Preliminary Results of New Modification of Vertical Muscle Transposition to Enhance Abducting Force in Sixth Nerve Palsy.

Introduction: Since 1907, multiple transposition procedures have been established for the treatment of abducens paralysis. The purpose of the study was to determine where the transposed muscle should be reattached in order to increase the tangential force necessary to improve abduction. Methods: Retrospective case review of 12 consecutive patients with abducens paralysis who underwent transposition procedures between 2016 and 2019 was conducted. Vertical rectus muscles are transposed to the insertion of lateral rectus muscle; the temporal parts are joined and sutured to the sclera on top of the lateral rectus muscle in the middle of the insertion. The nasal parts are sutured to the sclera following the spiral of Tillaux. The muscle junction suture is placed 8 mm from the insertion, with the temporal parts of the vertical muscles bellies joined and sutured to the lateral rectus muscle. A full-tendon transposition was performed on 11 patients, a half-tendon transposition procedure on 1 patient. The minimum follow-up was 3 months. Results: The mean preoperative deviation was ET of 37° (range: ET 24° to ET 51°). The mean preoperative abduction limitation was 5 mm from midline (range: 7 to 1 mm). The postoperative mean deviation was ET of 2° (range: 0° to ET 5°). The postoperative mean abduction improvement was 5 mm past midline (range: 2-6 mm). There were no complications or signs of anterior segment ischemia. Conclusion: To achieve the maximal abductive force from the transposed muscles, we suggest that the vertical muscles be reattached as close as possible to the middle of the lateral rectus insertion.

A genome-wide association study of blood cell morphology identifies cellular proteins implicated in disease aetiology.

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.

A signature of platelet reactivity in CBC scattergrams reveals genetic predictors of thrombotic disease risk.

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.

Genetic regulation of fetal hemoglobin across global populations.

Human genetic variation has enabled the identification of several key regulators of fetal-to-adult hemoglobin switching, including BCL11A, resulting in therapeutic advances. However, despite the progress made, limited further insights have been obtained to provide a fuller accounting of how genetic variation contributes to the global mechanisms of fetal hemoglobin (HbF) gene regulation. Here, we have conducted a multi-ancestry genome-wide association study of 28,279 individuals from several cohorts spanning 5 continents to define the architecture of human genetic variation impacting HbF. We have identified a total of 178 conditionally independent genome-wide significant or suggestive variants across 14 genomic windows. Importantly, these new data enable us to better define the mechanisms by which HbF switching occurs in vivo. We conduct targeted perturbations to define BACH2 as a new genetically-nominated regulator of hemoglobin switching. We define putative causal variants and underlying mechanisms at the well-studied BCL11A and HBS1L-MYB loci, illuminating the complex variant-driven regulation present at these loci. We additionally show how rare large-effect deletions in the HBB locus can interact with polygenic variation to influence HbF levels. Our study paves the way for the next generation of therapies to more effectively induce HbF in sickle cell disease and ß-thalassemia.

Incidence of Glaucoma-Related Adverse Events in the First 5 Years After Pediatric Lensectomy.

Importance: Glaucoma can develop following cataract removal in children. Objective: To assess the cumulative incidence of glaucoma-related adverse events (defined as glaucoma or glaucoma suspect) and factors associated with risk of these adverse events in the first 5 years after lensectomy prior to 13 years of age. Design, Setting, and Participants: This cohort study used longitudinal registry data collected at enrollment and annually for 5 years from 45 institutional and 16 community sites. Participants were children aged 12 years or younger with at least 1 office visit after lensectomy from June 2012 to July 2015. Data were analyzed from February through December 2022. Exposures: Usual clinical care after lensectomy. Main Outcomes and Measures: The main outcomes were cumulative incidence of glaucoma-related adverse events and baseline factors associated with risk of these adverse events. Results: The study included 810 children (1049 eyes); 443 eyes of 321 children (55% female; mean [SD] age, 0.89 [1.97] years) were aphakic after lensectomy, and 606 eyes of 489 children (53% male; mean [SD] age, 5.65 [3.32] years) were pseudophakic. The 5-year cumulative incidence of glaucoma-related adverse events was 29% (95% CI, 25%-34%) in 443 eyes with aphakia and 7% (95% CI, 5%-9%) in 606 eyes with pseudophakia; 7% (95% CI, 5%-10%) of aphakic eyes and 3% (95% CI, 2%-5%) of pseudophakic eyes were diagnosed as glaucoma suspect. Among aphakic eyes, a higher risk for glaucoma-related adverse events was associated with 4 of 8 factors, including age less than 3 months (vs ≥3 months: adjusted hazard ratio [aHR], 2.88; 99% CI, 1.57-5.23), abnormal anterior segment (vs normal: aHR, 2.88; 99% CI, 1.56-5.30), intraoperative complications at time of lensectomy (vs none; aHR, 2.25; 99% CI, 1.04-4.87), and bilaterality (vs unilaterality: aHR, 1.88; 99% CI, 1.02-3.48). Neither of the 2 factors evaluated for pseudophakic eyes, laterality and anterior vitrectomy, were associated with risk of glaucoma-related adverse events. Conclusions and Relevance: In this cohort study, glaucoma-related adverse events were common after cataract surgery in children; age less than 3 months at surgery was associated with elevated risk of the adverse events in aphakic eyes. Children with pseudophakia, who were older at surgery, less frequently developed a glaucoma-related adverse event within 5 years of lensectomy. The findings suggest that ongoing monitoring for the development of glaucoma is needed after lensectomy at any age.

On randomized sketching algorithms and the Tracy-Widom law.

There is an increasing body of work exploring the integration of random projection into algorithms for numerical linear algebra. The primary motivation is to reduce the overall computational cost of processing large datasets. A suitably chosen random projection can be used to embed the original dataset in a lower-dimensional space such that key properties of the original dataset are retained. These algorithms are often referred to as sketching algorithms, as the projected dataset can be used as a compressed representation of the full dataset. We show that random matrix theory, in particular the Tracy-Widom law, is useful for describing the operating characteristics of sketching algorithms in the tall-data regime when the sample size n is much greater than the number of variables d. Asymptotic large sample results are of particular interest as this is the regime where sketching is most useful for data compression. In particular, we develop asymptotic approximations for the success rate in generating random subspace embeddings and the convergence probability of iterative sketching algorithms. We test a number of sketching algorithms on real large high-dimensional datasets and find that the asymptotic expressions give accurate predictions of the empirical performance. Supplementary Information: The online version contains supplementary material available at 10.1007/s11222-022-10148-5.

Reactive infectious mucocutaneous eruption - repeat etanercept after intravenous immunoglobulin: A case report.

Reactive infectious mucocutaneous eruption is a recently distinguished mucosal-predominant blistering eruption triggered by respiratory infections. We describe a previously healthy 11-year-old Black female with rapidly progressive mucocutaneous blistering after prodromal respiratory infection symptoms. Reactive infectious mucocutaneous eruption was suspected and treated with systemic corticosteroids followed by etanercept. Twenty-four hours after etanercept, the diagnosis of multisystem inflammatory syndrome in children was raised and intravenous immunoglobulin was given. Rapidly worsening mucocutaneous disease ensued but was controlled by a second dose of etanercept. Our case highlights the following: (1) the novel observation of possible interaction/neutralization of etanercept by intravenous immunoglobulin, (2) the challenging differential diagnosis of multisystem inflammatory syndrome in children for reactive infectious mucocutaneous eruption patients in the Coronavirus disease 2019 (COVID-19) pandemic, and (3) the role of early treatment to prevent dyspigmentation.

G protein-coupled receptor kinase 5 regulates thrombin signaling in platelets via PAR-1.

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.

Familial pseudohyperkalemia induces significantly higher levels of extracellular potassium in early storage of red cell concentrates without affecting other standard measures of quality: A case control and allele frequency study.

BACKGROUND: Familial pseudohyperkalemia (FP) is characterized by an increased rate of potassium leakage in refrigerated red cells and is associated with the minor allele of the single nucleotide polymorphism rs148211042 (R723Q) in the ABCB6 gene. The study aims were to obtain the minor allele frequencies of ABCB6 variants and to measure supernatant potassium accumulation, and other red cell storage parameters, in red cell concentrates (RCC) from carriers of variant rs148211042 under standard blood bank conditions. STUDY DESIGN: Whole blood units were collected from 6 FP individuals and 11 controls and processed into RCC in additive solution. RCC were sampled and tested over cold storage for full blood count, extracellular potassium, glucose, lactate, microvesicle release, deformability, hemolysis, pH, adenosine triphosphate, and 2,3-diphosphoglycerate. RESULTS: Screening of genotyped cohorts identified that variant rs148211042 is present in 1 in 394 British citizens of European ancestry. FP RCC had significantly higher supernatant potassium at all time points from day 3 onwards (p < .001) and higher mean cell volume (p = .032) than controls. The initial rate of potassium release was higher in FP RCC; supernatant potassium reached 46.0 (23.8-57.6) mmol/L (mean [range]) by day 5, increasing to 68.9 (58.8-73.7) mmol/L by day 35. Other quality parameters were not significantly different between FP RCC and controls. CONCLUSION: These data suggest that if a blood donor has FP, reducing the RCC shelf-life to 5 days may be insufficient to reduce the risk of hyperkalemia in clinical scenarios such as neonatal large volume transfusion.

Advances in understanding the pathogenesis of hereditary macrothrombocytopenia.

Low platelet count, or thrombocytopenia, is a common haematological abnormality, with a wide differential diagnosis, which may represent a clinically significant underlying pathology. Macrothrombocytopenia, the presence of large platelets in combination with thrombocytopenia, can be acquired or hereditary and indicative of a complex disorder. In this review, we discuss the interpretation of platelet count and volume measured by automated haematology analysers and highlight some important technical considerations relevant to the analysis of blood samples with macrothrombocytopenia. We review how large cohorts, such as the UK Biobank and INTERVAL studies, have enabled an accurate description of the distribution and co-variation of platelet parameters in adult populations. We discuss how genome-wide association studies have identified hundreds of genetic associations with platelet count and mean platelet volume, which in aggregate can explain large fractions of phenotypic variance, consistent with a complex genetic architecture and polygenic inheritance. Finally, we describe the large genetic diagnostic and discovery programmes, which, simultaneously to genome-wide association studies, have expanded the repertoire of genes and variants associated with extreme platelet phenotypes. These have advanced our understanding of the pathogenesis of hereditary macrothrombocytopenia and support a future clinical diagnostic strategy that utilises genotype alongside clinical and laboratory phenotype data.

A genome-wide meta-analysis yields 46 new loci associating with biomarkers of iron homeostasis.

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.

Development and validation of a universal blood donor genotyping platform: a multinational prospective study.

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.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

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.

Whole-genome sequencing of patients with rare diseases in a national health system.

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 discovered1. 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 participants2, 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.

Genome-wide association study of eosinophilic granulomatosis with polyangiitis reveals genomic loci stratified by ANCA status.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare inflammatory disease of unknown cause. 30% of patients have anti-neutrophil cytoplasmic antibodies (ANCA) specific for myeloperoxidase (MPO). Here, we describe a genome-wide association study in 676 EGPA cases and 6809 controls, that identifies 4 EGPA-associated loci through conventional case-control analysis, and 4 additional associations through a conditional false discovery rate approach. Many variants are also associated with asthma and six are associated with eosinophil count in the general population. Through Mendelian randomisation, we show that a primary tendency to eosinophilia contributes to EGPA susceptibility. Stratification by ANCA reveals that EGPA comprises two genetically and clinically distinct syndromes. MPO+ ANCA EGPA is an eosinophilic autoimmune disease sharing certain clinical features and an HLA-DQ association with MPO+ ANCA-associated vasculitis, while ANCA-negative EGPA may instead have a mucosal/barrier dysfunction origin. Four candidate genes are targets of therapies in development, supporting their exploration in EGPA.

Author Correction: Discovery and refinement of genetic loci associated with cardiometabolic risk using dense imputation maps.

In the version of the article published, the surname of author Aaron Isaacs is misspelled as Issacs.

Risk thresholds for alcohol consumption: combined analysis of individual-participant data for 599 912 current drinkers in 83 prospective studies.

BACKGROUND: Low-risk limits recommended for alcohol consumption vary substantially across different national guidelines. To define thresholds associated with lowest risk for all-cause mortality and cardiovascular disease, we studied individual-participant data from 599 912 current drinkers without previous cardiovascular disease. METHODS: We did a combined analysis of individual-participant data from three large-scale data sources in 19 high-income countries (the Emerging Risk Factors Collaboration, EPIC-CVD, and the UK Biobank). We characterised dose-response associations and calculated hazard ratios (HRs) per 100 g per week of alcohol (12·5 units per week) across 83 prospective studies, adjusting at least for study or centre, age, sex, smoking, and diabetes. To be eligible for the analysis, participants had to have information recorded about their alcohol consumption amount and status (ie, non-drinker vs current drinker), plus age, sex, history of diabetes and smoking status, at least 1 year of follow-up after baseline, and no baseline history of cardiovascular disease. The main analyses focused on current drinkers, whose baseline alcohol consumption was categorised into eight predefined groups according to the amount in grams consumed per week. We assessed alcohol consumption in relation to all-cause mortality, total cardiovascular disease, and several cardiovascular disease subtypes. We corrected HRs for estimated long-term variability in alcohol consumption using 152 640 serial alcohol assessments obtained some years apart (median interval 5·6 years [5th-95th percentile 1·04-13·5]) from 71 011 participants from 37 studies. FINDINGS: In the 599 912 current drinkers included in the analysis, we recorded 40 310 deaths and 39 018 incident cardiovascular disease events during 5·4 million person-years of follow-up. For all-cause mortality, we recorded a positive and curvilinear association with the level of alcohol consumption, with the minimum mortality risk around or below 100 g per week. Alcohol consumption was roughly linearly associated with a higher risk of stroke (HR per 100 g per week higher consumption 1·14, 95% CI, 1·10-1·17), coronary disease excluding myocardial infarction (1·06, 1·00-1·11), heart failure (1·09, 1·03-1·15), fatal hypertensive disease (1·24, 1·15-1·33); and fatal aortic aneurysm (1·15, 1·03-1·28). By contrast, increased alcohol consumption was log-linearly associated with a lower risk of myocardial infarction (HR 0·94, 0·91-0·97). In comparison to those who reported drinking >0-≤100 g per week, those who reported drinking >100-≤200 g per week, >200-≤350 g per week, or >350 g per week had lower life expectancy at age 40 years of approximately 6 months, 1-2 years, or 4-5 years, respectively. INTERPRETATION: In current drinkers of alcohol in high-income countries, the threshold for lowest risk of all-cause mortality was about 100 g/week. For cardiovascular disease subtypes other than myocardial infarction, there were no clear risk thresholds below which lower alcohol consumption stopped being associated with lower disease risk. These data support limits for alcohol consumption that are lower than those recommended in most current guidelines. FUNDING: UK Medical Research Council, British Heart Foundation, National Institute for Health Research, European Union Framework 7, and European Research Council.

Incidental neuroblastoma with bilateral retinoblastoma: what are the chances?

A child with bilateral familial retinoblastoma underwent staging MRI brain and orbit which identified subtle leptomeningeal enhancement, thus prompting an MRI whole body, which revealed a retroperitoneal mass, confirmed on laparoscopic biopsy to be neuroblastoma. This is the first reported case of these two rare embryonal non-central nervous system tumors occurring concurrently. The cause of this concurrence is unknown despite their pathogenic similarities with a chance of 4 cases per 10 billion children aged 1-4 years. Incidental neuroblastomas in infants can regress spontaneously but this child underwent systemic chemotherapy for his retinoblastoma that may have caused regression of the neuroblastoma.

Nbeal2 interacts with Dock7, Sec16a, and Vac14.

Mutations in NBEAL2, the gene encoding the scaffolding protein Nbeal2, are causal of gray platelet syndrome (GPS), a rare recessive bleeding disorder characterized by platelets lacking α-granules and progressive marrow fibrosis. We present here the interactome of Nbeal2 with additional validation by reverse immunoprecipitation of Dock7, Sec16a, and Vac14 as interactors of Nbeal2. We show that GPS-causing mutations in its BEACH domain have profound and possible effects on the interaction with Dock7 and Vac14, respectively. Proximity ligation assays show that these 2 proteins are physically proximal to Nbeal2 in human megakaryocytes. In addition, we demonstrate that Nbeal2 is primarily localized in the cytoplasm and Dock7 on the membrane of or in α-granules. Interestingly, platelets from GPS cases and Nbeal2-/- mice are almost devoid of Dock7, resulting in a profound dysregulation of its signaling pathway, leading to defective actin polymerization, platelet activation, and shape change. This study shows for the first time proteins interacting with Nbeal2 and points to the dysregulation of the canonical signaling pathway of Dock7 as a possible cause of the aberrant formation of platelets in GPS cases and Nbeal2-deficient mice.

SNP in human ARHGEF3 promoter is associated with DNase hypersensitivity, transcript level and platelet function, and Arhgef3 KO mice have increased mean platelet volume.

Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.