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Immune thrombocytopenia (ITP) is an autoimmune disease characterized by an isolated decrease in platelet count and an increased risk of bleeding. The pathogenesis is complex, affecting multiple components of the immune system and causing both peripheral destruction of platelets and inadequate production in the bone marrow. In this article, we review the treatment of ITP from a historical perspective, discussing first line and second line treatments, and management of refractory disease.
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ABSTRACT: Fostamatinib, a recently approved Syk inhibitor used in adult primary immune thrombocytopenia (ITP), has been shown to be safe and effective in this disorder. However, clinical trial results may not be similarly reproduced in clinical practice. Here, 138 patients with ITP (both primary and secondary) from 42 Spanish centers who had been treated with fostamatinib were evaluated prospectively and retrospectively. The median age of our cohort (55.8% women) was 66 years (interquartile range [IQR], 56-80). The median time since ITP diagnosis at fostamatinib initiation was 51 months (IQR, 10-166). The median number of therapies before fostamatinib initiation was 4 (IQR, 2-5), including eltrombopag (76.1%), romiplostim (57.2%), and IV immunoglobulins (44.2%). Fifty-eight patients (42.0%) had signs/symptoms of bleeding in the month before treatment initiation. Seventy-nine percent of patients responded to fostamatinib with 53.6% complete responses (platelet count > 100 × 109/L). Eighty-three patients (60.1%) received fostamatinib monotherapy, achieving a high response rate (85.4%). The proportion of time in response during the 27-month period examined was 83.3%. The median time to platelet response was 11 days (IQR, 7-21). Sixty-seven patients (48.5%) experienced adverse events, mainly grade 1 to 2; the commonest of which were diarrhea (n = 28) and hypertension (n = 21). One patient had deep venous thrombosis, and one patient developed acute myocardial infarction. Fostamatinib was shown to be effective with good safety profile in patients with primary and secondary ITP across a wide age spectrum in this real-world study.
Subject(s)
Aminopyridines , Morpholines , Oxazines , Purpura, Thrombocytopenic, Idiopathic , Pyridines , Pyrimidines , Humans , Female , Male , Middle Aged , Purpura, Thrombocytopenic, Idiopathic/drug therapy , Aged , Oxazines/therapeutic use , Oxazines/adverse effects , Aged, 80 and over , Pyrimidines/therapeutic use , Pyrimidines/adverse effects , Morpholines/therapeutic use , Morpholines/adverse effects , Pyridines/therapeutic use , Pyridines/adverse effects , Aminopyridines/therapeutic use , Aminopyridines/adverse effects , Retrospective Studies , Treatment Outcome , Syk Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/adverse effects , Platelet Count , Prospective StudiesABSTRACT
Immune thrombocytopenia (ITP) is an autoimmune disease characterized by an isolated decrease in the platelet count and an increased risk of bleeding. The pathogenesis is complex, affecting multiple components of the immune system and causing both peripheral destruction of platelets and impaired central megakaryopoiesis and platelet production in the bone marrow. Here, we intend to contextualize the current knowledge on the pathophysiology, terminology, epidemiology, clinical manifestations, diagnosis, and prognosis of ITP from a historical perspective and the first references to the never-stopping garnering of knowledge about this entity. We highlight the necessity to better understand ITP in order to be able to provide ITP patients with personalized treatment options, improving disease prognosis and reducing the incidence or frequency of refractoriness.
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INTRODUCTION: The increase in the number of patients with hemoglobinopathies in Europe in recent decades highlights the need for more detailed epidemiological information in Spain. To fulfil this need, the Spanish Society of Pediatric Hematology and Oncology (SEHOP) sponsored the creation of a national registry of hemoglobinopathies known as REHem-AR (Spanish Registry of Hemoglobinopathies and Rare Anemias). Data from the transfusion-dependent (TDT) and non-transfusion-dependent (NTDT) ß-thalassemia cohorts are described and analyzed. METHODS: We performed an observational, multicenter, and ambispective study, which included patients of any age with TDT and NTDT, registered up to December 31, 2021. RESULTS: Among the 1741 patients included, 168 cases of thalassemia were identified (103 TDT and 65 NTDT-patients). Survival at 18 years was 93% for TDT and 100% for NTDT. Regarding management, 80 patients with TDT (77.7%) and 23 patients with NTDT (35.4%) started chelation treatment during follow-up, with deferasirox being the most widely used. A total of 76 patients within the TDT cohort presented at least 1 complication (73.8%), the most frequent being hemosiderosis and osteopenia-osteoporosis. Comparison of both cohorts revealed significant differences in the diagnosis of hepatic hemosiderosis (p = 0.00024), although these were not observed in the case of cardiac iron overload (p = 0.27). DISCUSSION: Our registry enabled us to describe the management of ß thalassemia in Spain and to analyze the morbidity and mortality of the cohorts of patients with TDT and NTDT. Complications related to iron overload in TDT and NTDT account for most of the morbidity and mortality of the disease, which is associated with a considerable social, psychological, and economic impact, although cardiac, osteopathy and endocrinological complications requiring more attention. The convenience and simplicity of online registries make it possible to homogenize variables and periodically update data, thus providing valuable information on these diseases.
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Hemosiderosis , Iron Overload , beta-Thalassemia , Humans , beta-Thalassemia/complications , beta-Thalassemia/epidemiology , beta-Thalassemia/therapy , Blood Transfusion , Demography , Iron Overload/etiologyABSTRACT
INTRODUCTION: Due to their low prevalence, rare bleeding disorders (RBDs) remain poorly characterized. AIM: To gain insight of RBDs through our clinical practice. METHODS: Retrospective study of the medical records of RBD patients followed up at the Central University Hospital of Asturias between January 2019 and December 2022. RESULTS: A total of 149 patients were included. Factor (F) VII (44 %) and FXI (40 %) deficiencies were the most common diagnosed coagulopathies. Most of the patients were asymptomatic (60.4 %) and the most frequent type of bleeding were mucocutaneous and after surgery. All replacement treatments were administered on demand and no patient was on a prophylaxis regimen. Currently available products were safe; allergic reactions after administration of plasma were the most frequent complication. Genetic analysis, carried out on 55 patients (37 %), showed that the most frequent mutations in RBDs are of missense type (71.9 %). We identified 11 different novel genetic alterations in affected genes. The c.802C > T (p.Arg268Cys) variant, previously described, was identified in 71 % (15 of 21) of the patients with FXI deficiency genotyped and none were related (probable founder effect). CONCLUSION: Our study on an unusual large single center cohort of RBD patients portrays location-dependent distinct genetic drives and clinical practice particularities.
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Blood Coagulation Disorders , Factor XI Deficiency , Humans , Retrospective Studies , Tertiary Care Centers , Blood Coagulation Disorders/epidemiology , Hemorrhage/diagnosis , Genotype , Rare Diseases/diagnosisABSTRACT
There are not many publications that provide a holistic view of the management of primary and secondary ITP as a whole, reflecting the similarities and differences between the two. Given the lack of major clinical trials, we believe that comprehensive reviews are much needed to guide the diagnosis and treatment of ITP today. Therefore, our review addresses the contemporary diagnosis and treatment of ITP in adult patients. With respect to primary ITP we especially focus on establishing the management of ITP based on the different and successive lines of treatment. Life-threatening situations, "bridge therapy" to surgery or invasive procedures and refractory ITP are also comprehensively reviewed here. Secondary ITP is studied according to its pathogenesis by establishing three major differential groups: Immune Thrombocytopenia due to Central Defects, Immune Thrombocytopenia due to Blocked Differentiation and Immune Thrombocytopenia due to Defective Peripheral Immune Response. Here we provide an up-to-date snapshot of the current diagnosis and treatment of ITP, including a special interest in addressing rare causes of this disease in our daily clinical practice. The target population of this review is adult patients only and the target audience is medical professionals.
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Purpura, Thrombocytopenic, Idiopathic , Thrombocytopenia , Adult , Humans , Purpura, Thrombocytopenic, Idiopathic/diagnosis , Purpura, Thrombocytopenic, Idiopathic/etiology , Purpura, Thrombocytopenic, Idiopathic/therapy , Platelet Count , Receptors, Thrombopoietin , Thrombopoietin/therapeutic useABSTRACT
OBJECTIVE: The aim of this study was to present the results obtained in the Newborn Screening Program (NSP) for sickle cell disease (SCD) in western Andalusia and the autonomous city of Ceuta in the first 3 years of implementation, and to describe the discrepancies found in the diagnosis of hemoglobinopathies between the screening method and the confirmatory tests. STUDY DESIGN: A descriptive and retrospective study was carried out, and the findings obtained in the newborns included in the NSP between November 2018 and December 2021 were analyzed. RESULTS: A total of 111,205 samples were screened by high-performance liquid chromatography (HPLC). The birth prevalence of SCD, sickle cell trait, hemoglobin C carriers, and the compound heterozygosity Hb C/ß-thalassemia was 1/12,356, 1/467, 1/1,278, and 1/55,602 newborns, respectively. Although there was a correlation between the first-line HPLC screening technique (VARIANTnbs HPLC analyzer, Bio-Rad) and the confirmatory tests in most cases, major discrepancies were found in detecting carriers of G-Philadelphia, D, E, and O-Arab hemoglobin variants, with the former having an incidence of 1/10,110 and the others 1/22,241. The carrier status of Hb G-Philadelphia produced an FAD pattern on the screening method that could be mistaken as Hb D, while Hb O-Arab was identified as an FA5 pattern. Hb D was initially recognized as Hb D in two cases. CONCLUSION: An NSP requires at least two different combined methods in order to identify the hemoglobin variant with sufficient certainty. Furthermore, even though software solutions for HPLC suggest a pattern, it must be confirmed with another technique to obtain a correct interpretation of the chromatograms. KEY POINTS: · The NSPs are an essential activity in preventive medicine.. · At least two different combined methods are required to correctly identify hemoglobin variants.. · Different variants can produce a similar or identical pattern by a single method..
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Hemophilia A (HA) is a rare bleeding disorder caused by factor VIII (FVIII) deficiency due to various genetic mutations in the F8 gene. The disease severity inversely correlates with the plasma levels of functional FVIII. The treatment of HA patients is based on FVIII replacement therapy, either following a prophylactic or on-demand regime, depending on the severity of the disease at diagnosis and the patient's clinical manifestations. The hemorrhagic manifestations are widely variable amongst HA patients, who may require monitoring and treatment re-adjustment to minimize bleeding symptoms. Notably, laboratory monitoring of the FVIII activity is difficult due to a lack of sensitivity to various FVIII-related molecules, including non-factor replacement therapies. Hence, patient management is determined mainly based on clinical manifestations and patient-clinician history. Our goal was to validate the ST Genesia® automated thrombin generation analyzer to quantify the relative hemostatic status in HA patients. We recruited a cohort of HA patients from the Principality of Asturias (Spain), following treatment and at a stable non-bleeding phase. The entire cohort (57 patients) had been comprehensively studied at diagnosis, including FVIII and VWF activity assays and F8 genetic screening, and then clinically monitored until the Thrombin Generation Test (TGT) was performed. All patients were recruited prior to treatment administration, at the maximum time-window following the previous dose. Interestingly, the severe/moderate patients had a similar TGT compared to the mild patients, reflecting the non-bleeding phase of our patient cohort, regardless of the initial diagnosis (i.e., the severity of the disease), treatment regime, and FVIII activity measured at the time of the TGT. Thus, TGT parameters, especially the peak height (Peak), may reflect the actual hemostatic status of a patient more accurately compared to FVIII activity assays, which may be compromised by non-factor replacement therapies. Furthermore, our data supports the utilization of combined TGT variables, together with the severity of patient symptoms, along with the F8 mutation type to augment the prognostic capacity of TGT. The results from this observational study suggest that TGT parameters measured with ST Genesia® may represent a suitable tool to monitor the hemostatic status of patients requiring a closer follow-up and a tailored therapeutic adjustment, including other hemophilia subtypes or bleeding disorders.
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BACKGROUND: Haemophilia A (HA) has been associated with poor health-related quality of life and a large economic burden, accentuated by severity, arthropathy, and inhibitors. To meet global standards of care, the management of HA should align with the principles of care outlined by the World Federation of Haemophilia. The aims of the present study were to establish a set of proposals to improve HA management within the Spanish National Health System (SNHS) and to estimate the impact its hypothetical implementation would generate from a clinical, healthcare, economic, and social perspective. METHODS: A multidisciplinary group of experts agreed on a set of 15 proposals to improve HA management within the SNHS. Thereafter, a forecast-type Social Return on Investment analysis was carried out to estimate the impact of implementing this set of proposals within the SNHS over a one-year timeframe, in relation to the required investment. RESULTS: This study estimated that the implementation of the complete set of 15 proposals would require a total investment of 2.34 M and have a total impact of 14.60 M. Accordingly, every euro invested in the complete set of 15 proposals would yield a social return of 6.23 (3.37 in the worst-case scenario and 9.69 in the best-case scenario) of both tangible and intangible nature in similar proportions (45.71 and 54.29%, respectively). CONCLUSIONS: These results can be used to inform policy and practice such that interventions that may potentially improve current public health challenges associated with the management of HA may be implemented.
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Hemophilia A , Delivery of Health Care , Hemophilia A/drug therapy , Hemophilia A/epidemiology , Humans , Investments , Quality of LifeABSTRACT
INTRODUCTION: Type 2N von Willebrand disease (VWD) is characterized by a decreased affinity of von Willebrand factor (VWF) for factor VIII (FVIII). Abnormal binding of FVIII to VWF (VWF:FVIIIB), results in low FVIII plasma levels, which can lead to a misdiagnosis of mild haemophilia A. Accurate diagnosis of type 2N VWD is essential for appropriate genetic counselling and therapy. This disease can be distinguished from haemophilia A by in vitro assays (measurement VWF:FVIIIB activity) and/or genetic analysis. AIM: To identify the current challenges in the diagnosis and treatment of this type of VWD and provide an in-depth description of the phenotypes and mutations identified. RESULTS: Twenty-eight patients had at least one type 2N mutation, and 13 of these had a type 2N mutation combined with other variations. Three type 2N mutations were detected: p.Arg816Trp, p.Arg854Gln, and p.Arg763Ser. Two of these are the most frequently described mutations worldwide. This mutational spectrum differs from the broad spectrum seen in neighbouring France, where at least eight distinct 2N mutations have been found. In the PCM-EVW-ES cohort, 11 asymptomatic type 2N carriers with borderline FVIII plasma levels would probably have been excluded if the evaluation had been based on clinical and laboratory data only. Likewise, three patients with a severe phenotype would have been classified as homozygous for a 2N mutation if only the phenotype study had been performed. CONCLUSION: The high detection yield and affordability of next-generation sequencing support the use of this technology as a first-line diagnostic tool in this setting.
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Hemophilia A , von Willebrand Disease, Type 2 , von Willebrand Diseases , von Willebrand Factor/genetics , Factor VIII/genetics , Heterozygote , Homozygote , Humans , von Willebrand Disease, Type 2/diagnosis , von Willebrand Disease, Type 2/geneticsABSTRACT
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.
Subject(s)
Hemophilia A , Aged , Autoantibodies , Factor VIII , Female , Hemophilia A/diagnosis , Hemophilia A/drug therapy , Hemophilia A/epidemiology , Humans , Male , Registries , Retrospective StudiesABSTRACT
The most severe clinical manifestations of the Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are due to an unbalanced immune response and a pro-thrombotic hemostatic disturbance, with arterial hypertension or diabetes as acknowledged risk factors. While waiting for a specific treatment, the clinical management of hospitalized patients is still a matter of debate, and the effectiveness of treatments to manage clinical manifestations and comorbidities has been questioned. In this study, we aim to assess the impact of the clinical management of arterial hypertension, inflammation and thrombosis on the survival of COVID-19 patients. The Spanish cohorts included in this observational retrospective study are from HM Hospitales (2035 patients) and from Hospital Universitario Central de Asturias (72 patients). Kaplan Meier survival curves, Cox regression and propensity score matching analyses were employed, considering demographic variables, comorbidities and treatment arms (when opportune) as covariates. The management of arterial hypertension with angiotensin-converting enzyme 2 (ACE2) inhibitors or angiotensin receptor blockers is not detrimental, as was initially reported, and neither was the use of non-steroidal anti-inflammatory drugs (NSAIDs). On the contrary, our analysis shows that the use on itself of corticosteroids is not beneficial. Importantly, the management of COVID-19 patients with low molecular weight heparin (LMWH) as an anticoagulant significantly improves the survival of hospitalized patients. These results delineate the current treatment options under debate, supporting the effectiveness of thrombosis prophylaxis on COVID-19 patients as a first-line treatment without the need for compromising the treatment of comorbidities, while suggesting cautiousness when administering corticosteroids.
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The clinical diagnosis of von Willebrand disease (VWD), particularly type 1, can be complex because several genetic and environmental factors affect von Willebrand factor (VWF) plasma levels. An estimated 60% of the phenotypic variation is attributable to hereditary factors, with the ABO blood group locus being the most influential. However, recent studies provide strong evidence that nonsynonymous single nucleotide variants (SNVs) contribute to VWF and factor VIII phenotypic variability in healthy individuals. This study aims to investigate the role of common VWF SNVs on VWD phenotype by analyzing data from 219 unrelated patients included in the "Molecular and Clinical Profile of von Willebrand Disease in Spain project." To that end, generalized linear mixed-effects regression models were fitted, and additive and epistatic analyses, and haplotype studies were performed, considering five VWD-related measures (bleeding score, VWF:Ag, VWF:RCo, factor VIII:C, and VWF:CB). According to these analyses, homozygotes: for p.Thr789Ala(C) would be expected to show 39% higher VWF:Ag levels; p.Thr1381Ala(C), 27% lower VWF:Ag levels; and p.Gln852Arg(C), 52% lower VWF:RCo levels. Homozygotes for both p.Thr789Ala(C) and p.Gln852Arg(T) were predicted to show 185% higher VWF:CB activity, and carriers of two copies of the p.Thr1381Ala(T)/p.Gln852Arg(T) haplotype would present a 100% increase in VWF:RCo activity. These results indicate a substantial effect of common VWF variation on VWD phenotype. Although additional studies are needed to determine the true magnitude of the effects of SNVs on VWF, these findings provide new evidence regarding the contribution of common variants to VWD, which should be taken into account to enhance the accuracy of the diagnosis and classification of this condition. ClinicalTrials.gov identifier: NCT02869074.
Subject(s)
Mutation, Missense , Polymorphism, Single Nucleotide , von Willebrand Diseases/blood , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , Adult , Computer Simulation , Factor VIII/genetics , Factor VIII/metabolism , Female , Haplotypes , Hemorrhage , Heterozygote , Homozygote , Humans , Male , Middle Aged , Phenotype , Prospective Studies , Registries , Regression Analysis , Spain , Young Adult , von Willebrand Factor/chemistryABSTRACT
BACKGROUND: Eltrombopag is useful for immune thrombocytopenia (ITP). However, results of clinical trials may not accurately mirror clinical practice reality. Here we evaluated eltrombopag for primary and secondary ITP in our ≥65-year-old population. METHODS: A total of 106 primary ITP patients (16 with newly diagnosed ITP, 16 with persistent ITP, and 74 with chronic ITP) and 39 secondary ITP patients (20 with ITP secondary to immune disorders, 7 with ITP secondary to infectious diseases, and 12 with ITP secondary to lymphoproliferative disorders [LPD]) were retrospectively evaluated. RESULTS: Median age of our cohort was 76 (interquartile range, IQR, 70-81) years. 75.9% of patients yielded a platelet response including 66.2% complete responders. Median time to platelet response was 14 (IQR, 8-21) days. Median time on response was 320 (IQR, 147-526) days. Sixty-three adverse events (AEs), mainly grade 1-2, occurred. The most common were hepatobiliary laboratory abnormalities (HBLAs) and headaches. One transient ischemic attack in a newly diagnosed ITP and two self-limited pulmonary embolisms in secondary ITP were the only thrombotic events observed. CONCLUSION: Eltrombopag showed efficacy and safety in ITP patients aged ≥65 years with primary and secondary ITP. However, efficacy results in LPD-ITP were poor. A relatively high number of deaths were observed.
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Benzoates/therapeutic use , Hydrazines/therapeutic use , Purpura, Thrombocytopenic, Idiopathic/drug therapy , Pyrazoles/therapeutic use , Age Factors , Aged , Aged, 80 and over , Benzoates/administration & dosage , Benzoates/adverse effects , Biomarkers , Combined Modality Therapy , Comorbidity , Drug Therapy, Combination , Female , Humans , Hydrazines/administration & dosage , Hydrazines/adverse effects , Male , Prognosis , Purpura, Thrombocytopenic, Idiopathic/blood , Purpura, Thrombocytopenic, Idiopathic/diagnosis , Pyrazoles/administration & dosage , Pyrazoles/adverse effects , Retrospective Studies , Treatment OutcomeABSTRACT
PURPOSE: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies. METHODS: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies. RESULTS: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) CONCLUSION: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.
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Exome Sequencing , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia/genetics , Genetic Predisposition to Disease , Cell Line , DNA Copy Number Variations/genetics , DNA Repair/genetics , DNA-Binding Proteins/genetics , Fanconi Anemia/pathology , Female , Gene Knockout Techniques , Humans , Male , Mutation, Missense/genetics , Polymorphism, Single Nucleotide/geneticsABSTRACT
Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074.
Subject(s)
Gene Silencing , Introns , Mutation, Missense , RNA Splicing , von Willebrand Factor/genetics , Alleles , Base Sequence , Blood Platelets/metabolism , Computational Biology , Exons , Female , Gene Frequency , Genotype , High-Throughput Nucleotide Sequencing , Humans , Leukocytes/metabolism , Male , RNA Splice Sites , RNA, Messenger/genetics , von Willebrand Diseases/geneticsABSTRACT
The multimeric analysis (MA) of plasma von Willebrand factor (VWF) evaluates structural integrity and helps in the diagnosis of von Willebrand disease (VWD). This assay is a matter of controversy, being considered by some investigators cumbersome and only slightly informative. The centralised study 'Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES)' has been carried out by including the phenotypic assessment and the genetic analysis by next generation sequencing (NGS) of the VWF gene (VWF). The aim of the present study was to evaluate the role of MA to the diagnosis of these patients and their potential discrepancies. Two hundred and seventy out of 480 patients centrally diagnosed with VWD had normal multimers, 168 had abnormal multimers and 42 a total absence of multimers. VWF MA was of great significance in the diagnosis of 83 patients (17.3%), it was also of help in the diagnosis achieved in 365 additional patients (76%) and was not informative in 32 cases (6.7%). With regard to discrepancies, 110 out of 480 (23%) patients centrally diagnosed with VWD presented some kind of discordance between VWF:RCo/VWF:Ag and/or VWF:CB/VWF:Ag ratios, multimeric study and/or genetic results. The VWF MA was key in the presence of novel mutations as well as in cases with phenotypic discrepancies. A comparison between the contribution of MA and VWF:CB showed a clearly higher contribution of the former in the diagnostic process. These data seem to reinforce the relevance of the VWF MA in VWD diagnosis, despite all its limitations.
Subject(s)
High-Throughput Nucleotide Sequencing , von Willebrand Diseases/diagnosis , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Genotype , Humans , Male , Middle Aged , Phenotype , Spain , Young AdultABSTRACT
OBJECTIVE: MLL gene is involved in more than 80 known genetic fusions in acute leukemia. To study the relevance of MLL partner gene and selected gene's expression, in this work, we have studied a cohort of 20 MLL-rearranged acute myeloid leukemia (AML). METHODS: Twenty MLL-rearranged AML patients along with a control cohort of 138 AML patients are included in this work. By RT-PCR and sequencing, MLL genetic fusion was characterized, and relative gene expression quantification was carried out for EVI1, MEIS1, MLL-3', RUNX1, SETBP1, HOXA5, and FLT3 genes. Risk stratification and association of MLL genetic partner and gene expression to overall survival, in the context of received therapy, were performed. RESULTS: MLLr cohort showed to have an OS more similar to intermediate-risk AML. Type of MLL genetic partner showed to be relevant in allo-HSCT response; having MLLT1 and MLLT3, a better benefit from it. Expression of MLL-3' region, EVI1 and FLT3, showed association with OS in patients undergoing allo-HSCT. CONCLUSION: We show that the MLL genetic partner could have implications in allo-HSCT response, and we propose three genes whose expression could be useful for the prognosis of this leukemia in patients undergoing allo-HSCT: 3' region of MLL, EVI1, and FLT3.
Subject(s)
3' Untranslated Regions , Biomarkers, Tumor , Gene Rearrangement , Histone-Lysine N-Methyltransferase/genetics , Leukemia, Myeloid, Acute/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , RNA, Messenger , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Gene Expression Regulation, Neoplastic , Hematopoietic Stem Cell Transplantation , Humans , In Situ Hybridization, Fluorescence , Infant , Kaplan-Meier Estimate , Karyotype , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/therapy , MDS1 and EVI1 Complex Locus Protein/genetics , Male , Middle Aged , Oncogene Proteins, Fusion/genetics , Prognosis , Proportional Hazards Models , Translocation, Genetic , Transplantation, Homologous , Treatment Outcome , Young Adult , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.