RESUMEN
Inherited platelet disorders are important conditions that often manifest with bleeding. These disorders have heterogeneous underlying pathologies. Some are syndromic disorders with non-blood phenotypic features, and others are associated with an increased predisposition to developing myelodysplasia and leukemia. Platelet disorders can present with thrombocytopenia, defects in platelet function, or both. As the underlying pathogenesis of inherited thrombocytopenias and platelet function disorders are quite diverse, their evaluation requires a thorough clinical assessment and specialized diagnostic tests, that often challenge diagnostic laboratories. At present, many of the commonly encountered, non-syndromic platelet disorders do not have a defined molecular cause. Nonetheless, significant progress has been made over the past few decades to improve the diagnostic evaluation of inherited platelet disorders, from the assessment of the bleeding history to improved standardization of light transmission aggregometry, which remains a "gold standard" test of platelet function. Some platelet disorder test findings are highly predictive of a bleeding disorder and some show association to symptoms of prolonged bleeding, surgical bleeding, and wound healing problems. Multiple assays can be required to diagnose common and rare platelet disorders, each requiring control of preanalytical, analytical, and post-analytical variables. The laboratory investigations of platelet disorders include evaluations of platelet counts, size, and morphology by light microscopy; assessments for aggregation defects; tests for dense granule deficiency; analyses of granule constituents and their release; platelet protein analysis by immunofluorescent staining or flow cytometry; tests of platelet procoagulant function; evaluations of platelet ultrastructure; high-throughput sequencing and other molecular diagnostic tests. The focus of this article is to review current methods for the diagnostic assessment of platelet function, with a focus on contemporary, best diagnostic laboratory practices, and relationships between clinical and laboratory findings.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas , Trastornos de las Plaquetas Sanguíneas/complicaciones , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Plaquetas/metabolismo , Citometría de Flujo , Hemostasis , Humanos , Pruebas de Función Plaquetaria/métodosRESUMEN
A single-center study was conducted on 120 patients with inherited disorders of primary hemostasis followed at our hematological center. These patients presented a variety of bleeding symptoms; however, they had no definitive diagnosis. Establishing a diagnosis has consequences for the investigation of probands in families and for treatment management; therefore, we aimed to improve the diagnosis rate in these patients by implementing advanced diagnostic methods. According to the accepted international guidelines at the time of study, we investigated platelet morphology, platelet function assay, light-transmission aggregometry, and flow cytometry. Using only these methods, we were unable to make a definitive diagnosis for most of our patients. However, next-generation sequencing (NGS), which was applied in 31 patients, allowed us to establish definitive diagnoses in six cases (variants in ANKRD26, ITGA2B, and F8) and helped us to identify suspected variants (NBEAL2, F2, BLOC1S6, AP3D1, GP1BB, ANO6, CD36, and ITGB3) and new suspected variants (GFI1B, FGA, GP1BA, and ITGA2B) in 11 patients. The role of NGS in patients with suspicious bleeding symptoms is growing and it changes the diagnostic algorithm. The greatest disadvantage of NGS, aside from the cost, is the occurrence of gene variants of uncertain significance.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas , Humanos , República Checa , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Trastornos de las Plaquetas Sanguíneas/genética , Pruebas de Función Plaquetaria , Secuenciación de Nucleótidos de Alto Rendimiento , Hemorragia , Proteínas Sanguíneas/genéticaRESUMEN
Platelets are generated and released into the bloodstream from their precursor cells, megakaryocytes that reside in the bone marrow. Though platelets have no nucleus or DNA, they contain a full transcriptome that, during platelet formation, is transported from the megakaryocyte to the platelet. It has been described that transcripts in platelets can be translated into proteins that influence platelet response. The platelet transcriptome is highly dynamic and has been extensively studied using microarrays and, more recently, RNA sequencing (RNA-seq) in relation to diverse conditions (inflammation, obesity, cancer, pathogens and others). In this review, we focus on bulk and single-cell RNA-seq studies that have aimed to characterize the coding transcriptome of healthy megakaryocytes and platelets in humans. It has been noted that bulk RNA-seq has limitations when studying in vitro-generated megakaryocyte cultures that are highly heterogeneous, while single-cell RNA-seq has not yet been applied to platelets due to their very limited RNA content. Next, we illustrate how these methods can be applied in the field of inherited platelet disorders for gene discovery and for unraveling novel disease mechanisms using RNA from platelets and megakaryocytes and rare disease bioinformatics. Next, future perspectives are discussed on how this field of coding transcriptomics can be integrated with other next-generation technologies to decipher unexplained inherited platelet disorders in a multiomics approach.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas , Megacariocitos , Trastornos de las Plaquetas Sanguíneas/metabolismo , Plaquetas/metabolismo , Humanos , Megacariocitos/metabolismo , ARN/metabolismo , Trombopoyesis/genética , TranscriptomaRESUMEN
Bernard-Soulier syndrome (BSS) is an autosomal-recessive bleeding disorder caused by biallelic variants in the GP1BA, GP1BB, and GP9 genes encoding the subunits GPIbα, GPIbß, and GPIX of the GPIb-IX complex. Pathogenic variants usually affect the extracellular or transmembrane domains of the receptor subunits. We investigated a family with BSS caused by the homozygous c.528_550del (p.Arg177Serfs*124) variant in GP1BB, which is the first mutation ever identified that affects the cytoplasmic domain of GPIbß. The loss of the intracytoplasmic tail of GPIbß results in a mild form of BSS, characterized by only a moderate reduction of the GPIb-IX complex expression and mild or absent bleeding tendency. The variant induces a decrease of the total platelet expression of GPIbß; however, all of the mutant subunit expressed in platelets is correctly assembled into the GPIb-IX complex in the plasma membrane, indicating that the cytoplasmic domain of GPIbß is not involved in assembly and trafficking of the GPIb-IX receptor. Finally, the c.528_550del mutation exerts a dominant effect and causes mild macrothrombocytopenia in heterozygous individuals, as also demonstrated by the investigation of a second unrelated pedigree. The study of this novel GP1BB variant provides new information on pathophysiology of BSS and the assembly mechanisms of the GPIb-IX receptor.
Asunto(s)
Síndrome de Bernard-Soulier/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Trombocitopenia/genética , Adulto , Síndrome de Bernard-Soulier/sangre , Síndrome de Bernard-Soulier/patología , Plaquetas/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Dominios Proteicos/genética , Trombocitopenia/patología , Factor de von Willebrand/metabolismoRESUMEN
We report peri- and post-operative management of haemostasis in a 11-year old girl with Glanzmann Thrombasthenia (GT) who had feminizing genitoplasty for genital ambiguity due to Congenital Adrenal Hyperplasia (CAH-21 Hydroxylase deficiency). A blend of Glanzmann Thrombasthenia (GT) and DSD 46XX due to CAH is not reported in literature. Surgery particularly genitourinary reconstruction in patients with GT is challenging due to risk of intra and post-operative bleeding. Haemostasis can successfully be achieved with platelet transfusions, antifibrinolytic (Tranexamic acid) and judicious use of recombinant factor VIIa (rFVIIa) even in a resource limited setting.
Asunto(s)
Trombastenia , Niño , Femenino , Hemostasis , Humanos , Transfusión de Plaquetas , Hemorragia Posoperatoria , Proteínas Recombinantes , Trombastenia/complicaciones , Trombastenia/terapiaRESUMEN
The area of women and inherited bleeding disorders has undergone quick expansion in recent years. More patients are being identified and expertise to diagnose and manage these patients is now essential for practising physicians. Programs to help educate and empower patients and caregivers are now in place. Common inherited bleeding disorders affecting women include von Willebrand disease (VWD), inherited platelet disorders, and rare inherited bleeding disorders such as factor VII (FVII) deficiency and factor XI (FXI) deficiency. Specific clinical tools have been developed to help clinicians and patients screen for the presence of these bleeding disorders in both adult and pediatric populations. Affected women can experience heavy menstrual bleeding and resulting iron deficiency anemia, postpartum hemorrhage, and hemorrhagic ovarian cysts which need to be properly managed. Excessive bleeding can adversely affect quality of life in these women. Front line therapy for bleeding in mild cases focuses on the use of non-specific hemostatic agents such as DDAVP ®, tranexamic acid and hormonal agents but specific factor replacement and/or blood products may be required in more severe cases, in severe bleeding or as second line treatment when bleeding is not responsive to first line agents. Iron status should be optimised in these women especially in pregnancy and use of an electronic app can now help clinicians achieve this. These patients should ideally be managed by a multidisciplinary team whenever possible even remotely. Although clinical research has closed some knowledge gaps regarding the diagnosis and management of these women, there remains significant variation in practise and lack of evidence-based guidelines still exists in many spheres of clinical care in which caregivers must rely on expert opinion. Ongoing efforts in education and research will continue to improve care for these women and restore quality of life for them.
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Hemorragia , Hemostáticos/uso terapéutico , Complicaciones Hematológicas del Embarazo , Calidad de Vida , Enfermedades de von Willebrand , Femenino , Hemorragia/sangre , Hemorragia/tratamiento farmacológico , Hemorragia/genética , Humanos , Embarazo , Complicaciones Hematológicas del Embarazo/sangre , Complicaciones Hematológicas del Embarazo/tratamiento farmacológico , Complicaciones Hematológicas del Embarazo/genética , Enfermedades de von Willebrand/sangre , Enfermedades de von Willebrand/tratamiento farmacológico , Enfermedades de von Willebrand/genéticaRESUMEN
The diagnosis of inherited platelet disorders (IPD) is a complex task. Indeed, due to their rarity, their wide clinical spectrum (intensity of hemorrhagic symptoms) and the need for specialized biological assays (only performed in reference centers) IPDs can be diagnosed very late. However, it is important to remember the crucial need for early diagnosis in order to avoid the use of unnecessary and potentially harmful treatments for the patient. A thorough personal and family history, a complete physical examination and a simple biological work up (blood count, blood smear and platelet occlusion time) will lead to the suspicion of an IPD. It will then be up to the physician to refer the patient to a specialist in order to complete the diagnostic work up and therefore establishing a definitive diagnosis. Here is a description of the most well-known IPDs and their diagnostic algorithms.
Le diagnostic des thrombopénies et thrombopathies constitutionnelles est une tâche complexe. En effet, leur caractère rare, leur hétérogénéité clinique (intensité des symptômes hémorragiques) et la nécessité d'examens complémentaires biologiques spécialisés (uniquement réalisés dans certains centres de référence) expliquent le diagnostic parfois tardif de ces pathologies. Cependant, il convient de rappeler l'importance cruciale d'un diagnostic correct précoce pour éviter le recours à des traitements inutiles et potentiellement néfastes pour le patient en cas de thrombopénie mal diagnostiquée. Une anamnèse personnelle et familiale fouillée, un examen clinique complet et un bilan biologique de base (hémogramme, frottis sanguin et temps d'occlusion plaquettaire) permettront de suspecter une origine congénitale à la thrombopénie que présente un patient. Il reviendra alors au médecin de référer ce dernier à un spécialiste pour la réalisation d'un bilan complet visant à obtenir un diagnostic précis. Nous vous proposons ici une description des thrombopénies et thrombopathies constitutionnelles ainsi que des algorithmes pour leur diagnostic.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas , Algoritmos , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Plaquetas , Hemorragia , Humanos , AnamnesisRESUMEN
The number of genes involved in the identification of macrothrombocytopenia (MTP) is growing but the clinical consequences for the affected patients are not well determined. Here, we report the management of the bleeding risk for a patient with the newly reported and rare DIAPH1-related disease during surgery for infertility and then during her subsequent pregnancy. The R1213* DIAPH1 variant responsible for a mild bleeding syndrome in six families was considered a potential risk factor for our patient. Preliminary laparoscopic surgery was followed by neosalpingostomy to open the obstructed fallopian tube that was followed by an ectopic pregnancy requiring further surgery, tranexamic acid was used on each occasion and no bleeding complications were observed. A second pregnancy proceeded to term; the mother's platelet count was controlled throughout the gestation period and remained close to her basal values. No bleeding occurred at delivery or during the postpartum period. In conclusion, with strict repeated assessments of blood parameters and maintenance of the platelet count, the bleeding risk in pregnancy in DIAPH1-related disease can be successfully controlled.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Complicaciones del Embarazo , Trombocitopenia/diagnóstico , Trombocitopenia/genética , Trombocitopenia/terapia , Sustitución de Aminoácidos , Biomarcadores , Transfusión Sanguínea , Manejo de la Enfermedad , Femenino , Forminas , Variación Genética , Edad Gestacional , Hemorragia/etiología , Hemorragia/prevención & control , Humanos , Mutación , Recuento de Plaquetas , Embarazo , Resultado del Embarazo , SíndromeRESUMEN
Platelet transmission electron microscopy (PTEM) is considered the gold standard test for assessing distinct ultrastructural abnormalities in inherited platelet disorders (IPDs). Nevertheless, PTEM remains mainly a research tool due to the lack of standardized procedures, a validated dense granule (DG) count reference range, and standardized image interpretation criteria. The aim of this study was to standardize and validate PTEM as a clinical laboratory test. Based on previously established methods, we optimized and standardized preanalytical, analytical, and postanalytical procedures for both whole mount (WM) and thin section (TS) PTEM. Mean number of DG/platelet (plt), percentage of plts without DG, platelet count (PC), mean platelet volume (MPV), immature platelet fraction (IPF), and plt light transmission aggregometry analyses were measured on blood samples from 113 healthy donors. Quantile regression was used to estimate the reference range for DG/plt, and linear regression was used to assess the association of DG/plt with other plt measurements. All PTEM procedures were standardized using commercially available materials and reagents. DG interpretation criteria were established based on previous publications and expert consensus, and resulted in improved operator agreement. Mean DG/plt was stable for 2 days after blood sample collection. The median within patient coefficient of variation for mean DG/plt was 22.2%; the mean DG/plt reference range (mid-95th %) was 1.2-4.0. Mean DG/plt was associated with IPF (p = .01, R2 = 0.06) but not age, sex, PC, MPV, or plt maximum aggregation or primary slope of aggregation (p > .17, R2 < 0.02). Baseline ultrastructural features were established for TS-PTEM. PTEM was validated using samples from patients with previously established diagnoses of IPDs. Standardization and validation of PTEM procedures and interpretation, and establishment of the normal mean DG/plt reference range and PTEM baseline ultrastructural features, will facilitate implementation of PTEM as a valid clinical laboratory test for evaluating ultrastructural abnormalities in IPDs.
Asunto(s)
Plaquetas/metabolismo , Microscopía Electrónica de Transmisión/métodos , Valores de Referencia , HumanosRESUMEN
BACKGROUND: Inherited platelet deficiency and/or dysfunction may be more common in the general population than has previously been appreciated. In 2013 the Israeli Inherited Platelet Disorder (IPD) Registry was established. METHODS: Clinical and laboratory data were collected to pre-specified registration forms. The study protocol was approved by the local hospital ethics committees. RESULTS: To date we have included in the registry 89 patients (male 52%) from 79 families. Most patients (74%) have a not-yet specified inherited thrombocytopenia (n=39) or non-specific platelet function disorder (n=27). Full clinical data were available for 81 (91%) patients. The median (range) age at presentation and time of follow-up were 1.8years (1day-17.8years) and 4.7 (0-26) years, respectively. The Pediatric Bleeding Questionnaire was available for 78patients; abnormal bleeding score (≥2) was recorded in 47 (52.8%, 95% CI 42%-63.5%) patients and was less frequent in patients followed for isolated thrombocytopenia. Abnormal score was associated with a longer time of follow-up, OR 1.19 (95% CI 1.04-1.36). CONCLUSION: Long term follow-up of patients with IPDs is important as bleeding risks may increase with time. We expect that clinical and laboratory information of patients/families with IPDs gathered in a systemic format will allow for better diagnosis and treatment of these patients.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas/complicaciones , Plaquetas/patología , Hemorragia/etiología , Adolescente , Adulto , Trastornos de las Plaquetas Sanguíneas/patología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Sistema de Registros , Trombocitopenia/complicaciones , Trombocitopenia/patología , Adulto JovenRESUMEN
BACKGROUND: RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and a predisposition to acute leukaemia. Platelets possess three distinct types of granules and secretory processes involving dense granules (DG), α-granules and vesicles or lysosomes containing acid hydrolases (AH). Dense granules and granule deficiencies have been reported in patients with RUNX1 mutations. Little is known regarding the secretion from AH-containing vesicles. METHODS AND RESULTS: We studied two related patients with a RUNX1 mutation, easy bruising, and mild thrombocytopenia. Platelet aggregation and 14 C serotonin in platelet-rich plasma (PRP) were impaired in response to ADP, epinephrine, collagen and arachidonic acid. Contents of DG (ATP, ADP), α-granules (ß-thromboglobulin) and AH-containing vesicles (ß-glucuronidase, ß-hexosaminidase, α-mannosidase) were normal or minimally decreased. Dense granules secretion on stimulation of gel-filtered platelets with thrombin and divalent ionophore A23187 (4-12 µmol L-1 ) were diminished. ß-thromboglobulin and AH secretion was impaired in response to thrombin or A23187. We studied thromboxane-related pathways. The incorporation of 14 C -arachidonic acid into phospholipids and subsequent arachidonic acid release on thrombin activation was normal. Platelet thromboxane A2 production in whole blood serum and on thrombin stimulation of PRP was normal, suggesting that the defective secretion was not due to impaired thromboxane production. CONCLUSIONS: These studies provide the first evidence in patients with a RUNX1 mutation for a defect in AH (lysosomal) secretion, and for a global defect in secretion involving all three types of platelet granules that is unrelated to a granule content deficiency. They highlight the pleiotropic effects and multiple platelet defects associated with RUNX1 mutations.
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Trastornos de las Plaquetas Sanguíneas/genética , Trastornos de las Plaquetas Sanguíneas/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Haploinsuficiencia , Hidrolasas/metabolismo , Adulto , Ácido Araquidónico/metabolismo , Trastornos de las Plaquetas Sanguíneas/sangre , Plaquetas/metabolismo , Estudios de Casos y Controles , Preescolar , Femenino , Humanos , Masculino , Mutación , Fosfolípidos/biosíntesis , Recuento de Plaquetas , Vesículas Secretoras/metabolismo , Serotonina/metabolismo , Tromboxanos/biosíntesisRESUMEN
Knowledge in the field of inherited thrombocytopenias (ITs) has greatly improved over the last 15 years. Several new genes responsible for thrombocytopenia have been identified leading to the definition of novel nosographic entities and to a much better characterization of the phenotypes of these diseases. To date, ITs encompass 22 disorders caused by mutations in 24 genes and characterized by different degrees of complexity and great variability in prognosis. Making a definite diagnosis is important for setting an appropriate follow-up, choosing the best treatments and providing proper counseling. Despite the abovementioned progress, diagnosis of ITs remains difficult and these disorders are still underdiagnosed. The purpose of this review is to provide an updated guide to the diagnosis of ITs based on simple procedures. Moreover, the currently available therapeutic options for these conditions are recapitulated and discussed.
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Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/terapia , Trombocitopenia/diagnóstico , Trombocitopenia/terapia , Diagnóstico Diferencial , HumanosAsunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Trastornos de las Plaquetas Sanguíneas/genética , Hipercolesterolemia/genética , Enfermedades Intestinales/genética , Errores Innatos del Metabolismo Lipídico/genética , Lipoproteínas/genética , Fitosteroles/efectos adversos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/sangre , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/sangre , Animales , Trastornos de las Plaquetas Sanguíneas/sangre , Femenino , Humanos , Lipoproteínas/sangre , Masculino , Ratones , Fitosteroles/genéticaRESUMEN
BACKGROUND: The International Society of Thrombosis & Hemostasis (ISTH) bleeding assessment tool (ISTH-BAT) is used to record bleeding symptoms in patients with possible bleeding disorders. AIM: To investigate the utility of the ISTH-BAT in predicting platelet dysfunction in individuals with suspected inherited platelet function disorders. METHOD: Individuals with clinical evidence of bleeding and suspected inherited platelet function disorder and healthy volunteers were included in the study. The ISTH-BAT questionnaire was applied prior to light transmission aggregometry (LTA). RESULTS: A total of 261 participants were included (100 healthy volunteers, and 161 with suspected inherited platelet function disorders). The ISTH-BAT score in participants with suspected inherited platelet function disorders (median 2; interquartile range [IQR] 5-1) was significantly higher than in healthy volunteers (median 0; IQR 2-0). There was also a significant difference between participants with suspected inherited platelet function disorders with a platelet defect detected by LTA (median 4; IQR 8-3) and those with normal platelet function (median 2; IQR 3-1) (p < 0.001). The ISTH-BAT score was associated with a demonstrable platelet defect on platelet function testing (area under the receiver operating characteristic curve = 0.8 [95% confidence interval 0.72-0.87, p = < 0.001] and odds ratio 3.25 [95% confidence interval 2.13-4.37, p = < 0.001]). CONCLUSION: The ISTH-BAT is a useful tool for documenting bleeding symptoms and the score obtained is also predictive of the presence of a platelet defect on LTA in patients with suspected inherited platelet dysfunction.
Asunto(s)
Trastornos de las Plaquetas Sanguíneas , Hemorragia/diagnóstico , Pruebas de Función Plaquetaria , Adolescente , Adulto , Trastornos de la Coagulación Sanguínea Heredados/diagnóstico , Trastornos de la Coagulación Sanguínea Heredados/genética , Trastornos de las Plaquetas Sanguíneas/diagnóstico , Trastornos de las Plaquetas Sanguíneas/genética , Niño , Preescolar , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Curva ROC , Índice de Severidad de la Enfermedad , EspectrofotometríaRESUMEN
This study evaluated 65 pregnancies in 34 women with five different inherited platelet function disorders. Gestation was similar to that of the general population. Severe bleeds requiring blood transfusions were observed in 50% of deliveries in Glanzmann thrombasthenia (GT), but not in the patients with delta storage pool disease, Hermansky-Pudlak syndrome, P2Y12 defect or defect of thromboxane A2 receptor. Of note, severe haemorrhage also occurred in women with GT who had received prophylactic platelet transfusions, suggesting that better preventive treatments are required. Diagnosis and degree of spontaneous bleeding tendency before pregnancy were reliable parameters to predict the delivery-related bleeding risk.
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Trastornos de las Plaquetas Sanguíneas/terapia , Hemorragia/prevención & control , Transfusión de Plaquetas , Complicaciones Hematológicas del Embarazo/terapia , Adolescente , Adulto , Femenino , Humanos , Persona de Mediana Edad , EmbarazoAsunto(s)
Trastornos de las Plaquetas Sanguíneas/genética , Proteínas Sanguíneas/genética , Enfermedades Genéticas Congénitas/genética , Mutación Missense , Mielofibrosis Primaria/genética , Adolescente , Adulto , Sustitución de Aminoácidos , Trastornos de las Plaquetas Sanguíneas/complicaciones , Femenino , Enfermedades Genéticas Congénitas/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Mielofibrosis Primaria/etiologíaRESUMEN
BACKGROUND: Genetic diagnosis of inherited platelet disorders (IPDs) is mainly performed by high-throughput sequencing (HTS). These short-read-based sequencing methods sometimes fail to characterize the genetics of the disease. OBJECTIVES: To evaluate nanopore long-read DNA sequencing for characterization of structural variants (SVs) in patients with IPDs. METHODS: Four patients with a clinical and laboratory diagnosis of Glanzmann thrombasthenia (GT) (P1 and P2) and Hermansky-Pudlak syndrome (HPS) (P3 and P4) in whom HTS missed the underlying molecular cause were included. DNA was analyzed by both standard HTS and nanopore sequencing on a MinION device (Oxford Nanopore Technologies) after enrichment of DNA spanning regions covering GT and HPS genes. RESULTS: In patients with GT, HTS identified only 1 heterozygous ITGB3 splice variant c.2301+1G>C in P2. In patients with HPS, a homozygous deletion in HPS5 was suspected in P3, and 2 heterozygous HPS3 variants, c.2464C>T (p.Arg822∗) and a deletion affecting 2 exons, were reported in P4. Nanopore sequencing revealed a complex SV affecting exons 2 to 6 in ITGB3 (deletion-inversion-duplication) in homozygosity in P1 and compound heterozygosity with the splice variant in P2. In the 2 patients with HPS, nanopore defined the length of the SVs, which were characterized at nucleotide resolution. This allowed the identification of repetitive Alu elements at the breakpoints and the design of specific polymerase chain reactions for family screening. CONCLUSION: The nanopore technology overcomes the limitations of standard short-read sequencing techniques in SV characterization. Using nanopore, we characterized novel defects in ITGB3, HPS5, and HPS3, highlighting the utility of long-read sequencing as an additional diagnostic tool in IPDs.
Asunto(s)
Síndrome de Hermanski-Pudlak , Trombastenia , Humanos , Homocigoto , Eliminación de Secuencia , Síndrome de Hermanski-Pudlak/genética , Análisis de Secuencia de ADN , Trombastenia/genética , Secuenciación de Nucleótidos de Alto Rendimiento , ADNRESUMEN
Hereditary platelet delta (δ)-storage pool deficiency is a rare condition in which there are fewer dense granules in platelets disrupting primary hemostasis. It can cause a mild-moderate bleeding tendency with normal coagulation studies; hence, it is an underdiagnosed diagnostic challenge. The authors present three patients with hereditary platelet delta (δ)-storage pool deficiency who had heavy menstrual bleeding, excessive bleeding following surgery, mucocutaneous bleeding, and a bleeding score greater than or equal to 6. These cases reveal the susceptibility of underdiagnosing platelet disorders and the significance of utilizing a bleeding assessment tool to help guide further workup with transmission electron microscopy to visualize the fewer dense granules in platelets. Although bleeding is typically moderate, it can be severe in certain scenarios, like after mucosal surgeries, and can lead to death, highlighting the importance of the condition's recognition and prophylactic treatment.
RESUMEN
INTRODUCTION: Rare Bleeding Disorders have a low population prevalence and may not be recognized by most clinicians. In addition, knowledge gaps of the indicated laboratory tests and their availability add to the potential for delayed diagnosis or misdiagnosis. The lack of widely available commercial, regulatory body approved esoteric tests limits them to reference laboratories, thus limiting easy access for patients. AREAS COVERED: A literature search of PubMed, Medline, and Embase and a review of international society guidelines were performed. Additional references from published articles were reviewed. A patient-centered approach to recognition and evaluation of RBD is discussed. EXPERT OPINION: Recognition of RBD relies on obtaining a detailed patient's personal and family hemostatic history. Inquiry into a history of involvement of other organ systems is important and, if present, should lead to suspicion of an inherited platelet disorder or a variant of Ehlers-Danlos Syndrome. Multiple factors contribute to the complexity of developing efficient algorithms for diagnostic testing. Limitations in diagnostic sensitivity and specificity of screening tests, diagnostic tests, and esoteric tests further compound the complexity of establishing a diagnosis. Educational efforts focusing on clinician awareness of RBDs and available testing options are vital for optimal management of such patients.
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Trastornos de la Coagulación Sanguínea , Trastornos de las Plaquetas Sanguíneas , Humanos , Hemostasis , Hemorragia , AlgoritmosRESUMEN
BACKGROUND: Excessive or abnormal mucocutaneous bleeding (MCB) may impact all aspects of the physical and psychosocial wellbeing of those who live with it (PWMCB). The evidence base for the optimal diagnosis and management of disorders such as inherited platelet disorders, hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD), Ehlers-Danlos syndromes (EDS), and von Willebrand disease (VWD) remains thin with enormous potential for targeted research. RESEARCH DESIGN AND METHODS: National Hemophilia Foundation and American Thrombosis and Hemostasis Network initiated the development of a National Research Blueprint for Inherited Bleeding Disorders with extensive all-stakeholder consultations to identify the priorities of people with inherited bleeding disorders and those who care for them. They recruited multidisciplinary expert working groups (WG) to distill community-identified priorities into concrete research questions and score their feasibility, impact, and risk. RESULTS: WG2 detailed 38 high priority research questions concerning the biology of MCB, VWD, inherited qualitative platelet function defects, HDS/EDS, HHT, bleeding disorder of unknown cause, novel therapeutics, and aging. CONCLUSIONS: Improving our understanding of the basic biology of MCB, large cohort longitudinal natural history studies, collaboration, and creative approaches to novel therapeutics will be important in maximizing the benefit of future research for the entire MCB community.
More people experience mucocutaneous bleeding (MCB), affecting tissues like skin and gums, than have hemophilia A or B. MCB is not understood as well as hemophilia. Common types of MCB include nosebleeds, bleeding gums, heavy menstrual bleeding, and digestive tract bleeding. Mucocutaneous inherited bleeding disorders include inherited platelet disorders, hereditary hemorrhagic telangiectasia (HHT), hypermobility spectrum disorders (HSD) and Ehlers-Danlos syndromes (EDS), von Willebrand Disease (VWD), and others. Diagnosing and treating MCB is complicated and sometimes medical providers dismiss the bleeding that patients report when they cannot find a medical explanation for it. Many people with mucocutaneous bleeding (PWMCB) do not receive the care they need; for example, women with VWD live with symptoms for, on average, 16 years before they are diagnosed in the US. This struggle to obtain care has important negative impacts on patients' physical and psychological health and their quality-of-life. The National Hemophilia Foundation (NHF), a large US bleeding disorders patient advocacy organization, set out to develop a National Research Blueprint for Inherited Bleeding Disorders focused on community priorities. They brought together a group of patients, providers, and researchers with MCB expertise to identify the research that would most improve the lives of PWMCB through targeted and accessible diagnostics and therapies. We report in this paper that research is needed to better understand the biology of MCB and to define the mechanisms of disease in these disorders. We also describe high priority research questions for each of the main disorders, novel therapeutics, and aging.