humans with the results from platelet function studies in Grk5-/- mice, we obtain evidence that GRK5 regulates the human platelet response to thrombin via the PAR-1 pathway., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Rare coding variation has historically provided the most direct connections between gene function and disease pathogenesis. By meta-analysing the whole exomes of 24,248 schizophrenia cases and 97,322 controls, we implicate ultra-rare coding variants (URVs) in 10 genes as conferring substantial risk for schizophrenia (odds ratios of 3-50, P < 2.14 × 10 -6 ) and 32 genes at a false discovery rate of <5%. These genes have the greatest expression in central nervous system neurons and have diverse molecular functions that include the formation, structure and function of the synapse. The associations of the NMDA (N-methyl-D-aspartate) receptor subunit GRIN2A and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor subunit GRIA3 provide support for dysfunction of the glutamatergic system as a mechanistic hypothesis in the pathogenesis of schizophrenia. We observe an overlap of rare variant risk among schizophrenia, autism spectrum disorders 1 , epilepsy and severe neurodevelopmental disorders 2 , although different mutation types are implicated in some shared genes. Most genes described here, however, are not implicated in neurodevelopment. We demonstrate that genes prioritized from common variant analyses of schizophrenia are enriched in rare variant risk 3 , suggesting that common and rare genetic risk factors converge at least partially on the same underlying pathogenic biological processes. Even after excluding significantly associated genes, schizophrenia cases still carry a substantial excess of URVs, which indicates that more risk genes await discovery using this approach., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro . Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system., Competing Interests: RKG acts as a consultant for UMOVIS lab and has received honoraria for educational activities from Johnson & Johnson, ViiV and GSK. J.N. holds an ITEN grant (10.13039/100004319Pfizer) to explore the role of deubiquitinating enzymes in oxygen-sensing unrelated to the current work. MT has received honoraria from GSK, Johnson & Johnson and Bayer unrelated to the current work, and is a member of the Scientific Advisory Board of MorphogenIX. JRB has received an honorarium from AstraZeneca for a presentation unrelated to the current work. DKM has held consultancies, research funding, or support for educational activities from NeuroTrauma Sciences LLC, Lantmannen AB, GlaxoSmithKline Ltd, PressuraNeuro Ltd, 10.13039/100009006Integra LifeSciences, and Cortirio Ltd. KGCS is a co-founder and/or consultant with PredictImmune, Rheos Medicines, GSK and Kymab, unrelated to the current work. A priority patent covering part of this work has been filed., (© 2022 The Authors.)

Each day, about 10 12 erythrocytes and platelets are released into the bloodstream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs among hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA counterpart. We found that only one out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of thousands of identified putative open reading frames, deep ribosome-footprinting sequencing, and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic., (© 2022 Nicolet et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Genetic association studies for blood cell traits, which are key indicators of health and immune function, have identified several hundred associations and defined a complex polygenic architecture. Polygenic scores (PGSs) for blood cell traits have potential clinical utility in disease risk prediction and prevention, but designing PGS remains challenging and the optimal methods are unclear. To address this, we evaluated the relative performance of 6 methods to develop PGS for 26 blood cell traits, including a standard method of pruning and thresholding (P + T) and 5 learning methods: LDpred2, elastic net (EN), Bayesian ridge (BR), multilayer perceptron (MLP) and convolutional neural network (CNN). We evaluated these optimized PGSs on blood cell trait data from UK Biobank and INTERVAL. We find that PGSs designed using common machine learning methods EN and BR show improved prediction of blood cell traits and consistently outperform other methods. Our analyses suggest EN/BR as the top choices for PGS construction, showing improved performance for 25 blood cell traits in the external validation, with correlations with the directly measured traits increasing by 10%-23%. Ten PGSs showed significant statistical interaction with sex, and sex-specific PGS stratification showed that all of them had substantial variation in the trajectories of blood cell traits with age. Genetic correlations between the PGSs for blood cell traits and common human diseases identified well-known as well as new associations. We develop machine learning-optimized PGS for blood cell traits, demonstrate their relationships with sex, age, and disease, and make these publicly available as a resource., Competing Interests: P.A. is a full-time employee of Regeneron Pharmaceuticals. A.S.B. has received grants (outside of this work) from AstraZeneca, Bayer, Biogen, BioMarin, Bioverativ, Merck, Novartis, Regeneron, and Sanofi. J.D. reports grants, personal fees, and non-financial support from Merck Sharp & Dohme (MSD); grants, personal fees, and non-financial support from Novartis; grants from Pfizer; and grants from AstraZeneca outside the submitted work. J.D. sits on the International Cardiovascular and Metabolic Advisory Board for Novartis (since 2010), serves on the Steering Committee of UK Biobank (since 2011), is an MRC International Advisory Group (ING) member, London (since 2013), an MRC High Throughput Science ‘Omics Panel Member, London (since 2013), a Scientific Advisory Committee member for Sanofi (since 2013), an International Cardiovascular and Metabolism Research and Development Portfolio Committee member for Novartis, and was a member of the Astra Zeneca Genomics Advisory Board (2018)., (© 2021 The Author(s).)

Cardiometabolic diseases are frequently polygenic in architecture, comprising a large number of risk alleles with small effects spread across the genome 1-3 . Polygenic scores (PGS) aggregate these into a metric representing an individual's genetic predisposition to disease. PGS have shown promise for early risk prediction 4-7 and there is an open question as to whether PGS can also be used to understand disease biology 8 . Here, we demonstrate that cardiometabolic disease PGS can be used to elucidate the proteins underlying disease pathogenesis. In 3,087 healthy individuals, we found that PGS for coronary artery disease, type 2 diabetes, chronic kidney disease and ischaemic stroke are associated with the levels of 49 plasma proteins. Associations were polygenic in architecture, largely independent of cis and trans protein quantitative trait loci and present for proteins without quantitative trait loci. Over a follow-up of 7.7 years, 28 of these proteins associated with future myocardial infarction or type 2 diabetes events, 16 of which were mediators between polygenic risk and incident disease. Twelve of these were druggable targets with therapeutic potential. Our results demonstrate the potential for PGS to uncover causal disease biology and targets with therapeutic potential, including those that may be missed by approaches utilizing information at a single locus., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Telomeres, the end fragments of chromosomes, play key roles in cellular proliferation and senescence. Here we characterize the genetic architecture of naturally occurring variation in leukocyte telomere length (LTL) and identify causal links between LTL and biomedical phenotypes in 472,174 well-characterized UK Biobank participants. We identified 197 independent sentinel variants associated with LTL at 138 genomic loci (108 new). Genetically determined differences in LTL were associated with multiple biological traits, ranging from height to bone marrow function, as well as several diseases spanning neoplastic, vascular and inflammatory pathologies. Finally, we estimated that, at the age of 40 years, people with an LTL >1 s.d. shorter than the population mean had a 2.5-year-lower life expectancy compared with the group with ≥1 s.d. longer LDL. Overall, we furnish new insights into the genetic regulation of LTL, reveal wide-ranging influences of LTL on physiological traits, diseases and longevity, and provide a powerful resource available to the global research community., (© 2021. The Author(s).)

The implementation of high-throughput sequencing (HTS) technologies in research and diagnostic laboratories has linked many new genes to rare bleeding, thrombotic, and platelet disorders (BTPD), and revealed multiple genetic variants linked to those disorders, many of them being of uncertain pathogenicity when considering the accepted evidence (variant consequence, frequency in control datasets, number of reported patients, prediction models, and functional assays). The sequencing effort has also resulted in resources for gathering disease-causing variants associated with specific genes, but for BTPD, such well-curated databases exist only for a few genes. On the other hand, submissions by individuals or diagnostic laboratories to the variant database ClinVar are hampered by the lack of a submission process tailored to capture the specific features of hemostatic diseases. As we move toward the implementation of HTS in the diagnosis of BTPD, the Scientific and Standardization Committee for Genetics in Thrombosis and Haemostasis has developed and tested a REDCap-based interface, aimed at the community, to submit curated genetic variants for diagnostic-grade BTPD genes. Here, we describe the use of the interface and the initial submission of 821 variants from 30 different centers covering 14 countries. This open-access variant resource will be shared with the community to improve variant classification and regular bulk data transfer to ClinVar., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Variation in adiposity is associated with cardiometabolic disease outcomes, but mechanisms leading from this exposure to disease are unclear. This study aimed to estimate effects of body mass index (BMI) on an extensive set of circulating proteins., Methods: We used SomaLogic proteomic data from up to 2737 healthy participants from the INTERVAL study. Associations between self-reported BMI and 3622 unique plasma proteins were explored using linear regression. These were complemented by Mendelian randomisation (MR) analyses using a genetic risk score (GRS) comprised of 654 BMI-associated polymorphisms from a recent genome-wide association study (GWAS) of adult BMI. A disease enrichment analysis was performed using DAVID Bioinformatics 6.8 for proteins which were altered by BMI., Results: Observationally, BMI was associated with 1576 proteins (P < 1.4 × 10 -5 ), with particularly strong evidence for a positive association with leptin and fatty acid-binding protein-4 (FABP4), and a negative association with sex hormone-binding globulin (SHBG). Observational estimates were likely confounded, but the GRS for BMI did not associate with measured confounders. MR analyses provided evidence for a causal relationship between BMI and eight proteins including leptin (0.63 standard deviation (SD) per SD BMI, 95% CI 0.48-0.79, P = 1.6 × 10 -15 ), FABP4 (0.64 SD per SD BMI, 95% CI 0.46-0.83, P = 6.7 × 10 -12 ) and SHBG (-0.45 SD per SD BMI, 95% CI -0.65 to -0.25, P = 1.4 × 10 -5 ). There was agreement in the magnitude of observational and MR estimates (R 2  = 0.33) and evidence that proteins most strongly altered by BMI were enriched for genes involved in cardiovascular disease., Conclusions: This study provides evidence for a broad impact of adiposity on the human proteome. Proteins strongly altered by BMI include those involved in regulating appetite, sex hormones and inflammation; such proteins are also enriched for cardiovascular disease-related genes. Altogether, results help focus attention onto new proteomic signatures of obesity-related disease., (© 2021. The Author(s).)

Transcriptional profiling of hematopoietic cell subpopulations has helped to characterize the developmental stages of the hematopoietic system and the molecular bases of malignant and non-malignant blood diseases. Previously, only the genes targeted by expression microarrays could be profiled genome-wide. High-throughput RNA sequencing, however, encompasses a broader repertoire of RNA molecules, without restriction to previously annotated genes. We analyzed the BLUEPRINT consortium RNA-sequencing data for mature hematopoietic cell types. The data comprised 90 total RNA-sequencing samples, each composed of one of 27 cell types, and 32 small RNA-sequencing samples, each composed of one of 11 cell types. We estimated gene and isoform expression levels for each cell type using existing annotations from Ensembl. We then used guided transcriptome assembly to discover unannotated transcripts. We identified hundreds of novel non-coding RNA genes and showed that the majority have cell type-dependent expression. We also characterized the expression of circular RNA and found that these are also cell type-specific. These analyses refine the active transcriptional landscape of mature hematopoietic cells, highlight abundant genes and transcriptional isoforms for each blood cell type, and provide a valuable resource for researchers of hematologic development and diseases. Finally, we made the data accessible via a web-based interface: https://blueprint.haem.cam.ac.uk/bloodatlas/.

Mitochondrial DNA (mtDNA) variants influence the risk of late-onset human diseases, but the reasons for this are poorly understood. Undertaking a hypothesis-free analysis of 5,689 blood-derived biomarkers with mtDNA variants in 16,220 healthy donors, here we show that variants defining mtDNA haplogroups Uk and H4 modulate the level of circulating N-formylmethionine (fMet), which initiates mitochondrial protein translation. In human cytoplasmic hybrid (cybrid) lines, fMet modulated both mitochondrial and cytosolic proteins on multiple levels, through transcription, post-translational modification and proteolysis by an N-degron pathway, abolishing known differences between mtDNA haplogroups. In a further 11,966 individuals, fMet levels contributed to all-cause mortality and the disease risk of several common cardiovascular disorders. Together, these findings indicate that fMet plays a key role in common age-related disease through pleiotropic effects on cell proteostasis., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Trait-associated genetic variants affect complex phenotypes primarily via regulatory mechanisms on the transcriptome. To investigate the genetics of gene expression, we performed cis- and trans-expression quantitative trait locus (eQTL) analyses using blood-derived expression from 31,684 individuals through the eQTLGen Consortium. We detected cis-eQTL for 88% of genes, and these were replicable in numerous tissues. Distal trans-eQTL (detected for 37% of 10,317 trait-associated variants tested) showed lower replication rates, partially due to low replication power and confounding by cell type composition. However, replication analyses in single-cell RNA-seq data prioritized intracellular trans-eQTL. Trans-eQTL exerted their effects via several mechanisms, primarily through regulation by transcription factors. Expression of 13% of the genes correlated with polygenic scores for 1,263 phenotypes, pinpointing potential drivers for those traits. In summary, this work represents a large eQTL resource, and its results serve as a starting point for in-depth interpretation of complex phenotypes., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Studies on the relationship between renal function and the human plasma proteome have identified several potential biomarkers. However, investigations have been conducted largely in European populations, and causality of the associations between plasma proteins and kidney function has never been addressed., Methods: A cross-sectional study of 993 plasma proteins among 2882 participants in four studies of European and admixed ancestries (KORA, INTERVAL, HUNT, QMDiab) identified transethnic associations between eGFR/CKD and proteomic biomarkers. For the replicated associations, two-sample bidirectional Mendelian randomization (MR) was used to investigate potential causal relationships. Publicly available datasets and transcriptomic data from independent studies were used to examine the association between gene expression in kidney tissue and eGFR., Results: In total, 57 plasma proteins were associated with eGFR, including one novel protein. Of these, 23 were additionally associated with CKD. The strongest inferred causal effect was the positive effect of eGFR on testican-2, in line with the known biological role of this protein and the expression of its protein-coding gene ( SPOCK2 ) in renal tissue. We also observed suggestive evidence of an effect of melanoma inhibitory activity (MIA), carbonic anhydrase III, and cystatin-M on eGFR., Conclusions: In a discovery-replication setting, we identified 57 proteins transethnically associated with eGFR. The revealed causal relationships are an important stepping stone in establishing testican-2 as a clinically relevant physiological marker of kidney disease progression, and point to additional proteins warranting further investigation., (Copyright © 2021 by the American Society of Nephrology.)

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

Background: Megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. They mature within the adult bone marrow and release platelets into the circulation. Until now, there have been no transcriptional studies of primary human bone marrow MKs., Objectives: To characterize MKs and HSCs from human bone marrow using single-cell RNA sequencing, to investigate MK lineage commitment, maturation steps, and thrombopoiesis., Results: We show that MKs at different levels of polyploidization exhibit distinct transcriptional states. Although high levels of platelet-specific gene expression occur in the lower ploidy classes, as polyploidization increases, gene expression is redirected toward translation and posttranslational processing transcriptional programs, in preparation for thrombopoiesis. Our findings are in keeping with studies of MK ultrastructure and supersede evidence generated using in vitro cultured MKs. Additionally, by analyzing transcriptional signatures of a single HSC, we identify two MK-biased HSC subpopulations exhibiting unique differentiation kinetics. We show that human bone marrow MKs originate from these HSC subpopulations, supporting the notion that they display priming for MK differentiation. Finally, to investigate transcriptional changes in MKs associated with stress thrombopoiesis, we analyzed bone marrow MKs from individuals with recent myocardial infarction and found a specific gene expression signature. Our data support the modulation of MK differentiation in this thrombotic state., Conclusions: Here, we use single-cell sequencing for the first time to characterize the human bone marrow MK transcriptome at different levels of polyploidization and investigate their differentiation from the HSC., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Objective: To compare four haemoglobin measurement methods in whole blood donors., Background: To safeguard donors, blood services measure haemoglobin concentration in advance of each donation. NHS Blood and Transplant's (NHSBT) customary method have been capillary gravimetry (copper sulphate), followed by venous spectrophotometry (HemoCue) for donors failing gravimetry. However, NHSBT's customary method results in 10% of donors being inappropriately bled (ie, with haemoglobin values below the regulatory threshold)., Methods: We compared the following four methods in 21 840 blood donors (aged ≥18 years) recruited from 10 NHSBT centres in England, with the Sysmex XN-2000 haematology analyser, the reference standard: (1) NHSBT's customary method; (2) "post donation" approach, that is, estimating current haemoglobin concentration from that measured by a haematology analyser at a donor's most recent prior donation; (3) "portable haemoglobinometry" (using capillary HemoCue); (4) non-invasive spectrometry (using MBR Haemospect or Orsense NMB200). We assessed sensitivity; specificity; proportion who would have been inappropriately bled, or rejected from donation ("deferred") incorrectly; and test preference., Results: Compared with the reference standard, the methods ranged in test sensitivity from 17.0% (MBR Haemospect) to 79.0% (portable haemoglobinometry) in men, and from 19.0% (MBR Haemospect) to 82.8% (portable haemoglobinometry) in women. For specificity, the methods ranged from 87.2% (MBR Haemospect) to 99.9% (NHSBT's customary method) in men, and from 74.1% (Orsense NMB200) to 99.8% (NHSBT's customary method) in women. The proportion of donors who would have been inappropriately bled ranged from 2.2% in men for portable haemoglobinometry to 18.9% in women for MBR Haemospect. The proportion of donors who would have been deferred incorrectly with haemoglobin concentration above the minimum threshold ranged from 0.1% in men for NHSBT's customary method to 20.3% in women for OrSense. Most donors preferred non-invasive spectrometry., Conclusion: In the largest study reporting head-to-head comparisons of four methods to measure haemoglobin prior to blood donation, our results support replacement of NHSBT's customary method with portable haemoglobinometry., (© 2020 The Authors. Transfusion Medicine published by John Wiley & Sons Ltd on behalf of British Blood Transfusion Society.)

Iron is essential for many biological functions and iron deficiency and overload have major health implications. We performed a meta-analysis of three genome-wide association studies from Iceland, the UK and Denmark of blood levels of ferritin (N = 246,139), total iron binding capacity (N = 135,430), iron (N = 163,511) and transferrin saturation (N = 131,471). We found 62 independent sequence variants associating with iron homeostasis parameters at 56 loci, including 46 novel loci. Variants at DUOX2, F5, SLC11A2 and TMPRSS6 associate with iron deficiency anemia, while variants at TF, HFE, TFR2 and TMPRSS6 associate with iron overload. A HBS1L-MYB intergenic region variant associates both with increased risk of iron overload and reduced risk of iron deficiency anemia. The DUOX2 missense variant is present in 14% of the population, associates with all iron homeostasis biomarkers, and increases the risk of iron deficiency anemia by 29%. The associations implicate proteins contributing to the main physiological processes involved in iron homeostasis: iron sensing and storage, inflammation, absorption of iron from the gut, iron recycling, erythropoiesis and bleeding/menstruation.

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored., (© 2021 by The American Society of Hematology.)

High levels of tissue factor pathway inhibitor (TFPI), caused by a longer TFPIα half-life after binding to a factor V splice variant and variants in the F5 gene, were recently identified in 2 families with an as-yet-unexplained bleeding tendency. This study aimed to investigate free TFPIα in a well-characterized cohort of 620 patients with mild to moderate bleeding tendencies and its association to genetic alterations in the F5 gene. TFPIα levels were higher in patients with bleeding compared with healthy controls (median [interquartile range], 8.2 [5.5-11.7] vs 7.8 [4.3-11.1]; P = .026). A higher proportion of patients had free TFPIα levels more than or equal to the 95th percentile compared with healthy controls (odds ratio [OR] [95% confidence interval (CI)], 2.82 [0.98-8.13]). This was pronounced in the subgroup of patients in whom no bleeding disorder could be identified (bleeding of unknown cause [BUC; n = 420]; OR [95% CI], 3.03 [1.02-8.98]) and in platelet function defects (PFDs) (n = 121; OR [95% CI], 3.47 [1.09-11.08]). An increase in free TFPIα was associated with a mild delay in thrombin generation (prolonged lag time and time to peak), but not with alterations in routinely used global clotting tests. We could neither identify new or known genetic variations in the F5 gene that are associated with free TFPIα levels, nor an influence of the single-nucleotide variant rs10800453 on free TFPIα levels in our patient cohort. An imbalance of natural coagulation inhibitors such as TFPIα could be an underlying cause or contributor for unexplained bleeding, which is most probably multifactorial in a majority of patients., (© 2021 by The American Society of Hematology.)

The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. This unusual clinical course is consistent with a contribution of antibodies to both viral clearance and progression to severe disease. In the absence of these confounders, we take an experimental medicine approach to examine the in vivo utility of remdesivir. Over two independent courses of treatment, we observe a temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients.

Restless legs syndrome (RLS) is a common neurological sensorimotor disorder often described as an unpleasant sensation associated with an urge to move the legs. Here we report findings from a meta-analysis of genome-wide association studies of RLS including 480,982 Caucasians (cases = 10,257) and a follow up sample of 24,977 (cases = 6,651). We confirm 19 of the 20 previously reported RLS sequence variants at 19 loci and report three novel RLS associations; rs112716420-G (OR = 1.25, P = 1.5 × 10 -18 ), rs10068599-T (OR = 1.09, P = 6.9 × 10 -10 ) and rs10769894-A (OR = 0.90, P = 9.4 × 10 -14 ). At four of the 22 RLS loci, cis-eQTL analysis indicates a causal impact on gene expression. Through polygenic risk score for RLS we extended prior epidemiological findings implicating obesity, smoking and high alcohol intake as risk factors for RLS. To improve our understanding, with the purpose of seeking better treatments, more genetics studies yielding deeper insights into the disease biology are needed.

Angiotensin-converting enzyme 2 (ACE2) and serine protease TMPRSS2 have been implicated in cell entry for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19). The expression of ACE2 and TMPRSS2 in the lung epithelium might have implications for the risk of SARS-CoV-2 infection and severity of COVID-19. We use human genetic variants that proxy angiotensin-converting enzyme (ACE) inhibitor drug effects and cardiovascular risk factors to investigate whether these exposures affect lung ACE2 and TMPRSS2 gene expression and circulating ACE2 levels. We observed no consistent evidence of an association of genetically predicted serum ACE levels with any of our outcomes. There was weak evidence for an association of genetically predicted serum ACE levels with ACE2 gene expression in the Lung eQTL Consortium ( p = 0.014), but this finding did not replicate. There was evidence of a positive association of genetic liability to type 2 diabetes mellitus with lung ACE2 gene expression in the Gene-Tissue Expression (GTEx) study ( p = 4 × 10 -4 ) and with circulating plasma ACE2 levels in the INTERVAL study ( p = 0.03), but not with lung ACE2 expression in the Lung eQTL Consortium study ( p = 0.68). There were no associations of genetically proxied liability to the other cardiometabolic traits with any outcome. This study does not provide consistent evidence to support an effect of serum ACE levels (as a proxy for ACE inhibitors) or cardiometabolic risk factors on lung ACE2 and TMPRSS2 expression or plasma ACE2 levels., Competing Interests: D.G. is a part-time employee of Novo Nordisk. A.S.B. reports grants outside of this work from AstraZeneca, Biogen, BioMarin, Bioverativ, Merck, Novartis, Pfizer and Sanofi and personal fees from Novartis. B.E.E. is on the scientific advisory boards of Freenome and Celsius Therapeutics and is a full-time employee at Genomics plc while on a leave of absence from Princeton University. J.E.P. has received travel and accommodation expenses and hospitality from Olink to speak at Olink-sponsored academic meetings. E.P. is a full-time employee of Pfizer. J.D. reports grants, personal fees and non-financial support from Merck Sharp & Dohme (MSD), grants, personal fees and non-financial support from Novartis, grants from Pfizer and grants from AstraZeneca outside the submitted work. J.D. sits on the International Cardiovascular and Metabolic Advisory Board for Novartis (since 2010); the Steering Committee of UK Biobank (since 2011); the MRC International Advisory Group (ING) member, London (since 2013); the MRC High Throughput Science 'Omics Panel Member, London (since 2013); the Scientific Advisory Committee for Sanofi (since 2013); the International Cardiovascular and Metabolism Research and Development Portfolio Committee for Novartis; and the AstraZeneca Genomics Advisory Board (since 2018). All other authors have no conflicts of interest to declare., (© 2020 The Authors.)

Both poly(A) enrichment and ribosomal RNA depletion are commonly used for RNA sequencing. Either has its advantages and disadvantages that may lead to biases in the downstream analyses. To better access these effects, we carried out both ribosomal RNA-depleted and poly(A)-selected RNA-seq for CD4 + T naive cells isolated from 40 healthy individuals from the Blueprint Project. For these 40 individuals, the genomic and epigenetic data were also available. This dataset offers a unique opportunity to understand how library construction influences differential gene expression, alternative splicing and molecular QTL (quantitative loci) analyses for human primary cells.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease., (© 2020 by The American Society of Hematology.)

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT., (© 2020 by The American Society of Hematology.)

Tissue factor (TF) is the primary initiator of blood coagulation in vivo and the only blood coagulation factor for which a human genetic defect has not been described. As there are no routine clinical assays that capture the contribution of endogenous TF to coagulation initiation, the extent to which reduced TF activity contributes to unexplained bleeding is unknown. Using whole genome sequencing, we identified a heterozygous frameshift variant (p.Ser117HisfsTer10) in F3, the gene encoding TF, causing premature termination of TF (TFshort) in a woman with unexplained bleeding. Routine hematological laboratory evaluation of the proposita was normal. CRISPR-edited human induced pluripotent stem cells recapitulating the variant were differentiated into vascular smooth muscle and endothelial cells that demonstrated haploinsufficiency of TF. The variant F3 transcript is eliminated by nonsense-mediated decay. Neither overexpression nor addition of exogenous recombinant TFshort inhibited factor Xa or thrombin generation, excluding a dominant-negative mechanism. F3+/- mice provide an animal model of TF haploinsufficiency and exhibited prolonged bleeding times, impaired thrombus formation, and reduced survival following major injury. Heterozygous TF deficiency is present in at least 1 in 25,000 individuals and could limit coagulation initiation in undiagnosed individuals with abnormal bleeding but a normal routine laboratory evaluation.

Most loci identified by GWASs have been found in populations of European ancestry (EUR). In trans-ethnic meta-analyses for 15 hematological traits in 746,667 participants, including 184,535 non-EUR individuals, we identified 5,552 trait-variant associations at p < 5 × 10 -9 , including 71 novel associations not found in EUR populations. We also identified 28 additional novel variants in ancestry-specific, non-EUR meta-analyses, including an IL7 missense variant in South Asians associated with lymphocyte count in vivo and IL-7 secretion levels in vitro. Fine-mapping prioritized variants annotated as functional and generated 95% credible sets that were 30% smaller when using the trans-ethnic as opposed to the EUR-only results. We explored the clinical significance and predictive value of trans-ethnic variants in multiple populations and compared genetic architecture and the effect of natural selection on these blood phenotypes between populations. Altogether, our results for hematological traits highlight the value of a more global representation of populations in genetic studies., Competing Interests: Declaration of Interests Competing financial interests are declared in Table S1F., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Blood cells play essential roles in human health, underpinning physiological processes such as immunity, oxygen transport, and clotting, which when perturbed cause a significant global health burden. Here we integrate data from UK Biobank and a large-scale international collaborative effort, including data for 563,085 European ancestry participants, and discover 5,106 new genetic variants independently associated with 29 blood cell phenotypes covering a range of variation impacting hematopoiesis. We holistically characterize the genetic architecture of hematopoiesis, assess the relevance of the omnigenic model to blood cell phenotypes, delineate relevant hematopoietic cell states influenced by regulatory genetic variants and gene networks, identify novel splice-altering variants mediating the associations, and assess the polygenic prediction potential for blood traits and clinical disorders at the interface of complex and Mendelian genetics. These results show the power of large-scale blood cell trait GWAS to interrogate clinically meaningful variants across a wide allelic spectrum of human variation., Competing Interests: Declaration of Interests Adam Butterworth has received grants (outside of this work) from AstraZeneca, Biogen, BioMarin, Bioverativ, Merck, Novartis, and Sanofi; James Floyd has consulted for Shionogi; Qi Guo is a full-time employee of BenevolentAI; Joanna Howson is a full-time employee of Novo Nordisk. Parsa Akbari is a full-time employee of Regeneron Pharmaceuticals., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non-self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.

Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered 1 . Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants 2 , we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.

Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies 1-3 . Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed 4 ) identified multiple new candidate PID-associated genes, including IVNS1ABP. We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of-and interplay between-novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans.

Background: The MNS blood group system is defined by three homologous genes: GYPA, GYPB, and GYPE. GYPB encodes for glycophorin B (GPB) carrying S/s and the "universal" antigen U. RBCs of approximately 1% of individuals of African ancestry are U- due to absence of GPB. The U- phenotype has long been attributed to a deletion encompassing GYPB exons 2 to 5 and GYPE exon 1 (GYPB*01N)., Study Design and Methods: Samples from two U-individuals underwent Illumina short read whole genome sequencing (WGS) and Nanopore long read WGS. In addition, two existing WGS datasets, MedSeq (n = 110) and 1000 Genomes (1000G, n = 2535), were analyzed for GYPB deletions. Deletions were confirmed by Sanger sequencing. Twenty known U- donor samples were tested by a PCR assay to determine the specific deletion alleles present in African Americans., Results: Two large GYPB deletions in U- samples of African ancestry were identified: a 110 kb deletion extending left of GYPB (DEL&#95;B&#95;LEFT) and a 103 kb deletion extending right (DEL&#95;B&#95;RIGHT). DEL&#95;B&#95;LEFT and DEL&#95;B&#95;RIGHT were the most common GYPB deletions in the 1000 Genomes Project 669 African genomes (allele frequencies 0.04 and 0.02). Seven additional deletions involving GYPB were seen in African, Admixed American, and South Asian samples. No samples analyzed had GYPB*01N., Conclusions: The U- phenotype in those of African ancestry is primarily associated with two different complete deletions of GYPB (with intact GYPE). Seven additional less common GYPB deletion backgrounds were found. GYPB*01N, long assumed to be the allele commonly encoding U- phenotypes, appears to be rare., (© 2020 AABB.)