Real-time single-exposure ROI-driven HDR adaptation based on focal-plane reconfiguration

Proc. SPIE 9400, Real-Time Image and Video Processing 2015This paper describes a prototype smart imager capable of adjusting the photo-integration time of multiple regions of interest concurrently, automatically and asynchronously with a single exposure period. The operation is supported by two interwined photo-diodes at pixel level and two digital registers at the periphery of the pixel matrix. These registers divide the focal-plane into independent regions within which automatic concurrent adjustment of the integration time takes place. At pixel level, one of the photo-diodes senses the pixel value itself whereas the other, in collaboration with its counterparts in a particular ROI, senses the mean illumination of that ROI. Additional circuitry interconnecting both photo-diodes enables the asynchronous adjustment of the integration time for each ROI according to this sensed illumination. The sensor can be recon gured on-the- y according to the requirements of a vision algorithm.España MINECO (FEDER) TEC2012-38921-C02 IPT-2011-1625-430000 IPC-20111009 CDTIJunta de Andalucía TIC 2338-2013 CEIC

Characterization of the platelet phenotype caused by a germline RUNX1 Variant in a CRISPR/Cas9-generated murine model

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01311, PI17/01966, and CB15/00055), Fundación Séneca (19873/GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundación Mutua Madrileña (FMM, AP172142019), and Sociedad Española de Trombosis y Hemostasia (SETH-FETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2019). The authors' research on IPDs is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, which is supported by the Hemorrhagic Diathesis Working Group of the Spanish Society of Thrombosis and Haemostasis. A.M.-Q., C.F.-I., and L.H.-C. were supported by predoctoral grants from the Junta de Castilla y León, Spain. E.V. was supported by the predoctoral grant from the University of Salamanca, Spain. IG-T and RB were supported by "Contratos postdoctorales Programa II) from the University of Salamanca, Spain

Molecular dissection of structural variations involved in antithrombin deficiency

Inherited antithrombin deficiency, the most severe form of thrombophilia, is predominantly caused by variants in SERPINC1. Few causal structural variants have been described, usually detected by multiplex ligation-dependent probe amplification or cytogenetic arrays, which only define the gain or loss and the approximate size and location. This study has done a complete dissection of the structural variants affecting SERPINC1 of 39 unrelated patients with antithrombin deficiency using multiplex ligation-dependent probe amplification, comparative genome hybridization array, long-range PCR, and whole genome nanopore sequencing. Structural variants, in all cases only affecting one allele, were deleterious and caused a severe type I deficiency. Most defects were deletions affecting exons of SERPINC1 (82.1%), but the whole cohort was heterogeneous, as tandem duplications, deletion of introns, or retrotransposon insertions were also detected. Their size was also variable, ranging from 193 bp to 8 Mb, and in 54% of the cases involved neighboring genes. All but two structural variants had repetitive elements and/or microhomologies in their breakpoints, suggesting a common mechanism of formation. This study also suggested regions recurrently involved in structural variants causing antithrombin deficiency and found three structural variants with a founder effect: the insertion of a retrotransposon, duplication of exon 6, and a 20-gene deletion. Finally, nanopore sequencing was determined to be the most appropriate method to identify and characterize all structural variants at nucleotide level, independently of their size or type.Supported by the National Institute for Health Research (NIHR) for the NIHR BioResource project (grant numbers RG65966 and RG94028), by the Instituto de Salud Carlos III grant; Fondo Europeo de Desarrollo Regional (FEDER) grant PI18/00598; and Fundación Séneca 19873/GERM/15. M.E.d.l.M.-B. has a postdoctoral contract from University of Murcia, Murcia, Spain. C.B.-P. has a Río Hortega fellowship. B.d.l.M.-B. has a postdoctoral fellowship from Fundación Séneca. J.C.-G. has a predoctoral fellowship from the Ministry of Universities FPU19/03662

Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature β1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and β1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.This work was supported by grants from Instituto de Salud Carlos III (ISCIII) & Feder (PI17/01966, PI20/00926) and cofunded by European Union (ERDF/ESF, “Investing in your future”), Gerencia Regional de Salud (GRS2061/A/2019, GRS2135/A/2020, GRS2314/A/2021), Fundación Mutua Madrileña (FMM, AP172142019), Sociedad Española de Trombosis y Hemostasia (SETH-FETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2020 and 2021), Fundación Castellano Leonesa de Hematología y Hemoterapia (FUCALHH 2020), Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD12/0036/0069), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233). Progetti di ricerca di rilevante interesse Nazionale (PRIN 2017Z5LR5Z), and the European Commission (H2020-FETOPEN-1-2016-2017-SilkFusion ID 767309). The author´s research on Inherited Platelet Disorders is conducted in accordance with the aims of the multicentric project “Functional and Molecular Characterization of Patients with Inherited Platelet Disorders” of Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC). A.M.-Q. is fully supported by an “Ayuda predoctoral de la Junta de Castilla y León” by the Fondo Social Europeo (JCYL- EDU/556/2019 PhD scholarship) and received an “Ayuda para breves estancias formativas” from the Sociedad Española de Hematología y Hemoterapia (SEHH-FEHH), and from the Sociedad Española de Trombosis y Hemostasia (SETH-FETH); E.V. is fully supported by an “Ayuda para contratos predoctorales de la Universidad de Salamanca cofinanciadas por el banco Santander,” programa propio III convocatoria 2018; I.S.-G. is supported by a contract from the University of Salamanca cofinanced by the Junta de Castilla y León (Council of Education) and FEDER-European Union [ref. SA0118P20 (2)]; S.S.-M. and C.M.-G. received funding from the European Research Council (ERC) under the ERA-Per-Med programme (ERAPERMED2018-275) SYNtherapy and ISCIII (AC18/00093) cofunded by ERDF/ESF, “Investing in your future”; I.G.-T. and R.B. are supported by a grant from the Universidad de Salamanca (“Contrato postdoctoral Universidad de Salamanca programa propio II, 2019”)Peer reviewe

Spanish guidelines for the use of targeted deep sequencing in myelodysplastic syndromes and chronic myelomonocytic leukaemia

The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies

Molecular Characterization of the Region 7q22.1 in Splenic Marginal Zone Lymphomas

Splenic marginal zone lymphomas (SMZL) are an uncommon type of B-cell non-Hodgkin's lymphoma (NHL-B) in which no specific chromosomal translocations have been described. In contrast, the most frequent cytogenetic abnormality is the loss of the long arm of chromosome 7 (7q). Previous reports have located this loss in the 7q32 region. In order to better characterize the genomic imbalances in SMZL, molecular studies were carried out in 73 patients with SMZL. To gain insight into the mapping at 7q a tiling array was also used. The results confirmed the loss of 7q as the most frequent change. In addition, several abnormalities, including 4q22.1, 1q21.3–q22, 6q25.3, 20q13.33, 3q28, 2q23.3–q24.1 and 17p13, were also present. A loss of 7q22.1 at 99925039–101348479 bp was observed in half of the cases. The region of 7q22.1 has not previously been characterised in SMZL. Our results confirmed the presence of a new region of loss on chromosome 7 in these NHL

Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders

Inherited platelet disorders are a heterogeneous group of rare diseases, caused by inherited defects in platelet production and/or function. Their genetic diagnosis would benefit clinical care, prognosis and preventative treatments. Until recently, this diagnosis has usually been performed via Sanger sequencing of a limited number of candidate genes. High-throughput sequencing is revolutionizing the genetic diagnosis of diseases, including bleeding disorders. We have designed a novel high-throughput sequencing platform to investigate the unknown molecular pathology in a cohort of 82 patients with inherited platelet disorders. Thirty-four (41.5%) patients presented with a phenotype strongly indicative of a particular type of platelet disorder. The other patients had clinical bleeding indicative of platelet dysfunction, but with no identifiable features. The high-throughput sequencing test enabled a molecular diagnosis in 70% of these patients. This sensitivity increased to 90% among patients suspected of having a defined platelet disorder. We found 57 different candidate variants in 28 genes, of which 70% had not previously been described. Following consensus guidelines, we qualified 68.4% and 26.3% of the candidate variants as being pathogenic and likely pathogenic, respectively. In addition to establishing definitive diagnoses of well-known inherited platelet disorders, high-throughput sequencing also identified rarer disorders such as sitosterolemia, filamin and actinin deficiencies, and G protein-coupled receptor defects. This included disease-causing variants in DIAPH1 (n=2) and RASGRP2 (n=3). Our study reinforces the feasibility of introducing high-throughput sequencing technology into the mainstream laboratory for the genetic diagnostic practice in inherited platelet disorders.This study was supported by research grants from the Gerencia Regional de Salud (GRS 1370/A/16), ISCIII & Feder (PI14/01956), CIBERER CB15/00055, Fundación Séneca (19873/GERM/15) and Sociedad Española de Trombosis y Hemostasia (SETH). SPW holds a British Heart Foundation chair.Peer Reviewe

A novel genetic variant in PTGS1 affects N-glycosylation of cyclooxygenase-1 causing a dominant-negative effect on platelet function and bleeding diathesis.

During platelet activation, arachidonic acid (AA) is released from membrane phospholipids and metabolized to thromboxane A2 (TXA2) through the actions of cyclooxygenase-1 (COX-1) and TXA2 synthase. Note, TXA2 binds to the platelet TXA2 receptor, causing shape change, secretion and platelet aggregation.1 Also, COX-1 (599aa; 70 kDa) has cyclooxygenase and peroxidase activities and it is functionally active as a homodimer, with each COX-1 monomer consisting of four highly conserved domains: an N-terminal signal peptide, a dimerization domain, a membrane-binding domain (MBD) and a large C-terminal catalytic domain2 (Figure 1A). Irreversible COX-1 inhibition by aspirin is a widely established anti-platelet therapy in cardiovascular disease.Fundación Mutua Madrileña, Grant/Award Number: AP172142019; Fundación Séneca, Grant/Award Number: 19873/GERM/15; Gerencia Regional de Salud, Grant/Award Numbers: 1647/A/17, 2061A/19; Instituto de Salud Carlos III (ISCIII) & Feder, Grant/Award Numbers: CB15/00055, PI17/01966, PI18/00598, PI20/00926, PI17/01311; Junta de Castilla y León; British Heart Foundation, Grant/Award Number: PG/17/40/33028; Ayuda a Grupos de Trabajo en Patología Hemorrágica; Premio López Borrasca 2019; Sociedad Española de Trombosis y Hemostasia

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality