A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

A genetic association study of circulating coagulation factor VIII and von Willebrand factor levels.

ABSTRACT: Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

Crista Galli Morphometry and Morphology: An Anatomical, Radiological, and Machine Learning Application Study / Crista Galli Morfometría y Morfología: Un Estudio de Aplicación Anatómica, Radiológica y de Aprendizaje Automático

SUMMARY: The study purposed to examine the morphometry and morphology of crista galli in cone beam computed tomography (CBCT) and apply a new analysis, supervised Machine Learning techniques to find the answers to research questions "Can sex be determined with crista galli morphometric measurements?" or "How effective are the crista galli morphometric measurements in determining sex?". Crista galli dimensions including anteroposterior, superoinferior, and laterolateral were measured and carried out on 200 healthy adult subjects (98 females; 102 males) aged between 18-79 years. Also, crista galli was classified with two methods called morphological types and Keros classification. In this study, the Chi-square test, Student's t-test, and Oneway ANOVA were performed. Additionally, Machine Learning techniques were applied. The means of the CGH, CGW, and CGL were found as 14.96 mm; 3.96 mm, and 12.76 mm in males, respectively. The same values were as 13.54 mm; 3.51 mm and 11.59±1.61 mm in females, respectively. The CG morphometric measurements of males were higher than those of females. There was a significant difference between sexes in terms of morphological classification type. Also, when the sex assignment of JRip was analyzed, out of 102 male instances 62 of them were correctly predicted, and for 98 female instances, 70 of them were correctly predicted according to their CG measurements. The JRip found the following classification rule for the given dataset: "if CGH<=14.4 then sex is female, otherwise sex is male". The accuracy of this rule is not high, but it gives an idea about the relationship between CG measurements and sex. Although the issue that CG morphometric measurements can be used in sex determination is still controversial, it was concluded in the analysis that CG morphometric measurements can be used in sex determination. Also, Machine Learning Techniques give an idea about the relationship between CG measurements and sex.

En el estudio se propuso examinar la morfometría y la morfología de la crista galli del hueso etmoides usando tomografía computarizada de haz cónico (CBCT) y aplicar un nuevo análisis, técnicas de aprendizaje automático supervisado para encontrar las respuestas a las preguntas de investigación "¿Se puede determinar el sexo con mediciones morfométricas de la crista galli?" o "¿Qué tan efectivas son las medidas morfométricas de la crista galli para determinar el sexo?". Las dimensiones de la crista galli, incluidas los diámetros anteroposterior, superoinferior y laterolateral, se midieron y realizaron en 200 sujetos adultos sanos (98 mujeres; 102 hombres) con edades comprendidas entre los 18 y los 79 años. La crista galli se clasificó con dos métodos llamados tipos morfológicos y clasificación de Keros. En este estudio, se realizaron la prueba de Chicuadrado, la prueba t de Student y ANOVA de una vía. Adicionalmente, se aplicaron técnicas de Machine Learning. Las medias de CGH, CGW y CGL se encontraron en 14,96 mm; 3,96 mm y 12,76 mm en hombres, respectivamente. Los mismos valores fueron 13,54 mm; 3,51 mm y 11,59 ± 1,61 mm en mujeres, respectivamente. Las medidas morfométricas del CG de los hombress fueron más altas que las de las mujeres. Hubo una diferencia significativa entre sexos en cuanto al tipo de clasificación morfológica. Además, cuando se analizó la asignación de sexo de JRip, de 102 instancias masculinas, 62 de ellas se predijeron correctamente, y de 98 instancias femeninas, 70 de ellas se predijeron correctamente de acuerdo con las mediciones de CG. El JRip encontró la siguiente regla de clasificación para el conjunto de datos dado: "si CGH<=14.4, por tanto el sexo es femenino, de lo contrario, el sexo es masculino". La precisión de esta regla no es alta, pero da una idea de la relación entre las medidas del CG y el sexo. Aunque la pregunta si las medidas morfométricas CG se pueden usar en la determinación del sexo sigue aún siendo controvertida. Se concluyó en el análisis que las medidas morfométricas CG se pueden usar en la determinación del sexo. Además, las técnicas de aprendizaje automático dan una idea de la relación entre las medidas de CG y el sexo.

Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

BACKGROUND: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. METHODS: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. RESULTS: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. CONCLUSIONS: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.

A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared to its predecessor. Gene annotations are now more complete, significantly improving the mapping precision of genomic, transcriptomic, and proteomics data sets. We jointly analyzed 163 short-read whole genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ~20.0 million sequence variations, of which 18.7 thousand are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

Prevalence of Polypharmacy and Potential Drug-Drug Interactions Associated with Risk Factors in the Era of HIV Integrase Inhibitors: A Prospective Clinical Study.

People living with human immunodeficiency virus (PLWH), with the availability of modern antiretroviral drugs, have multiple comorbidities, which increase the risk of polypharmacy and potential drug-drug interactions (PDDIs). This is a particularly important issue for the aging population of PLWH. This study aims to review the prevalence and risk factors for PDDIs and polypharmacy in the era of HIV integrase inhibitors. A cross-sectional, two-center, prospective observational study was conducted on Turkish outpatients between October 2021 and April 2022. Polypharmacy was defined as the use of ≥5 non-HIV medications, excluding over-the-counter (OTC) drugs, and PDDIs were classified using the University of Liverpool HIV Drug Interaction Database (harmful/red flagged and potentially clinically relevant/amber flagged). The median age of the 502 PLWH included in the study was 42 ± 12.4 years and 86.1% were males. Most individuals (96.4%) were given integrase-based regimens (unboosted 68.7% and boosted 27.7%). In total, 30.7% of individuals were taking at least one OTC drug. The prevalence of polypharmacy was 6.8% (9.2% when OTC drugs were included). During the study period, the prevalence of PDDIs was 1.2% for red flag PDDIs and 16% for amber flag PDDIs. CD4+ T cell count >500 cells/mm3, number of comorbidities ≥3, comedication with drugs affecting blood and blood-forming organs, cardiovascular drugs, and vitamin/mineral supplements were associated with red flag or amber flag PDDIs. Drug interaction prevention is still important in HIV care. Individuals with multiple comorbidities should be closely monitored for non-HIV medications to prevent PDDIs.

Increased soluble urokinase plasminogen activator levels modulate monocyte function to promote atherosclerosis.

People with kidney disease are disproportionately affected by atherosclerosis for unclear reasons. Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived mediator of kidney disease, levels of which are strongly associated with cardiovascular outcomes. We assessed suPAR's pathogenic involvement in atherosclerosis using epidemiologic, genetic, and experimental approaches. We found serum suPAR levels to be predictive of coronary artery calcification and cardiovascular events in 5,406 participants without known coronary disease. In a genome-wide association meta-analysis including over 25,000 individuals, we identified a missense variant in the plasminogen activator, urokinase receptor (PLAUR) gene (rs4760), confirmed experimentally to lead to higher suPAR levels. Mendelian randomization analysis in the UK Biobank using rs4760 indicated a causal association between genetically predicted suPAR levels and atherosclerotic phenotypes. In an experimental model of atherosclerosis, proprotein convertase subtilisin/kexin-9 (Pcsk9) transfection in mice overexpressing suPAR (suPARTg) led to substantially increased atherosclerotic plaques with necrotic cores and macrophage infiltration compared with those in WT mice, despite similar cholesterol levels. Prior to induction of atherosclerosis, aortas of suPARTg mice excreted higher levels of CCL2 and had higher monocyte counts compared with WT aortas. Aortic and circulating suPARTg monocytes exhibited a proinflammatory profile and enhanced chemotaxis. These findings characterize suPAR as a pathogenic factor for atherosclerosis acting at least partially through modulation of monocyte function.

Clinical characteristics and risk factors associated with severe disease and outcome of patients with COVID-19.

INTRODUCTION: Since the beginning of the pandemic, factors associated with mortality in patients with corona virus infection disease 2019 (COVID-19) have been investigated. Comorbidities and increased age have been frequently reported to be associated with mortality. We aimed to evaluate the factors associated with unfavorable outcome of patients with COVID-19 at an early period of the pandemic. METHODOLOGY: This single center, retrospective, observational study was conducted among laboratory confirmed COVID-19 patients hospitalized between March 11 and May 5, 2020, at Umraniye Training and Research Hospital, Istanbul, Turkey. The effects of the severity of illness, comorbidities, symptoms, and laboratory findings on the clinical outcome were evaluated. Factors associated with unfavorable outcome (necessity of mechanical ventilation or death) were examined using Cox proportional hazards models. RESULTS: Out of a total of 728 patients, 53.8% were men and median age 54 years. The 30-day mortality rate was 4.9% among all hospitalized patients. A logistic regression model identified six predictors of unfavorable clinical outcome: age, severity of illness, the numbers of comorbidities, lymphopenia, high levels of C-reactive protein, and procalcitonin. CONCLUSIONS: The mortality rate was lower among the patients with COVID-19, hospitalized during the early period of the pandemic. Older age, higher severity score on admission, the numbers of comorbidities, higher levels of C-reactive protein, procalcitonin, and lymphopenia were identified to be associated with unfavorable outcome of the hospitalized patients with COVID-19.

Hepatitis B immunization data of patients living with HIV/AIDS: a multi-centre study.

OBJECTIVES: Human immunodeficiency virus (HIV) and hepatitis B virus (HBV) are the two leading viruses that cause the greatest number of virus-related morbidities in the world. HIV/HBV coinfection is correlated with high morbidity and mortality. For this particular reason hepatitis B vaccination is crucial for people living with HIV. METHODS: Patients who are being followed-up for HIV/AIDS and who have received a hepatitis B vaccine in 4 HIV clinics over a 5-year time period have been studied. Our multi-centered, retrospective, cross-sectional and observational study investigates factors that affect hepatitis B vaccination immune response of individuals living with HIV. The patients have been studied for the parameters such as age, sex, CD4 count at the time of diagnosis or vaccination, HIV-RNA levels, comorbidities, vaccine dosage, success of immunization after vaccination, and the demographics of the patients who have and have not developed immunity. RESULTS: Of 645 patients that are being followed-up in our clinics, 158 received hepatitis B vaccine; 39 of these 158 patients have been excluded from the study because they did not fulfil the inclusion criteria. Finally, 119 patients were evaluated in the study, 17 of the patients (14.3%) were females and 102 (85.7%) were males. The median age was 41.11 ± 10.09 (min-max: 18-75). Twenty-three of the patients (19.3%) were at the stage of AIDS during diagnosis while 80.7% were at the stage of HIV infection. Ninety-one of the patients (76.5%) have been administered a single dose hepatitis B vaccine on the standard 0, 1st, 6th month vaccination schedule, whereas 23.5% were administered a double dose on the same vaccination schedule. When further evaluated to find whether the patient was able to develop sufficient immunity (anti-HBs ≥ 10), it was found that the immune response was statistically significantly higher in the patients whose CD4 count was greater than 200 at the time of the first diagnosis and vaccination (p = 0.05 and p = 0.001, respectively). The patients have also been evaluated according to the number of doses they received (1 vs. 2). The immune response of the patients who received two doses was statistically significantly higher (p = 0.041). CONCLUSION: We can conclude that in the patients with CD4 count less than 200 at the time of their diagnosis and vaccination a high dose recombinant hepatitis B vaccine should definitely be administered as the normal dose and higher dose have similar side effect profiles and the higher dose provides greater immunity.

Appropriate Use of Tocilizumab in COVID-19: Early Use is Beneficial.

Objective: Interleukin-6 inhibitor Tocilizumab (TCZ) is effective to prevent the mortality of severe COVID-19 by suppressing the cytokine storm, however, its appropriate use needs to be detailed. We aimed to describe the appropriate use of TCZ in severe to critical cases with COVID-19 pneumonia in the early phase of the pandemic. Materials and Methods: This single-center, retrospective, observational study was conducted in a polymerase chain reaction (PCR) positive COVID-19 patients who received TCZ between April 01, 2020 and June 30, 2020, in Ümraniye Research and Training Hospital Istanbul, Turkey. The factors affecting mortality were compared. Results: A total of 67 patients met the inclusion criteria during the study period. Overall, 76% of those patients were male, with a median age of 61 years. The 28-day mortality rate was 51% among all patients who were hospitalised for COVID-19 pneumonia. A logistic regression model identified the predictors of 28-day fatality; the number of comorbidities, high levels of C-reactive protein (CRP) before initiation of TCZ, initiation of TCZ in the intensive care unit (ICU) and not receiving an additional dose of TCZ. Conclusion: The number of comorbidities, high levels of CRP, initiation of TCZ in the ICU and not receiving the additional dose of TCZ were significant risk factors for fatality among patients with COVID-19 who received TCZ. Early initiation of TCZ when cytokine storm is suspected is appropriate for the prevention of fatality.

Allelic Variants in Established Hypopituitarism Genes Expand Our Knowledge of the Phenotypic Spectrum.

We report four allelic variants (three novel) in three genes previously established as causal for hypopituitarism or related disorders. A novel homozygous variant in the growth hormone gene, GH1 c.171delT (p.Phe 57Leufs*43), was found in a male patient with severe isolated growth hormone deficiency (IGHD) born to consanguineous parents. A hemizygous SOX3 allelic variant (p.Met304Ile) was found in a male patient with IGHD and hypoplastic anterior pituitary. YASARA, a tool to evaluate protein stability, suggests that p.Met304Ile destabilizes the SOX3 protein (ΔΔG = 2.49 kcal/mol). A rare, heterozygous missense variant in the TALE homeobox protein gene, TGIF1 (c.268C>T:p.Arg90Cys) was found in a patient with combined pituitary hormone deficiency (CPHD), diabetes insipidus, and syndromic features of holoprosencephaly (HPE). This variant was previously reported in a patient with severe holoprosencephaly and shown to affect TGIF1 function. A novel heterozygous TGIF1 variant (c.82T>C:p.Ser28Pro) was identified in a patient with CPHD, pituitary aplasia and ectopic posterior lobe. Both TGIF1 variants have an autosomal dominant pattern of inheritance with incomplete penetrance. In conclusion, we have found allelic variants in three genes in hypopituitarism patients. We discuss these variants and associated patient phenotypes in relation to previously reported variants in these genes, expanding our knowledge of the phenotypic spectrum in patient populations.

The phenotypic spectrum associated with OTX2 mutations in humans.

Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development. Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

FGL1 as a modulator of plasma D-dimer levels: Exome-wide marker analysis of plasma tPA, PAI-1, and D-dimer.

BACKGROUND: Use of targeted exome-arrays with common, rare variants and functionally enriched variation has led to discovery of new genes contributing to population variation in risk factors. Plasminogen activator-inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), and the plasma product D-dimer are important components of the fibrinolytic system. There have been few large-scale genome-wide or exome-wide studies of PAI-1, tPA, and D-dimer. OBJECTIVES: We sought to discover new genetic loci contributing to variation in these traits using an exome-array approach. METHODS: Cohort-level analyses and fixed effects meta-analyses of PAI-1 (n = 15 603), tPA (n = 6876,) and D-dimer (n = 19 306) from 12 cohorts of European ancestry with diverse study design were conducted, including single-variant analyses and gene-based burden testing. RESULTS: Five variants located in NME7, FGL1, and the fibrinogen locus, all associated with D-dimer levels, achieved genome-wide significance (P < 5 × 10-8 ). Replication was sought for these 5 variants, as well as 45 well-imputed variants with P < 1 × 10-4 in the discovery using an independent cohort. Replication was observed for three out of the five significant associations, including a novel and uncommon (0.013 allele frequency) coding variant p.Trp256Leu in FGL1 (fibrinogen-like-1) with increased plasma D-dimer levels. Additionally, a candidate-gene approach revealed a suggestive association for a coding variant (rs143202684-C) in SERPINB2, and suggestive associations with consistent effect in the replication analysis include an intronic variant (rs11057830-A) in SCARB1 associated with increased D-dimer levels. CONCLUSION: This work provides new evidence for a role of FGL1 in hemostasis.

The Endoplasmic Reticulum Cargo Receptor SURF4 Facilitates Efficient Erythropoietin Secretion.

Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent single guide RNAs (sgRNAs) resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the endoplasmic reticulum (ER) compartment and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

Whole-exome sequencing identifies rare variants in STAB2 associated with venous thromboembolic disease.

Deep vein thrombosis and pulmonary embolism, collectively defined as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Common genetic variants conferring increased varying degrees of VTE risk have been identified by genome-wide association studies (GWAS). Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 result in perinatal lethal thrombosis in homozygotes and markedly increased VTE risk in heterozygotes. However, currently described VTE variants account for an insufficient portion of risk to be routinely used for clinical decision making. To identify new rare VTE risk variants, we performed a whole-exome study of 393 individuals with unprovoked VTE and 6114 controls. This study identified 4 genes harboring an excess number of rare damaging variants in patients with VTE: PROS1, STAB2, PROC, and SERPINC1. At STAB2, 7.8% of VTE cases and 2.4% of controls had a qualifying rare variant. In cell culture, VTE-associated variants of STAB2 had a reduced surface expression compared with reference STAB2. Common variants in STAB2 have been previously associated with plasma von Willebrand factor and coagulation factor VIII levels in GWAS, suggesting that haploinsufficiency of stabilin-2 may increase VTE risk through elevated levels of these procoagulants. In an independent cohort, we found higher von Willebrand factor levels and equivalent propeptide levels in individuals with rare STAB2 variants compared with controls. Taken together, this study demonstrates the utility of gene-based collapsing analyses to identify loci harboring an excess of rare variants with functional connections to a complex thrombotic disease.

Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.

Genome-wide linkage analysis and whole-exome sequencing identifies an ITGA2B mutation in a family with thrombocytopenia.

Hereditary thrombocytopenias can be subclassified based on mode of inheritance and platelet size. Here we report a family with autosomal dominant (AD) thrombocytopenia with normal platelet size. Linkage analysis and whole exome sequencing identified the R1026W substitution in ITGA2B as the causative defect. The same mutation has been previously reported in 7 Japanese families/patients with AD thrombocytopenia, but all of these patients had macrothrombocytopenia. This is the first report of a family with AD thrombocytopenia with normal platelet size resulting from mutation in ITGA2B. ITGA2B mutations should therefore be included in the differential diagnosis of this latter disorder.

Extended regions of suspected mis-assembly in the rat reference genome.

We performed whole-genome sequencing for eight inbred rat strains commonly used in genetic mapping studies. They are the founders of the NIH heterogeneous stock (HS) outbred colony. We provide their sequences and variant calls to the rat genomics community. When analyzing the variant calls we identified regions with unusually high levels of heterozygosity. These regions are consistent across the eight inbred strains, including Brown Norway, which is the basis of the rat reference genome. These regions show higher read depths than other regions in the genome and contain higher rates of apparent tri-allelic variant sites. The evidence suggests that these regions may correspond to duplicated segments that were incorrectly overlaid as a single segment in the reference genome. We provide masks for these regions of suspected mis-assembly as a resource for the community to flag potentially false interpretations of mapping or functional results.

Colistin nephrotoxicity in critically ill patients after implementation of a new dosing strategy.

INTRODUCTION: Intravenous colistin is increasingly used to treat multidrug-resistant Gram-negative infections. Highly variable nephrotoxicity rates have been reported. Recent PK/PD studies propose a loading dose and a maintenance dose for better efficacy, but data on the renal toxicity of such regimens are rare. This study aimed to evaluate the incidence and risk factors for nephrotoxicity related to colistin after implementation of a new dosing regimen including a loading dose. METHODOLOGY: This was a prospective observational study that was made between adult patients who received a minimum of 48 hours of intravenous colistin from December 2012 to January 2014 at the medical and surgical intensive care units (ICU) of a university hospital. The severity of acute kidney injury (AKI) was defined by the RIFLE criteria. RESULTS: Fifty-nine patients met the inclusion criteria, and 31 (52.5%) developed nephrotoxicity. The APACHE-II score was > 15 in 81% of patients. The median time to nephrotoxicity was 7 days. Patients with AKI were in risk (10.2%), injury (16.9%), failure (25.4%), and none of the patients developed permanent renal insufficiency. A logistic regression model identified three predictors of colistin-associated nephrotoxicity: age; the number of days that estimated target plasma concentrations of colistin were ≥ 3.5 mg/L in the first week of therapy; and baseline creatinine level. CONCLUSION: In this cohort of severely ill ICU patients, colistin led to a relatively high rate of nephrotoxicity. Further studies are needed to identify the optimal dose for both efficacy and safety.

Genome-Wide Association Transethnic Meta-Analyses Identifies Novel Associations Regulating Coagulation Factor VIII and von Willebrand Factor Plasma Levels.

BACKGROUND: Factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are associated with risk of arterial and venous thrombosis and with hemorrhagic disorders. We aimed to identify and functionally test novel genetic associations regulating plasma FVIII and VWF. METHODS: We meta-analyzed genome-wide association results from 46 354 individuals of European, African, East Asian, and Hispanic ancestry. All studies performed linear regression analysis using an additive genetic model and associated ≈35 million imputed variants with natural log-transformed phenotype levels. In vitro gene silencing in cultured endothelial cells was performed for candidate genes to provide additional evidence on association and function. Two-sample Mendelian randomization analyses were applied to test the causal role of FVIII and VWF plasma levels on the risk of arterial and venous thrombotic events. RESULTS: We identified 13 novel genome-wide significant ( P≤2.5×10-8) associations, 7 with FVIII levels ( FCHO2/TMEM171/TNPO1, HLA, SOX17/RP1, LINC00583/NFIB, RAB5C-KAT2A, RPL3/TAB1/SYNGR1, and ARSA) and 11 with VWF levels ( PDHB/PXK/KCTD6, SLC39A8, FCHO2/TMEM171/TNPO1, HLA, GIMAP7/GIMAP4, OR13C5/NIPSNAP, DAB2IP, C2CD4B, RAB5C-KAT2A, TAB1/SYNGR1, and ARSA), beyond 10 previously reported associations with these phenotypes. Functional validation provided further evidence of association for all loci on VWF except ARSA and DAB2IP. Mendelian randomization suggested causal effects of plasma FVIII activity levels on venous thrombosis and coronary artery disease risk and plasma VWF levels on ischemic stroke risk. CONCLUSIONS: The meta-analysis identified 13 novel genetic loci regulating FVIII and VWF plasma levels, 10 of which we validated functionally. We provide some evidence for a causal role of these proteins in thrombotic events.