OBJECTIVETo study the clinical feature and alpha II b beta 3 gene mutations of three Glanzmann thrombasthenia (GT) pedigrees.

METHODSPlatelet counts (BPC), blood film, bleeding time, platelet aggregation and flow cytometry were used for phenotype diagnosis of all the patients. All the exons of alpha II b and beta 3 genes were amplified by polymerase chain reaction (PCR) and direct sequencing was performed for mutational screening. One hundred and three healthy blood donors were as normal controls.

RESULTSThree probands showed normal BPC, defective platelets aggregation, prolonged bleeding time and significantly reduced platelet aggregation to ADP, epinephrine, and collagen, while relatively normal aggregation to ristocetin. Flow cytometry showed platelet surface expressed alpha II b beta 3 was strongly reduced in proband 1 and proband 3 and mildly reduced in the amount of surface expressed alpha II b beta 3 (63%) in proband 2. Sequencing results showed that proband 1 had a G10A homozygous mutation in alpha II b, and a G1412T homozygous mutation in beta3. Compound heterozygous mutations in beta3, G1199A and 1525delC were identified in proband 2. No mutations in alpha II b beta 3 gene were identified in proband 3.

CONCLUSIONSCompound homozygous mutations, GI0A in alpha II b and G1412T in beta3, lead to GT in proband 1. Compound heterozygous mutations in beta3, G1199A and 1525delC, lead to GT in proband 2. The mutations of G10A, G1412T and 1525delC were reported for the first time in GT patients.

Exons ; genetics ; Female ; Humans ; Male ; Mutation ; Pedigree ; Platelet Membrane Glycoprotein IIb ; genetics ; Thrombasthenia ; genetics

BACKGROUNDGlanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by the tendency to hemorrhage and the inability of platelets to aggregate in response to agonists. GT is caused by a defect of the platelet glycoprotein IIb/IIIa complex. The objective of this study was to describe the clinical features and the genetic cause of GT in a 6-year-old girl from south China.

METHODSA three-generation family was studied. The proband patient aged 6 years and her parents undertook examinations of platelet counts, blood film, bleeding time, platelet aggregation, and flow cytometry. All coding exons of the ITGA2B and ITGB3 genes were amplified by polymerase chain reaction (PCR), and direct sequencing was performed for mutational screening on the patient and normal controls consisted of 52 healthy blood donors. Reverse transcription PCR was conducted to test for exon skipping.

RESULTSThe proposita patient showed dispersing platelets, prolonged bleeding time, and severely reduced platelet aggregation in response to the physiological agonists adenosine diphosphate (ADP), epinephrine, collagen, and ristocetin. Flow cytometric measurements showed that the contents of alphaIIb and beta3 were significantly decreased. Sequencing results demonstrated two different types of heterozygous mutations existed in the alphaIIb gene (c.2930delG and IVS15-1delG). The compound mutations were also confirmed in the patient's mother and father separately.

CONCLUSIONSThe alphaIIbbeta3 deficiency of the proband was caused by two compound ITGA2B mutations, which were first reported in Chinese GT patients. The IVS15-1delG was first confirmed to cause an exon skipping.

Asian Continental Ancestry Group ; Child ; Female ; Flow Cytometry ; Heterozygote ; Humans ; Integrin alpha2 ; genetics ; Integrin beta3 ; genetics ; Mutation ; Pedigree ; Reverse Transcriptase Polymerase Chain Reaction ; Thrombasthenia ; genetics ; metabolism ; pathology

OBJECTIVETo explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation.

METHODSAll exons and exon-intron boundaries of alpha II b and beta3 gene were amplified by PCR and analyzed by direct DNA sequencing. Gene polymorphisms were excluded by direct DNA sequencing. Alpha II b L721R and Q860X mutants expressing vectors were constructed by in vitro site-directed mutagenesis. The expression of alpha II b L721R and Q860X mutants on transfected cell membrane were analyzed by flow cytometry and the whole expression level was confirmed by Western blot. The subcellular localizations of alpha II b L721R and Q860X mutants were determined by immunofluorescent confocal scanning microscopy.

RESULTSThe alpha II b compound heterozygous mutations, T2255G (L721R) and C2671T (Q860X), were identified in the proband, the former being inherited from the maternal side and the latter the paternal side. The 293T cells cotransfected with mutated alpha II b L721R and wild-type beta3 expression plasmids expressed 2.1% of normal amount of alpha II b on the cell surface as shown by FACS, in contrast to 31.9% of normal amount of alpha II b on the cells cotransfected with cDNAs of mutated alpha II b Q860X and wildtype beta3 expression plasmids. Western blot of the cell lysates showed no detectable mature alpha II b in cells lysates with L721R mutant. While, truncated alpha II b protein was detected in cell lystes with Q860X mutant. Immunofluorescence studies demonstrated that both L721R and Q860X mutant pro-alpha II bbeta33 complex colocalized in endoplasmic reticulum, but a little in Golgi.

CONCLUSIONSThe L721R and Q860X mutations of alpha II b prevent transport of the pro-alpha II bbeta3 complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression. The impaired alpha II bbeta3 transport is responsible for the thrombasthenia.

Animals ; CHO Cells ; Child, Preschool ; Cricetinae ; Cricetulus ; Female ; Genetic Vectors ; Heterozygote ; Humans ; Integrin alpha2beta1 ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutation ; Thrombasthenia ; genetics ; Transfection

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.
Dependovirus ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Platelet Glycoprotein GPIIb-IIIa Complex ; biosynthesis ; genetics ; Platelet Membrane Glycoprotein IIb ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thrombasthenia ; metabolism ; therapy

OBJECTIVE: To explore the molecular mechanism of Glanzmann thrombasthenia (GT). METHODS: All 45 exons of alphaIIb and beta3 subunit genes as well as their splicing sites were amplified by polymerase chain reaction(PCR) with 40 primer pairs, and then the PCR products were used to screen the gene mutation by single strand conformation polymorphism-polyacrylamide gel electrophoresis (SSCP-PAGE). The mutation was further confirmed by direct DNA sequencing. RESULTS: A DNA band alterated migration was detected after SSCP-PAGE. DNA sequencing showed that a base deletion within the band at the site of 540 in GPIIb gene(540A) was found. CONCLUSION The frame-shift mutation caused by the deletion of 540A in GPIIb gene is a novel mutation which is a genetic defect in patients with GT.
Base Sequence ; Child, Preschool ; Exons ; genetics ; Frameshift Mutation ; genetics ; Gene Deletion ; Humans ; Integrin beta3 ; genetics ; Male ; Molecular Sequence Data ; Platelet Membrane Glycoprotein IIb ; genetics ; Sequence Analysis, DNA ; Thrombasthenia ; genetics

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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 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.
Animals ; CRISPR-Cas Systems ; Codon, Nonsense ; Disease Models, Animal ; Fibrinogen/genetics* ; Humans ; Integrin alpha2/genetics* ; Mice ; Oligonucleotides ; Platelet Glycoprotein GPIIb-IIIa Complex/genetics* ; RNA, Guide ; Thrombasthenia/genetics* ; Thrombin/genetics*

OBJECTIVEMutation screening was performed in a pedigree of Glanzmann's thrombasthenia (GT) and prenatal diagnosis was performed.

METHODSIn this study, reverse transcription-PCR-sequencing and PCR-sequencing, as well as restriction fragment length polymorphism(RFLP) and A/T-cloned-sequencing, were used to screen the ITGA2B and ITGB3 mutation in a pedigree with Glanzmann's thrombasthenia in the RNA and DNA level. Prenatal diagnosis was performed for this pedigree.

RESULTSDeletion of 99 bps was found in the cDNA of the patient in the pedigree, leading to deletion of 33 codons (from codon 160 to 192). After genomic analysis, the patient was found to be a compound heterozygote of c.374C to G mutation and intron 4(IVS-4) + 5 G to C mutation. The two mutations were inherited from the parents. IVS-4 + 5 G to C mutation was a point mutation in the splice site, while c.374C to G mutation was out of the splice site. But both of them resulted in the same splice pattern in RNA. The two mutations were novel mutations which have not been reported in Human Gene Mutation Database (HGMD) and the mutation data base of Glanzmann's thrombasthenia. The results of ITGB3 gene screening is normal in the proband and his parents.

CONCLUSIONTwo novel mutation, c.374C to G and IVS-4 + 5 G to C were found in this study, which might be the cause of GT in the pedigree.

Base Sequence ; Child, Preschool ; Female ; Gene Order ; Genetic Testing ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Mutation ; genetics ; Pedigree ; Platelet Glycoprotein GPIIb-IIIa Complex ; chemistry ; genetics ; Pregnancy ; Prenatal Diagnosis ; Protein Conformation ; Thrombasthenia ; diagnosis ; genetics