Diagnosis Of Bernard-Soulier Syndrome Research Articles

Elevated CD9 expression as a potential biomarker for diagnosis of Bernard-Soulier syndrome.

Diagnosis of inherited platelet glycoprotein disorders is based on specific laboratory techniques such as aggregometry and flow cytometry. Flowcytometry is a powerful method, but equivocal results are produced in some cases. New cluster of differentiation markers could resolve the diagnostic dilemmas. Abnormal expression of CD9 in Bernard-Soulier syndrome (BSS) is recently reported. We aimed to determine the diagnostic significance of CD9 expression in a cohort of Iranian patients with inherited platelet glycoprotein defects. Twelve BSS, 21 Glanzmann thrombasthenia and 16 healthy controls were included in the present study. Flowcytometric diagnosis of BSS and Glanzmann thrombasthenia was made by analysis of CD41/61 and CD42a/42b CD markers. Moreover, phycoerythrin-labelled anti CD9 was examined in patients and healthy controls. The mean fluorescence intensity (MFI) of CD9 among the three groups was compared using suitable statistical methods and a P value of less than 0.05 considered statistically significant. Mean MFI of CD9 was 990.0 in BSS patients versus 421.2 and 317.3 in individuals with Glanzmann thrombasthenia and healthy controls, respectively (P < 0.05). Between the two-group comparison of means by the Mann-Whitney test revealed a P value of less than 0.001 for BSS group versus GT (2.4-fold) and BSS versus healthy controls (2.9-fold). CD9 molecule also expressed differently in patients with Glanzmann thrombasthenia in comparison with healthy controls (P < 0.001), although with a less magnitude (1.3-fold). According to our findings, CD9 is a potential biomarker for laboratory diagnosis of inherited glycoprotein defects, especially to elucidate the ambiguous results in BSS cases.

Novel mutation in the glycoprotein Ibβ in a patient with Bernard–Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive disorder characterized by a prolonged skin-bleeding time and thrombocytopenia with giant platelets. The hallmark of BSS is an abnormal platelet attachment to the vessel wall due to reduced or abnormal glycoprotein Ib/IX/V complex. We present a case of BSS in a 14-month-old boy caused by a novel genetic mutation. The patient has the typical clinical findings of BSS, but he was misdiagnosed for a long period. Evaluation of the peripheral blood smear revealed giant platelets and genetic testing confirmed the diagnosis of BSS. The child was found to be homozygous for a nonsense mutation (c.423C > A) in the glycoprotein Ibβ (GPIbβ) gene. Knowing that we are dealing with a very rare syndrome, the detected mutation in our patient was homozygous. Although the parents were nonconsanguineous, we believe that they were related in a distant parental connection, which the parents and their family were not aware of.

Six Novel Mutations Identified in the Glycoproteins Ib Alpha, Ib Beta and IX Genes Among Twenty-Two Unrelated Patients with Bernard-Soulier Syndrome in Brazil

Abstract 1156▪▪This icon denotes a clinically relevant abstract Introduction:Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder, caused by mutations within the glycoprotein (GP) Ib alpha, GP Ib beta and GP IX genes that encode three of four subunits of the platelet GP Ib-V-IX adhesion receptor. In the present study we evaluated the mutations involved in the diagnosis of BSS from twenty-two unrelated patients. Patients and Methods: All patients were followed in two large bleeding disorder reference centers in Brazil. The diagnosis of BSS was established based on the presence of mucocutaneous bleeding and macrotrombocytopenia, and was confirmed by platelet ristocetin aggregation and flow cytometry for the platelet GP Ib alpha (CD 42b), GP Ib beta (CD 42c), and GP IX (CD 42a). Available first- and second-degree relatives were also contacted for clinical, laboratory and molecular evaluation. Genomic DNA from all index cases was used for sequence analysis of the three genes, GP1BA, GP1BB and GP9, and the results were confirmed in relatives when available. Results: Twenty-two unrelated patients with the confirmed diagnosis of BSS were enrolled in this study. Among twenty-two index cases, twenty-one had one or two mutations identified, including six novel mutations. We also identified two mutations that have been previously reported, GP1BA C209S (Simsek, et al., 1994), and GP9 A140T (Wang, et al., 2004). The six novel mutations correspond to conserved regions, and they consist of two mutations in the GP1BA (L51R, and L99P), three in GP1BB (M-25A, L72R, and L112P), and one in GP9 (P52Q). One of these novel mutations, the GP1BB L112P was observed in twelve of twenty-two unrelated cases. PCR-restriction fragment length analysis of genomic DNA from a hundred normal unrelated controls were performed to evaluate the presence of GP1BA L99P and GP1BB L112P mutations by using the AlwN I and Alu I restriction enzymes, respectively. All controls were negative for both mutations. Interestingly, when we analyzed the relatives of the index cases indentified with the mutations GP1BA L99P and GP1BB M-25A, we found evidence of mild macrotrombocytopenia in the heterozygote carriers of these mutations. The relatives heterozygous for GP1BB L112P showed normal platelet count and morphology. However, in three index cases with mild macrotrombocytopenia, GP1BB L112P in heterozygosity was the only mutation identified. Furthermore, site-directed mutagenesis was carried out to evaluate the expression of the novel mutations GP1BA L51R, and GP1BA L99P. Compared to the GP1BA wild-type, both mutations were only minimally expressed in CHO bIX cells, which stably express GP Ib beta and GP IX. Conclusions: We identified in this study eight distinct mutations among twenty-two unrelated SBS patients, including six novel mutations. The GP1BB L112P mutation was found in twelve of the twenty-two index cases, suggesting that this could be due to a founder effect. Identifying such a frequent mutation in this population of BSS patients will be helpful for genetic diagnosis of this condition in Brazil. Furthermore these mutations significantly add to the mutation database of BSS and will inevitably provide insights into the function of GP Ib-V-IX. Disclosures:No relevant conflicts of interest to declare.

Novel Mutation in the Glycoprotein Ibβ in a Patient with Bernard-Soulier Syndrome: Possibility of Distant Parental Consanguinity

Abstract 4689 Introduction:Bernard-Soulier syndrome (BSS) is a rare autosomal recessive disorder characterized by a prolonged skin-bleeding time and thrombocytopenia with giant platelets. The hallmark of BSS is an abnormal platelet attachment to the vessel wall due to reduced or abnormal glycoprotein (GP) Ib/IX/V complex. We present a case of BSS in a 14-month-old boy caused by a novel genetic mutation. Patient and methods:A 14-month-old Syrian boy was referred to our medical center with a history of easy bruisability, epistaxis, and low platelet counts since the age of 5 months. He was the second child of non-consanguineous parents. His parents and siblings were negative for a history of bleeding disorders. His nephew was reported to have low platelets counts. The patient was hospitalized in Syria several times and was initially diagnosed as having idiopathic thrombocytopenic purpura. He received corticosteroids and high-dose intravenous immunoglobulin both of which were ineffective. He also received platelet transfusion for recurrent epistaxis, which improved as per the parents. The possibility of a platelet disorder was raised and he was referred to our center for further investigation.Physical examination showed bruises over the face and extremities. Abdominal examination was negative for hepatosplenomegaly. There were no congenital abnormalities. His platelet count was 100 × 109/l. The presence of giant platelets was noted on blood film inspection. Other blood cell counts, blood chemistry and coagulation studies, including von Willebrand factor (vWF) antigen and activity, were within the normal range. A clinical diagnosis of BSS was made. Peripheral blood was obtained for genetic testing.DNA sequencing and analysisThe initial extraction of the DNA material was done using the PEL-FREEZ extraction kit (PEL-FREEZ, DYNAL, USA) and genomic material stored at −80°C. We performed direct sequencing of the entire coding regions including exon-intron boundaries of the GPIbα and GPIbβ genes (GenBank: NM_000173.5 and NM_000407.4; mutation numbering + 1 corresponds to the A of the ATG-translation initiation codon, respectively). Genomic DNA from patient was amplified by PCR with oligonucleotide primers complementary to flanking intronic sequences. Primers were designed using the Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) (primer sequences and PCR conditions are available on request). PCR products were sequenced employing ABI BigDye chemistry (Applied Biosystems, Darmstadt, Germany). The same primers as for PCR were used as sequencing primers. Samples were run and analyzed on an ABI PRISM 3130 genetic analyzer (Applied Biosystems). Conclusion:We present a case of BSS in a 14-month-old boy caused by a novel nonsense mutation (c.423C>A) in the GPIbβ gene. Knowing that we are dealing with a very rare syndrome, the detected mutation in our patient was homozygous. Although the parents were non-consanguineous, we believe that they were related in a distant parental consanguinity which the parents and their family were not aware of. Disclosures:No relevant conflicts of interest to declare.

Flow cytometry as a tool in the diagnosis of Bernard-Soulier Syndrome in Brazilian patients

Bernard-Soulier Syndrome (BSS) is an inherited recessive bleeding disorder. In some instances, diagnosis might be restricted to routine blood exams, including bleeding time, prothrombin time (PT), and partial thromboplastin time (APTT). Exams such as platelet aggregation, and testing for expression of ristocetin cofactor, or von Willebrand factor may not be commonly performed. This leads to misdiagnosis in a number of patients, which are subsequently treated erroneously. Flow cytometry has been used widely as a tool in the diagnosis of leukemias, lymphomas, and many other immuno-hematological diseases. The purpose of this study was to assess whether flow cytometry could be helpful in the diagnosis of Bernard-Soulier Syndrome in Brazilian patients. For this, we examined a selected group of 15 patients with suspected BSS based on classical diagnosis. We used a panel of antibodies to detect the expression of glycoproteins GPIbα, GPIIb, GPIIIa, GPIX, as well as CD9. Abnormalities of GPIb and GPIX were observed in nine of the 15 patients, which included severe reduction of both antigens, of one or the other, or normal levels but weak expression. Strikingly, this abnormality correlated with severely reduced expression of CD9 in all cases. We discuss the implications for flow cytometric diagnosis of BSS.

Schwere Thrombozytopenie und sieben problemlose Geburten

Summary Severe thrombocytopenia and seven problem-free births We present four patients from two Swiss families with a rare thrombocytopathy, Bernard-Soulier syndrome (BSS), with varying haemorrhagic diatheses. The diagnosis of BSS was suspected on the basis of a low platelet count and morphological changes to the thrombocytes. The platelets showed an isolated defect on ristocetin-induced agglutination. Immunophenotyping of the thrombocytes demonstrated lack of GP Ib expression of varying degree in comparison to normal controls. The combination of these findings confirmed the diagnosis of BSS. Differential diagnosis and treatment strategies are discussed. A congenital thrombocytopathy should always be considered in patients with thrombocytopenia of unknown origin and abnormal platelet morphology.

Use of recombinant factor VIIa in the management of severe bleeding episodes in patients with Bernard–Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare congenital platelet disorder characterized by defective platelet adhesion and manifested by spontaneous and often profuse bleeding. Recombinant factor VIIa (rFVIIa) is a haemostatic agent licensed for the treatment of bleeding episodes in patients with haemophilia and inhibitors, which may represent a low-risk alternative to existing therapies in the management of patients with BSS. Here, we describe the use of rFVIIa for the treatment of three severe bleeding episodes in two patients with BSS. Data were extracted by automated searching of the international, Internet-based registry http://www.haemostasis.com . Patient 1, a 24-year-old woman, was admitted with severe epistaxis and hypotension. The diagnosis of BSS was confirmed by macrothrombocytopenia, absence of ristocetin-induced platelet agglutination (RIPA) and absence of glycoprotein (GP) Ibalpha and IX on the platelet surface. Epsilon aminocaproic acid (EACA; two 50-mg/kg doses), packed red blood cells (PRBCs, 2 U) and platelets (30 U) failed to control the bleeding and, after 13 h, three bolus doses of rFVIIa (90 microg/kg body weight) and a third dose of EACA were administered; bleeding stopped after the third dose of rFVIIa. Patient 2, a 15-year-old girl, initially presented with severe menorrhagia. A lack of RIPA and severe deficiency of GPIbalpha on the platelet surface confirmed the diagnosis of BSS. EACA and fresh-frozen plasma did not control the haemorrhage, but two bolus doses of rFVIIa (98 microg/kg body weight) resulted in a marked decrease in bleeding. On second admission, patient 2 had severe epistaxis and mild menorrhagia. Two rFVIIa doses (98 and 122.5 microg/kg body weight) were given, and the bleeding stopped. No adverse events were reported in these cases. These three admissions highlight the potential of rFVIIa for the treatment of severe bleeds in patients with BSS.

Syndrome de Bernard-Soulier révélé par une thrombopénie sévère en période néonatale

Bernard–Soulier syndrome (BSS) is a congenital autosomal recessive bleeding disorder characterised by giant platelets, the lack of thrombocytopenia or a moderate one, prolongation of skin bleeding time, and absent platelet aggregation in response to ristocetin. We report a case of BSS revealed by major neonatal thrombocytopenia. A newborn was admitted for thrombocytopenic purpura initially believed to be due to a maternal auto-immune thrombocytopenia. Because of the persistence of the thrombopenia till the age of 7 months despite therapy by corticosteroids and immunoglobulins, and because of the detection of anti-1b antiplatelets antibodies after transfusion, BSS diagnosis was evoked. In such a situation of major thrombocytopenia, the main therapeutic measure is prevention. Therapy by DDAVP may be used after the age of 3 years in situations of high haemorrhagic risk. This case report underlines the importance of a precise diagnosis in front of a maternal thrombocytopenia and the possibility of antenatal diagnosis of BSS.

Flow Cytometric Analysis of Platelet Surface Glycoproteins in the Diagnosis of Bernard-Soulier Syndrome

The use of flow cytometry in the diagnosis of Bernard-Soulier syndrome (BSS) in patients with giant platelets and thrombocytopenia was investigated as an adjunct to ristocetin-induced platelet aggregation (RIPA) studies because of problems experienced with aggregation studies, particularly at the time of presentation, in the pediatric age group. Eight patients suspected of having BSS were studied with respect to platelet expression of glycoprotein Ibα (CD42b) and glycoprotein Ilia (CD61) using an EPICS Profile II flow cytometer. Twelve patients with normal platelet morphology and platelet counts were used as normal controls. One patient with Alport's syndrome, four patients with immune thrombocytopenic purpura (ITP), and one patient with Glanzmann thrombasthenia were also studied. In all eight patients suspected of having BSS, deficiency of glycoprotein Iba was demonstrated. Normal expression was demonstrated in 12 control patients, in one patient with giant platelets with Alport's syndrome, and in four patients with ITP. Absence of glycoprotein Ilia was demonstrated in Glanzmann thrombasthenia. In the pediatric age group one is able to demonstrate abnormalities of platelet membrane glycoprotien in patients with thrombocytopathias using flow cytometry. This method has the advantage of being rapid and can be performed on small volumes of blood suitable for pediatric practice.

Bernard-Soulier Syndrome: Whole Blood Diagnostic Assays of Platelets

Diagnosing Bernard-Soulier syndrome (BSS), a congenital hemorrhagic disorder of blood platelets, is complicated by the difficulty of separating the giant platelets from other blood cells to allow studies of platelet function and structure. We report on the use of three whole blood assays for diagnosing BSS. Whole blood platelet aggregation responses studied with an electrical impedance aggregometer were equivalent to those more laboriously obtained by using platelet-rich plasma prepared by unit gravity sedimentation and studied with an optical light transmittance aggregometer. Platelet aggregation responses were normal with adenosine diphosphate or collagen stimulation but absent with ristocetin or bovine plasma stimulation. Whole blood radioimmunoassay of platelet glycoprotein (GP) expression was performed by using iodinated murine monoclonal antibodies HP1-1D (anti-GP IIb/IIIa) and 6D1 (anti-GP Ib). After incubation with citrated whole blood, centrifugation was used to separate cell-bound antibody that was quantitated with a gamma counter. The patient's whole blood had a normal level of cell-bound GP IIb/IIIa but a substantially reduced level of cell-bound GP Ib (5% of normal mean). Whole blood smear immunocytochemical staining with monoclonal antibodies and qualitative analysis by light microscopy revealed a considerable reduction of GP Ib expression by the patient's giant platelets, whereas GP IIb/IIIa expression was normal. These data helped establish the diagnosis of BSS. We conclude that these three relatively simple assays of platelets in whole blood should be of particular value in the clinical laboratory differential diagnosis of patients with congenital thrombocytopenias and giant platelet syndromes.

Diagnosis of Bernard-Soulier syndrome and Glanzmann's thrombasthenia with a monoclonal assay on whole blood.

Two hereditary platelet disorders, Bernard-Soulier syndrome and Glanzmann's thrombasthenia, are characterized by selective deficiencies of platelet membrane glycoproteins. Murine monoclonal antibodies were developed against platelet membrane glycoprotein Ib and against the glycoprotein IIb/IIIa complex. A rapid whole blood assay for the deficiency of these glycoproteins was developed and used to study whole blood samples from six patients with Glanzmann's thrombasthenia and three patients with Bernard-Soulier syndrome. Patients with type I and type II Glanzmann's thrombasthenia were easily detectable with this assay. This permits the diagnosis of these disorders on 200 microliters of whole blood within 2 h of blood sampling.