Bernard-Soulier Syndrome Research Articles

Platelets, glycoprotein Ib-IX, and von Willebrand factor are required for FeCl3-induced occlusive thrombus formation in the inferior vena cava of mice

Venous thromboembolism is a leading cause of death from cardiovascular disease. Despite the importance of the glycoprotein (GP) Ib-IX/von Willebrand factor (vWF) axis in arterial thrombosis, its requirement in venous, not venule thrombosis in response to endothelial injury (not stenosis or stasis) is uncharacterized. GPIbα-vWF participation in FeCl3-induced thrombus formation was evaluated in the inferior vena cava (IVC). Stable, occlusive thrombus formation in response to FeCl3-induced injury of the IVC was studied. FeCl3 (20% FeCl3, 10 minutes)-induced occlusive thrombosis required platelets as confirmed by a lack of occlusion in thrombocytopenic mice, and stable occlusion in control animals. No IVC occlusion was observed using GPIbα-deficient animals, a model of the human Bernard-Soulier syndrome (BSS). Transgenic IL-4 R/GPIbα mice (lack murine GPIbα, but express the extracellular domain of the human interleukin (IL-4 receptor fused to the transmembrane and cytoplasmic domains of human GPIbα) were studied to determine if the absence of IVC occlusion in the BSS mouse was caused by GPIbα extracellular domain deficiency rather than platelet BSS phenotype associated abnormalities. As with GPIbα knock-out mice, no occlusion was observed in the IVC of IL-4 R/GPIbα mice. The IVC of vWF-deficient mice also failed to occlude in response to FeCl3 treatment. The chimeric protein GPIbα(2V)-Fc prevented occlusion, demonstrating that GPIbα-vWF A1 domain interaction is required for FeCl3-induced stable thrombus formation in the IVC. Therefore, FeCl3-induced stable, occlusive thrombus formation in the IVC is platelet, and apparently GPIbα-vWF interaction dependent, despite the large diameter and low venous flow rate in the IVC.

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.

Inherited platelet disorders and oral health

Platelets play a key role in thrombosis and hemostasis. Accumulation of platelets at the site of vascular injury is the first step in the formation of hemostatic plugs, which play a pivotal role in preventing blood loss after injury. Platelet adhesion at sites of injury results in spreading, secretion, recruitment of additional platelets, and formation of platelet aggregates. Inherited platelet disorders are rare causes of bleeding syndromes, ranging from mild bruising to severe hemorrhage. The defects can reflect deficiency or dysfunction of platelet surface glycoproteins, granule contents, cytoskeletal proteins, platelet pro-coagulant function, and signaling pathways. For instance, Bernard-Soulier syndrome and Glanzmann thrombasthenia are attributed to deficiencies of glycoprotein Ib/IX/V and GPIIb/IIIa, respectively, and are rare but severe platelet disorders. Inherited defects that impair platelet secretion and/or signal transduction are among the most common forms of mild platelet disorders and include gray platelet syndrome, Hermansky-Pudlak syndrome, and Chediak-Higashi syndrome. When necessary, desmopressin, antifibrinolytic agents, and transfusion of platelets remain the most common treatment of inherited platelet disorders. Alternative therapies such as recombinant activated factor VII are also available for a limited number of situations. In this review, we will discuss the management of patients with inherited platelet disorders in various clinical situations related to dental cares, including surgical intervention.

Myocardial infarction in two cousins heterozygous for ASN41HIS autosomal dominant variant of Bernard–Soulier syndrome

Bernard-Soulier Syndrome is characterized by thrombocytopenia with large platelets and defective aggregation to ristocetin. The bleeding tendency is variable but may be severe. The syndrome is due to genetic defects of the GPIb-V-IX complex and it has been maintained to be protective from thrombotic events. Here we present the first two cases of documented M.I. in two cousins, heterozygous for the Arg41His mutation which is responsible for a dominant form of Bernard-Soulier Syndrome. In one of the two patients an aneurysm of the aorta was also present. The patients had a mild bleeding tendency which was severely aggravated by treatment with antiplatelet drugs. These clinical observations are in contrast with experimental studies which demonstrate that Bernard-Soulier-like strains of mice show a decreased thrombus generation in several experimental settings.

A novel ELISA for diagnosis of Glanzmann’s thrombasthenia and the heterozygote carriers

A sensitive and specific sandwich ELISA was developed for the diagnosis of Glanzmann's thrombasthenia (GT) and the heterozygote carriers of the disease using whole blood platelets. The assay used anti-CD36 antibody to capture platelets from platelet-rich plasma which was subsequently treated with a bioengineered disintegrin/alkaline phosphatase hybrid protein specific for GP IIb/IIIa. The test allows large number of samples to be typed and can also be used on stored samples. The assay correctly diagnosed 40 normal healthy individuals, 10 GT cases, 10 heterozygotes, 3 Bernard-Soulier syndrome cases and 2 type 3 GT cases. ELISA plates were stable at room temperature up to 3 weeks without any loss of activity. This novel and simple test can be widely used for heterozygote detection besides diagnosing GT cases without using a sophisticated flow cytometer or a platelet aggregometer and has wide applicability in countries like India where many of these cases remain undiagnosed due to the lack of diagnostic facilities.

Whole Blood Impedance Aggregometry: A New Tool for Severe Inherited Platelet Disorder Diagnosis?

Abstract 5266Diagnosis and characterization of platelet function disorders may be challenging. It requires multiple laboratory data including the assessment of platelet functions. Platelet function analysis is most commonly performed using light transmission aggregometry (LTA). LTA is a time-consuming method requiring centrifugation steps and large blood volumes. It is difficult to perform in children and in cases of thrombocytopenia. In contrast, platelet aggregation in whole blood using impedancemetry (WBI) is a fast method, allows omission of centrifugation steps and performance of platelet function studies under more physiological conditions with small samples size. It is based on the change of resistance proportional to the amount of platelets sticking to two electrodes where an alternating current is applied. Multiplate® (for “multiple electrode aggregometry”, Dynabite Medical) is a new generation of WBI aggregometer using diluted blood and single-use test cells containing twin electrodes that reduce the variation of results. We have already showed the good Multiplate® performance concerning ristocetin-induced platelet aggregation in a population of 30 patients with characterized von Willebrand disease (Valarche et al, 2011). Our aim in this ongoing study was to assess the performance of WBI in patients with inherited platelet function disorders. We tested 8 patients including 2 unrelated patients with Glanzmann Thrombasthenia (GT), 2 unrelated patients with Bernard-Soulier Syndrome (BSS), 1 patient with Gray Platelet Syndrome (GPS) and 3 patients from the same family with a platelet type von Willebrand disease (PTVWD). GT, BSS, and PTVWD diagnosis were confirmed using genotyping. BSS and GPS patients had chronic thrombocytopenia. GT, BSS, GPS and 1/3 PTVWD had platelet function tests with LTA in parallel. WBI was performed on heparinized whole blood diluted at ½ in NaCl at 37°C and triggered using high (0.77 mg/mL, WBI RH) and low (0.5 mg/mL, WBI RL) final ristocetin concentrations, ADP (6.5 Âμ Mol, WBI ADP) and collagen (3.2 Âμg/mL, WBI Coll). Results were expressed in arbitrary unit (AU) corresponding to the area under the aggregation curve observed during 6 min. Normal ranges indicated in brackets were based on the mean +/− 2 SD of 30 healthy volunteers’ results. Results highlighted in grey are those out of the normal ranges (Table 1).Table 1:Results of the 8 patients with inherited platelet disorders.PatientsPlatelet count (109/L)WBI RH (AU) [>500]WBI RL (AU) [<150]WBI ADP (AU) [>550]WBI Coll (AU) [>500]GT 116923441443GT 224955417ND7BSS 134371119129BSS 230254733582GPS7916217ND42PTVWD22099493ND338PTVWD231116560ND1092PTVWD2341174168ND852All patients except those with PTVWD had decreased results with WBI. However, as expected, patients with GT had flat traces using WBI ADP and WBI Coll but normal or only decreased curves (234 – 554 AU) using WBI RH. On the opposite, BSS patients had flat traces using WBI RH but detectable curves using WBI ADP (191 – 335 AU) despite decreased platelet count. The thrombocytopenic GPS patient has a flat trace using WBI Coll and decreased WBI RH (162 AU). Members of the PTVWD family had normal results except a slightly increased result with WBI RH in 1/3 patients. Finally, LTA results performed in 6/8 patients were all in accordance with those of the WBI. In conclusion, in 8 patients with well characterized inherited platelet disorders, WBI was able to detect all abnormalities except PTVWD. In such cases, different ristocetin concentrations use might be critical to increase sensitivity. In our hands, WBI was able to discriminate disorders involving platelet glycoprotein (GP) IIb-IIIa from GP Ib-IX-V: GT patients exhibited flat traces using WBI ADP and WBI Coll, whereas patients with BSS exhibited flat traces with ristocetin. These preliminary results need to be confirmed on a larger population of patients with various characterized platelet function disorders. They suggest that WBI using the Multiplate® analyzer, which is a fast, easy and blood-preserving test, could be a valuable extra step before or in addition to the classic LTA for the diagnosis of severe inherited platelet disorders. Disclosures:No relevant conflicts of interest to declare.

Successful Outcome of Two Pregnancies in a Woman with Bernard Soulier Syndrome

Abstract 4676 INTRODUCTION:Bernard Soulier Syndrome (BSS) is a rare qualitative and quantitative autosomal recessive platelet disorder molecularly characterized by defective GPIb-IX-V membrane glycoprotein that is related to platelet adhesion and interaction with both Von Willebrand factor and endothelium. It is usually expressed by an enlarged bleeding time and decreased yet giant sized platelets, and can cause severe hemorrhagic complications during pregnancy. CASE REPORT:We present two successful pregnancies and labour management on a 33-year-old woman diagnosed in her childhood with BSS. Ristocetin induced platelet agglutination test and membrane glycoprotein study were performed to get the diagnosis. Furthermore, in the last year we have completed the diagnosis with platelet receptor expression flow citometry analysis, finding out depleted antiGPIbα and antiGPIX antibodies. Changes which prevent GPIbα for correct expression and processing were observed. Also integrin αIIbβ3 levels were risen due to the big sized platelets. A PCR product was obtained from genomic DNA using the GPIbα oligonucleotide pair; sense and antisense, and direct DNA sequencing of the amplification was performed in a model ABIprism 377 DNA sequencer (Perkin-Elmer Cetus). The direct sequencing of the sense strand of the 5x fragment shows in the patient the simultaneous presence of G and C nucleotids in 688 position, and the T and A nucleotids in 715 position. These punctual mutations give Ala200Pro substitutions and Cys209Ser in heterozygosis in mature peptide, respectively, Figure 1. The Ala200Pro mutation has not been reported in others cases of Bernard-Soullier, previously. The Cys209Ser has been described in other two Spanish patients: one in homozygosis and the other in heterozygosis associated to the mutation.Our patient presented hemorrhagic symptoms from 18 month old with an average of 40×103/μ l platelet count and increased platelet volume who required multiple platelet transfusions during her childhood. In the menarche main hemorrhagic manifestations consisted on methrorragia and epistaxis which were controlled with tranexamic acid. At the age of 26 a spontaneous ovarian cyst rupture required urgent hospital admission and two platelet pool transfusion. On December 2007 she consulted for epistaxis, and on September 2008 cesarean delivery was planned at 38th week of pregnancy; platelet count was 40×103/μ l, antiplatelet antibodies were negative for membrane glycoproteins and antiHLA-1 was positive against 98% donors tested. As a result, an HLA-compatible donor was searched, and two platelet apheresis were transfused one hour before surgery and every 12 hours after the procedure. The next 4 days one apheresis every 48 hours were transfused with no hemorrhagic symptoms. On January 2010, she presented with her second pregnancy; the patient had severe anemia, with high transfusion requirements; neither hemorrhage or hemolysis was detected, and even the possibility of a Munchausen syndrome was questioned. Unplanned caesarean was performed at 32th week because of the clinical instability of the patient, and this time we could not find on time an HLA-compatible donor, so two random platelet pools were transfused one hour before surgery and one platelet apheresis every day during 4 days after caesarean deliver, without any hemorrhagic complication. The newborn was asymptomatic with normal platelet count. The patientxs haemoglobin level returned to normal after five days of delivery. CONCLUSIONS:We present the successful labour management of two consecutive pregnancies on a young woman diagnosed with BSS. The low prevalence of this syndrome (less than one case per million population) explains the absence of well-established protocols for the management of pregnancy in this disease. Up to date there are only twelve reported cases of pregnancy and delivery in BSS patients, most of them presenting with severe hemorrhagic complications. Correct platelet transfusion is the mainstay of treatment, and screening for anti-platelet antibodies study is mandatory due to its high prevalence in these patients. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

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.

A Novel Missense Mutation Asp69Gly in the Platelet Glycoprotein Ib(beta) Gene in Two Families with Bernard-Soulier Syndrome,

Abstract 3300▪▪This icon denotes a clinically relevant abstractBernard-Soulier Syndrome (BSS) is characterized by macrothrombocytopenia and impaired platelet function due to defects of the GpIb-IX-V complex. Patients with Bernard-Soulier Syndrome are rare and only few disease causing mutations have been identified in the platelet gylcoprotein Ib(alpha), Ib(beta) or glycoprotein IX genes, respectively.We characterized 2 independent families with Bernard-Soulier Syndrome. Affected members of both families presented with a bleeding history including gross hematoma after minimal trauma, recurrent epistaxis, menorrhagia and prolonged bleeding after surgery. Routine laboratory tests showed reduced platelet numbers and increased platelet volume. Consistent with the diagnosis of Bernard-Soulier syndrome FACS analysis demonstrated reduced GpIb-IX expression on the platelet surface. Platelet agglutination in the presence of ristocetin (RIPA) was impaired.Using standard techniques we sequenced the GpIb(alpha), GpIb(beta) and GpIX genes. We identified a novel mutation Asp69Gly in the GpIb(beta) gene in both families. The mutation was homozygous in two children of the first family and one child of the second family. As expected, the parents of the affected index patients were heterozygous mutation carriers. To further investigate the impact of the Asp69Gly mutation on GpIb expression we used structure prediction software and homology-based protein modeling. Asp69Gly lies within an extracellular region flanking a leucin-rich element and cysteine-cluster. It affects the highly conserved center aspartic acid of a CDC motif that is thought to form one of two cystein-loops close to the transmembrane domain. We propose that this mutation disrupts the regular conformation of the cystein loop, potentially leading to abnormal protein folding and protein decay in the endoplasmic reticulum.In addition to a mere defect of biosynthesis our data hint at additional functional consequences of the Asp69Gly mutation. The father and the mother of the index patient in the first family, who are heterzygous for the Asp69Gly mutation, express only slightly reduced levels of platelet GpIb-IX based on FACS analysis. Nevertheless both of them suffer from epistaxis and menorrhagia, respectively. Expression studies are underway to clarify the influence of the Asp69Gly mutation on GpIb expression and function. Preliminary data show reduced cell surface expression of the GPIb-IX complex and reduced binding to von Willebrand-factor (VWF).While the functional and structural effects of mutations in the GpIb(alpha) and GpIX genes have been thoroughly studied, the impact of mutations in the GpIb(beta) gene is less clear. Our results could help to elucidate how the GpIb(beta) protein contributes to the assembly and surface expression of the GpIb-IX-V complex. 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.

Application of a Diagnostic Algorithm for Inherited Thrombocytopenia Patients in the Setting of a Developing Country

Abstract 1163▪▪This icon denotes a clinically relevant abstractInherited thrombocytopenias are rare genetic disorders which frequently represent a diagnostic challenge, even after extensive diagnostic work-up. This is due to the requirement of specialized laboratory tests and to the fact that the underlying disease-causing mutation remains in many cases still unknown. Increasing knowledge regarding the molecular etiology of inherited thrombocytopenias contributes both to patient diagnosis, prognosis and genetic counseling and may lead to the identification of novel regulators of platelet production. Recently, mutations in the ANKRD26 gene have been described, which underlie inherited thrombocytopenias with normal platelet size. We applied a previously proposed algorithm (Haematologica 2003; 88: 582–592) and developed a twining program in collaboration with a specialized center to improve the diagnostic yield in a cohort of patients in Argentina. Based on this algorithm, patients were classified according both to the presence or absence of clinical or laboratory features other than thrombocytopenia and to platelet size. Simple laboratory tests, such as platelet function studies, peripheral blood smear and flow cytometry were performed in all cases, while confirmatory tests were performed according to initial diagnostic suspicion, and included analysis of non-muscle myosin heavy chain IIA distribution in neutrophils by immunofluorescence and mutational screening of candidate genes, such as MYH9, RUNX1, MPL, WASP, GPIBA and ANKRD26. Studies unavailable at the local institution were performed in a specialized center in the setting of the twining program. Thirty-five patients, belonging to 14 pedigrees, were included, age 32 (4–72) years old, 20 were female. Platelet count was 83 (5–170)×109/L, 49% were classified as macrothrombocytopenia, 46% had normal platelet size while 6% harboured small platelets. In the latter, a diagnosis of X-linked thrombocytopenia secondary to WASP mutation was made. Four pedigrees with macrothrombocytopenia had MYH9-related disease, one had Bernard-Soulier syndrome, while in four other, no molecular diagnosis was reached. Among pedigrees with normal platelet size, one had FPD/LMA due to RUNX1 mutation, whereas the c.-127A>G mutation in 5’UTR region of the ANKRD26 gene was detected in another family. In addition, a working diagnosis of familial ITP was made in a three-generation pedigree who had autosomal dominant thrombocytopenia and normal platelet size, based on the finding of shortened platelet life-span and complete response to splenectomy. Overall, a molecular diagnosis was established in 9 of 14 (64%) pedigrees, and MYH9-RD comprised the most frequent single disorder. Application of this diagnostic algorithm proved feasible in a resource-limited setting, as data provided by careful medical history and simple laboratory tests allowed to identify patients in whom further, specialized studies were justified. Collaboration of a specialized center was essential for molecular diagnosis and proved invaluable to build local capacities. Disclosures:No relevant conflicts of interest to declare.

Quaternary Organization of GPIb-IX Complex and Insights Into Bernard-Soulier Syndrome Revealed by the Crystal Structures of GPIbβ Ectodomain and a GPIbβ/GPIX Chimera

Abstract 2195Platelet glycoprotein (GP)Ib-IX receptor complex contains three subunits, GPIbα, GPIbβ and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome (BSS). We have crystallized the GPIbβ ectodomain (GPIbβE) and determined the structure to reveal a single leucine-rich repeat with N- and C-terminal disulfide bonded capping regions. The central region of the structure can be divided into concave parallel β-sheet and convex loops. The crystal structure of a GPIbβE/GPIXE chimera that contains three non-continguous convex loops of GPIX and retains a GPIbβ-binding site of GPIX (Mo et al. J. Thromb. Haemost. 7:1533–40, 2009) was also determined. The chimera, but not GPIbβE, forms a homotetramer in the crystal, revealing a quaternary interface between GPIbβ and GPIX ectodomains. Central to this interface is residue Tyr106 from GPIbβ that inserts into a shallow and largely hydrophobic pocket generated by two convex loops from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIbβ and GPIX in the full-length complex. Eight GPIbβ missense mutations identified from BSS patients were examined in transiently transfected Chinese hamster ovary cells for changes to the GPIb-IX complex surface expression. Six of the eight mutations lead to secretion defect and/or misfolding of GPIbβE. In contrast, the other two mutations, A108P and P74R, were found to maintain normal secretion and folding of GPIbβE but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIbβE interface with GPIX indicates that residues Ala108 and Pro74 in GPIbβ are located at the GPIbβE/GPIXE interfaces. Based on the tetrameric arrangement of the chimera structure, we propose a structural model for the GPIb-IX complex that embodies its organizing principles and helps to provide mechanistic insights on its assembly, function and regulation. Disclosures:No relevant conflicts of interest to declare.

International Consortium for the Study of Clinical and Molecular Aspects of Bernard-Soulier Syndrome

Abstract 707▪FN2▪This icon denotes a clinically relevant abstractBernard-Soulier syndrome (BSS) is an extremely rare inherited bleeding disorder characterized by a defect of the GPIb/IX/V complex, which is essential for hemostasis, as the GPIbα subunit binds to subendothelial von Willebrand factor. Since the identification of the first mutation in 1990, almost one hundred cases carrying mutations in the GP1BA, GP1BB, and GP9 genes have been described. Most of the mutations prevent the coordinated association of the complex or binding to the von Willebrand factor. BSS is usually transmitted as a recessive trait with giant platelets and severe bleeding tendency. However, there are families with a dominant mild form, in which the affected individuals have only moderate macrothrombocytopenia and bleeding tendency.A correct definition of the clinical and laboratory features, together with accurate genotype/ phenotype correlation studies, remains essential for understanding the molecular basis of the disease and managing patients appropriately. Moreover, it is important to understand the variability of clinical manifestations. Since BSS is rare with an estimated prevalence of 1:1,000,000, an International Consortium has recently been established to collect a large series of cases and families worldwide.At present, the Consortium has been compiling data from 165 unrelated families, of which 50% have not been previously described. In this cohort, the molecular genetic testing reveals more than 30 novel mutations, confirming the wide spectrum of alterations responsible for the disease. Data from 65 unrelated families (69 patients) mainly from France, Italy and Japan show that 23 have mutations in GP9 and 29 in GP1BB. In the remaining 13 families, the defective gene is GP1BA. In agreement with the view that BSS is an extremely rare disease, 53 probands carried homozygous mutations, 10 are compound heterozygous, and 2 hemizygous because of a 22q11 deletion of the DiGeorge syndrome.The mean age of patients at diagnosis was 18 years (range 0–75 years) of which 27 were males and 38 females. Misdiagnosis of autoimmune thrombocytopenia was frequent and 26 patients were previously treated with steroids, intravenous immunoglobulins and/or splenectomy. Except two Japanese cases without any bleeding manifestations, patients presented with a variable bleeding diathesis measured by the World health Organization bleeding scale: grades 1, 2, 3 and 4 in 9, 18, 19 and 10 patients, respectively.The mean platelet count was 64×109/L (range 24–130) as determined by microscopy. In contrast, using a cell counter, thrombocytopenia was more severe (45×109/L; range 5–125). The mean platelet mean diameter was larger than in controls and varied from 2.9 to 7.5 mm. Ristocetin-induced platelet agglutination was absent or lower than 22% of normal response in all patients.Flow cytometry revealed a defective expression of the GPIb/IX/V expression in all patients. Correlating between expression data and gene affected, we found that the expression of GP1ba was often undetectable in patients with GP1BA mutations whereas it was higher, 8% and 17%, in patients with mutations of GP1BB and GP9, respectively. Instead, the GPIX mean level was 14%, 8% and 25% in patients with GP9, GP1BB and GP1BA mutations. The expression of GPV was higher than that of the other subunits, being more than 30% regardless of which gene was mutated.This is the largest cohort of BSS patients characterized to date. These patients together with the other 100 cases not yet included in the BSS database will enable correlations of the molecular genetic defects, receptor expression and clinical manifestations observed in BSS patients. Disclosures:Zieger:CSL Behring Hattersheim: Research Funding.

Quaternary organization of GPIb-IX complex and insights into Bernard-Soulier syndrome revealed by the structures of GPIbβ and a GPIbβ/GPIX chimera

AbstractPlatelet GPIb-IX receptor complex has 3 subunits GPIbα, GPIbβ, and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome. We have crystallized the GPIbβ ectodomain (GPIbβE) and determined the structure to show a single leucine-rich repeat with N- and C-terminal disulphide-bonded capping regions. The structure of a chimera of GPIbβE and 3 loops (a,b,c) taken from the GPIX ectodomain sequence was also determined. The chimera (GPIbβEabc), but not GPIbβE, forms a tetramer in the crystal, showing a quaternary interface between GPIbβ and GPIX. Central to this interface is residue Tyr106 from GPIbβ, which inserts into a pocket generated by 2 loops (b,c) from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIbβ and GPIX in the full-length complex. Eight GPIbβ missense mutations identified from patients with Bernard-Soulier syndrome were examined for changes to GPIb-IX complex surface expression. Two mutations, A108P and P74R, were found to maintain normal secretion/folding of GPIbβE but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIbβE interface with GPIX indicates they disrupt the quaternary organization of the GPIb-IX complex.

Correction of murine Bernard-Soulier syndrome by lentivirus-mediated gene therapy.

Bernard-Soulier syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib-IX-V complex. The main treatment for BSS is platelet transfusion but it is often limited to severe bleeding episodes or surgical interventions due to the risk of alloimmunization. We have previously reported successful expression of human GPIbα (hGPIbα) in human megakaryocytes using a lentiviral vector (LV) encoding human GP1BA under control of the platelet-specific integrin αIIb promoter (2bIbα). In this study, we examined the efficacy of this strategy for the gene therapy of BSS using GPIbα(null) as a murine model of BSS. GPIbα(null) hematopoietic stem cells (HSC) transduced with 2bIbα LV were transplanted into lethally irradiated GPIbα(null) littermates. Therapeutic levels of hGPIbα expression were achieved that corrected the tail bleeding time and improved the macrothrombocytopenia. Sequential bone marrow (BM) transplants showed sustained expression of hGPIbα with similar phenotypic correction. Antibody response to hGPIbα was documented in 1 of 17 total recipient mice but was tolerated without any further treatment. These results demonstrate that lentivirus-mediated gene transfer can provide sustained phenotypic correction of murine BSS, indicating that this approach may be a promising strategy for gene therapy of BSS patients.

A A386G biallelic GPIb gene mutation with anomalous behavior: a new mechanism suggested for Bernard-Soulier syndrome pathogenesis

Platelet glycoprotein GPIbα mutations are the basic defect behind Bernard-Soulier syndrome, a rare inherited macrothrombocytopenia characterized by anomalies of the GPIbα, GPIbβ and GPIX subunits of von Willebrand factor receptor. A 32-year old man was investigated for suspected Bernard-Soulier syndrome. Ristocetin induced agglutination was absent. Flow cytometry and Western blot analysis showed a severe reduction in GPIbα, but sequencing revealed only a biallelic c.386A>G substitution, theoretically leading to a p.Asn110Glu variation. To further clarify the data, megakaryocyte cultures were set. Though the maturation of megakaryocytes was normal, proplatelet formation was defective and GPIbα mRNA was not detectable. GPIX protein was slightly reduced and GPIbβ polypeptide almost absent. Computational analysis showed that the c.386A>G mutation disrupted an exon splicing enhancer motif involved in the proper maturation of the GPIbα transcript. The c.386A>G mutation suggests a unique mutational mechanism causing the virtual absence of GPIbα without creating a stop codon.

Clinical and laboratory features of 103 patients from 42 Italian families with inherited thrombocytopenia derived from the monoallelic Ala156Val mutation of GPIb (Bolzano mutation)

Bernard-Soulier syndrome is a very rare form of inherited thrombocytopenia that derives from mutations in GPIbα, GPIbβ, or GPIX and is typically inherited as a recessive disease. However, some years ago it was shown that the monoallelic c.515C>T transition in the GPIBA gene (Bolzano mutation) was responsible for macrothrombocytopenia in a few Italian patients. Over the past 10 years, we have searched for the Bolzano mutation in all subjects referred to our institutions because of an autosomal, dominant form of thrombocytopenia of unknown origin. We identified 42 new Italian families (103 cases) with a thrombocytopenia induced by monoallelic Bolzano mutation. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy. Although the clinical expression was variable, patients with this mutation typically had a mild form of Bernard-Soulier syndrome with mild thrombocytopenia and bleeding tendency. The most indicative laboratory findings were enlarged platelets and reduced GPIb/IX/V platelet expression; in vitro platelet aggregation was normal in nearly all of the cases. Our study indicates that monoallelic Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy, affecting 20% of patients recruited at our institutions during the last 10 years. Because many people from southern Italy have emigrated during the last century, this mutation may have spread to other countries.

Inherited Hematologic and Oncologic Syndromes

Inherited Hematologic and Oncologic Syndromes

Rare and Unusual Bleeding Manifestations in Congenital Bleeding Disorders

Epistaxis, superficial and deep hematomas, hemarthrosis, gastrointestinal bleeding, hematuria represent the most frequent hemorrhagic events in congenital coagulation disorders. Occasionally, bleeding manifestations occur in unusual sites or are peculiar. A clotting defect may alter the clinical aspect of skin conditions or infections (hemorrhagic scabies or varicella). Hemobilia may occur as a complication of transjugular liver biopsy in hemophilia or Bernard-Soulier syndrome. Hemarthrosis of small joints of feet and hands occur in patients with hemophilia treated with protease inhibitors. Intramedullary hematomas of long bones have been described in α2-plasmin inhibitor or fibrinogen deficiencies. Spleen fracture with consequent hemoperitoneum has been reported in patients with fibrinogen deficiency. Rectus muscle sheath hematoma may occur in patients with factor VII (FVII)or FX deficiency. Acute or subacute intestinal obstruction may be caused by intramural wall hematomas in hemophilia and von Willebrand (vW)-disease. Physicians should always keep in mind that a congenital hemorrhagic disorder may cause bleeding in any tissue of the body and therefore alter the normal clinical features of a given disease.

Molecular basis of Bernard–Soulier syndrome in 27 patients from India

Bernard-Soulier syndrome (BSS) is an extremely rare (1:1 million) bleeding disorder of platelet adhesion, caused by defects in the glycoprotein (GP)Ib/IX/V complex. The diagnosis in 27 patients was based on low platelet count, presence of giant platelets and aggregometry studies. Flow cytometry to assess the surface GPIb/IX/V complex showed reduced (7.7-57%) expression. gDNA was screened for mutations in the GPIBA, GPIBB, GP9 genes using PCR-conformation sensitive gel electrophoresis (CSGE). Thirteen different disease-causing mutations, including missense (54%), frameshifts (38%) and nonsense (8%) mutations, were identified in 27 patients. Eleven of them were novel including five novel frameshifts (GPIbα: p.Gln97_98fsX113, p.Pro402_403fsX52; GPIbβ: p.Arg17fsX14; GPIX: p.Gly24fsX43, p. Pro130fs, a nonsense mutation (GPIX, p.94, Gln>X) and five novel missense mutations (GPIbα: p.492, Tyr>His; GPIbβ: p.65, Pro>Arg, p.129, Gln>His, p.132, Leu>Pro; GPIX: p.55, Phe>Cys). Interestingly, four common mutations, Cys8Arg (n = 6) and Phe55Ser (n = 2), Phe55Cys (n = 2) in GPIX and a novel 22-bp deletion in the GPIBB gene predicting p.Arg17fsX 14 (n = 10) were seen in 20 patients. The molecular data presented here is the largest series of BSS patients to be reported so far, adding significantly to the mutation database of this condition and also useful for its genetic diagnosis in India.