ABSTRACT
Objective: The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored. Methods: The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate, collagen, epinephrine, arachidonic acid, and ristocetin. The expression levels of CD41 (αâ ¡b), CD61 (ß3), and CD42b (GPâ b) on the platelet surface was detected by flow cytometry. Gene sequencing technology was used for the genetic identification of the family. RT-PCR was used in the detection of mRNA splicing, and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. The expressions of total αâ ¡b and ß3 in platelets were analyzed by Western blot. Results: Except ristocetin, the other four inducers could not induce platelet aggregation in the proband. Flow cytometry showed that the expression levels of αâ ¡b and ß3 were only 0.25% and 9.76%, respectively, on the platelet surface of the proband, whereas GPâ b expression was relatively normal. The expression levels of glycoproteins in the other family members were almost normal. c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing. The c.480C>G mutation was inherited from his mother, and the c.2929C>T mutation was inherited from his father. The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother, resulting in the deletion of 99 bases in c.476G-574A (p.S160-S192). qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father. Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5'SS splice site. The three-dimensional structural model of the αâ ¡b subunit showed that the ß-propeller domain of the p.S160-S192 deletion lost two ß-strands and one α-helix in blade 2. The c.2929C>T nonsense mutation caused premature translation termination and produced a truncated protein with the deletion of p.R977-E1039, including the cytoplasmic domain, transmembrane domain, and a ß chain of the extracellular Calf-2 domain. The total αâ ¡b expression of the proband was absent, and the relative expression of ß3 was 11.36% of the normal level. Conclusion: The compound heterozygous mutation c.480C>G in exon 4 and c.2929C>T in exon 28 of the ITGA2B gene probably underlies Glanzmann thrombasthenia in this pedigree.
Subject(s)
Heterozygote , Integrin alpha2 , Mutation , Pedigree , Thrombasthenia , Humans , Integrin alpha2/genetics , Thrombasthenia/genetics , Male , Female , Platelet Aggregation , Genotype , AdultABSTRACT
BACKGROUND: Glanzmann's thrombasthenia (GT) is a congenital platelet disorder affecting approximately 1:1 000 000 people globally and characterized by impaired platelet aggregation and clot retraction. Autosomal recessive, loss-of-function, variants in ITGA2B or ITGB3 of the αIIbß3 receptor cause the disease in humans. A cat affected by Glanzmann's and macrothrombocytopenia was presented to the UC Davis VMTH. HYPOTHESIS/OBJECTIVES: Severe thrombopathia in this cat has an underlying genetic etiology. ANIMALS: A single affected patient, 2 age-matched clinically healthy controls, and a geriatric population (n = 20) of normal cats. METHODS: Physical examination and clinical pathology tests were performed on the patient. Flow cytometry and platelet aggregometry analyses for patient phenotyping were performed. Patient and validation cohort gDNA samples were extracted for Sanger sequencing of a previously identified ITGA2B (c.1986delC) variant. Reverse transcriptase PCR was performed on patient and healthy control PRP samples to verify ITGA2B variant consequence. RESULTS: A novel c.1986_1987insCC autosomal recessive variant in ITGA2B was identified. This variant was absent in a population of 194 unrelated cats spanning 44 different breeds. Complete loss of ITGA2B transcript and protein expression was verified by RT-PCR and flow cytometry, explaining the underlying etiology of GT, and likely macrothrombocytopenia, in this cat. CONCLUSIONS AND CLINICAL IMPORTANCE: This study emphasizes the role of precision medicine in cardiovascular disease of cats and identified yet another variant that may be of utility for screening in the feline population. This study provides a small-volume, standardized, successful protocol for adequate platelet RNA isolation and subsequent molecular assessment of gene expression in cats.
Subject(s)
Cat Diseases , Frameshift Mutation , Integrin alpha2 , Thrombasthenia , Animals , Cats , Thrombasthenia/veterinary , Thrombasthenia/genetics , Cat Diseases/genetics , Integrin alpha2/genetics , Male , FemaleABSTRACT
INTRODUCTION: Poor response to platelet and recombinant factor VII administration is a major problem in patients with Glanzmann Thrombasthenia (GT). The risk factors associated with poor response to treatment in these patients are unknown. Some genetic variations of cytokines may contribute to therapy resistance. AIMS: We evaluated, for the first time, whether genetic polymorphisms on cytokine genes are related to poor treatment response in GT patients. METHODS: We enrolled 30 patients with GT (15 resistant and 15 non-resistant) and 100 healthy controls. Gene polymorphisms of IL-10 and TNF-α were analysed using TaqMan Realtime PCR, and IL-1, IL-1R1 and IL-1RN were investigated with the RFLP method. In-silico analyses were performed to predict the potential impact of these polymorphisms. RESULTS: In the resistant group, all patients had a variant of the IL-10 gene at the -1082 position (rs1800896), with a GG genotype that was significantly more frequent than the non-resistant group. Analysis between healthy controls and GT patients revealed a probable correlation between rs3783550, rs3783553, rs3917356 and rs2234463 and GT. The In-silico study indicated that TNF-α rs1800629 and IL-10 rs1800896 polymorphisms result in different allelic expressions which may contribute to poor response to therapy. CONCLUSIONS: These findings suggest that polymorphisms in the IL-10 and IL-1 receptor antagonist genes may play a role in poor therapy response in GT patients. In addition, some polymorphisms in IL-1α, IL1-ß, IL-1R1 and IL-R antagonists might be involved in the GT progression.
Subject(s)
Interleukin 1 Receptor Antagonist Protein , Thrombasthenia , Female , Humans , Male , Case-Control Studies , Genotype , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin 1 Receptor Antagonist Protein/therapeutic use , Interleukin-10/genetics , Polymorphism, Genetic , Polymorphism, Single Nucleotide , Receptors, Interleukin-1 Type I/genetics , Recombinant Proteins/therapeutic use , Thrombasthenia/genetics , Thrombasthenia/drug therapy , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Hermanski-Pudlak Syndrome , Thrombasthenia , Humans , Homozygote , Sequence Deletion , Hermanski-Pudlak Syndrome/genetics , Sequence Analysis, DNA , Thrombasthenia/genetics , High-Throughput Nucleotide Sequencing , DNASubject(s)
Thrombasthenia , Humans , Thrombasthenia/diagnosis , Thrombasthenia/genetics , Integrin beta3/genetics , MutationABSTRACT
BACKGROUND/AIM: Recently, it was reported that the non-synonymous c.1431C > T (p. G477=) mutation of the integrin subunit ß3 (ITGB3) gene is the cause of Glanzmann's thrombasthenia (GT). However, the functional consequences of this mutation on the ITGB3 gene and protein expression remain to be elucidated. Therefore, this study was conducted to cover this scientific shortage. METHODS: Peripheral blood samples were collected from Chinese family members (parents and proband and his sister), and DNA was extracted and sequenced using whole-exome and Sanger sequencing. The effect of c.1431C > T mutation on the splicing of mRNA was verified by the in vitro minigene assay and the three variants that resulted from the mutation were cloned into a phage vector and pEGFP-C1 vector, and ITGB3 gene and protein expression was detected in the transfected 293 T cells using qPCR and Western blotting. RESULTS: Minigene splicing assay showed that c.1431C > T mutation causes three kinds of alternative splicing; (1) a 95 bp deletion in the middle of exon10, (2) a 155 bp deletion (95 bp deletion in the middle of exon10 plus a 60 bp deletion in the right side of exon10), and (3) a 261 bp deletion in the right side of exon10. The in vitro expression assay showed that the c.1431C > T variant did not affect the ITGB3 mRNA levels, but directly led to protein truncation and declined expression. CONCLUSION: Due to its significant impact on protein expression, c.1431C > T mutation in ITGB3 could be considered a pathogenic variant of GT. This could enrich the ITGB3 mutation spectrum and provide a base for the genetic diagnosis of GT.
Subject(s)
Thrombasthenia , Humans , Thrombasthenia/genetics , Thrombasthenia/diagnosis , Mutation , RNA Splicing , Base Sequence , RNA, Messenger/genetics , Integrin beta3/geneticsABSTRACT
BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive platelet aggregation disorder caused by mutations in ITGA2B or ITGB3. OBJECTIVES: We aimed to assess the phenotype and investigate the genetic etiology of a GT pedigree. METHODS: A patient with bleeding manifestations and mild mental retardation was enrolled. Complete blood count, coagulation, and platelet aggregation tests were performed. Causal mutations were identified via whole exome and genome sequencing and subsequently confirmed through polymerase chain reaction and Sanger sequencing. The transcription of ITGB3 was characterized using RNA sequencing and reverse transcription polymerase chain reaction. The αâ ¡b and ß3 biosynthesis was investigated via whole blood flow cytometry and in vitro studies. RESULTS: GT was diagnosed in a patient with defective platelet aggregation. Novel compound heterozygous ITGB3 variants were identified, with a maternal nonsense mutation (c.2222G>A, p.Trp741∗) and a paternal SINE-VNTR-Alu (SVA) retrotransposon insertion. The 5' truncated SVA element was inserted in a sense orientation in intron 11 of ITGB3, resulting in aberrant splicing of ITGB3 and significantly reducing ß3 protein content. Meanwhile, both the expression and transportation of ß3 were damaged by the ITGB3 c.2222G>A. Almost no αâ ¡b and ß3 expressions were detected on the patient's platelets surface. CONCLUSION: Novel compound heterozygous ITGB3 mutations were identified in the GT pedigree, resulting in defects of αâ ¡bß3 biosynthesis. This is the first report of SVA retrotransposon insertion in the genetic pathogenesis of GT. Our study highlights the importance of combining multiple high-throughput sequencing technologies for the molecular diagnosis of genetic disorders.
Subject(s)
Thrombasthenia , Humans , Thrombasthenia/diagnosis , Thrombasthenia/genetics , Retroelements , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Phenotype , Blood Platelets/metabolism , Integrin beta3/genetics , Integrin beta3/metabolismABSTRACT
Glanzmann thrombasthenia (GT) is a genetic bleeding disorder characterised by severely reduced/absent platelet aggregation in response to multiple physiological agonists. The severity of bleeding in GT varies markedly, as does the emergency situations and complications encountered in patients. A number of emergency situations may occur in the context of GT, including spontaneous or provoked bleeding, such as surgery or childbirth. While general management principles apply in each of these settings, specific considerations are essential for the management of GT to avoid escalating minor bleeding events. These recommendations have been developed from a literature review and consensus from experts of the French Network for Inherited Platelet Disorders, the French Society of Emergency Medicine, representatives of patients' associations, and Orphanet to aid decision making and optimise clinical care by non-GT expert health professionals who encounter emergency situations in patients with GT.
Subject(s)
Emergency Medicine , Thrombasthenia , Humans , Thrombasthenia/genetics , Thrombasthenia/therapy , Consensus , Health PersonnelABSTRACT
Mutations in the αIIb ß-propeller domain have long been known to disrupt heterodimerization and intracellular trafficking of αIIbß3 complexes leading to diminished surface expression and/or function, resulting in Glanzmann thrombasthenia. Our previous study on three ß-propeller mutations, namely G128S, S287L, and G357S, showed variable defects in protein transport correlated with the patient's clinical phenotypes. Pulse-chase experiments revealed differences in αIIbß3 complex maturation among the three mutations. Hence, the current study aims to correlate conformational changes caused by each one of them. Evolutionary conservation analysis, stability analysis, and molecular dynamics simulations of the three mutant structures were carried out. Stability analysis revealed that, while G128S and G357S mutations destabilized the ß-propeller structure, S287L retained the stability. Wild-type and mutant ß-propeller structures, when subjected to molecular dynamics simulations, confirmed that G128S and G357S were both destabilizing in nature when compared with the wild-type and S287L based on several parameters studied, like RMSD, RMSF, Rg, FEL, PCA, secondary structure, and hydrogen bonds. In our previous study, we demonstrated that mutant S287L αIIbß3 complexes were more stable than the wild-type αIIbß3 complexes, as evidenced in pulse-chase experiments. These findings corroborate variable intracellular fates of mutant αIIbß3 complexes as a result of these ß-propeller mutations.
Subject(s)
Integrin alpha2 , Integrin beta3 , Platelet Glycoprotein GPIIb-IIIa Complex , Thrombasthenia , Humans , Integrin beta3/genetics , Molecular Dynamics Simulation , Mutation , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Protein Structure, Secondary , Thrombasthenia/genetics , Thrombasthenia/metabolism , Integrin alpha2/genetics , Integrin alpha2/metabolismABSTRACT
BACKGROUND: Glanzmann thrombasthenia (GT) is a rare bleeding disorder with a high prevalence in communities where consanguineous marriages are mainstream. Endometriosis is a chronic inflammatory disease, and its risk increases in women with menstrual periods of longer than six days. The phenotypic expression of endometriosis is determined by the frequency and rate of the menstrual flow, as well as genetic and environmental factors. RESULT AND CASE PRESENTATION: 14-year-old monozygotic twin sisters with GT who developed ovarian endometriosis were referred to Hazrat Rasoul Hospital due to severe dysmenorrhea. In ultrasonic examination, endometrioma cysts were reported in both patients. They both went under endometrioma cystectomy, and the bleeding was managed using antifibrinolytic drugs, followed by recombinant activated coagulation factor VII. Both were discharged after 3 days. In the ultrasound examination performed one year after the surgery, ovaries were normal in the first twin, while the second twin had a 28 × 30 hemorrhagic cyst in the left ovary. DISCUSSION AND CONCLUSION: Menstrual bleeding and genetic factors are two theories that could be related to GT and endometriosis association, and GT could be considered a risk factor for endometriosis.
Subject(s)
Cysts , Endometriosis , Ovarian Neoplasms , Thrombasthenia , Humans , Female , Adolescent , Thrombasthenia/genetics , Endometriosis/genetics , Endometriosis/complications , Endometriosis/diagnosis , Twins, Monozygotic , Hemorrhage , Cysts/complicationsABSTRACT
This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate ex vivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate ex vivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of αIIbß3 on quiescent platelets that spontaneously bind/store fibrinogen, and activation-dependent antibodies (ligand-induced binding site-319.4 and PAC-1) report ß3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153Sß3 substitution within the ßI-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G>A splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153ß3. F153 is completely conserved among ß3 of several species and all human ß-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of αIIb-F153Sß3 also displays reduced levels of a constitutively activated αIIb-S153ß3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153ß3 is critical for maintaining the resting conformation of α2- and α1-helices of the ßI-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the α2- and α1-helices of the ßI-domain toward the constitutively active αIIbß3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains αIIbß3 activation. The data collectively demonstrate that disruption of F153ß3 can significantly alter normal integrin/platelet function, although reduced expression of αIIb-S153ß3 may be compensated by a hyperactive conformation that promotes viable hemostasis.
Subject(s)
Platelet Glycoprotein GPIIb-IIIa Complex , Thrombasthenia , Humans , Amino Acids/genetics , HEK293 Cells , Mutation , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Thrombasthenia/genetics , Thrombasthenia/metabolismABSTRACT
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 αIIbß3 (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 αIIbß3 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 αIIbß3 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.
Subject(s)
Thrombasthenia , Humans , Thrombasthenia/genetics , Receptors, Fibrinogen , Ristocetin , Pakistan , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolismABSTRACT
OBJECTIVE: To construct a mouse model of Glanzmann's thrombasthenia (GT) with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation by CRISPR/Cas9 technology, and then further explore the expression and function of glycoprotein αIIbß3 on the surface of platelet membrane. METHODS: The donor oligonucleotide and gRNA vector were designed and synthesized according to the ITGA2B gene sequence. The gRNA and Cas9 mRNA were injected into fertilized eggs with donor oligonucleotide and then sent back to the oviduct of surrogate mouse. Positive F0 mice were confirmed by PCR genotyping and sequence analysis after birth. The F1 generation of heterozygous GT mice were obtained by PCR and sequencing from F0 bred with WT mice, and then homozygous GT mice and WT mice were obtained by mating with each other. The phenotype of the model was then further verified by detecting tail hemorrhage time, saphenous vein bleeding time, platelet aggregation, expression and function of αIIbß3 on the surface of platelet. RESULTS: The bleeding time of GT mice was significantly longer than that of WT mice (P<0.01). Induced by collagen, thrombin, and adenosine diphosphate (ADP), platelet aggregation in GT mice was significantly inhibited (P<0.01, P<0.01, P<0.05). Flow cytometry analysis showed that the expression of αIIbß3 on the platelet surface of GT mice decreased significantly compared with WT mice (P<0.01), and binding amounts of activated platelets to fibrinogen were significantly reduced after thrombin stimulation (P<0.01). The spreading area of platelet on fibrinogen in GT mice was significantly smaller than that in WT mice (P<0.05). CONCLUSION: A GT mouse model with ITGA2B c.2659 C>T (p.Q887X) nonsense mutation has been established successfully by CRISPR/Cas9 technology. The aggregation function of platelet in this model is defective, which is consistent with GT performance.
Subject(s)
Codon, Nonsense , Integrin alpha2 , Thrombasthenia , Animals , CRISPR-Cas Systems , Disease Models, Animal , Fibrinogen/genetics , Humans , Integrin alpha2/genetics , Mice , Oligonucleotides , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , RNA, Guide, Kinetoplastida , Thrombasthenia/diagnosis , Thrombasthenia/genetics , Thrombin/geneticsABSTRACT
The objective of this study was to elucidate the molecular characteristics of a Chinese family with Glanzmann's thrombasthenia (GT). The proband was diagnosed with GT based on clinical manifestations, platelet aggregation, and the expression of CD41 and CD61 in platelets. Whole-exome and Sanger sequencing were used to detect genetic defects related to GT in the proband and the family of the pedigree. Whole-exome sequencing showed a c.1784-1802delinsGTCACA, p. S595Cfs*70 homozygous mutation in exon 11 of the ITGB3 gene in the proband. Heterozygous mutations were found in the proband's parents, grandmother, uncle, aunt, and younger brother. This novel p. S595Cfs*70 ITGB3 gene mutation is not present in the 1000 Genomes and ExAC databases.
Subject(s)
Databases, Nucleic Acid , Exons , Frameshift Mutation , Homozygote , Integrin beta3 , Thrombasthenia/genetics , Adolescent , Female , Humans , Exome SequencingABSTRACT
Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterized by impaired platelet aggregation due to defects in integrin αIIbß3, a fibrinogen receptor. Platelet phenotypes and allelic variations in 28 Turkish GT patients are reported. Platelets αIIbß3 expression was evaluated by flow cytometry. Sequence analyzes of ITGA2B and ITGB3 genes allowed identifying nine variants. Non-sense variation effect on αIIbß3 expression was studied by using transfected cell lines. 3D molecular dynamics (MDs) simulations allowed characterizing structural alterations. Five new alleles were described. αIIb:p.Gly423Asp, p.Asp560Ala and p.Tyr784Cys substitutions impaired αIIbß3 expression. The αIIb:p.Gly128Val substitution allowed normal expression; however, the corresponding NM_000419.3:c.476G>T variation would create a cryptic donor splicing site altering mRNA processing. The ß3:p.Gly540Asp substitution allowed αIIbß3 expression in HEK-293 cells but induced its constitutive activation likely by impairing αIIb and ß3 legs interaction. The substitution alters the ß3 I-EGF-3 domain flexibility as shown by MDs simulations. GT variations are mostly unique although the NM_000419.3:c.1752 + 2 T > C and NM_000212.2:c.1697 G > A variations identified in 4 and 8 families, respectively, might be a current cause of GT in Turkey. MD simulations suggested how some subtle structural variations in the ß3 I-EGF domains might induce constitutive activation of αIIbß3 without altering the global domain structure.
Subject(s)
Integrin alpha2 , Integrin beta3 , Platelet Glycoprotein GPIIb-IIIa Complex , Thrombasthenia , Epidermal Growth Factor , HEK293 Cells , Humans , Integrin alpha2/genetics , Integrin beta3/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Thrombasthenia/genetics , Thrombasthenia/metabolism , TurkeyABSTRACT
Glanzmann's thrombasthenia (GT) is an autosomal recessive congenital bleeding disorder of platelet aggregation. Mutations in ITGA2B and ITGB3 genes result in quantitative and/or qualitative abnormalities of the glycoprotein receptor complex IIb/IIIa (integrin αIIbß3), which in turn impairs platelet aggregation and lead to GT. In this study, whole genome single nucleotide polymorphism (SNP) genotyping as well as whole exome sequencing was performed in a large family segregating GT. Analysis of the genotypes localized the disease region to chromosome 17q21.2-q21.3. Filtration of whole exome data and candidate variants prioritization identified a pathogenic variant in the ITGB3 gene. The single nucleotide deletion variant (c.2113delC) in exon 13 of the ITGB3 gene is predicted to cause a frameshift and absence of vital C-terminal domains including the transmembrane helix and the cytoplasmic domain. Clinical variability of the bleeding phenotype in affected individuals with the same mutation suggests that other genetic and nongenetic factors are responsible for determining GT features.
Subject(s)
Integrin beta3 , Thrombasthenia , Humans , Exons , Frameshift Mutation , Integrin beta3/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Saudi Arabia , Thrombasthenia/geneticsABSTRACT
The diagnosis of inherited platelet function disorders (IPFDs) is challenging owing to the unavailability of essential testing methods, including light transmission aggregometry and flow cytometry, in several medical centers in Korea. This study, conducted by the Korean Pediatric Hematology Oncology Group from March 2017 to December 2020, aimed to identify the causative genetic variants of IPFDs in Korean patients using next-generation sequencing (NGS). Targeted exome sequencing, followed by whole-genome sequencing, was performed for diagnosing IPFDs. Of the 11 unrelated patients with suspected IPFDs enrolled in this study, 10 patients and 2 of their family members were diagnosed with Glanzmann thrombasthenia (GT). The variant c.1913+5G>T of ITGB3 was the most common, followed by c.2333A>C (p.Gln778Pro) of ITGB2B. Known variants of GT, including c.917A>C (p.His306Pro) of ITGB3 and c.2975del (p.Glu992Glyfs*), c.257T>C (p.Leu86Pro), and c.1750C>T (p.Arg584*) of ITGA2B, were identified. Four novel variants of GT, c.1451G>T (p.Gly484Val) and c.1595G>T (p.Cys532Phe) of ITGB3 and c.1184G>T (p.Gly395Val) and c.2390del (p.Gly797Valfs*29) of ITGA2B, were revealed. The remaining patient was diagnosed with platelet type bleeding disorder 18 and harbored two novel RASGRP2 variants, c.1479dup (p.Arg494Alafs*54) and c.813+1G>A. We demonstrated the successful application of NGS for the accurate and differential diagnosis of heterogeneous IPFDs.
Subject(s)
Integrin alpha2/genetics , Integrin beta3/genetics , Polymorphism, Single Nucleotide , Thrombasthenia/genetics , Child, Preschool , Female , Gene Frequency , Humans , Infant , Infant, Newborn , Male , Republic of KoreaABSTRACT
Background/aim: Glanzmann thrombasthenia (GT) is a rare autosomal recessively inherited bleeding disorder characterized by the quantitative (type 1 and type 2) or qualitative (type 3) deficiency in platelet membrane glycoprotein (GP) IIb/IIIa (CD41a/CD61) fibrinogen receptors. In type 1, 2, and 3, CD41a/CD61 expression is 5%, 5%20% and above 20%, respectively. In this study, diagnosis of GT was confirmed and subgroups were identified in 32 Turkish patients by flow cytometry analysis. Materials and methods: CD41a/CD61 expression levels in platelet-rich plasma (PRP) obtained from peripheral venous EDTA blood samples were analyzed with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). GT subgroup analysis was performed by counting 50,000 events in the BD FACSDiva Software v6.1.3 program of the instrument. Results: In the present study, in blood samples of 32 patients from 23 families with GT and 22 healthy controls, co-expression levels of CD41a and CD61 in PRP was analyzed. 12 out of 23 families were consistent with type 1 GT (52.2%), 4 were consistent with type 2 GT (17.4%), and 7 were consistent with type 3 GT (30.4%). Conclusion: Especially due to consanguineous marriages, GT with various glycoprotein levels may be detected. As a result of the flow cytometry analysis of the present study with the highest GT patient population in Turkey, type 1 GT patients were the most common subgroup. In the determination of the GT subgroups; especially in the detection of type 3 GT, flow cytometry is the most sensitive glycoprotein analysis method. In addition to light transmission aggregometry, CD41a/CD61 study by flow cytometer confirms diagnosis when mutation analysis cannot be performed.