Diagnóstico de las diátesis hemorrágicas hereditarias mediante el análisis genético por secuenciación masiva

[ES] Las diátesis hemorrágicas hereditarias (DHH) constituyen un grupo amplio y heterogéneo de trastornos hereditarios y/o congénitos poco frecuentes, relacionados con la alteración en la hemostasia primaria y en la hemostasia secundaria. Como consecuencia, el paciente experimenta episodios de sangrado con distintos grados de gravedad. No es infrecuente encontrar pacientes con estas características en la práctica clínica habitual. La gran heterogeneidad de las DHH, principalmente en los trastornos plaquetarios hereditarios, proviene de que las alteraciones en diferentes genes pueden producir un fenotipo clínico similar para cada una de ellas. Para su correcto diagnóstico es preciso, en la actualidad, combinar varias metodologías, lo que requiere un esfuerzo considerable en tiempo y en dinero. A pesar de ello, en bastantes ocasiones no se llega a un diagnóstico de certeza. Cuando los síntomas de sangrado hacen sospechar la existencia de una DHH (especialmente en niños, pero también en adultos en los que la patología no se haya manifestado en la infancia), se aplica un algoritmo que consiste en la caracterización clínica, fenotípica y funcional o del laboratorio del paciente. Todos estos estudios son necesarios para poder dirigir el análisis molecular a un gen candidato y, así, poder confirmar el diagnóstico. Esta estrategia supone un reto diagnóstico debido a que, en bastantes ocasiones, el diagnóstico de estas enfermedades es difícil, sobre todo en los casos con fenotipos hemorrágicos leves o indeterminados. También requiere una gran cantidad de pruebas, una validación interna e interlaboratorial y la repetición del estudio para que desaparezcan posibles factores intercurrentes adquiridos. Por tanto, las principales limitaciones para la detección de las DHH son: a) La falta de acuerdo en su clasificación y en su estandarización; b) la presencia de fenotipos clínico-biológicos coincidentes y su gran heterogeneidad clínica y molecular; y c) la dificultad para incorporar las herramientas diagnósticas a la rutina diaria y su complejidad técnica. Así, por ejemplo, en los trastornos plaquetarios hereditarios (TPHs), la heterogeneidad clínica, el solapamiento de fenotipos o la dificultad para definirlo obstaculizan la búsqueda de mutaciones en un gen candidato y, por tanto, el diagnóstico molecular. Por ello, sólo es factible identificar el gen afectado en el 40-50% de los TPHs. Por otro lado, en los trastornos de la hemostasia secundaria, elgen candidato a estudio suele estar bien definido, aunque pueden existir deficiencias combinadas. Sin embargo, como ocurre en la enfermedad de von Willebrand (52 exones codificantes) o, principalmente en la hemofilia A (26 exones y 186 kb de tamaño), tras descartar las mutaciones recurrentes (inversión del intrón 22; intrón 1), la secuenciación de un gran número de exones supone otra dificultad en tiempo y coste. En estos casos, también, la secuenciación convencional sería un método inapropiado para la rutina clínica diaria. La carencia de métodos diagnósticos sencillos aplicados en la práctica clínica habitual, motiva que, en ocasiones, al clínico le resulte complicado, a veces imposible, emitir un diagnóstico certero. En consecuencia, existe el riesgo de la prescripción de tratamientos inapropiados que no solucionarán su patología, por lo que el paciente no mejorará y podrá tener efectos secundarios no deseados. Además, se multiplicarán los gastos inherentes a su tratamiento y a la identificación de la anomalía causante del trastorno, al tener que emplear métodos gravosos y complejos desde el punto de vista clínico y dilatados en el tiempo. Hasta hace poco tiempo, la búsqueda de información genética a gran escala aplicada al diagnóstico clínico era, desde el punto de vista técnico, compleja, y, desde el económico, poco viable. Sin embargo, en la actualidad se dispone de un nuevo conjunto de métodos de alto rendimiento denominados secuenciación masiva o Next- Generation Sequencing (NGS), que son herramientas valiosas tanto para la investigación como para la evaluación de trastornos clínicos causados por mutaciones genéticas. Estas plataformas de alto rendimiento, en sus distintas modalidades como paneles de genes customizados (targeted genes panel), exoma completo (Whole-Exome Sequencing) o incluso, el genoma completo (Whole-Genome Sequencing) tienen la capacidad de secuenciar gran cantidad de información genética de manera paralela, simultánea y de forma precisa para identificar las mutaciones causantes de las enfermedades de base genética. Esta tecnología ha evolucionado rápidamente durante la última década hasta el punto de constituir hoy una alternativa atractiva, tanto a nivel práctico como económico, con la que abordar el diagnóstico de patologías con alta complejidad. De este modo, en el momento actual es razonable plantearse el uso de la NGS tanto para explorar cambios en todos los genes que con alta probabilidad pueden causar un fenotipo clínico y de laboratorio concreto, como para buscar la causa no conocida de patologías plaquetarias de fenotipo inespecífico que son, por otra parte, las más comunes y difícilmente abordables. Además, como bien es conocido, si los fenotipos de estas entidades pueden solaparse, es lógico pensar que el diseño de un panel que incluya a todos aquellos genes conocidos que se relacionan con cualquiera de las DHH descritas hasta el momento podría contribuir a un mejor diagnóstico de estas enfermedades. Incluso, a igualdad de tiempo y coste, estas plataformas pueden rastrear mutaciones en muchos más genes que la secuenciación clásica de Sanger. Por lo tanto, la secuenciación masiva parece una herramienta idónea para avanzar en el diagnóstico de estas enfermedades. Así, hemos planteado el análisis simultáneo de un gran número de genes y exones mediante dos tipos de paneles de genes, uno encaminado a los trastornos de la hemostasia primaria y otro, a la secundaria. Como veremos más adelante, hemos empleado la captura de secuencia que es idónea para secuenciar alteraciones genéticas hereditarias que ocurren en el 50-100% de las células, de manera precisa y asequible económicamente. De hecho, existen precedentes que demuestran cómo la tecnología NGS no sólo es capaz de identificar mutaciones ya descritas con alta eficiencia, sino que también ha logrado encontrar nuevas mutaciones responsables de los síntomas observados en el diagnóstico de las DHH, pretendiendo su incorporación al algoritmo diagnóstico de la ISTH. Como conclusión de lo expuesto, proponemos como hipótesis de trabajo que la NGS permitirá la búsqueda sistemática de mutaciones incluidas en un extenso panel de genes relacionados con alguna de las múltiples manifestaciones de las DHH, lo que contribuirá al diagnóstico correcto de estos pacientes. La finalidad de este panel es su aplicación rutinaria en el diagnóstico de todos los pacientes con sospecha de DHH. De este modo, se incrementará notablemente la posibilidad de obtener un diagnóstico correcto de estos enfermos

Two novel variants of the ABCG5 gene cause xanthelasmas and macrothrombocytopenia: a brief review of hematologic abnormalities of sitosterolemia

[EN] Background: Sitosterolemia (STSL) is a recessive inherited disorder caused by pathogenic variants in the ABCG5 and ABCG8 genes. Increased levels of plasma plant sterols (PSs) usually result in xanthomas and premature coronary atherosclerosis, although hematologic abnormalities may occasionally be present. This clinical picture is unfamiliar to many physicians, and patients may be at high risk of misdiagnosis. Objectives: To report two novel ABCG5 variants causing STSL in a Spanish patient, and review the clinical and mutational landscape of STSL. Patient/Methods: A 46-year-old female was referred to us with lifelong macrothrombocytopenia. She showed familial hypercholesterolemia-related xanthomas. Molecular analysis was performed with high-throughput sequencing. Plasma PS levels were evaluated with gas–liquid chromatography. The STSL landscape was reviewed with respect to specific online databases and all reports published since 1974. Results: A blood smear revealed giant platelets and stomatocytes. Novel compound heterozygous variants were detected in exons 7 (c.914C>G) and 13 (c.1890delT) of ABCG5. The patient showed an increased plasma level of sitosterol. These findings support the diagnosis of STSL. In our review, we identified only 25 unrelated STLS patients who presented with hematologic abnormalities including macrothrombocytopenia

Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression

Article number: 127[EN]BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation

CRISPR/Cas9-generated models uncover therapeutic vulnerabilities of del(11q) CLL cells to dual BCR and PARP inhibition

[EN]The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality

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

Performance of Screening Strategies for Latent Tuberculosis Infection in Patients with Inflammatory Bowel Disease: Results from the ENEIDA Registry of GETECCU

(1) Aims: Patients receiving antitumor necrosis factor (anti-TNF) therapy are at risk of developing tuberculosis (TB), usually due to the reactivation of a latent TB infection (LTBI). LTBI screening and treatment decreases the risk of TB. This study evaluated the diagnostic performance of different LTBI screening strategies in patients with inflammatory bowel disease (IBD). (2) Methods: Patients in the Spanish ENEIDA registry with IBD screened for LTBI between January 2003 and January 2018 were included. The diagnostic yield of different strategies (dual screening with tuberculin skin test [TST] and interferon-gamma-release assay [IGRA], two-step TST, and early screening performed at least 12 months before starting biological treatment) was analyzed. (3) Results: Out of 7594 screened patients, 1445 (19%; 95% CI 18-20%) had LTBI. Immunomodulator (IMM) treatment at screening decreased the probability of detecting LTBI (20% vs. 17%, p = 0.001). Regarding screening strategies, LTBI was more frequently diagnosed by dual screening than by a single screening strategy (IGRA, OR 0.60; 95% CI 0.50-0.73, p < 0.001; TST, OR 0.76; 95% CI 0.66-0.88, p < 0.001). Two-step TST increased the diagnostic yield of a single TST by 24%. More cases of LTBI were diagnosed by early screening than by routine screening before starting anti-TNF agents (21% [95% CI 20-22%] vs. 14% [95% CI 13-16%], p < 0.001). The highest diagnostic performance for LTBI (29%) was obtained by combining early and TST/IGRA dual screening strategies in patients without IMM. (4): Conclusions: Both early screening and TST/IGRA dual screening strategies significantly increased diagnostic performance for LTBI in patients with IBD, with optimal performance achieved when they are used together in the absence of IMM

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

Effect of viral storm in patients admitted to intensive care units with severe COVID-19 in Spain: a multicentre, prospective, cohort study

Background: The contribution of the virus to the pathogenesis of severe COVID-19 is still unclear. We aimed to evaluate associations between viral RNA load in plasma and host response, complications, and deaths in critically ill patients with COVID-19. Methods: We did a prospective cohort study across 23 hospitals in Spain. We included patients aged 18 years or older with laboratory-confirmed SARS-CoV-2 infection who were admitted to an intensive care unit between March 16, 2020, and Feb 27, 2021. RNA of the SARS-CoV-2 nucleocapsid region 1 (N1) was quantified in plasma samples collected from patients in the first 48 h following admission, using digital PCR. Patients were grouped on the basis of N1 quantity: VIR-N1-Zero ([removed]2747 N1 copies per mL). The primary outcome was all-cause death within 90 days after admission. We evaluated odds ratios (ORs) for the primary outcome between groups using a logistic regression analysis. Findings: 1068 patients met the inclusion criteria, of whom 117 had insufficient plasma samples and 115 had key information missing. 836 patients were included in the analysis, of whom 403 (48%) were in the VIR-N1-Low group, 283 (34%) were in the VIR-N1-Storm group, and 150 (18%) were in the VIR-N1-Zero group. Overall, patients in the VIR-N1-Storm group had the most severe disease: 266 (94%) of 283 patients received invasive mechanical ventilation (IMV), 116 (41%) developed acute kidney injury, 180 (65%) had secondary infections, and 148 (52%) died within 90 days. Patients in the VIR-N1-Zero group had the least severe disease: 81 (54%) of 150 received IMV, 34 (23%) developed acute kidney injury, 47 (32%) had secondary infections, and 26 (17%) died within 90 days (OR for death 0·30, 95% CI 0·16–0·55; p<0·0001, compared with the VIR-N1-Storm group). 106 (26%) of 403 patients in the VIR-N1-Low group died within 90 days (OR for death 0·39, 95% CI 0·26–0·57; p[removed]11 página

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Management of acquired hemophilia A: results from the Spanish registry

The Spanish Acquired Hemophilia A (AHA) Registry is intended to update the status of AHA in Spain. One hundred and fifty-four patients were included and retrospectively followed for a median of 12 months. Patients were predominantly male (56.3%), with median age at diagnosis of 74 years. AHA was more frequently idiopathic (44.1%) and autoimmune disorder-associated (31.7%). Thirty-four percent of patients were on antithrombotic therapy at diagnosis. Hemostatic treatment was used in 70% of patients. Recombinant activated factor VII was more frequently infused (60.3% vs 20.6% activated prothrombin complex concentrate). Only 1 patient did not achieve control of hemorrhage. Complete remission (CR) was achieved by 84.2% of cases after immunosuppressive therapy. Steroids alone were less efficient than the other strategies (68.2% vs 87.2%, P = .049), whereas no differences existed among these (steroids/cyclophosphamide, 88.5%, vs steroids/calcineurin inhibitors, 81.2%, vs rituximab-based regimens, 87.5%). Female sex and high inhibitor levels influenced CR negatively. Thirty-six deaths (23.8%) were reported. Main causes of death were infection (15 patients, 9.9%) and hemorrhage (5 patients, 3.3%). All hemorrhage-related and half the infection-related deaths occurred within 2 months of diagnosis. Prior antithrombotic therapy was inversely associated with survival, irrespective of age. Median age of nonsurvivors was significantly higher (79 vs 73 years in survivors). Patients dying of infection were older than the other nonsurvivors (85 vs 78 years). In summary, fatal infection in the first months is common in our series. Antithrombotic therapy is associated with mortality. Particular care should be taken to avoid misdiagnosis