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The molecular basis of Glanzmann thrombasthenia (GT) was studied in 40 families from southern India. Of 23 identified mutations (13 in the alphaIIb (ITGA2B) gene and 10 in the beta3 (ITGB3) gene), 20 were novel and three were described previously. Three mutations in the beta3 gene-p.Leu143Trp (Leu117Trp), p.Tyr307Stop (Tyr281Stop), and p.Arg119Gln...
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... and splicing of mRNA have been extensively and Baralle, 2004], the effect of a lack of a stop codon was investigated in this study. Reverse transcription of plateletderived mRNA and amplification of a cDNA fragment encompassing exons 27-29 were performed and yielded two PCR products: a major product of 216 bp and a minor product of 323 bp ( Fig. 2A). Nucleotide sequence analysis of the isolated major PCR product revealed skipping of exon 28, whereas the length of the minor product was consistent with splicing-in of exon 28. ...
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... acceptor splice site precedes the five intronic nucleotides (aggtgag). Thus, theoretically, two splicing alternatives leading to identical mRNA sequences are possible. According to the annotated splicing pattern, the GTGAG sequence belongs to the 3 0 end of exon 9, while according to Genscan prediction, it belongs to the 5 0 end of exon 10 (Fig. 2B, upper panel). If the annotated exon definition is correct, then the 1261G4A mutation occured 5 bp upstream from the 3 0 end of exon 9, coding for Val395Met. If, however, the Genscan prediction is correct, then the 1261G4A mutation occurred at the donor splice site of IVS 9, giving rise to the formation of an extra exon of 28 bp within IVS 9 and the ...
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... then the 1261G4A mutation occured 5 bp upstream from the 3 0 end of exon 9, coding for Val395Met. If, however, the Genscan prediction is correct, then the 1261G4A mutation occurred at the donor splice site of IVS 9, giving rise to the formation of an extra exon of 28 bp within IVS 9 and the addition of five nucleotides to the 5 0 end of exon 10 (Fig. 2B, lower panel). Thus, the predicted alternatively spliced mRNA is 33 nucleotides longer than the wildtype mRNA and codes for 11 extra amino acid residues in addition to the Val395Met substitution. Interestingly, an adjacent c.1260G4A silent mutation (located in the last nucleotide of exon 9 according to Genscan prediction) was previously shown to ...
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Tyrosine phosphorylation of the β3 subunit of the major platelet integrin αIIbβ3 has been shown to occur during thrombin-induced platelet aggregation (1). We now show that a wide variety of platelet stimuli induced β3 tyrosine phosphorylation, but that this phosphorylation occurred only following platelet aggregation. Several lines of evidence
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... Leu143Trp (c.428T > G, rs121918452) identified in the siblings pt.13 and pt.14 affects the protein domain of the beta subunit in the N-terminal region. This results in the intracellular retention of misfolded αIIbβ3 heterodimers and has been described in a Pakistani child whose platelets express less than 10% of the total amount of αIIbβ3 [14] and in the Indian population [15]. Peretz et al suggest that this variant is an Indian founder mutation [15]. ...
... This results in the intracellular retention of misfolded αIIbβ3 heterodimers and has been described in a Pakistani child whose platelets express less than 10% of the total amount of αIIbβ3 [14] and in the Indian population [15]. Peretz et al suggest that this variant is an Indian founder mutation [15]. Interestingly, the patient who was investigated in this study has parents who migrated from India to Pakistan. ...
... Leu143Ser (c.428T > C) identified in pt. 15, an alteration at the same position has not been described so far and therefore, we classified this variant as likely pathogenic. Grantham score between the wild-type amino acid leucine (hydrophobic) and serine (polar uncharged) is 145 (0-215) and therefore higher than in exchange with tryptophan. ...
Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited platelet disorder occurring frequently in populations with high incidence of consanguineous marriages. GT is characterized by quantitative and/or qualitative defect of the platelet αIIbβ3 (GPIIb/IIIa) receptor caused by pathogenic variants of the encoding genes: ITGA2B and ITGB3. Patients present with a moderate to severe bleeding tendency with normal platelet count. Platelets show reduced/absent aggregation for all agonists except ristocetin in light transmission aggregometry and reduced/absent αIIbβ3 expression in flow cytometry (FC). In this study, we investigated a cohort of 20 Pakistani patients and 2 families collected from the National Institute of Blood Disease, Karachi and Chughtai’s Lab, Lahore. Platelet aggregation studies, FC (platelet CD41, CD61, CD42a, CD42b) and direct sequencing of the candidate genes were performed. All patients showed altered platelet aggregation, but normal agglutination after stimulation with ristocetin. Absent/reduced αIIbβ3 receptor expression was present in the platelets of 16 patients, in 4 patients expression was borderline/normal. Candidate gene sequencing identified pathogenic/likely pathogenic variants in 15 patients. Seven variants are novel. One patient with absent receptor expression remained without genetic finding. 13 (86.7%) of 15 patients stated consanguinity reflected by homozygosity finding in 14 (93.3%) patients.
... [9] Research regarding the predictive presentation of hereditary hemostatic disorders showed trauma as the commonest presenting factor, followed by surgery and menorrhagia. [10][11] However, clinical symptomatology is complex phenomenon. A great deal is written about how different bleeding disorders may be identified and treated, however, an aspect that merits just as much attention is the how an recognition of these disorders can be made possible by looking at type of routine bleeding symptoms along gynecologic bleeding among women. ...
Objective: To study the spectrum of bleeding complaints among women with bleeding disorders. Methodology: This observational study was done at Dept. of Pathology – Liaquat University Hospital, Hyderabad from January 2019 to July 2019 upon a sample of 121 women, selected via non-probability, consecutive sampling). Women presenting to study setting with complaints of non-traumatic bleeding were included in the study. After taking written informed consent, the data was obtained from patient interviews and laboratory investigations was. The data obtained was analyzed using SPSS v. 21.0. Results: The mean age of the women was 28.13 years (±5.21 SD). Among the 121 women studied, 73.55% hailed from urban areas, while 26.45% were from rural residential background. The most common presenting complaint was menorrhagia (30.58%), followed by bruising (17.36%) and epistaxis (15.7%). Among the underlying hemostatic pathologies, VWD was the most common (15.7%), followed by other platelet dysfunctions comprising the second most common finding (6.6%). Mean duration of presence of symptoms was 34 months (±17 SD). Conclusion: As per the findings of this study, menorrhagia, occasional bruising and epistaxis are reported to be the most common non-traumatic bleeding complaints among women with bleeding disorders. The presence of these symptoms may serve as potential indicators of the probable presence such as bleeding disorders and help in early referral, timely diagnosis and appropriate treatment. Keywords: menorrhagia, epistaxis, Hemostatic Dysfunction, Bleeding Disorder, Non traumatic bleeding complaints
... India and showed that there were no correlations between bleeding severity and genotype or αIIbβ3 expression. 3 In addition, we previously showed that the nonsense mutation ITGA2B c.2671C > T (p.Q891X) recurred in five patients from four unrelated families (three patients encoded GT3, GT2, and GT1 were homozygotes). We hypothesized that this was a hot-spot mutation in GT patients in China. ...
Glanzmann's thrombasthenia (GT) is a severe hemorrhagic disease. It is caused by mutations in ITGA2B or ITGB3, which are the respective genes encoding integrin αIIb and β3. Despite widespread mutational analysis, the mechanisms underlying the extensive variability in bleeding severity observed among affected individuals remains poorly understood. In order to explore the mechanisms conferring for bleeding heterogeneity, three GT patients with ITGA2B c.2671C > T (p.Q891X) who possessed different bleeding scores were studied. Analysis showed that there was significant difference in nonsense‐mediated mRNA decay (NMD) efficiency among the three patients. These differences positively correlated with their bleeding score. Next, a knock‐in mouse model (KI mice) with the ITGA2B c.2659C > T (p.Q887X) was generated using CRISPR/Cas9. Importantly, this mutation is homologous to ITGA2B c.2671C > T (p.Q891X) in humans. The bleeding time of KI mice was significantly in comparison to the wide‐type mice. Interestingly, bleeding was stopped after treatment with caffeine, which is a known NMD inhibitor. This suggests that NMD efficiency potentially influences bleeding severity in ITGA2B c.2659C > T (p.Q887X) KI mice.
... Hence unsurprisingly, dysfunction of b-integrins appears to cause defective platelet aggregation and HBM in mice models (250). Autosomal recessive mutations in ITGA2B lead to reduced production of either glycoprotein IIb or IIIa, resulting in Glanzmann thrombasthenia (MIM273800) which is characterized by excessive bleeding (251). Only one case of Glanzmann thrombasthenia has been reported with a bone phenotype, with generalized and skull base osteosclerosis observed on plain radiographs of a 5 day old baby (252), termed osteopetrosis and thought due to impaired osteoclast function (253). ...
The phenotypic trait of high bone mass (HBM) is an excellent example of the nexus between common and rare disease genetics. HBM may arise from carriage of many ‘high bone mineral density [BMD]’-associated alleles, and certainly the genetic architecture of individuals with HBM is enriched with high BMD variants identified through genome-wide association studies of BMD. HBM may also arise as a monogenic skeletal disorder, due to abnormalities in bone formation, bone resorption, and/or bone turnover. Individuals with monogenic disorders of HBM usually, though not invariably, have other skeletal abnormalities (such as mandible enlargement) and thus are best regarded as having a skeletal dysplasia rather than just isolated high BMD. A binary etiological division of HBM into polygenic vs. monogenic, however, would be excessively simplistic: the phenotype of individuals carrying rare variants of large effect can still be modified by their common variant polygenic background, and by the environment. HBM disorders—whether predominantly polygenic or monogenic in origin—are not only interesting clinically and genetically: they provide insights into bone processes that can be exploited therapeutically, with benefits both for individuals with these rare bone disorders and importantly for the many people affected by the commonest bone disease worldwide—i.e., osteoporosis. In this review we detail the genetic architecture of HBM; we provide a conceptual framework for considering HBM in the clinical context; and we discuss monogenic and polygenic causes of HBM with particular emphasis on anabolic causes of HBM.
... La thrombasthénie de Glanzmann (TG) est un trouble hémorragique héréditaire rare [1], transmis sur un mode autosomique récessif [2]. Décrite pour la première fois en 1918 par Glanzmann, son incidence est très faible de 1/1.000.000 ...
... La TG est une maladie héréditaire rare transmise sur un mode autosomique récessif [1] affectant la lignée des mégacaryocytes et caractérisée par un défaut d'agrégation plaquettaire [2]. Cette pathologie est caractérisée par la présence d'anomalies quantitatives et/ou qualitatives de l'intégrine αIIbβ3 qui joue le rôle de récepteur au fibrinogène lors de l'agrégation plaquettaire [7,8,9]. ...
... Les deux gènes ITGA2B et ITGB3 codant respectivement pour les GP IIb et GP IIIa sont localisés sur le chromosome 17q21-23. Les mutations rencontrées lors de la TG sont le plus souvent sporadiques avec quelques cas de mutations familiales dans les populations où le taux de mariages consanguins est élevé [2]. ...
La Thrombasthénie de Glanzmann (TG) est un trouble hémorragique héréditaire rare mais grave. Nous rapportons 68 épisodes hémorragiques chez 9 cas de TG. Bien que la transfusion de plaquettes soit le traitement standard des épisodes hémorragiques, la prise en charge thérapeutique de la TG reste lourde et émaillée de complications transfusionnelles. Parmi les 68 épisodes hémorragiques, 58 étaient sévères. La transfusion de plaquettes a été utilisée dans 57 épisodes. Le facteur rVIIa a été administré dans 6 épisodes. Nous avons noté 3 accidents transfusionnels aux plaquettes dans 2 cas et une inefficacité transfusionnelle dans 3 cas. Des anticorps anti-HLA de classe 1 positifs ont été notés dans 1 cas. Aucun décès n'a été rapporté. Il est intéressant de montrer le rôle du facteur rVIIa qui constitue une alternative thérapeutique en cas d'absence de réponse aux transfusions de plaquettes et/ou d'allo-immunisation anti-plaquettaire.
... Genomic variations associated with Glanzmann thrombasthenia are highly variable, and mutations can be family-specific and previously uncharacterized. Thus, different DNA screening methods can be used, including single-strand conformation polymorphism analysis (SSCP) [21][22][23][24], denaturing gradient gel electrophoresis (DGGE) [25], conformation sensitive gel electrophoresis (CSGE) [23], and high-resolution melting analysis (HRM) [26]. These screening approaches are usually followed by Sanger sequencing. ...
: Glanzmann thrombasthenia is an inherited severe bleeding disease. Mutations associated with Glanzmann thrombasthenia are highly heterogeneous and occur across the two genes coding for the platelet αIIbβ3 integrin. This study was aimed at identifying Glanzmann thrombasthenia-associated novel mutations in Tunisian patients. Seven unrelated Glanzmann thrombasthenia patients issued from high consanguineous families (86%; 6/7 of the patients) were studied. Glanzmann thrombasthenia diagnoses were based on patients' bleeding histories and platelet aggregation tests. Screening of ITGA2B and ITGB3 genes was performed by denaturing high-performance liquid chromatography (DHPLC) analysis. Amplicons with abnormal elution profiles were subjected to direct sequencing. DHPLC/sequencing analysis identified a pathogenic homozygous mutation in exon 26 at position c.2702C>A, inducing a substitution of a serine to a stop codon (p.S901*) in the ITGA2B gene, in three patients. This mutation was only previously reported in a Glanzmann thrombasthenia patient of a Tunisian origin and not in other populations. We diagnosed a pathogenic Glanzmann thrombasthenia mutation in ITGA2B screened by DHPLC that appears to be specific to individuals of Tunisian heritage and that deserves to be investigated in first intention. As a result, we determined that performing prenatal diagnosis and setting a prevention strategy via counselling for affected heterozygote individuals will be helpful for Tunisian Glanzmann thrombasthenia families where there is still a high rate of consanguinity.
... Defining the affected molecular defect has helped to ascribe specific function to individual platelet proteins. Classic examples of inherited platelet diseases are: Bernard-Soulier syndrome, where platelets fail to adhere to vWF due to GPIb-IX-V mutations [9]; gray platelet syndrome, which is typified by platelets having an α-granule deficiency due to mutations in the endoplasmic reticulum protein, NBEAL2 [10]; and Glanzmann thrombasthenia, which is consistent with abnormal αIIbβ3 leading to impaired platelet aggregation [11,12]. While important strides have been made utilizing state-of-the-art proteomics and deep genomic sequencing technologies, multiple functions have been assigned to most platelet proteins, currently creating a barrier to accurately predicting phenotype from genotype. ...
Hemostasis is the physiological control of bleeding and is initiated by subendothelial exposure. Platelets form the primary vascular seal in three stages (localization, stimulation and aggregation), which are triggered by specific interactions between platelet surface receptors and constituents of the subendothelial matrix. As a secondary hemostatic plug, fibrin clot formation is initiated and feedback-amplified to advance the seal and stabilize platelet aggregates comprising the primary plug. Once blood leakage has been halted, the fibrinolytic pathway is initiated to dissolve the clot and restore normal blood flow. Constitutive and induced anticoagulant and antifibrinolytic pathways create a physiological balance between too much and too little clot production. Hemostatic imbalance is a major burden to global healthcare, resulting in thrombosis or hemorrhage.
... Fifteen patients were homozygotes, 21 patients were compound heterozygotes, while 7 patients were identified only 1 heterozygous variant and 2 patients harbored no pathogenic variant on ITGA2B or ITGB3 (Figure 2). These results stand in consensus with other reports that did not identify pathogenic variants in some patients(Table 2).16,17,23 Here, our 25 novel pathogenic variants included 14 missense (ITGA2B: n = 9, ITGB3: n = 5), 4 nonsense (ITGA2B: n = 2, ITGB3: n = 2), 3 splicing (ITGA2B), and 4 frameshift (ITGA2B: n = 1, ITGB3: n = 3). ...
Glanzmann's thrombasthenia (GT) is a rare bleeding disorder characterized by spontaneous mucocutaneous bleeding. The disorder is caused by quantitative or qualitative defects in integrin αIIbβ3 (encoded by ITGA2B and ITGB3) on the platelet and is more common in consanguineous populations. However, the prevalence rate and clinical characteristics of GT in nonconsanguineous populations have been unclear. We analyzed 97 patients from 93 families with GT in the Han population in China. This analysis showed lower consanguinity (18.3%) in Han patients than other ethnic populations in GT‐prone countries. Compared with other ethnic populations, there was no significant difference in the distribution of GT types. Han females suffered more severe bleeding and had a poorer prognosis. We identified a total of 43 different ITGA2B and ITGB3 variants, including 25 previously unidentified, in 45 patients. These variants included 14 missense, four nonsense, four frameshift, and three splicing site variants. Patients with the same genotype generally manifested the same GT type but presented with different bleeding severities. This suggests that GT clinical phenotype does not solely depend on genotype. Our study provides an initial, yet important, clinical and molecular characterization of GT heterogeneity in China.
... A) to Arg551, this competes for H-bonds with aIIb residues Asp542, Ser594 and Asp591 changing interatom distances and creating an additional H-bond within con- necting loops extending from the lower region of the thigh (Figure 1a). A homozygous Pro507Arg (P476R) thigh domain substi- tution was detected in a patient from India ( Peretz et al., 2006). Pro507 is poorly conserved (Figs S1 and S2) but the physical and chemical deviation between Pro and Arg is important (Grantham score: 103). ...
Background:
Studies on the inherited bleeding disorder, Glanzmann thrombasthenia (GT), have helped define the role of the αIIbβ3 integrin in platelet aggregation. Stable bent αIIbβ3 undergoes conformation changes on activation allowing fibrinogen binding and its taking an extended form. The αIIb genu assures the fulcrum of the bent state. Our goal was to determine how structural changes induced by missense mutations in the αIIb genu define GT phenotype.
Methods:
Sanger sequencing of ITGA2B and ITGB3 in the index case followed by in silico modeling of all known GT-causing missense mutations extending from the lower part of the β-propeller, and through the thigh and upper calf-1 domains.
Results:
A homozygous c.1772A>C transversion in exon 18 of ITGA2B coding for a p.Asp591Ala substitution in an interconnecting loop of the lower thigh domain of αIIb in a patient with platelets lacking αIIbβ3 led us to extend our in silico modeling to all 16 published disease-causing missense variants potentially affecting the αIIb genu. Modifications of structuring H-bonding were the major cause in the thigh domain although one mutation gave mRNA decay. In contrast, short-range changes induced in calf-1 appeared minor suggesting long-range effects. All result in severe to total loss of αIIbβ3 in platelets. The absence of mutations within a key Ca2+-binding loop in the genu led us to scan public databases; three potential single allele variants giving major structural changes were identiffied suggesting that this key region is not protected from genetic variation.
Conclusions:
It appears that the αIIb genu is the object of stringent quality control to prevent platelets from circulating with activated and extended integrin.
... These diverse genetic defects result in deficiency or dysfunction of the GPIIb/IIIa complex on platelets that were presented as qualitative and/or quantitative abnormalities of aIIbb3 integrin [1,4,5]. The first mutation causing Glanzmann's thrombasthenia was described by Bray and Shuman in 1990, and more than 150 mutations have been reported since then [9]. Currently, Human Gene Mutation Database (HGMD) lists more than 250 and 150 mutations in aIIb and b3 genes, respectively [10]. ...
... Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Due to large number of ITGA2B and ITGB3 exons, first, all exons and splice sites of ITGA2B (30 exons) and ITGB3 (15 exons) were amplified using PCR with a high-fidelity Taq polymerase, and the primers which were derived from Nair and Mitchell studies (Table 1) [9,15]. The PCR mixture consisted of 1.5-ml DNA sample, 46.5-ml Taq DNA polymerase 2Â master mix (Ampliqon, Amsterdam, Netherlands), and 1 ml of each forward and reverse primers. ...
: Quantitative and/or qualitative defects of the platelet membrane glycoprotein IIb/IIIa complex lead to the clinical entity of Glanzmann's thrombasthenia. A large variety of mutations and polymorphisms are responsible for the aberrant expression and defective activity of this heterodimeric complex. The current study aimed to determine the pattern of mutations among Iranian population with Glanzmann's thrombasthenia. A total of 20 patients with Glanzmann's thrombasthenia have been evaluated. All exons and splice sites of ITGA2B and ITGB3 genes were amplified using touchdown PCR. Mutation screening was analyzed using conformation sensitive gel electrophoresis heteroduplex PCR, and DNA sequencing. In addition to finding one previously identified mutation and polymorphism, the experimenters explored 3 and 2 novel mutations and polymorphisms, respectively. One substitution mutation, two deletions of a single nucleotide, one insertion of a single nucleotide, two synonymous polymorphisms, and one missense polymorphism were found using Sanger sequencing method. All detected mutations were homozygous which will most likely contribute to the pathogenesis of Glanzmann's thrombasthenia. Furthermore, it suggested ITGB3 as the mainly affected gene impaired in the patients with Glanzmann's thrombasthenia. As expected, the molecular results were consistent with the phenotypic findings so that GPIIb/IIIa complex was disrupted due to mutations in all type-I Glanzmann's thrombasthenia patients. It is concluded that intronic alterations or epigenetic regulations could be responsible for aberrant expression and/or defective activity of GPIIb/IIIa complex among other patients.
